BIOPHYSICAL

CHEMISTRY
PRINCIPLES AND TECHNIQUES

BIOPHYSICAL
CHE vIISTRY
PRINCIPLES AND TECHNIQUES
AVINASH UPADHYAY, M.Sc., Ph.D.
Department of Biochemistry,
Hislop College,
Nagpur (M.S.)
KAKOLI UPADHYAY, M.Sc., Ph.D.
Department of Biochemistry,
Lady Amritabal Dga College, Shankarnagar,
Nagpur (M.S.)
"
NIRMALENDU NATH, M.Sc., Ph.D.
Retired Professor,
Department of Blchemistr,
Nagpur University, LIT Premises,
Nagpur (M.S.)
%Iimalaya PublishingHouse
MUMBAI

DELHI 0 NAGPUR

BANGALORE

HYDERABAD

Nag u r
Bangalore
AUTHORS
No part ofthis book shall be reproduced, reprinted or translated for any purpose
whatsoever withou
prior permission of the publisher in writing.
First Edition
: 1993
Second Revised Edit/on
: 1997
Reprint
:
lkprkt
:
2000
Rprlnt
:
00.
eprint
:
Third Revised Edition
:
2002 (May)
Repr/nt
:
2003
Published by
:
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for HIMALAYA PUBLISHING HOUi,
"Ramdoot, Dr.Bhalerao Marg, Girgaon,
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PROF. CHANDRA NTH

PIONEER OF IDIAN BIOCHEM/STRY

PREFACE TO THE THIRD REVISED EDITION

One of the problems facing the writers of textbooks in fast moving subjects is t
he
obsolescence of old information and sprouting of new knowledge. Luckily, for us,
even
with tremendous growth in biochemistry, the basic techniques of study have remai
ned
unaltered. Yet, variations and improvi.ations of the old techniques have been pe
rfected
since the book was last revised. The present resion is an attempt to take into a
ccount
these changes.
The Achilles' heel of the previous edition was the chapter on centrifugation. Th
is
was brought to our notice time and again by students and teachers of the subject
from
diverse corners of the nation. We are happy to state that the chapter has been t
horoughly
revised, almost rewritten, and the scope of the discussion has been greatly expa
nded.
Another aspect that has been completely revised is infrared spectrometry. Again
it
was brought to our eyes by people who have taken critical notice of the book. Th
e current
discussion of the aspect is again much wider in its scope and care has been take
n to
give several examples of its use in biochemistry and allied sciences.
New techniques such as fluorescence energy transfer, fluorescence polarization,
non-radioactive labeling, etc. have been added. Also, radioimmunoassay has been
discussed more extensively along with a good discussion of its variant,
radioimmunometry.
A sore point with the previous editions was the lack of anindex. This shortcom,
g
has been addressed and the book now has a detailed index.
The above are the major additions. Numerous small changes in virtually all the
will become visible to the teachers of the subject who have seen the first two
editions. New problems have also been added at the end of the chapters.
Late in the day, when we had already submitted the revised draft for publication
,
we received the UGC syllabi for Biochemistry, Microbiology, Botany, Zoology,
Biotec.hnology, and otherallied life science subjects We were pleasantly surpris
ed to
draftnot only covered all major points but had more to offer leaving
in the syllabi. The. current edition will be of good use to students
any life science subject.

In its revised form, we feel confident that the book win be much more useful to
all
concerned.
We would like to thank the great number of teachers and students who have praise
d
our book; they provided us the support that eyery author nxis so much. We are eq
ually
to those who criticized the book for providing the motivation to revise it.

ACKNOWLEDGEMENTS
A book of this expanse does not bcome possible without contribution of
several willing souls. We have been lucky that several colleagues and students
helped us in our endeavour in whichever way they could, sometimes even
going out of the way to do it.
We would like to thank Prof. H. F. Daginawala, former Head of the
Department of Biochemistry, Nagpur University, for his constant
encouragement and help. We are also highly indebted to Dr. N. V. Shastri,
Head of the Department of Biochemistry, Nagpur University, for his
suggestions regarding the changes to be made in the revised edition. The
fact that he taught this very subject to two of the authors (AU & KU) has
!.everything to do with the writing of the textbook.
. Heartfelt thanks are due to Ms. Ragi Radhakrishnan, one of our
,cherished students, for designing the cover for the 3rd edition and for
going through much of the 2rid edition in search of mistakes, typographical
otherwise.
Dr. Rajnish Kaushik and Dr. Shibani Mitra Kaushik, St. Louis, Missouri,
out of the way to provide us as much current literature as they could
of the fact that both of them were laden with their own research work.
cannot really express our feeling of gratitude towards them.
We are indebted to Dr. Saraswati Sukumar (Johns Hop.kins), and Dr.
Runnebaum, The Salk Institute, La Jolla, USA, who provided us
of much of the recent literature.
Thanks are also due to several of our colleagues, friends and students
Dr. Irfan Rahman, Dr. Ashish Bhelwa, Dr. Saibal Biswas, Dr. S.N.
Ms.
Sadhana Naidu, Ms, Ramfla Bhojwani, Dr. Raymond Andrew, Dr.
Deshpande, Mr. Amol Amin, Mr. S. Wankhede, Dr. Shyam Biswal,
Ms. Anjali Gadkari.
We record our appreciation of Mrs. Harsha Dave who was involved in
,g the Ist edition.
Finally, we would like to thank Shri Gokul Pandey and Shri D. P.
of Himalaya Publishing House for their constant help and valuable

1 - 65
66- 74
75 - 99

CONTENTS
ACIDS AND BASES
Electrolytic Dissociation and Electrolytes -- Ionization: Basis of
Acidity and Basicity w Bronsted-Lowry Theory: Acid is a Proton
Donor, Base is a Proton Acceptor -- Strength of Acids and Bases -Acid-Base Equilibria in Water -- Function and Structure of
Biomolecules is pH Dependent w Measurement of pH: Use of
Indicators m Electrometric Determination of pH -- Buffers: Systems
which Resist Changes in pH -- Titrations: The Interaction of an
Acid with a Base.
ION SPECIFIC ELECTRODES
Ion Selective Electrodes Measure the Activity of Metal Long m Glass
Membrane Electrodes m Solid-State Ion Exchanger Electrodes Solid-State Crystal E
lectrodes Liquid-Membrane Electrodes w Gas-Sensing Electrodes.
3.
THE COLLOIDAL PHENOMENA
Classification of Colloids -- Properties of Colloids -- Donnan
Equilibrium. . .
DIFFUSION AND OSMOSIS
A Molecular-Kinetic approach to Diffusion m Methods of
Determination of Diffusion Coefficient Significance of Diffusion Coefficient w D
iffusion of Electrolytes -- Diffusion of Water Across
Membranes: Osmosis Measurement of Osmotic Pressure Van't HoiTs Laws of Osmotic P
ressure --Theories of Osmotic Pressure
and Semipermeability m Osmotic Behaviour of Cells m Molecular
Weight Determination from Osmotic Pressure Measurements -Significance of Osmosis in Biology.
VISCOSITY
Factors Affecting Viscosity Measurement of Viscosity Applications of Viscornetry

Significance of Viscosity in Biological Systems.

I00- 121
122- 144

SURFACE TENSION
Factors Affecting Surface Tension w Measurement of Surface
Tension.
145-156

ADSORFtlON
Kinds of Adsorption Interactions -- Adsorption Characteristics -Molecular Orientation Adsorption Isotherms: Quantitative
157- 174

Relationships -- Adsorption from Solutions n The Importance of

Adsorption Phenomena.
8.
SPECTROPHOTOMETRY
Basic Principles -- The Laws of Absorption n Significance of
Extinction Coefficient (Box) Problems (Box) -- Preparation of Standard Graph (Bo
x) Deviations From Beer's Law -- Absorption Spectrum -- Why is Absorption Spectr
um Specific For A Substance?
-- The Chromophore Concept -- Instrumentation For UV-Visible
And Infrared Sprectrophotometry -- Radiant Energy Sources -Wavelength Selectors -- Detection Devices -- Amplification And
Readout Double Beam Operation -- Double wavelength Spectrophotometer Application
s of UV-Visible Spectrophotometry Qualitative Aalysis -- How to Interpret Absorp
tion Spectra of Biological Macromolecules (Box) -- Quantitative Analysis Enzyme
Assay n Molecular Weight Determination -- Study of Cis-trans
Isomerism Other Physicochemical Studies -- Control of Purification Difference Sp
ectrophotometry Turbidimetry and Nephelometry -- Theory and Applications of Infr
ared
Spectrophotometry -- Calculation ofVibrational Frequencies -Modes of Vibration Infrared Spectra of Common Functional Groups -- The Carbon Sk
eleton -- Carbonyl Group -- Hydroxy
Compounds Nitrogen Compounds -- Infrared Spectrophotometer: Mode of Operation Sa
mpling Techniques -- Applications of Infrared Spectrophotometry -- Disadvantages
of Infrared
Spectrophotometry -- Spectro.fluorimetry Structural Factors Which
give Rise to Fluorescence -- Fluorescence and Phosphorescence (Box)
-- Fluorometry: Theory and Instrumentation -- Applications -Fluorescence Spectra and Study of Protein Structure -- Extrinsic
Fluorescence -- Fluorescence Energy Transfer -- Fluorescence
Polarization n Luminometry -- Flame Spectrophotometry -Instrumentation for Emission Flame Photometry Instrumentation for Atomic Absorpt
ion Spectrophotometry -- Atomic Fluorescence
-- Nuclear Magnetic Resonance Spectrophotometry -- Magnetic
Properties of the Nucleus -- Nuclear Resonance Chemical Shifts: Position of Sign
als '-- Hyperfine Splitting -- Instrumentation Applications Electron Spin Resona
nce Spectrometry n Applications -- Spin Labeling -- Mossbauer Spectrophotometry
-Applications Some Solved Problems
9.
OTHER OPTICAL TECHNIQUES FOR MOLEC
CHARACTERIZATION
Circular Dlchrolsm and Optical Rotatory Dispersion -- Rotational
Diffusion -- Flow Bircfringence D E1ectrlc Birefringence D
Polarization of Fluorescence -- Light Scatterlng-- X-ray Diffraction.
175-270
271-300

12.

10. CENTRIFUGATION
Basic Principles of Centrlfugatlon m Relative Centrifugal Force (RCF)
-- Other Factors Affecting Sedimentation -- Instrumentation -Desktop Centrifuge -- High Speed.Centrifuge The Ultracentrifuge
-- Analytical Ultracentrifuge Fixed-angle Rotors -- Vertical-tube rotors Swingin
g-bucket Rotors n Wall Effects Preparative Centrifugation -- Differential Centri
fugation -- Density Gradient
Centrifugation -- Rate Zonal Centrifugation -- Isopycnic
Centrifugation Gradient Materials Preparation of Density Gradients --Choice of R
otors Centrifugation in Zonal Rotors --Centrifugation Analytical Basic Principle
s of Centrifugation --Factors Affecting Sedimentation Velocity -- Sedimentation
Coefficient
-- Factors Affecting Standard Sedimentation Coefficient Measurement of Sedimenta
tion Coefficient Concentration Distribution -- Applications Of Boundary Sediment
ation -- Band
Sedimentation Determination of Molecular Weights
301-343

II. CHROMATOGRAPHY
344-421

Survey of Chromatographic Procedures -- Techniques of Chromatography

-- i. Plane Chromatography -- A. Paper Chromatography
B. Thin-Layer Chromatography 2. Column Chromatography --Types of Chromatography
-- 1. Chromatography 2.
Partition Chromatography A. Liquid-Liquid Chromatography B. Gas-Liquid Chromatog
raphy (GLC} -- 3. Gel Permeation
Chromatography 4. Ion Exchange Chromatography 5. Affinity
Chromatography High Performance Liquid Chromatography Some Specialized Technique
s -- Hydroxyapatite Chromatography
-- An Affinity System for Base Dependent Fractionation of DNA -An Affinity System for Fractionating supercoiled and NonSupercoiled DNA -- DNA-Cellulose Chromatography.

ELECTROPHORESIS
422-478

Migration of an Ion in an Electric Field m Factors Affecting

Electrophoretic Mobility -- Types of Electrophoresis 1. Free Electrophoresis 2.
Zone Electrophoresls. General Techniques of Zone
Electrophoresis -- 1. Paper Electrophoresis 2. Cellulose Acetate
Electrophoresis 3. Gel Electrophoresis. Specialized Electrophoretic
Techniques I. Discontinuous (Disc) Gel Electrophoresis 2. Gradient Electrophores
is 3. High Voltage Electrophoresis (H.V.E.)
4. Isoelectric Focussing 5. Two-Dimensional Gel Electrophoresis
6. Immunoelectrophoresis 7. Pulse-Field Gel Electrophoresis
8. Electrophoresis on Cellular Gels. Electrophoresis in Genetic
Analysis 1. Restriction Mapping. 2. Southern Transfer. 3. Gel Retardation or Ban
d Shift Assay. 4. DNA Sequencing. 5. DNA Foot
printLng. -

14.
13.
ISOTOPES IN BIOLOGY
,Radioactive Decay =- Production of Isotopes -- Synthesis of Labeled
Compounds -- Interaction of Radioactivity with Matter m
Measurement of Radioactivity -- 1. Methods Based Upon Gas
Ionization --A. Ionization Chambers B. Proportional Counters
C. Fundamentals of Geiger Counters 2. Photographic Methods
3. Methods Based Upon Excitation w A. Liquid Scintillation
Counting Use of Stable Isotopes in Biology -- The TracerTechnique
-- Use of Isotopes as Tracers in Biological Sciences -- Some
Information About Commonly Used Isotopes -- Safety Aspects -Dosimetry.
CERTAIN PHYSICOCHEMICAL TECHNIQUES USEFUL
IN BIOCHEMISTRY
Polymerase Chain Reaction -- Enzyme-Linked Immunosorbent
Assay (ELISA) -- Flow Cytometry.
479 - 54,
546 - 56!

-- APPENDICES
-- INDEX
567593 - 60:

1
ACIDS AND BASES
A history of the quest to understand the molecular basis of acid - base properti
es ,m.akes
for a very amusing reading. For instance, in 1773 Doctor Samuel Jhonson averred
that acids
iare composed of pointed particles which affect the taste in a sharp and piercin
g manner".
iAnother attempt to explain the nature of acids was made by Lavoisier when he pr
oposed that
i
the characteristic behaviour of acids was due to the presence of oxygen. Stimula
ted by this
observation, Sir Humphrey Davy went to great lengths to show that hydrochloric a
cid also
contalns oxygen. He, of course, failed in his attempt thereby disproving the the
ory of Lavoisier.
Even the later history of acid - base research is not without its share of amuse
ment, albeit in
:a manner different to the above described instances. In 1884 Svante August Arrh
enius in his
dissertation proposed the theory of electrolytic dissociation and ionization on
which
current understanding of acid - base character is based. The doctoral dissertati
on was,
greeted by the lowest possible pass-mark by the University of Uppsala, Sweden. F
or
, Arrhenius was awarded Nobel Prize in Chemistry in 1903.
ELECTROLYTIC DISSOCIATION AND ELECTROLYTES
Let us consider a simple experiment. A pair
1.1.
F.Jcperlmental system for determining
electrical conductivity of a solutWn. The
bulb does not light when there is a nonbulb l@hts when the beaker cohtalns
eteces n
of electrodes is connected in series to a light bulb
and to a source of electricity (Figure 1.1). As long
as the electrodes hang separated in the air, no
electric current flows through the circuit, and the
bulb does not light. If however, the two electrodes
are touched to each other, the circuit is completed
and the bulb lights. If the electrodes are dipped
into a beaker containing water purified by
repeated distillations, the bulb does not light. This
tella us that water is not a good conductor of
electricity and is not capable of completing the

circuit. If we dissolve an acid, a base, or a salt in

water in which the electrodes are dipped, the bulb
lights up. Obviously, these substances are able
to carry the current and thereby complete the
circuit. Substances produc/ng solutions capable
of conductlng electric'.tty are called electrolytes. On
the other hand, substances producin9 solutions
incapable of conducting electricity are known as
non-electrolytes. Table 1.1 provides a few
examples of electrolytes and non-electrolytes.

2
Bophs. Chemis
What is the mechanism by which, electrolytes conduct electricity? Arrhenius' the
ory proving
an answer. The theory proposes that acids, bases, and salts undergo dissociation
in water
varying degrees, each molecule giving rise to oppositely charged long. For examp
le, if gase
hydrogen chloride is bubbled into water, virtually all the hydrogen chloride mol
ecules re.
with .water (Figure1.2) giving rise to a hydronium ion (positively charged) and
a chloride i
{negatively charged}. These long can now be carried to the cathode and the anode
respectiv
thereby completing the circuit. This theory of Arrhenius is known as the theory
of electrol3
dissociation.

Water
Hydrogen
Chloride
Collision 'Complex'
Hydronium Ion
Chloride Io:

Figure 1.2. When gaseous hydrogen chloride is bubbled in water, HCI mo/ecu/es co
llide with water molecu
Collisions of sufficient energy and proper orientation produce hydronlum long an
d chloride long.
Going back to the experiment we discussed, a diligent observer would note that c
erta
substances cause the bulb to be brightly lit, whereas other substances cause the
bulb to
only dimly lit. This experimental observation permits us to subdivide the electr
olytes into groups. Substances that dissociate almost completely and produce sol
utions that are very go
conductors of electricity are known as strong electrolytes; substances which dis
sociate only part
and produce solutions which are poor conductors of electricity are known as weak
electroly
The difference between strong and weak electrolytes was attributed by him to a d
ifference in t
degree of ionization.
IONIZATION : BASIS OF ACIDITY AND BASIClTY

Arrhenius Theory : H Ion is the Acid, OH- Ion is the bae

Fom the experiment that we have discussed above, one can safely conclude that ac
i
base reactions are a function of ionization p-nciple. Thus, based on ionization
princip
nhenius defined acids and bases. These definitions are elaborated below.
Acls : Acids were described by Arrhenlus as compounds containing hydrogen will
upon addition to water become ionized to yield 14+ long. Nitric acid (14N03), wh
ich is a solut
strong electrolyte or srong ac [Le,, it dissociates completely in water to produ
ce t4+ long), m
be cited as an example.
HNO3 H + NO
Nitrous acid (HNO2) , a weak electrolyte {Le., dissociates only partially to pro
duce H+ iont
may be cited as an example of a weak acid.
HNO2 # H+ +NO.
(A single arrow ----> denotes reactions that go completely to
the right; a double arrow x--- denot
re.actions that go only partially to the right).

3
Acids and Bases
Table 1.1 Examples of Electrolytes and Nonelectrolytes
Strong Electrolytes

Hydrochloric acid, HCI [H+ + Cl-] .

Nitric acid, HNOa [H+ + NO]
Sulfuric acid, H2SO4 [H+ + HSO]
Sodium hydroxide, NaOH [Na+ + OH-]
Potassium chloride, KCI [K+ + CI-]
Silver nitrate, AgNO3, [Ag+ + NO ]
Sodium chloride, NaCI [Na + CI-]
Copper fib sulphate, CuSO4 [Cu2+ + SO-]

Weak Electrolytes
Nonelectrolytes

Acetic acid CH3COOH [CHaCOOH]

Lactic acid, CHaCHOHCOOH [CHaCHOHCOOH]
Ammonia, NH3 [NHa]
Hydrogen sulphide, H2S [H2S]
Mercury (II} chloride, HgC12 [HgCI2 ]
Glucose C6H1206 [C6H1206 ]
Sucrose C12H22011 [C12H22011 ]
Ethyl alcohol, C2HsOH [C2H5OH ]
Methyl alcohol, CH3OH [CH3OH ]
Acetone CH3COCH3 [CHaCOCHa ]

Species in parentheses are predominant in solution. The difference between weak

and
nonelectrolyte is that weak electrolytes dissociate very lltfle (not shown in th
e table) whereas the
nonelectrolytes do not dissociate at all.
Bases : According to the Arrhenius definition, bases are compounds which upon io
nization
in water yield OH- (hydroxide) long. Sodium hydroxide, which dissociates complet
ely to produce
OH- long, may be cited as an example.
NaOH Na+ + OHThe Arrhenius concept is important in that it has provided us with the first mec
hanistic
approach to acid - base behaviour and has been instrumental for the development
of more
sophisticated theories. There are, however, two major shortcomings in the Arrhen
ius model.
"- (0 In the AIThenius model the acid-base reactions are limited to aqueous solu
tions (this
is not a problem as far as biological systems are concerned since all reactions
must
take place in aqueous solutions).
(//)
The theory limits bases to hydroxide compounds. This is very unsatisfact
ory because
it is well known that many organic compounds which are not hydroxides, for examp
le
ammonia, show basic properties in their chemistry.
In the year 1923, two more theories defining acid-base character were proposed.
The first
theory, Bronsted and Lowry theory, is very satisfactory for understanding physio
logical processes
and will therefore form the basis of all further discussions. The second theory,
proposed by
G. N. Lewis is much more general than the Bronsted - Lowry concept. A brief disc
ussion of this
theory is given in Box 1.1.

4
Biophysical Chemistr
Bronsted - Lowry Theory :' Acid is a Proton Donor, Base is a Proton Acceptor
This theory defines an acid as any compound that yields protons (H+ long) and a
base
as any compound that combines with a proton. In other words, acids are proton do
nors and
bases are proton acceptors. It should be noted that as-far as acids arc concerne
d, Arrhenius
and Bronsted - Lowry theories are similar ; in both cases acids give off H+ long
. However, the
concept of a base is much broader in the Bronsted theory, hydroxyl ion being jus
t one of the
possible bases. Cited below are a few examples which will illustrate the point m
uch better.

general equation
H2SO4
H+ HSO
HC1
H+ "
+
C1HsPO4
H+ [
+
HuPO
CHaCOOH
H+[
+
CHaCOOHCOa
H+ [
+
HCO
HCO

H+ I
+
CO -

HsO+
H+{
+
H.O
HA
H+ I
+
A-

Concept of conjugate ac/d and conjugate base : Each of the compounds listed abov
e as acid,
pon ionization, produces. H+ long. Their ionization also produces long or molecu
les which can
ombine with a proton (HSO , Cl-, H2PO , CHsCOO-, etc). According to the definiti
on, these
which can combine with a proton are bases: Thus, we can say that every acid diss
ociates to a proton and a base (if the reaction is reversed, a base can combine
with a proton to
an acid}. The Bronsted -Lowry theory thus conceives of an acid base 'pair'. An acid
its corresponding base are said to be 'conjugate', i.e., 'joined in a pair'. Thu
s, CI- is the
of HCI, likewise H20 is the conjugate base of H30+.
An acid is a proton donor. Its strength would depend upon the ease with which it
can
a proton. An acid will yield a proton with comparative ease if its conjugate bas

e is weak.
HCI as an example. Its conjugate base, CI-, is a weak base; it is not a very goo
d
In solutions, therefore, HCI is completely ionized to produce H and CI-. HCI IS
i strong acid because/ts conjugate base/s wea/ Let us consider another example,
that of
Its conjugate base CH COO- is stronger base compared to CI-. The acetate ion,
binds the proton much more tenaciously with the result that in solution acetic a
cid is
' ionized. CHCOOH Is a weak ac/d because/ts conjugate base Is strong. Similar co
ncepts
e drawn for bases also and their strength would epend upon the strength of their
conjugate
The Bronsted Lowry theory gives us the following reciprocal relations :
-- ff an acid is strong, its conjugate base is weak:
-- if an acid is weak, its conjugate base is strong.
-- if a base is strong, its conjugate acid is weak.
if a base is weak, its conjugate acid is strong.
Concept of an a/ka/i : In the previous pages NaOH was regarded as an Arrhenius b
ase
ionized to produce OH- long. NaOH, however, is not a Bronsted base because, as a
it has little ability to accept a proton. NaOH can act as a base solely because
upon
it gives rise to OH- long which are very good proton acceptors. NaOH and other
hydroxides like KOH, therefore act as bases by proxy. Such compounds, under the
theory, are known as alkalies.

6
Biophysical Chemisl
Amphoteric substances : Substances which can behave both as an acid and as a bas
e
referred to as amphoteric. Thus, under the Bronsted concept, liquid ammonia qual
ifies as
acid
NH3 NH + H+
and as a base too
-.
NH3 + H+ NH
Similar is the case with water which behaves as an acid
HOH x- H + OHand as a base
HOH + H+ --- I-IO*
Sa/ts : Under this tleory salts are thought to be compounds which are formed by
replaci
the ionizable hydrogen with a metal ion or with any other positively charged gro
up. Thu
CH3COONa is the sodium salt of CH-COOH formed by replacement of the proton by th
e N
ion. KCI is a salt of HCI formed by replacement of the proton by K+ ion.
CH3CO0 CH3CO0
Acid
Ionizable
Salt
Metal
Hydrogen
STRENGTH OF ACIDS AND BASES
(Throughout the dlscussiori, acids will be treated as examples. However, the dis
cussic
applies equally well to bases, albeit, in a reverse manner).
In a preceding section we have said that the strength of an acid depends upon th
e strength
weakness of its conjugate base. This, however, is not the only determinant of st
rength. Apm
from strength of conjugate base, the strength of an acid depends upon (i) the ba
sic strength
the solvent, and (ii) the dielectric constant of the solvent. Both these factors
are discusse

below.
The Basic Strength of the Solvent
So far we have been writing the ionization reaction of HCI as
HCI .--x H+ +
and the general ionization reaction of acids as
HA --- H++A
It is, however, well known that H+ long do not exist in acid solutions. This is
because th
H+ long combine with the solvent molecules to give rise to 'lyonlum long'. Let u
s illustrate th
caseby considering a specific example, that of water, as a solvent. In water, th
e H long {formec
due to ionization of an acid) are known to combine with water molecules to give
rise to H30+
the. hydronlum long (also known as the oxonium or hydroxonium ionsl.
H++H20 ---- H30+

iAds m Boes 7
Recall that Bronsted - Lowry concept states that a base is a proton acceptor, Th
us water
the above cae (and solvents in general} is acting as a base.
We can now rewrite the general lonition reaction of an acid In water
HA+H20H30+A- '
The strerth of the acid, HA, now is a function of the competition between the tw
o bases,
, and H20 to accept the Ionlzable hydrogen.
Coe I : A= is strorer than H20, In th-is cae A- Is a stronger base and bind to t
he
bI hydrogen much more tenaciously than H20. A a consequence, the dissociation of
acld, HA, will be less and it not be a stror acld in water.
Case 2 : A- is weaker than H-O. In this case, once the acid is dissolved in wate
r, A= Mill 10se
ionlzable hydrogen to water Zhich is a stronger base. The dissociation of the ac
id, HA, Mill
and the acid may even be completely dissociated. The acid, HA, will be a strong
in water.
We can now generalize the above observations./f the basic strength of the solven
t is less
base, the ac will be v,,,eak in that solvent. If the basic strength
greater tbn that of the conjugate base, the ackl will be strong in that solvent.
To drive the point home, let us consider the strength of the same acid in two so
lvents.
Case
I : Acetic acid in water. The acetate ion is a stronger base than water.
Therefore,
is a weak acid in water.
0
O
CH3-C-OH+H-OH CH--C--O:+H30+ "
Case
2 : Acetic acid in liquid ammonia. Acetate ion is a weaker base as compa
red to
Therefore, acetic acid which was a weak acid in water, is a strong acid in liqui
d
O
O

CH3-C-O-H+NH3 CH3-C-O-+NH
The above examples show the relative nature of the designations strong and weak.
The
that an acid is strong does not convey much sense unless we know in relation to
directlon of proton transfer and its extent depend upon these relative proton donating proton-bindlng abilities of the potential acids and the solvent. It can
thus be said that the
ran acid s always relative to.the basic strength of the solvent used.
Constant of the Solvent
Upon ionization the acid splits into two oppositely charged long, H+ and A-. The
se long
attract each other and recombine. However, solvents of high dielectric constant
greatly
attraction between oppositely charged partlcles dissolved in them. This action o
f the
acid and consequently is important for the strength of acid.
acid in a solvent of high dielectric constant Mill dissociate greatly and will t
herefore be
The same acid, in a solvent which has a low dielectric constant, will not dissoc
iate much

+ H+
8
B/ophysicat Chemts
and will consequently be weak.Water is a solvent which has a very high dielectri
c eonstan'
room temperature, almost 80. On the other hand, petroleum ether has a very low d
ielee
constant, just 2.2. A given acid can therefore dissociate to a much greater exte
nt in water tt
in petroleum ether. The dielectric constant is thus of great importance in deter
mining
stre.ngth of an acld.
Effectof Structure on the Strength of -cids
It is a commonly accepted fact that carboxylic acids are stronger than other org
anic aci
Why is that so? The reason usually given is that the carboxylate anion (the conj
ugate ba
formed upon dissociation is stabilized by resonance (two equivalent resonance st
ructures]
such a manner that it is more stable than the original acid molecule.
o
l
R--C//
u" +
R--C
\oH
Resonance stabilized anion
On the other hand, in the alkoxide ion, TO-, the negative charge is not delocali
zed an(
concentrated on the single oxygen atom. This anion, therefore, is not as stable
as the resonar
stabilized carboxylate anion. The resonance stabilization promotes dissociation
in the carboxy
acids making them stronger in relation to the organic acids where lack of resona
nce stabfllzati
decreases dissociation.
If resonance stabilization were the only factor all carboxylic acids would have
had t
same strength. This is not so. Carboxylic acids which contain strong electron at
tracting grou
(halogens) on the alpha - carbon are stronger than the unsubstituted acids. On t
he other ha
carboxylic acids bearing electron releasing groups (methyl) on the alpha - carbo

n atom a
weaker than the unsubstituted acids. These electrostatic factors, in which elect
rons are eith
attracted to or repelled from one atom or group of atoms with respect to another
are known
inductive effects. Electron attracting groups withdraw electrons from the carbox
ylate grou
This weakens the oxygen - hydrogen bond thereby facilitating ionization and rele
ase of a proto
Moreover, these groups also help stabilization of the conjugate base by resonanc
e.
C1
0
CI
O
(I) CI-----C<---- C
Cl<---- C <------ C
C1
O <--- H
CI
O

Acids and Bases

9
Inductive effects are additive and increase with the number of substitutions by
electron
withdrawing or electron releasing groups. These effects also are sensitive to di
stance. Thus,
substitution by a halogen on the beta carbon of a carboxylic acid is not as effe
ctive as one on
the alpha - carbon.
What Do We Mean by 'Strength of an Acid'?
So far we have not reviewed this term critically. We, however, have been using t
he term
loosely to convey in essence the H ion concentration [H+] . Thus, when we said th
at HCI is a
strong acid, what we meant was that HCI ionizes t give a high [H]. When we said th
at CH,COOH
is a weak acid, we meant that CH3COOH ionizes to only a little extent giving a l
ow [H+]. AIIhough
this is the way we are going to use this term subsequently in this chapter, we m
ight as well
understand its actual meaning.
The concept ofactW/ty : The long in soluUon, are separated from one another by s
hielding
layers of solvent and thus have little attraction for each other. If, however, w
e increase the
concentration of the solution, the intervening distances between different long
start decreasing.
In a dilute solution, the long move about freely without the hindrance of attrac
tive forces from
oppositely charged long. In a concentrated solution, however, the long can not m
ove freely
because they are closer to each other and therefore are affected by oppositely c
harged long. The
long then assume a certain degree of orientation. Each ion is surrounded by an '
ion atmosphere'
of opposite charge which reduces its movement. Thus the effective concentration
of the long is
sllghfly less than its absolute concentration. This effective concentration is k
nown bya better
term, activity. Thus, in true sense, the strength of an acid is a measure of the
activity of H long
and not of its concentration. It may be said that the activity and concentration
of H long might
be identical in dilute solutions. It is only in the concentrated solutions that
they start to differ.
Activity coejc/ent : Activity is a measure of the effective concentration of the
solutes in
The activities might be related to the absolute concentrations by a proportional

ity
called activity coefficient. The symbol used for activity coefficient is . The eq
uation for
the relationship is as follows
a=C
a is the activity and C the concentration. Units of both a and C are moles per l
itre. The
r coefficient approaches unity at infinite dilution.
Does Not Reflect The Strength of an Acid
We know that HCI is a considerably stronger acid as compared to CH-COOH. HCI ion
izes
'and almost all the hydrogen of HCl is present as H+ at any point of tim. HCI al
so conducts
current much better than CHsCOOH. The two acids, however, have similar titration
25 ml of 0.01 N NaOH are required to fully tltrate 25 ml of 0.01 N HCI. The same
of 0.0 IN NaOH is required to fully titrate 25 m. of 0.0 IN CH3COOH. Both acids
give
titration response because of the following scheme of events. Acetic acid is wea
kly
1.3% of its hydrogen is present as H+. When we add alkali to this solution, the
long liberated from it combine with the H present and remove them in the form of
water.
CHsCOOH --- CH3CO0- + H+

NaOH Na +
OHH20

in which two reactants A and B interact to form two products, C and D. Note that
th
reaction is reversible. According to the law of mass action the rate of the reac
tion to the righ
will depend upon the molar concentrations of A and B (throughout the discussion
we assum
that the solution is dilute and thus activity is equal to concentration) Thus
I 0
Bophyslcal Chemls
To achieve equilibrium with respect to dissociation, more acetic acid molecules
dissocia
to give rise to another 1.3 % H*. These too are removed as water in the manner d
escribed abov
The process continues till all acetic acid has ionized to give up protons which
get removed =
water. A similar process takes place with HCI also
' HC1 C1- +

H+

+ ,
NaOH -------> Na +
OHHO
The two acids therefore end up giving similar titration profiles. The acidity me
asured titration is known as the total or the titratable acidity and reflects th
e concentration of an a
in solution. It does not, however, reflect the strength of an acid or its actual
acidity.
ACID-BASE EQUILIBRIA IN WATER
The free hydronium ion concentration, [H3 O]' doininates chemical reactions In ph
ysiologic
systems. Since all physiological fluids are aqueous based, the concentration of
hydronium for
may determine the extent to which the reaction proceeds, the rate at which it go
es, or tt
detailed mechanism of how it takes place in such solutions. For example, in both
, living and t
vitro physiological systems, specific enzyme activity is often quite dependent o
n the effectiv
concentration of the hydronium ion.
Adjusting and controlling the free hydronium ion concentration is a necessity in
an
biochemical experiment. To understand the complex equilibria which are always pr
esent in acid - base system is therefore of paramount importance.

The Law of Mass Action

The law of mass action, evolved mainly by Guldberg and Waage, states that the ra
te of
chemical reaction at a given time is proportional to the active masses of reacti
ng substances prese
at that time. The active mass for molecules is essentially equal to their molar
concentration
However, for long, as one would recall, the active mass means the effective conc
entration or t13
activity (which might be equal to molar concentration in dilute solutions).
Let us consider the reaction
A+Bx--C+D
Vr a [Al. IBI
where [A] and [B] are expressions of molar concentrations of A and B, and V is t
h
Apart from the molecular concentration, the'chemical afllfities
reaction velocity to the right,
r
the reactants should also be taken into account. The chemical affinities are con
stant at a give:
temperature and other reaction conditions. In the above equation, therefore, we
might introduc
a proportionality constant which corrects for the particular chemical affinity.

10
Bophgsical Chemistry
To achieve equilibrium with respqqt to dissociation, more acetic acid molecules
dissociatl
to give rise to another 1.3 % H+. These too are removed as water in the manner d
escribed above
The process continues till all acetic acid has ionized to give up protons which
get removed a
water. A stmflar process takes place with HC] also
'HC1
C1- +

Na+
OH-

H*
NaOH
+

HeO
The two acids therefore end up giving similar titration profiles. The acidity me
asured b]
titration is known as the total or the titratable acidity and reflects the conce
ntration of an acia
in solution. It does not, however, reflect the strength of an acid or its actual
acidity.
ACID-I-.,Bi .,UILRIA WATER
The free hydronium ion concentration, [H30], dominates chemical reactions in phys
iological systems. Since all physiological fluids are aqueous based, the concent
ration of hydronium long
may determine the extent to which the reaction proceeds, the rate at which it go
es, or the
detailed mechanism of how it takes place in such solutions. For example, in both
. living and h
vitro physiological systclns, specific enzyme activity is often quite dependent
on the effective
concentration of the hydronium ion.
Adjusting and controlling the free hydronlum ion concentration is a necessity in
any
biochemical experiment. To understand the complex equilibria which are always pr
esent in an
acid - base system is therefore of paramount importance.
The I,w of Ma ,tion
The law of mass action, evolved mainly by Guldberg and Waage, states that the ra
te of a

chemical reaclon at a 9hen time s proportlonoJ to the active masses of reactj su

bstances present
at that time. The active mass for molecules is essentially equal to their molar
concentrations.
However, for long. as one would recall, the active mass means the effective conc
entration or the
activity (which might be equal to molar concentration in dilute solutions).
Let us consider the reaction
A+B-C+D
in which two reactants A and B interact to form two products, C and D. Note that
the
reaction is reversible. According to the law of mass action the rate of the reac
tion to the right
will depend upon the molar concentrations of A and B (throughout the discussion
we assume
that the solution is dilute and thus activity is equal to concentration} Thus
V a [A]. [BI
r
where [A] and [B] are expressions of molar concentrations of A and B, and V is t
he
reaction velocity to the right. Apart from the molecular concentration, thechemi
cal aiIties of
the reactants should also be taken into account. The chemical affinities are con
stant at a given
temperature and other reaction conditions. In the above equation, therefore, we
might introduce
a proportionality constant which corrects for the particular chemical affinity.

AddsandBases
11
Thus
vr = K1 IA). IB]
Since the reaction is reversible, the products C and D will react to give A and
B. Writing
expression for the velocity of the reaction to the leR
V! = K2 It). [D] ....
At equilibrium, the rate of the reaction to the right and that to the left will
be equal. So
that
and / [A]. [BI = K2 [Cl. [D]
[C].[D] KI
[A].tB] K,. =
where ,.is the equilibrium constant and is an expression of the chemical affinit
ies of
obvious from the above equilibrium equation that if K is large the
the right predominates, which means that affinity between A and B is higher than
that at equilibrium the concentration of C and D is higher than that of A and B.
when Kis small.
The equilibrium equation may be stated in words : at equilibrium the product of
the
of the substances formed in a chemical reaction divided by the product of
of the reactants in that reaction is a constant referred to as the equilibrium
/v We may stress again that the activities of the reacting species will give the
and not the molar concentrations which are used only for the sake of simplicity.
The law of chemical equilibrium may be applied to virtually all reversible react
ions and
including the ionization of acids and bases.
of Water
As per the collision theory, it is expected that water molecules constantly coll
ide with
water molecules. It may further be expected that at any instant a minute fractio
n
collisions might give rise to the following change:

This reaction is better illustrated in Figure 1.3.

Biophysical Chemistry
Figure 1.3. A collision between two water molecules can result n the formation o
f a hydronlwn ion and a hydrox
ion. The collision should be of su.clent energy and proper orlentation ,
It is obvious from the above equation that for every single hydronium ion formed
, a hydros.
ion is also produced. Thus ionization of water forms these two long in equal num
bers therek
ensuring that pure water is essentially neutral. The dissociation of water has b
een confirme
by electrical conductivity experiments. These experiments also tell us that at e
quilibrium a
small percentage of water molecules becomes ionized; actually Just slightly more
tha
10-7%. This means that water is almost a nonelectrolyte. At higher temperatures
the number
collisions between water molecules will be higher producing a sllghtly higher nu
mber hydronium long. But the water will stay essentially neutral because an equa
lly higher numb
of hydroxyl long will also form.
The Equilibrium Constant and Ionization Constant of Water
We have seen that water has only a very slight tendency to ionize, However, the
produc
of ionization, H30+ and OH- have very profound biological effects. It is therefo
re necessary th
we express the extent of ionization of water quantitatively.
We can represent the ionization of water simply as
H--OH H++OH(although we have said before that H long do not exist as such and the correct re
presentatic
would be H,O, we can think that hydronium ion is the hydrated form of H and take t
he liber
to express tas H for the sake of simplicity). The equilibrium constant of such a
reactic
according to the law of mass action would be
-]
=
We have seen that water has a very slight tendency to ionize. This means that t!
.. concentration of water should be virtually unchanged by ionization. T
he concentration of wat.
per lit-re in pure water is equal to the number of grams of H20 in 1 L d
ivided by the gra

molecular weight, Le. 1000/18 = 55.5

2
M or 0.55 x 10 M. Substituting this value in
equilibrium constant expression we get

13
[H+] [OH-]
From electrical conductivity measurements of water thevalue of Keq has been calc
ulated
il very carefully and has been,found to be 1.8.-x 10-16 at 25C. Substituting this
value for K in
the above equation, we get
[H+l [OH-]
',
1.8 x 10-16 55.5
" Rean'angg
(55.5)
(1.8 x 10-Ie = [H*] [OH-]
99.9 x 10-18 = [H+] [OH-]
1.0 x I0-14 ffi [H*] [OH-]
The product of the equilibrium constant of water He. and concentration of water,
55.5,
is taken to be constant, is known as ion7,atlon constant or the dssocfation cons
tant or
ion product of water and is symbolically denoted as K Thus,
/ = 1.0 x I0=14 = [HI [OH-] at 25"C,
The above equation is substantially true for water and for dilute aqueous soluti
ons. In
olutions the product of hydrogen ion concentration and hydroxyl ion concentratio
n is
constant value 10-I,4 (at 25"C) whether the solution is acidic, basic or neutral
. Value of Kw
widely with temperature as shown in Table 1.2. When concentrations of [H+] and [
OH-]
as in pure water, the solution is said to be neutraL. Under such conditions,
of the value of Kw allows us to calculate the concentration of H+ and OH-.
I I0-14-- [I-gl [OH-]
I X 10-14-- [H+]2
solving for H+, we get
[H+I = I x 10-7

[H+]= [OH-] = 1 x 10-TM

Ionization Constant of Water at Var/ous Temperatures
Temperature ('C}
Ionization Constant (K
I0
2.92
x
10-s
18
0.8
x
10-14
24
1.0

10-14
25
1.2
X
I0-14
30
1.47

10-14
37 (Normal body temperature)
3,13
x
10-14
Thus when a solution is neutral, the concentrations of H and OH- are both 10-7 M.
On
ther hand, ff the solution is acidic, the concentration of H+ would be higher th
an 10-7 and

I 4 Bphysmd Chem
that of OI-I- would be less than 10-7 M. Furthermore, when the solution is basic
, the concen
of H should be less than 10-7 M while the concentration of OH- would be higher th
an
Thus, the ionization constant of water, Kw, is of great help to calculate the co
ncentration
ff the concentration of OH- is known, and vice versa.
The relationship [H*] [OH-] = Kw = 1 x 10"4 helps in elcution of [H*] if[OH-] is
known and
Vke versa. The following examples demonstrate it.
(1) Calculate the [H+] of the solution which is 0,01 Nfor NaOH at 24=C,
Ans. NaOH is a strong alkali and by definition dissociates fully. Thus a solutio
n which is 0.01 N
with respect to NaOH is also 0.01 Nwith respectto OH-. Therefore [OH-] of the so
lution is 0.01
g tool per litre or 1 x 10" g tool per litre. Putting this value into the equati
on
[H*] [OH"] = K=, = 1 x 10-4
we get
[H+].I X 10-= = I x 10"4
Therefore
0-14
IH* =

= 1 x 1 Og tool per Iltre

1
X
1
12

lx10-=
(2) Calculate the [OH'] of the solution which is 0.001 N for HCI at 24'C.
Ans. HCI is a strong acid and by definition dissociates fully
respect to HCI is also 0.001 Nwith repect to H+. Therefore, [H+]of the solution
is 0.001
litre or I x 10"a g tool per lltre. The [OH-] of this solution will be

I
OH- = lx10"
lx10'a
=1x10"11 g rnol per litre

(3) Calculate the [OH-] for each of the following

neutral. The temperature is 24'C.

(/) [H+] = 1 x 10"e mole/litre.

(ii) [H+] = 4 x i0-e mole/litm.
(iii) [H7 = 2.5 x 10"mole/litre.
(iv) [H+] = 2 x 10-2mole/litre.
Arts. (i) lx10-s mole/litre. (ii) 2.5x10-s
(iv) 5 x 10-13 mole litre.
.
mole/litre. (ill) 4x 10-e

Simple Way of Denoting H+ and OH- Concentrations: The Concept of pH

Because of the importance of trace concentrations of hydrogen and hydroxyl long,
scientist
routinely have to make hundreds and thousands of measurements. The manipulation
of sucl

and Bases

15

wkward figures as negative exponents (e.g., 10-7) or even their decimal equivale
nts (0.000000 I)
cumbersome and tedious. As a matter of simple convenience, the chemists, chiefly
Sorensen, ng ago devised a shortcut. This shortcut is thepH scale which is a co
nvenient tool to designate
the actual concentration of H (and therefore of OH-) in any aqueous solution in t
he range of
acidity between 1.0 MH and 1.0 M OH-. Mathematically, the term pH is def'med by t
he equation
Let us see how convenient is the pH scale. We know that the hydrogen ion concent
ration
n a neutral solution at 25 is 1 10-7 M. The pH of this solution would be given by
I
pH = log-lx10-7
= log (1 x 107) = log 1.0 + log 107
=0+7
pH= 7
Thus the cumbersome figure of neutrality, hydrogen ion concentration of 10-7 M,
is
into the simple pH value of 7. Solutions which are acidic will have pH values le
ss
7, and conversely, the solutions which are alkaline will have pH values larger t
han 7.
ion product of water (1 x 10-), forms the basis for the pH scale, the scale rang
es from
to 14 (Table 1.8).
1.3 The pH Scale
pH
[H*], M
pOH
[OH-], M
0
1
2
3
4

5
6
7
8
9
10
11
12
13
14
1.0
I0-l
10-2
10-3
io-4
10-5
10-6
10-7
10-8
10-9
i0-0
i0-I
10-12
10-13
i0-14
14
13
12
Ii
i0

9
8
7
6
5
4
3
2
l
0
I0-4
10-13
10-12
10-t
10-10
10-9
10-8
10-7
10-6
10-5
10-4
10-3
102
i0-I
1.0

It is necessary to understand that the pH scale is logarithmic and not arithmati

c. Thus,
is said that two solutions differ from each other by 1 pH unit, it ineans that o
ne solution
I0 times the hydrogen ion concdntration of the other. Thus, vinegar (pH 3.0) has

H
approximately 10,000 times greater than that of blood (pH 7.4). Table 1.4 lists
values of some important and commonly used aqueous fluids.

16
Table 1.4
Place of Various Materials in the pH Scale
Biophysical Chem

Mater/al
pH value
Household bleach
Household ammonia
Baking soda
Sea Water
Egg white
Hepatic duct bile
Intestinal Juice
Pancreatic Juice
Blood (human}
Tears (human)
Cerebrospinal fluid
Saliva
Urine
Milk
Kupffer cells (intracellular)
Black coffee
Beer
Tomato Juice (ripe)
Orange juice
Vinegar
Cola
Lemon Juice
Pure gastric juice

12.7
12.0
9.0
8.0
8.0
7.4 7.5 7.5 7.357.4

8.5
8.0
8.0
7.45

7.4
6.354.8 6.6 6.4 5.0

6.85
7.5
6,9
6.5

4.2 - 4.9
4.3
2.6 - 4.3
3.0
3.0
2.0
0.9

If we take the negative logarithm of the equation

Kw = [H+] JOWl = 10-4
we get
log IHI - log IOH-] = - log Kw = 14
but, - log [H] = pH, slmflarly - log [OH-] = pOH, and - log Kw = pKw
Thus
pH + pOH = pKw (at 24"C)
Sometimes the expression pOH is used to denote basicity (OH- concentration) of a
giv
solution.
It is important to note that the pH scale is applicable accurately only to solut

ions
ordinm-y temperature (approximately 24"C) where the value for pKw is 14. Only at
this temperatu
pH of neutral solutions will be 7.
Measurement of pH is of utmost importance to biologists in general and to bioche
mists
particular. This is so since pH determines not only the activity of blomolecules
such as enzyme
but may also be important for the stability of their structures. Moreover, measu
rement ofpH blood and urine can give us important diagnostic information.

It must be emphasized that the actual meaning of pH is the negative log of hydro
gen ion
r and not the hydrogen ion concentration. However, in dilute solutions with whic
h we
[ deal, activity is essentially equal to the concentration.
Measurement of pH of an aqueous solution can be-performed by using such indicato
r dyes
Isee later) as phenolphthalein, phenol red, litmus, etc. Accuratemeasurements, h
owever, require
specially glass electrodes which are very accurate (see later).
of Weak Acids
A biologist is more concerned with the behaviour of weak acids which are not com
pletely
when dissolved in water. Weak acids (and weak bases) occur commonly in biologlca
l
are responsible for metabolic regulation.
The law of mass action can be applied to formulate equilibrium equations for the
dissociation
If weak acids are given the genera/formula HA, their dissociation equation can
as

18
Bophystcal Chem
According to the law of mass action the equilibrium expression for this dissocia
tion
be written as "
where Ka is the dsn cot or the nan t ofwe acid. A hler vue
obviously means higher degree of ionization. Thus, lactic acid with a Ka of I.
I 0 is much more iod aeflc acid wch has a vue of I. 74 x I 0-s. s aut0mafl prodes
e oaon at lacc acid is a songer acid as comped to acec acid.
dlssociaon constt us deles e tendency of y acid, , to lose its proton.
A discussed eer in the secon on pH, it is cumbersome to hdle negave eo
vues. ese vues c be better hdl if e conveed to e negative log us,
og = p
fle mng use of p, it should be remembered at s vue would be less stronger ad d m
ore for a weber acid. us, lactic acid wch is songer acetic has a p vue of 3.86 a
s comped to 4.76 of acetic acid. Table 1.5 ts e d pv of some coon we acids.
Acid
pKa
Phosphoric acid (H3PO4)
Formic acid (HCOOH)
Lactic acid (CHaCHOHCOOH)
Acetic acid (CH^COOH)
Propionic acid CHaCH2COOH)
Carbonic acid (H2CO3]
Ammonium ion (NH )
7.25 x 10-3
1.78 x 10-4
1.38 x 10-4
1.74 x 10-3
1.35 x 10-s
7.9 x 10-7
5.62 x 10-1
2.14
3.75
3.86
4.76
4.87

6.1
9.25

We have so far considered the dissociation of monobasic acids only. However, the
re
certain polybasic acids, like H2C03, H3PO4 etc., whose dissociation should also
be conside
Polybasic acids dissociate in stages and an equilibrium expression for each stag
e, involvi
dissociation constant for each stae, may be written. We will take H2CO3 as an ex
ample. '
dissociation of H2CO3 takes place as follows:
H2CO3 H + HCO
2HCO xH+ + CO 3
We can write equilibrium expressions for each of the two stages
-- =K2-- 6.31 x 10-11
where K and K are the first and the second dissociation constant of the acid. Th
e dissociat
i
2
of the first hydrogen ion from H2CO3 IS opposed by the force of attraction of it
s linkage to I

Rgds and Bases

19.
molecule. The dissociation of the second proton is more difficult. It is held no
t only by the
primary union with the molecule but also by the attraction of the negative charg
e left on the
molecule by the dissociation of the first proton. The value of K2 is therefore l
ess than KI.
The dissociation constant of a weak acid may be employed to calculate the pH of
its
01ution of a known concentration. The procedure for such calculation is enumerat
ed below.
For the dissociation of a weak acid HA
HA : H+ + A
flae equilibrium expression may be written as
H+ A-]
le encentrations of H A- would be equal as they are formed In equal amounts.
v.erefore, [H+ 12 = Ka [HA] or [H+ ] = -/-a [HA]
We know that weak acids are only slightly ionized. Thus, we can safely assume th
at not
!note than 1% exists as H+ and A-. One can therefore assume that the concentrati
on of the
imdissociated acid, HA, is equal to the normality of the acid. This value can th
en be substituted
the above equation and the hydrogen ion concentration can be calculated. The val
ue of
.[!ydrogen ion concentration so calculated will be approximate as we have not co
rrected it for
slight dissociation which the weak acid has undergone.

20
1
Biolhysal Chemis
Ionization of Strong Acids
Strong acids (and bases) dissociate completely or almost completely in dilute aq
ue
solutions (HCI, H2S04). Thus, a 0.1 N solution of HCI is essentially 0.1 N in hy
drogen
coneentration also. It iS therefore nonsense to write equflibrlum equations and
equilibri,
constants for their dissociation. On the other hand, the higher number of long p
roduced by
strong acids promotes interactior between these long and ttlerefore, the activit
y of strong ac
might be less than the hydrogen ion concentration produced by ionization. Th pH
of str
acids of a given concentration is therefore slightly differerit than what is pre
dicted by th
concentration.
Hydro13is of lts ,
.. There are numerous salts which, from an Inspect.ion of their chemical formula
e, cannot i
any possible way provide either hydrogen long or hydroxyl long in water, Yet, in
solution, many
of these salts test either acld/c or basic: Thus sodium, acetate, CHsCOONa, test
s basic when-ll

1,4,
and Bases

21

. relydissolved in water. Obviously sodium acetate solution has more OH- long th
an it has H30 + .
, ammonium chloride, NH4CI, tests acidic in water which means that this solution
has
3 long than itlhas OH-. Inspection of the formulae of these salts tells us that th
ey can
jt podkibly provide H30* or OH" ions..What then is the mechanism by which extra
OH- or
tr. a H3O long are being produced in such salt solutions?
H' To .answer this qqestion let us find out which long will be present i
n a solution of these
lalts tn water. First let us consider sodium acetate: Upon dissolution i
n water, sodium acetate
mpletely dissociates, into the Na+ !ons and acetate, CH3COO-, long. Apa
from these two
S, some H,O* and OH- will also be present through dissociation of water.
This inspection
us that t.e only way sodium acetate can test basic is by decreasing the
HsO+ concentration
thus relatively increasing the oH- concentration, Let us find out whetle
r any of the two
COO" and Na, produced by sodium acetate, have the ability to combine with
protons
effectively reduce their concentration. Can Na+ long bind H30+ ? Obvious
ly no. Can
* long combine with OH, long ? No. Because NaOH is a strong base (Ikali)
, and by definition
ium long readily relgase OH- long. The answer, thus, does not lie in Na+
long. If now we
nsider CH3COO- long, we will see that this ion is derived from a weak ac
id, the acetic acid. We
.ve already seen that weak acids are weak because their conjugate bases
are strong and bind
th protons rather tenaciously reducing the extent of dissociation. The s
trong conjugate base,
ion in this cse, can. therefore bind with protons (H3O+) and effectively

remove them
solution leaving an excess of OH- long. This makes a so/llum acetate sol
ution test basic
1.4). Is i that all salts of weak acids and strong bases (sodium acetate
is an example)
basic in soltion? Yes, by definition all of them have strong conjugate b
ases which can
protons iom solution. We therefore can generalize the situation. Sa/ts o
f weak acids
bases test basic when dissolved in water.
.O
Long from the sal : Na CH3CwO-

Long from water:

H
OH' Tenden'y to combine
No posslbility of Interaction

Hydrolysis of sodgun acetate (salt of strong base and weak acid). Na and OH" do n
ot have a tendency
to combffe because they are derWed from strong alkali. CHo CO0-, however, has a
tendency to combine
w[tn H* of water becaus [t s a base covate to a weak ad. Th depletes long of wat
er wh{le OH"
rerna unchared. The olution #:Lcomes alkal{ne. (HO shown as H'for the sake of stl
ctty)

22
Biophysico2 Cherr
The same train of logic can be adapted to find out what will happen when
salts of st
acid and weak base {Figure 1.5}, and salts of strong acid and strong base {Figur
e 1.6}
dissolved in water. We can generalize these situations also and state that (0 sa
lts of strong c
and.weak bases test acidic when dissolved in water; and (il) salts of strong aci
ds and strong I
test neutral when dissolved in water.
Long from salt : NH
CILong from salt : Na
CI-

Long from water

Long from water ;
OHH
OH-

Figure 1.5. Hydrolysis of ammonium chloride (salt of

strong acid and weak base). Cl- and I-I* have
no tendency to combine. NH on the other
hand can combine with OH- long. This
depletes OH- tons of water while-14+ tons
remain constant. The solution becomes acidic
Figure 1.6. Hydrolysis of sodium chloride (salt of s
acid and strong base). As evldent fror
figure, none of the salt lons has any tenc
to combine with either l-I* or OH-.pro
by tonlzatton of water. The solution re
neutral.

If any ion from the salt interacts with water in such a manner as to change its
pH, hydro:
is said to occur.

The Effect of Salts Upon the Dissociation of Acids

Let us see what happens when a salt of a weak acid is mixed with the weak acl
solution. Experimental results with such mixed solutions tell us that the pH of
such solui
increases as compared to the pH when only the acid was present. This obviously m
eans
the addition of salt to acid solution has decreased the dissociation of the acid
. If we appl)
same principles which we considered in the above section, we can provide an answ
er for
decrease in dissociation. Let us consider the solution of acetic acid and its sa
lt, sodium ace
as an example. From our discussion above we know that sodium acetate is complete
ly dissoci
in solution, whereas acetic acid is only weakly dissociated. We, therefore, have
the follo
dissociation equations :
,
CH3COOH CH3COO- + H+
CH3COONa ---) CH3COO- + Na+
We have seen that acetic acid is a weak acid and dissociates very little. This i
s becaus
conjugate base, CH3COO', is very strong and binds protons tenaciously. Since sod
ium ace
dissociates completely, by adding this salt we are further increasing the concen
tration o!
conjugate base, /.e., the acetate ion. These extra long then combine with the sm
all numb
protons dissociated from acetic acid. Thus, the H ion concentration gets reduced
and the
increases. We can therefore say that the pH of a solution of weak acid and its s
alt is determ
-by the ratio of salt to acid in the solution. The higher the salt concentration
, the higher the
Table 1.6 elaborates the effect of changing salt to acid ratio on the pH of salt
-acid solution

23
tdds and Bases
sble 1.6 Effect ofChanging Salt/Acid Ratio on the pH of Salt-Acid Solution
Sodium Acetate
Acetic Acid
Rio
pH
(Molar)
(Normal)
Salt/Acid

0.05
0.10
0.15
0.20
0.2
0.2
0.2
0.2
0.2
0.00
0.25
0.50
' 0.75
1.0
2.7
4.6
4.4
4.6
4.7

The same set of principles discussed above apply in the case of solutions of wea
k hydroxides
d their salts also. We might cite the example of NH OH and NH4CI, where the diss

ociations
4
NH4OH NH + OHNH4CI --> NH + ClUe NH4OH is only partially dissociated, NHCI dissociates completely, The extra N
H long
ue to the dissociation of NH CI depress the dissociation of NH.OH thereby decrea
sing OHcentration
and thus a drop in pH restflts. The higher the sal concentration the lower the
BUFFERS : SYSTEMS WHICH RESIST CHANGES IN pH
Solutions which contain both weak acids and their salts are known as buffer solu
tions Ooy
same logic, solutions containing weak bases and their salts are also buffer solu
tions) because
[]/have the capacity to resist changes in pH when confronted with either an acid
or a base.
..]he principle behind this resistance of pH by buffers remains the same as desc
ribed in the
section. However, we will consider it once again in a more explicit manner. Let
us
a system of acetic acid and sodium acetate. From the discussion in the previous
know that sodium acetate dissociates fully and acetic acid dissociates only a li
ttle.
solution therefore contains undissociated acetic acid molecules, CHsCOOH, acetat
e long,
and Na+ long. Let us now see what happens when an acid or a base is added to thi
s
When an acid (HCI) is added :
CH3COO- + H+ + CI- ---> CH3COOH + CIIn solution HCI dissociates completely to produce hydrogen long and chloride lon
g. The
could have decreased the pH, combine with the strong conjugate base
are thus removed. The pH of the solution does not decrease appreciably (it fails
to the change in ratio of salt to acid in solution).
When an alkali (NaOH) is added :
CH3COOH + Na + OH- ---> CH3COO- + Na + H20
The strong alkali, NaOH, dissociates completely into its constituent long Na + a
nd OH-.
have increased the pH, bUt in the buffer solution they react with CH3COOH to giv
e
to water and acetate long. The pH does not increase appreciably (it increases on
ly in

to the change in the ratio of acid to salt in the solution).

pH = pKa - log
24
Biophysical Chemlstr
To what extent can a buffer solution resist change in pH ? A simple example will
be citec
If 10 ml of 0.1 N HCI is added to 990 ml of pure,water (pH 7.0), the pH of water
drops 4 unil
and becomes 3. Similarly, if 10 ml of 0.1 N NaOH is added to 990 ml of pure wate
r, the p]
increases by 4 points and becomes 11. However, if 10 ml f 0.1 N HCI is added to
990 ml of
buffer consisting 0.1 N acetic acid and 0.1 M sodium acetate (pH 4.76), the drop
in pH is on]
0.01 points. The pH changes merely to 4.75. Similarly, addition of 10 ml of 0.1
NNaOH to 99
ml of above buffer solution elicits a rise of merely 0.01 units on the pH scale.
The pH become
4.77. We thus see that buffer solutions resist chariges in pH to a very signific
ant extent (we wi
consider the same example quantitatively a little later).
We have seen that the conjugate base provided by salt dissociation is actually i
nvolved i
the buffering action. The metal long (like Na in sodium acetate) are not involved
. We shoul
therefore rewrite the definition of buffer solutions. Buffers are mixtures of we
ak acids and the
conjugate bases.
The Henderson-Hssselbslch Equation
Henderson-Hasselbalch equation is important for understanding buffer action and
aci
base balance in the blood and tissues of the mammalian system. The equation is d
erived in following way. Let us denote a weak acid by the general formula HA, an
d its salt by the genen
formula BA (B being the metal ion and A- being the conjugate base). The salt diss
ociate
completely, while the weak acid dissociates only partly. We can write the equtli
brium reactior
for the dissociation of HA and BA in the buffer solution as follows :
HA -I-I + ABA -B+ + AWe will soon find that Henderson-Hasselbalch equation is simply another way or w
ritir
the expression for the dissociation constant of a weak acid.
Solving for {H], we get

Taking the negative logarithm of both sides, the equation becomes

However, - log [H+] = pH, and - log Ka = pKa. Therefore,

or
also
[conjugate base]
or
pH = pKa + log
[acid]
necessary because the values of pK and activities vary
strength. The value of pKa on the basis of activities can be calculaled with the
help of
relationship :
and Bases
25
the negative sign, we invert - log [HA]/[A-] and obtain
pH = pKa + log
This is Henderson-Hasselbalch equation'Now, the weak acid, HA, is only slightly
dissociated
the absence of the salt. Thus very little of the A- long come through the dissoc
iation of
bak acid. On the other hand, we have seen that the salt BA is completely dissoci
ated and gives
Ehlgh concentration of A- long. It can, therefore, be safely assumed that the co
ncentration of
he undissociated acid [HA] is equal to the total acid concentration. We can also
assume that all
." has dissociated from BA and therefore the concentration of the conjugate base
, [A-] is equal
the concentration of the salt, [BA]. Taking into consideration these assumptioBs
, he equation can take many differont forms.
Isalt]
pH = pKa + Iog [acid]
[proton acceptor] "'..
pH = pKa + log [prot.0n donor] . .?w .
As with all the equations considered so far, the
more accurately when concentrations are converted to

pK (activity) = pK (concentration) - 1.018 f

Is the ionic strength of the solution. For most calculations, however, concentra
tions
fairly accurate results. Now, that we have derived an equation which relates pH
to
concentration (conjugate base Concentration) and the weak acid concentration,
;us see the quantitative basis of buffer solutions rslsting a large change in pH
. We have seen
addition of 10 ml 0.1 NHCI to 990 ml of pure water brings its pH down from 7 to
3. Let us
add this acid to 990 ml of 0.1 N acetic acid and 0.1 M sodium acetate
long disgociating from HC1 are neutralized by the acetate long.
CH3COO- +H+
CH3COOH
The addition of HCI therefore lowers the concentration of the acetate ion slight
ly and
the concentration of acetic acid by the same amount. If we assume that all I-I+
long have
neutralized, the drop in acetate ion concentration will be 10-3 mole/litre. The
concentration
acid would rise by the same amount. -

26
0.0999
0.101
pH = pKa + Iog-pKa of acetic acid is 4.76. Therefore,
pH = pKa + log 1.0
pH = pK + 0
pH = pKa
Thus to calculate the pK of any acid one only needs to dissolve that acid and it
s salt equal concentrations and thenaexperimentally determine the pH of the solu
tion. It will be equ to the pK of the acid. Some extremely important problems ab
out buffers which can be solw using Hederson-Hasselbalch equation are provided f
or in Box 1.6.
Henderson-Hasselbalch equation makes it clear that the pH of a buffer solution d
epen{
upon the pKa of the acid and upon the salt to acid concentration ratio. The lowe
r the pK oft]
acid the lower will be the pH. The buffer pH will increase with increasing salt
concentratio
Again, according to Henderson-Hasselbalch relationship, the actual salt and acid
concentratior
can be varied widely without any change in pH if the ratio between the two is un
ity. Thus,
lactate buffer containing 0.01 M lactate and 0.01 N lactic acid will have the sa
me pH even if fl
buffer is diluted 10 times or even 20 times. In actual cases, however, the pH of
the dilut
buffer increases slightly. This increase is not signii2cant enough.
Biophysical Chemistr
[CH3COO- i
mole
mole
mole
-- 0.1---0.001
= 0.0999-NAL
litre
litre
litre

mole
mole
mole
j[CH3COOH]FINAL = 0.1 -- + 0.001 -- = 01101
litre
litre
litre
Substituting the final salt and acid concentrations in the Henderson-Hasselbalch
equatio
we get
pH = 4.76 + log 0.0999
0.I01
= 4.75
The pH of the buffer solution after addition of I0 ml of 0.1 N HCI changes from
4.76 to 4.751
drop of merely 0.01 units of pH.
Henderson-Hasselbalch equation gives a very important relationship which makes
possible to calculate the pK 0f-any given acid with extreme ease. The relationsh
ip is, that if tt
molecular ratio oLIt to acid is unity in a solution, the pH of that solution wil
l be equal to tt
pK of flleacl tTind "<
: ::- r the ;.
[salt]
!i.: ".
pH = pKa + log [acid]

28
Btophys/cal Chel
Buffer Capacity
By buffer capacity we mean the capacity of the buffer to resist changes in pH. T
he ca
to resist changes in pH depends upon (i) the actual concentrations of salt and a
cid pre
the buffer, and (//) the salt to acid concentration ratio.
' First, let us consider the effect of actual salt and acid concentration on the
buffer ca
Let us add 1 ml of 0.1 N HCI to a lactate buffer solution containing 10 ml of 0.
1 M lactal
10 rnl of 0.1 N lactic acid {pH of this buffer will be equal to the pK- of lacti
c acid, 3.86, sin
ratio of salt to acid is unity). What will be the change in pH of thebuffer solu
tion ? The H
convert 1 ml of the salt to 1 ml of acid. The pH of the solution will therefore
b
9
pH = 3.86 + log = 3.86 + log 9-1og 11
II
= 3.86 + (0.9542 1.0414) = 3.77 (App.)
the change in pH of the buffer solution is therefore 3.86 - 3.76 = 0.09. Thus, o
ne ml of
HCI causes a decrease of about 0.09 pH unit.
Suppose we add 1 ml of 0.1 N HCI to a buffer solution containing I0 ml of 0.025
M I
and I0 ml of 0.025 N lactic acid. What will be the change in pH ? The HCI, in th
is cas
convert 4 ml of salt to acid. The pH of the solution will therefore be
pH = 3.86 + log -6 = 3.86 + log 6-1og 14
14
= 3.86 + (0.7782 - 1.1461) = 3.49
the change in pH of the buffer solution is therefore 3.86 - 3.49 = 0.37. Thus, i
n this c
ml of 0.1 N HCI causes a decrease of about 0.37 pH unit.
The above example tells us that the first buffer has a higher buffer capacity th
a
second. This means that buffers containing higher concentrations of salt and aci
d have a buffer capacity as compared to solutions with lower salt and acid conce
ntratns.

Let us now consider the effect of salt to acid concentration ratio upon the buff
er cap
To understand this, let us consider a lactate buffer composed of 15 ml of 0.1 Ml
actate ano
of 0.1 N lactic acid. The pH of this buffer would be
pH = 3.86 + log "" = 3.86 + (1.176-0.6989) = 4.34
5
Let us now add 1 rnl of 0.1 N HCI to this buffer. The HCI would convert 1 ml of
salt to 1
acid. The pH of the buffer will be
pH= 3.86+ log 14 = 3.86+ (1:1461-0.7782)= 4.23
6
Thus the pH of the buffer is lowered by 0.1 . pH unit. As shown previously, 1 ra
l of 0.1 h
added to a buffer composed of 10 ml of 0.1 M lactate and 10 ml 0.1 Nlactic acid
changes it
by 0.09 pH unit. This example elaborates the effect of salt to acid concentratio
n ratio on b
capacity. The generalized statement based on the above example can be that when
the ra
salt to acid concentration is unity, the buffer has maximum efficiency.

and Bases
29
The buffer range of any given buffer is about 2 pH units. It consists of one pH
unit on
side of the pK of the buffer acid. Thus, lactate buffer should be a good buffer
in the pH
. 2.86 -- 4.86. Iwe increase the concentration of buffer solution, we can also i
ncrease its
to a little extent. The selection of a proper buffer system for a given experime
ntal
a common problem. Some examples arc provided. For the pH range 3 to 4, phthalic
acid phthalate can be used; for the pH range 4-6, acetic acid-sodium acetate
is satisfactory; for the pH range 6 t-0 8, monosodium dihydrogen phosphate (acid
) monohydrogen phosphate (salt) buffer is useful (see Appendix).
How important buffers are for normal functioning of a body can be .understood fr
om the
.that the pH of blood is maintained strictly within the range 7.3 to 7.5. Death
is more or less
pH of 7.0 and above a pH of 7.9. In the laboratory, buffers are used for two mai
n
: (/) as reference standards for pH determination, and (//) to maintain optimum
acid reaction of a medium such as bacteria or tissue culture or an enzymatic rea
ction mixture. discuss more about some important biological buffers in a later s
ection.
PRECAUTIONARY INFORMATION ABOUT COMMONLY USED BUFFERS
As mentioned earlier, it is the pK value that is of utmost importance when decid
ing about
buffer has to be used. However, each buffer has other.chemical characteristics p
eculiar which must be borne in mind. Several buffers may fit the pH range one is
working in.
a few of them may have characteristics that are detrimental to the experimental
This becomes even more important considering the fact that most of the commonly
used
designed for biochemical use. The most common problems that plague .these
inhibition of some enzymes, precipitation of polyvalent cations, toxicity, absor
ption
light, strong effect of concentration and temperature on pH, and lack of good
activity in the most used pH range in biochemistry. A few most commonly used
are discussed below individually.
Buffer
The advantages of phosphate buffers are numerous. They have a high buffe
ring capacity.
Na and K salts are very highly soluble and thus any ratio of Na and K long can be
Because the long are highly charged, high ionic strength can be obtained without
the

molarity.
The last named advantage can become a disadvantage too, It is impossible to prep
are a
buffer with a high buffering capacity and a low ionic strength!
The actual disadvantage of the phosphate buffers are as follows. They ma
y bind polyvalent
2+
+
Chiefly, they bind Ca , and to a lesser extent, Mg2 . More importantly,
phosphate
are known to be toxic to mammalian cells. Another disadvantage isthe lac
k of buffering
in the range 7.5 to 8.0.
They are good buffers between the pH range 12.0 - 12.5.
The principal disadvantages of these buffers result because of relative
insolubility of most
of the sensitivity of pH to temperature chauges. High temperatures
extreme pH changes due to loss of CO2.
The buffering range in which these buffers work well is 10 - 10.8.
Buffer
This buffer is probably the most used in biochemistry and for obvious reasons. C
onsider
; advantages. (1) Since the pKa is , it has a high buffering capacity between 7.
5 and 8.5.

30
Biophysical
(2)Very low toxicity. (3) Does not intei-fere with most biochemical reactions. (
4)
pure forms.
. The disadvantages are as follows: (1) Like carbonate buffers, its
to a very high extent. (2) Like phosphate buffers, it reacts with a few metal lo
ng like
Ni2+, Ag+ etc. (3) It reacts with some glass electrodes and thus may lead to err
oneous
EDTA Buffers
EDTA (ethylenediaminetetraacetate) is not normally used for its buffering. It is
a
chelating agent of divalent cations and is added to other buffers mainly to redu
ce
concentrations of the divalent cations. Thus, one finds that EDTA buffers are us
ed very
when working with nucleic acids; the reason is that Mg2 is a cofactor for nucleas
es
of EDTA therefore abolishes the activity of these enzymes. This is exactly why E
DTA buffer
used when nucleic acids are to be stored. One precaution here. EDTA suffers from
disadvantage of absorbing very highly in the UV range. As such, if nucleic acid
has to be estimated, the concentration of this buffer should be kept yery low (0
.001 M).
Another buffer that suffers from high absorbance in the UV range is the barbitur
ate
Boric Acid and Glycine Buffers
Borate has weak toxicity and glycine, of course, has none. Both these buffers ha
ve a I
UV absorption. Borate is good between pH range 8.7 to 9.7 and glycine between 9.
5 to 10.
Additionally, borate is chosen for work with bacteriophages since it stabilizes
Glycylglyinc Buffer
This is often a buffer of choice for enzymo10gical work and works well in the pH
range
to 8.0. It also has very low UV absorbance. This is another major plus since enz
yme
assays in the UV range will not be impeded. One more great advantage is that it
has
for the divalent cations Ca2+ and Mg2+. These are precisely the cations that are
used very
for enzymological work.
The disadvantage with glycylglycine springs from its being a peptide : it is

proteases and as such cannot be used with these enzymes. Additionally, it cannot
be
crude protein preparations since such preparations may have protease contaminati
on.
Triethanolamine Buffer
This is another favorite for enzymological work. It has all the advantages
glycylglycine. It buffers at the same pH range and it doesn't suffer from the li
mitation
glycylglycine, namely protease susceptibility. Also, it is a volatile buffer and
therefore may
chosen for purification work where the buffer is to be subsequently removed.
The Good Buffers
These buffers are so named after their discoverer, Norman Good. Because of sever
problems with the buffers just discussed, Good looked at a large number of zwitt
erionic buffer
The buffers that he found good lack the drawbacks mentioned above. They are not
toxic, the,
do not absorb appreciably in the UV range, they do not precipitate divalent cati
ons, their pH
not sensitive to temperature changes, and they are quite soluble. These buffers
are given bel
in a tabulated form (Table 1.7). Since they have very long names, they are usual
ly known
their abbreviations. However, their full names are being provided in the table.

31
ethane acid
ADA
6.62
6.2 - 7.2
and Bases
1.7. Good's Buffers
Buffer Abbreviation
pKa
pH range where
(20C)
best used
-2- ACES 6.88 6.4 - 7.4
acid;
acid
iminodlacetic
[(carbamoylmethyl) imino]acid

acid
,lycine
propaneacid
-hydroxyethyl)- I -piperazineacid
ethanesulfonlc

BES
Bicine
CAPS
CHES
HEPES
HEPPS*
MES
MOPS
PIPES
TAPS
7.15
8.35
10.40
9.55
7.55
81o
6.15
7.20
6.80
8.40
6.6 - 7.6
7.8 - 8.8
9.7- 11.1
9.O
7.O
7.6
5.8
6.5
6.4
7.8

10.1
8.O
8.6
6.5
7.9
7.2
8.8

acid
1-bisglycine
TES
Tricine
7.50
8.15
7.0 - 8.0
7.6 - 8.8

known as EPPS.
TITRATIONS THE INTERACTION OF AN ACID WITH A BASE
The old definition of neutralization states that an acid and a base react with e
ach other to
water. The Bronsted-Lowry concept offers a much broader view 3333of the process
of
to this concept, neutralization is a process of proton transfer from an
a base. Neutralization need not result in the formation of a recognizable salt a
nd may
involve water.

32
HA
B
(acid)
(base)
Biophysical Chemistn.
BH+

+
A-

(conjugate
(conjugate
acid)
base)
Although, in the following pages, we shall be considering acid-base interactions
in aqueot
media, the above discussion will help us in identifying the conjugate acid and b
ase produced:
any neutralization process.
Titration is normally used to determine the amount of an acid in a given solutio
n. In th
procedure a known volume of an acid is titrated with a base (usually NaOH) whose
concentrati(
is accurately known. Small aliquots of the base are added till the acid is total
ly neutralized. T,
titration can be followed by adding an indicator to the acid solution or by cont
inuo
measurement of the pH by a pH meter. The concentration of the base and the volum
e requir
for fully neutralizing the acid are sufficient for calculations which will revea
l the concentratic
of the acid in solution.
Titration Curves of Weak Acids
Let us again take the example of acetic acid. Figure 1.7 represents the characte
rist
titration curve of acetic acid when it is titrated against a strong alkali. The
figure traces fl
course of titration of a 0.1 N solution of acetic acid with 0o I N NaOH at 25C.
Before tl
titration is started (i.e. before any NaOH is added), the acetic acid is slightl
y ionized and the I:
of the solution is due to acid alone. When successive aliquots of NaOH are added
, the OH= fro]
dissociation of NaOH will combine with the free H+ in solution to form water. As
soon as the fr
H+ is neutralized by OH- to water, some of the undissociated acetic acid immedia

tely dissociat(
further to satisfy its dissociation constant. Thus with each addition of NaOH, m
ore water
formed and more and more acetic acid gets converted to the acetate anion.
CH3COOH + Na+ + OH- ----) CH3COO- + H20 + Na
As the titration progresses, the concentration of acetate ion increases
continuously am
that of acetic acid decreases. Have we come across this situation before
? Yes. We know th
solutions of weak acids and their conjugate bases are known as buffers.
With the progress
titration, the solution is fast becoming a mixture of the conjugate base
, acetate, and the we
acid, acetic acid. The pH of this solution will now change in accordance
with the Hendersor
Hasselbalch equation, i.e., at any stage of titration, we should be able
to calculate the pH ofth
solution using the Henderson-Hasselbalch relationship. If we plot the pH
values against th
volume of alkali added we get the characteristic curve shown in Figure 1
.7. The titration curve
of all weak acids have similar shape (Figure 1.7). They differ only in t
heir location on the pl
scale. The position of the curve on the pH scale depends upon the pK of
the acid being titrate(
While dealing with the Henderson-Hasselbalch relationship, we havre alre
ady considered th
the pK, of an acid is equal to the pH of the solution containing equal c
oncentrations of both
salt anl the acid. Such a situation will clearly be present at the mid-p
oint of the titration. Th
the pH of the solution when the acid is half titrated represents the pK
of the acid being titrate
(Figure 1.7).
a
The titration curve of a weak acid is usually spread over about 4 pH units. Thus
, for
weak acid whose pK is 5, the titration begins at around pH 3. This acid will be
half titrated pH 5 and will stand c%mpletely titrated at around pH 7. If the pK
of the acid being titrated is the titration begin's at pH 5, is half completed,b
y pH 7, aad is complete at around pH 9. Th
tiI4".ation curves of these two acids will be displaced along the pH scale accor

ding to their respectiv

pkg.

and Bases
pH 7
33

!4
13
12
11
10
9
I
Midpoint
titration
Efficient
: 9-2S
buffering
;
zones:

Acetate

3
2
1
O
0.5
1.0

Equlvalente of OH------1.7.
Characteristic titration curves of weak acids. The midpoints of the titr
a(-ions have been indicated. Also
indicated are the predominant ionic species at the beginning, midpoint and end o
f the titrations. The
buffering zones have been shown.
From Figure 1.7, we note that the titration curves are relatively flat in their
centre sections.
zones are the buffering regions of the acid-conjugate base pair (Figure 1.7). On
the
of these curves one can select the salt acid concentrations that will give a goo
d buffer
One can see that the titration curve assumes greatest degree of flatness at its
pK
the acid to conjugate base concentration ratio is unity. This ratio obviously ha
s the
capacity. This is the proof for what we have already considered mathematically
: the buffer is most efficient in resisting pH changes when the ratio of salt
is unity. Figure 1.7 also shows that at both the. ends the titration curve
sharply. This means that the composition of the acid-conjugate base solution in
these
is not good foi- a buffer. It is obvious that at both the ends the ratio of conj
ugate base to
is far removed from unity.
Titration curves of weak bases follow the same pattern as seen for weak acids, b
ut in a
order as evident from Figure I.8.

34
2
I
of titration
11
I
10
NH4CI
pkw" pkb = 9.26 =
PH 7
r
I
1
10
0 0.5 1.0
------ HCI
Figure 1.8. Characteristic titration curve of weak bases.
Titration of a Strong Acid with a Strong Base

4
pH 3
OI
Acid
0.5 1.0
Equivalents of OH100%
0.1N NaOH Salt

lgure 1.9. Titration of a strong acid with strong base (0.1 N HCI against 0.1 N

NaOH).
Figure 1.9 represents the titration curve of 0.1 N HCI titrated against 0.1 N Na
OH. The
Striking thing about this titration curve {in general for titration curves of al
l strong acids) is the
very sluggish change in pH as successive aliquots of NaOH are added. To change t
he pH by one
unit, almost 80% of the NaOH is required. However, at the later stages the titra
tion curve shows
a sharp break and the pH changes rapidly. Thus the HCI solution has a good buffe
ring capacity
between pHI and 3. As this pH range is seldom used in biology, titration curves
of strong acids
are not so important to a biochemist.
Titration Curves of Polybasic Acids
Let us now consider titration curves of polybasic acids which can donate more th
an one
proton and can consequently possess more than one pK, corresponding to the succe
ssive
dissociation of each of the protons. A good example is affored by phosphoric aci
d, H3PO4, for
which three ionization steps and there corresponding pKa are

and Bases
35
I-13PO4 H2PO +H+, pKa = 2.24
H2PO HPO+ H+. pKa - 7.2
HPO- PO--'+ H+, pKa = 12.4
Thus, in a titration of phosphoric acid the first stage consists of titration of
H3PO to
the second in the titration of H2PO to HPO -, and the third in titration of HPO
: to phosphoric acid the three pK are much separated from each other. The titrat
ion,
therefore, shows a sharp break after each pK and at these regions the buffering
capacity
'the solutions is very poor (Figure 1. I0). However, there are three different p
H zones at which
hosphoric acid system can act as a very good buffer. This example of phosphoric
acid has
r chosen because many biological molecules contain phosphate-related groups.
groups enter into multistep acid-base processes closely analogous to those of ph
osphoric
11:[ orHPO--- PO +Hp/G=12.4 .
8t ,----for H,P4 HPO- + H+p = 7.2
* p 2 24
0
25 -.50 . .75
ml of 0.1N NaOH
Titration of 25 mls of O.1 N H3PO4 solution by 0.1 N NaOH solution, pKa of three
different stages are
shown. Three buffering zones, although not shown, are self evldent.
What happens if the polybasic acid happens to have different pKa values very clo
se to each
Let us answer this question with the help of an example of citric acid. Citric a
cid has
values which are relatively close to each other (pK1 = 3.1, pK2 = 4.7, pK3 = 6.4
). In
, case what will happen is that by the time the first H* is fugy titrated the se
cond H* also
titrating. Likewise, the titration of the second H* is not complete before the t
hird H*
In such cases there are no sharp breaks in the titration curve between successiv
e
and one observes a relatively flat curve throughout. Such systems are well buffe
red over a
range of pH. This is evident from the titration curve of citric acid shown in Fi
gure 1.11. If
acids having pK= values not far away from each other are mixed together, one wou

ld
similar type of curve for such a mixture also. This is what happens in the body.
The
pKa relaUvely close to each other. Their titration curves therefore
thereby enhancing their efficiency in the pH range maintained by the body fluids
.

36
Biophysical Chemt
13
12
II
i0
0
NaOH
Figure I. I I. Titration of citric acid by NaOH equivalent strength. Compare thi
s cun with Fig. I, I O. Citrate ti
gives a characterlstlc jlat curve because of overlapping flrst, second, and thir
d stages of hydrog
dissociation. Citric acid therefore has a large buffering zone.
FUNCTION AND STRUCTURE OF BIOMOLECULES IS pH DEPENDENT
The death of a human being below a blood pH of 7,0 and above a pH of 7.9 is en
testimony for the importance of pH to life in general. Examples may also be cite
d of dea,
tissue cultures and bacterial cultures in inadequately buffered media. It is the
refore a
obvious conclusion that biomolecules are profoundly affected by changes in pH. I
n any c
most of the important conponents of the living cell are acidic, basic, or amphot
eric and
alteration in the pH of the environment profoundly affects their state of ioniza
tion and the:
their conformation and biological activity.
In this section we will deal with pH-dependent properties of proteins and their
buff
blocks, amino acids. We will also discuss in brief the pH-dependent properties o
f o
biomolecules.
Ionization of Amino Acids is pH-Depenflent
All amino acids are amphiprotic compounds and can be denoted by the general forn
R
I
H -- C k NH2
COOH
Their a-amino group is weakly basic and has a pK in the range 9-I0.5.

a
a-carboxyl is acidic with the pK in the range 1.7 to 2.4. All amino acids are th
erefore ioniz
a
an aqueous solution depending on the prevailing pH.
The amino acids which do not possess any dissociable group in the side chain exi
three ionic forms :
R
R
R
H-- C --NH ,r
a.. H--C -- NH r
- H -- C--NH2
COOH
CO0COOCATION
ZWITrERION
ANION

pH=
2
E/iv of O H--curve for alanine.
pKal is for -COOH andpl
is for the a-NH .
At a low pH only the a-amino group is ionized and the amino acid is a cation. If
the pH is
a-carboxyl group starts dissociating. This process leaves a hgative charge on th
e
which already has a positive charge due to the amino group. The charges cancel o
ut
the amino acid possesses no net charge. This state is known as the zwitterionic
state and
; amino acid may be called a zw/tter/on. If the pH is still further raised, the
hydrogen ion from
group dissociates. This leaves only the negative charge on the amino acid due to
the
dissociation and the amino .acid behaves as an anion.
Thus, on the basis of the principles we have discussed earlier (Henderson-Hassel
balch
it can be said that at a pH equal to the pKa of the carboxyl group (PKal) the am
ino
as partly cation, partly zwitterion. Similarly, at pH equal to the pK of the ami
no
the amino acid will exist partly as anion and partly as zwitterion. In a solutio
n in
water the amino acid exists mostly as a zwitterion. Let us take the example of a
lanine.
CH3
H-- C -- NH2
COOH
zwitterion form in solution will be
CH3
H-C --NH
COO-

acid, HCI, to this solution of alanine in water it will behave as a base. The re
action
can be represented by the equation
"
CH3
CH3
+H3N- CH- COO- + H + Cl- +H3N- CH- COOH + Clthe other hand, if an alkali is added, alanine solution behaves as an acid. The
reaction
can be expressed by the equation
HaN--CHCOO- +Na+ OH- H2N--CH--COO- + Na + H20
in the zwitterionic alanine, a-amino group behaves as an acid and the
,
a-carboxyl group as a base.
IK,
What would the titration curve of alanine look like?
Figure 1.12 shows that the titration curve for a
lanine looks
like that of a diprotic weak acid. Prom the midp
oint of the
first titration curve we can calculate the PKal
(for the
|
dissociation of carboxyl group) and from the mid-point of
1
the second titration curve we can calculate the pKa2 [fo
r the
*
dissociation of the amino group). From these two pKavalu
es
we can calculate the pH of the solution of alani
ne in its
zwitterionic form by the equation

CATION
ACID

38
Thus for alanine
Biophysical Chern

2.34 + 9.69
pH =
= 6.02
2
Thus, when we dissolve crystalline alanine in pure water, the pH of the solution
is 6..
Or if an alanine solution is brought to the pH 6.02, alanlne wI exist as a zwitt
erion. This p}
known as the/so/on/c po/nt of alanine as alanlne at this pH does not possess any
net chin
This pH is also known as the tsoelectrtc pH of alanine as at this pH alanine swi
ll
electrophoretically immobile. In general the pH at which an amino acid exists as
a zwitterio]
known as the isoionic or isoelectric point of that amino acid.
We may now write the ionization of alanine indicating the specific pK
'

CH3
H -- C NH3+
COOH
PK =2.34
ak-------.x
CHa
CHa
=9.69
PKa
H C -- NH

C --' NH3
COOCOOzwrITERION
ANION
pH 6.02
ALKALI
ISOIONIC POINT

There are many amino acids which have side chains containing dissociable groups.
The,,
groups might be acidic or basic in character. Some of them might contain an extr
a carboxy]
group in their side chain (e.g., aspartic acid, glutamic acid) while some others
might contain extra amino group (e.g. lysine}..Their titration curves look like
those of polyprotic acids. Titrati(
curves for aspartic acid and lysine are given in Figure 1.13 and 1.14 respective
ly. Their isoior
points and different pKa are also shown in the figures. The student can thus wri
te th<
dissociation sequence from the data provided in the figures ( the sequence would
be much li
that of alanine represented above). It may be mentioned, as evident from the fig
ures, that tt
Isoionic pH of dicarboxylic amino acids are quite low somewhere around 3, while
the isoion
pH for the diamino-monocarboxylic amino acids such as lysine is quite high, some
where arour
10. Table 1.8 lists the pKa and the pI values of important amino acids.

Equivaleflla of OH:
EClUiValenl of OH-

Figure 1.13. Tltratlon cw for aspartlc ac pK is for

a-COOH, pK,,, for -COOH, and pl for
Figure 1.14. Titration curve forlysi+ne, pK= is fo=
-COOH, pl for a-NH 3 ' and pK

39
Cysteine
Isoleucine
Leucine
Lyse
Proline
PKa
2.35
9.69
2.17
9.04
12.48
2.09
3.86
9.82
1.71
8.33
10.78
2.19
4.25
9.67
2.34
9.6
1.82
6.0
9.17
2.36
9.68
2.36
9.60
2.18
8.95

10.53
2.28
9.21
1.83
9.13
1.99
10.60
2.21
9.15
pI
6.02
10.76
2.98
5.02
3.22
5.97
7.58
6.02
5.98
5.75
5.98
6.10
5.68
and Bases

1.8 pKa and pl Values of Some Common Am/no Acids

Conjugate acid
Aspartic acid
Glutamic acid
Glycine
Hisdine
Methionine
Phenylalanine
a,-COOH
a-NH
a-COOH ,
a-NH
Guanldtnlum-NH;
a-COOH
-COOH
a-NH a-COOH
a-NH
-SH
a-COOH
-COOH
a-NH a-COOH
a-NH
a-COOH
Imidazole-NH+
a-NH -COOH a-NH a-COOH
a-COOH
a-NH
-NH a-COOH
a-NH a-COOH
a-NH a-COOH
a-NH a-COOH
a-NH

4O
Threonine
Valine
6.53
5.88
5.65
5.97
Trytophan
Tyrostne
a-COOH
a-NH
ct-COOH
a-NH
a-COOH
a-NH
--OH
a-COOH
a-NH
2.63
10.43
2.38
9.39
2.20
9.11
10.07
2.32
9.62

What is it that we learn from the acid-base properties of these amino acids?
1.8 it becomes clear that the only amino acid which has a p/Q near to the pH of
blood
body fluids is histidine. Its imidazole is half ionized at that pH. We have alre
ady considered
importance of pKa for the buffering action of solutions. Because it has a pKa ne
ar to the

PH, histidine possesses a significant buffering action in blood.

protein of erythrocytes is a unique protein in that it contains a large number o
fhistidine
in its structure. These histldine residues impart considerable buffering power t
o haemo
near pH 7, which is important to the role of red blood cells in the transport of
oxygen
carbon dioxide by the blood.

Bases
41
of Amino Acids are pH-Dependent
"All the properties of amino acids which depend upon their ionization would natu
rally be
prevailing pH. We can cite the example of solubility of amino acids. Amino acids
least soluble at their isoionic pHs. They are much more soluble at pHs lower and
higher
pHs. Thus, at a given pH different amino acids will have different solubilities
upon how far removed is the pH of the solution from their own isoionic points.
Other properties which are affected by the extent of ionization are the. metal c
helating
optical rotation, the ultraviolet absorption at a given wavelength and even the
biological
Can be Separated on the Basis of Charge
Can we utilize this pH-dependent ionization of amino acids for their separation?
A small
are given a mixture of three amino acids, alanine, aspartic
It is required thatwe separate these three amino acids from each other and
/hem in a more or less pure state. From Table 1.8 we can find out that the pl of
alanine,

COOI
Aepertio Imid
CHNI+
Net hargo zero
Itet charge- 1
COO"
ginin
Cim
Figure 1.15. pH dependent separutWn of amlno acids. If the pH of a solutWn of th
ree arnlno acids, vz.,
aspar acld and argIne s brought to 6.0, due to ther different pK values alantne
becomes zwltterlon
the anode; argnlne becomes positively charged and moves toword the cathode.
If a potential difference is now applied across the vessel containing this solut
ion, aspart
acid will move towards the anode by virtue of its negative charge. Lysine on the
other hand wi
move towards the cathode and alanlne will remain immobile because it contains no
net char
(Figure 1.15). We can thus effectively separate a mixture of amino acids by elec
trophoresis o.
the basis of the differential charge they possess at different pHs. Amino acids
are usual.
separated by paper electrophoresis using high voltages (see chapter 12).
A better method to separate amino acids .on the basis of the charge they possess
at differen
pHs is ion exchange chromatography. We, however, defer a discussion on this tech
nique to later Chapter (chapter 1 I) where it is discussed in reasonable details
.
Ionization of Proteins i pH-Dependent
We have seen that ionization of amino acids is pH-dependent. It is therefore a c
oro.a.r)
that ionization of proteins should also be pH-dependent since proteins are amino
acids Itnke
together by peptide bonds. There are, however, significant differences. Since th
e a-amlno an
a-carboxyl groups of.successive amino acids are involved in the formation of pep
tide bon
(Figure I, 16) they ar unavailable for ionization. Thus, only the terminal, free
a-amino group a
the amino end of the protein, and the free a-carboxyl group at the c-termlnal en
d of the protek
remain ionizable. But since a protein consists of manyamino acids possessing dis

sociable side
chain groups, these dissociable groups will provide a protein with its character
istic charge
pattern at a given pH. As we have seen that the dissociation of these side chain
groups is also
pH-dependent, the ionization of proteins also is pH-dependent.
42
Biophys.d Chemist
aspartic acid, and arglnine are6.02, 2.98, and 10.76 respectively. Now if we bri
ng the pH off
solution containing a mixture of these three amino acids to 6.0, what will be th
e state ofionizatl,
of these amino acids? Since the pH is extremely near the pI of alanine, this ami
no acid will
present as a zwitterion with no net charge. Aspartic acid on the other hand will
be negativ(
charged at this pH and arginine will bear a positive charge.
I
I

and Bases
43
(ore-)
pH7
-1
protein
NH=
- (CH2).NH.C,NH2
- NH
CO0CH2.COOH
- (CH2),I.N
-Nll
tl
(CH2)-NH.C-NH
imidazole
pH 12
- NH2
-COO-

44
Biophysical Chemts
The Three Dimensional Protein Structure is pH-Dependent
The conformation of a protein molecule in space is a result of a specillc coilin
g and fok
of the polypeptide chains. The molecule may be very large with molecular weight
extendin
several million daltons. The stability of the three dimensional structure is not
entirely du
the peptide bonds. Other types of bonds such as the electrostatic bonds between
ionized gro
of opposite charges are important for maintaining this stability. Since the numb
er of the ioni
groups and the extent of ionization both are a function of changes in pH, the st
ability of
three dimensional structure of proteins is also pH-dependent (also see Box 1.10)
.
Most globular proteins fold themselves up in a manner that most of the amino aci
ds v
potentially ionizable polar side chains are clustered together in one part of th
e molecule, wl
other amino acids with non-polar side chains are clustered together in another p
art of
molecule. Example may be cited of myoglobin in which all the polar side chain am
ino acids
clustered at the periphery of the molecule and the non-polar amino acids are clu
stered centr
The core of the molecule is therefore hydrophobic while the periphery is hydroph
ilic.
Proteins Can Act as Buffers
Figure 1.17 shows the titration curve of protein taking gelatin as an example.'
characteristic feature of the curve is that it does not break sharply at any pH
and is relati,
flat at many pH ranges. This type of a titration curve is a hallmark of a good b
uffer with a I
buffering range. The absence of sharp breaks and the relative flatness at many p
H range,
due to the multi dissociab!e character of the protein. There are many ionlzable
groups wh
ionize successively and simultaneously throughout most of the pH range. We will
discuss ra
about proteins as buffers a little later.
123456
78 910111213
pH
Figure I. 17. Dissociation curve of standard gelatin preparatR)n in hydrochloric

acid or sodium hydroxide solutio

30C.
The isoelectric pH of a protein is the pH at which the protein is immobile in an
elecl
field. At this pH, the protein exists as a zwitterion having equal number of pos
itive and negal
charges, the net charge being zero. The isoionic point of a protein is defined a
s the pH at wh
the total number of H taken up by a protein is equal to the total number of H diss
ociated fr
it. Isoionic and isoelectric point of a protein will essentially be the same if
the protein does
bind long other than H. Since this is an ideal condition and not usually obtainab
le, the isoio
and isoelectric points of a given protein differ.
.Separation Methods for Proteins Depend on Ionization
Just as discussed for amino acids, proteins are also separated on the basis of t
he extenl
their ionization, i.e., on the basis of the charge density they possess. Thus, i
n an electrophore

and Bases
45
the mobility of a protein is a funcUon of its charge density, crcnt proteins
:with differing mobflities because their charge density is different and become
separated
each other. The process is discussed in details in Chapter 12.
A much better technique of protein purification is that of isoelectxic focussing
. Use is
e of the isoelectric point of a protein. Suppose that the isoelectric point of a
protein is 6.0.
a pH gradient in a column so that the pH varies from 1-10 and place all the
the sample on top of the column and start the current, all the proteins will mig
rate
the charge that they possess. At the space where pH of the gradient is 6, while
r proteins will continue moving, the desired protein will become immobile since
it possesses
charge; the pH is isoelectric for it. The method is discussed in reasonable deta
ils in
12.
Another powerful tool for protein purification which also depends upon the charg
e density
ion-exchange chromatography. We defer a discussion of this technique to Chapter
11.
of Proteins is pH-Depenflent
We have already seen that the three dimensional structure of proteins is depende
nt upon
stability. Biological activity of proteins is known to be an attribute of one sp
ecific
of the protein molecules (also see Box 1.10), Thus, any change in pH, which is
to the three dimensional structure of protein molecules is definitely detrimenta
l to
We know that enzyme activities are a function of pH of the medium. Large
from optimal pH alter the activity of an enzyme drastically. At extreme pHs the
might become denatured losing its function completely. Even slight changes in pH
,
r not be of a destabilizing nature, alter the activity of an enzyme in particula
r, and
in general, This was primarily the evidence which led to the suggestion that the
activity
an attribute of a small part .of the whole molecule, the active site (the site
is engaged in catalysis). Small pH changes which might not alter the structure o
f a
substantial enough to alter the ionization of a single dissociable group within
this
site. This is the reason why slight alterations in pH cause a change in the acti
vity of a
may also be mentioned that the effect of pH on the biological activity of protei
ns may
evidence about the dissociable groups present in an active site.

46
Biophy

Bases
47
Catalysis
In reactions catalyzed by general acids or bases the catalyst functions as an ac
ceptor or
protons. Often the critical proton transfer step is to or from a carbon atom of
the
state species. How can we ascertain that a proton transfer is the mechanism behi
nd
can carry out the reaction (which we suspect to be acid-base catalyzed)
(heavy water) rather than H20. Deuterium is an isotope of hydrogen and has twice
as
mass as hydrogen. Due to isotope effect (see Chapter 13) deuterium ion will reac
t much
' than the hydrogen ion. Thus, "ff the reaction is catalyzed by a proton transfe
r step,
!take place at a much slower rate in heavy water. Using such strategies many enz
ymatic
have been shown to be acid-base catalyzed. Many times both a general acid and a
base are involved in catalysis; the acid donates a proton to the reaction interm
ediate
he base accepts one.
be expected, the strength of the general acid or base, i.e., its proton dissocia
tion
of reactions catalyzed by proton transfer steps. The most significant
at physiological pH is the imidazole group of histidine which has a pKavalue
6.0. This pKa enables it to act both as a proton donor and a proton acceptor at
pH. Another factor which can affect the rate of acid-base catalyzed reactions is
or accepts a proton. The imidazole
is particularly effective in this case also. It has an equal rate of protonation
and
physiological pH with a half-time of less than 0.1 us. There are several enzymes
catalytic properties to the tmidazole group of histidine.
general acid-base catalysis is, can be proved by a simple example. To hydrolyze
bonds without enzymatic mediation, very high concentrations ofH+ and OH- are req
uired
long reaction periods. Chymotrypsin achieves this hydrolysis
pH and at body temperature fairly quickly.
of Most Biomolecules is pH-Dependent

which pos.sess multiple dissociable groups will be affected by changes in

similar to the proteins described above. Thus, nucleic acids, mucopolysaccharide
s,
will be affected by changes in pH. Their three dimensional structures and
their biological activity would be affected by changes in pH.
t only biopolymers but even smaller metabolites may be affected by pH. One of th
e main
t of the cellular metabolites being charged is that the plasma membrane is
lmpex?meable to charged species. It is therefore easier for the cell to retain t
hese
the cell. Important ionizable groups possessed by cellular metabotites

48
Biophysical
are carboxyl and amino groups, and the phosphate group. Even the rate of catalys
is
metabolites may be dependent upon their extent of ionization and will therefore
be
upon pH.
Biologically Important Buffers
We have seen how all major biomolecules are affected by pH changes. The conclusi
on
we have derived is that the biological activity of most biomolecules is dependen
t on pH. We
therefore grasp the importance of maintenance of the pH of physiological fluids
within
limits conducive for normal functioning of cellular processes. The control of pH
in biolo
systems is achieved by the action of efficient buffering systems whose chemical
nature
that they can resist pH changes due to the metabolic production of acids such as
lactic a
and bases such as ammonia. The major buffering systems found in cellular fluids
are I
bicarbonate buffers, phosphate buffers and protein/proteinate buffers.
Buffering of Blood
An interesting example of buffering in biological systems is provided by what oc
curs
blood. The pH here is maintained within extremely narrow limits of 7.36-7.4 for
venous bl
and 7.38-7.42 for arterial blood. Three distinct buffering systems are responsib
le for maintair
the pH within these narrow limits : (/) H2CO3 and HCO , (//) the acid and base s
pecies
oxygenated haemoglobin, and (///) the acid and base species of deoxygenated haem
oglobin.
A detailed description of haemoglobin is uncalled for here, but what can be ment
ioner
that this iron containing protein resides within the red blood cells in relative
ly abundant quant
14-16 grams/I00 ml of whole blood. The predominant function of haemoglobin is to
tran,
oxygen from lungs to respiring tissues. Within the lungs, oxygen from the incomi
ng air ent
the erythrocytes and becomes bound to the iron atoms of haemoglobin. The oxygena
haemoglobin, while it is being carried by the blood, donates its oxygen to vario
us respi
tissues. In this process the oxyhaemoglobin becomes deoxygenated and via venous
blood retu
to the lungs to indulge in the oxygen transport cycle again. While engaging thus
in its prim

role of oxygen transport, haemoglobin also contributes importantly to the buffer

ing of bl
How? Let us consider the following points before we elaborate the whole mechanis
m.
(/)
It is known that oxygen binding to haemoglobin is weakened by decreasing
the pH.
other words, oxygen dissociation from haemoglobin is promoted by increasing hydr
o
ion concentration. This phenomenon is known as the Bohr effect after its discove
(//)
CO2 (diffusing from the tissues into the erythrocytes) does not reside i
n the erythroc
as such. It is converted by the enzyme carbonic anhydrase to H2COa.H2C0a ionize
HCO and H so that the ratio of HCO /H2CO3 becomes 20/1 (substitute this r in Hend
erson-Hasselbalch equation along with 6.1 as the pKa for H2CO3 and calct
the pH; it comes to 7.4, the pH of blood l). The ionization of H2CO8 can cau
decrease in pH because it increases the hydrogen ion concentration. In allwe may
that input of CO2 can decrease the pH if H is not eliminated.
BLOOD. VII= 7.4 .
. CARBONIC

HC03 +H+

CO2 1-12J 1-12J3

How is the H+ generated by incoming CO2 quenched? To answer this question,
have to consider the acid-base properties of haemoglobin.

49
Acids and Bases
We have already mentioned that haemoglobin is rich in histidine, an amino acid
which has a pK of about 6.0 (imidazole group) making it predominantly ionlzable
at
the blood pH. We denote haemoglobin by the symbol Hb. We can include another H i
n
this symbol to denote the ionlzable hydrogen. Experimental determination proves
that oxygenated haemoglobin (HHbO2) has a pK of 6.-62, while deoxygenated
haemoglobin (HHb) has a pK, of about 8.18. This automatically means that oxygena
ted
haemoglobin is a stronger acid as compared to deoxygenated haemoglobin. Thus,
at pH of blood, 7.4, the base form of oxygenated haemoglobin would predominate
(HbO . ). On the contrary at the same pH, the acid form of deoxygenated haemoglo
bin
(HHb) would be predominant.
HHbO
(pKg. = 6.62)
BLOOD. pH = 7.4
HbO. + H+
BLOOD, pH = 7.4
HI-Ib

Hb- + H

(pK = 8.18)
With these points in background, let us consider the bufferLng of btood. We have
seen that
incoming C02 causes an increase in H ion concentration which has to be eliminated
. This H
reacts with the base form of oxygenated haemoglobin, HbO . (which is predominant
at pH 7.4)
converting it to its acid form HHbO2, At this point the Bohr effect comes into p
lay and oxygen
dissociates from haemoglobin and diffuses into the tissues. The acid form of oxy
haemoglobin
becomes deoxy. We have already seen that deoxyhaemoglobin has a higher pK and do
es not
dissociate to a great extent. It thus remains as HHb. What has taken place now i
s that not only
the H released by incoming CO2 has been buffered, but in the process the objectiv
e of oxygen
transport to the tissues has also been achieved.
The CO2 in the form of HCO now leaves the erythrocyteS and is carried to lungs t
hrough the venous blood. To counterbalance the exit of HCO , CI- moves into the
erythrocytes from
plasma. This is known as the chloride shift.

When the venous blood reaches the lungs, HCO and 02 enter erythrocytes and CI- e
xits. 02 binds to HHb converting it to HHbO2. The Bohr effect, however, does not
take place because
the ionizable hydrogen is quickly donated to HCO to convert it to H2CO3.HHb02 ge
ts converted to HbO .HbO . can now indulge in transporting oxygen while it is ca
rried by the blood to different tissues. H2CO3, by the reverse action of carboni
c anhydrase is converted to H20 and

50
,:-'.::::::::::::
::::i:?
"::::) .::::::::::i!!ii!!!i!
H,CO= H+ + HCO= :!:!:!:::: 7,
n :::::::::::
i::i::i:i
:':':':':':"
.:.:.:-.. o
:i:i:i:!:i:!
.:::::::::: u
I=:: ::::::.
" :::::::::::!
(A} Events in enjthrocyte when arterial blood reaches the respiring tissue.
CARBONIC
: :Z ::;:::::::
(B) Events "in enjthrocyte when venous blood reaches the lungs.
Figure I, 18. Buffering of blood. Role of HCO / H 2 CO3 and HbO / HHbO2 buffers,
From the foregoing discussion we can conclude that the main buffering systems
are the HCO ;/I-I2CO3, HbO ;/HHbO., and Hb-/HHb. In addition, blood contains
proteins and m'ay substances possessin phosphate groups. Thus, two additional
systems which operate in blood are HPO4-/HPO and proteinate-/protein systems. Th
e
systems, however, contribute less to the buffering of blood than the main system
s discusse
above.
Buffers of Tissue Fluids and Tissues
The main buffering system present in spinal fluid, lymph, and exudates are simil
ar t
those present in blood. The HCO /H2CO system is much more predominant in these f
lui(
As compared to this the contribution of potein buffers is much smaller.

Adds ar Bases
51
In tissues, the buffering systems are mainly the HCO /H2CO3, and the pro
teinate-/protin.
The contribution of the latter, however, is much more here as compared t
o their contribution in
tissue fluids. Within the cell many organic acids and their conjugate ba
ses also exert buffering
actions. We have already seen citrate in this light.
MMMMT OF pH : USE OF INDICATORS
Indicators are substances which demonstrate characteristic color properties. The
y show
one color above a characteristic pH and another color below that pH or pH range.
They are often
used for pH measurement when high levels of accuracy are not required (their use
has decreased
in modern days and the pH meter is the most often used instrument). The indicato
r with which
we are all familiar is litmus which is red when acidic and blue when basic. Othe
r indicator
substances also exhibit characteristic change in color with change in pH; their
colors might,
however, be different from those of litmus. What is the mechanism behind such co
lor change by
indicators? The indicators act like a weak acid dissociating in the following ma
nner :
Him H + In(acid)
(conjugate base)
In acid solutions where [H] is high, the indicator dye does not dissociate apprec
iably. As
pH is raised and [H+] decreases, the dissociation of dye becomes predominant til
l a stage comes
when the dye is predominantly dissociated. The dye exhibits different colors in
the dissociated
and undissociated states.
We can write an equation for the dissociation constant of indicators.
When the dye is half dissociated
[HInl=1 Thus [H+] = KI.

LIn- .J
The dissociation constant at equilibrium for an indicator is therefore the value
of H at
which the color change of dye is half complete. A negative log of this value wil
l give us the pKa
of indicator.
Table 1.9 gives a list of the commonly used indicators. By using two or more ind
icators we
determine the pH of a given sample within one to two units. For example, a given
sample is
colorless for p, henolphthalein, The pH of the sample must be less than 9. The s
ame sample
however tests blue for bromothymol blue. Its pH, therefore: can not be below 8.
The pH of the
given sample, therefore, must be within pH 8 and 9.

52
Table 1.9 Some common Acid-Base Indicators
Ind/cator
pH Range
Acid Color
Base Color

Methyl Violet
Thymol Blue
Methyl Yellow
Methyl Orange
O.5- 1.5
1.2 - 2.8
2.9 - 4.0
3.1 - 4.4
Yellow
Red
Red
Red
Blue
Yellow
Yellow
Yellow

Bromophenol Blue
Bromocresol Green
Methyl Red
Chlorophenol Red
Bromothymol Blue
Paranitrophenol
Phenol Red
Cresol Red
Thymol Blue
Phenolphthalein

Thymolphthalein
Alizarin Yellow R
3.0 - 4.6
3.8 - 5.4
4.2 - 6.3
4.8 - 6.4
6.0 - 7.6
6.2 - 7.5
6.4 - 8.0
7.2 - 8.8.
8.0 - 9.6
8.0 - 9.8
9.3- 10.5
10.1 - 12,0
Yellow
Yellow
Red
Yellow
Yellow
Colorless
Yellow
Yellow
Yellow
Colorless
Colorless
Yellow
Blue-Violet
Blue
Yellow
Red
Blue
Yellow
Red
Red
Blue
Red
Blue
Violet

Papers impregnated with several dyes are commercially available. The change in t
heir
colors when dipped into a sample iscompared with a color-pH code on the dispense
r. These
papers are very efficient and can determine the pH of a given sample to about 0.
1 of a pH unit.
These papers are, however, useless when the test solutions are highly colored or
when very
high accuracy is needed. For such measurements, pH meters are used. These are di
scussed
below.
ELECTROMETRIC DETERMINATION OF pH
Earlier on we have seen that indicators are unsatisfactory for measuring pH of s
olutions
which are colored. We have also seen that for works which require absolute accur
acy, indicators
are not a good tool for pH measurement. Such situations call for electrometric d
etermination of
pH. For a student of biochemistry, probably the most familiar instrument is the
pH-meter. Its
operation is quite simple; it consists of a glass electrode which when dipped in
to a solution
develops an electrical potential depending upon the hydrogen ion concentration a
nd this potential
is read off the display which is calibrated into a pH scale. Let us try to under
stand the basic
principles underlying the working of a pH meter.
Electrode Potentials
When a strip of metal (electrode) is dipped into water, it tends to dissolve owi
ng to its
solution pressure, P. While the atoms of metal go into solution, they leave behi
nd their loosely
bound valence electrons as a negative charge on the electrode. Since the electro
de has developed
a negative charge, it now attracts the positively charged metal long which have
already gone
into solution. Some of the positively charged metal long might reattach themselv
es to the
negatively charged electrode. The tendency of the atoms to leave the metal and t
he tendency of
metal ion.s to reattach themselves, finally becomes balanced and an equilibrium
is reached. At

Acids and Bases

53
equilibrium some positively charged metal long remain in solution and the opposi
te charge of
the electrode and the metal long gves rise to a potential difference.
Let us now alter the conditions a bit by dipping the metal int( a solution of on
e of its salts.
This is an altogether different situation because there are many metal long alre
ady in solution.
These metal long will oppose the separation of more metal long from the electrod
e and an
equilibrium will be achieved early..The point of'equilibrium in this case will b
e dependent upon
the relative values of the two opposing forces: the solution pressure (P) of the
metal and the
osmotic pressure (p) of the metal ions-in solution. Three possibilities arise (F
igure 1.19):
P>p
P=p
"Figure I. 19. Diagram indicating how electrode potentials arise when a metal st
rip is dipped into a solution of one of
its salts.
P = Solution pressure of the metal; p = Osmotic pressure of the metal..
(t)
P > p. Since the solution pressure is higher, the atoms from the metal s
trip continue
to dissolve as positive long till the accumulated charge is strong enough to opp
ose
further dissolution. The metal strip in this situation acquires a negative charg
e as
compared to the solution which becomes relatively positive.
(//)
P < p. The opposite takes place. Since the osmotic pessure of metal long
in solution is
higher, they attach themselves to the metal strip increasing the positive charge
on the
electrode. The solution becomes relatively negative.
(///)
P = p. Since both the opposing forces are equal, metal long neither leav
e the electrode
nor the metal long in solution get attached to the electrode. No potential devel
ops.
Taking thermodynamic reasoning into consideration, the potential difference betw
een a
metal and a solution of one of its salts is given by Nernst equation

RT P
E = --In-... (1)
nF p
where E = electrode potential, R = gas constant (8.316joules per degree), T= abs
olute temperature,
n = valency of metal ion, F= faraday (96,500 coulombs), P= solution pressure, an
d p -- osmotic
pressure of metal long in solution. The natural logarithm is denoted by '/n'.
We know that RT/F is constant for any given temperature. Therefore, the magnitud
e and
the sign of the electrode potential, E ,will be dependent upon n, P and p. If we
use the same
metal, the electrode potential will depend solely upon osmotic pressure, p, of t
he solution in
which the electrode is dipped. Since osmotic pressure can be regarded as proport
ional to
concentration, we can say that the electrode potential is a function of concentr
ation of the
metal long in solution (actually the electrode potential is dependent upon activ
ity and not
concentration). We may therefore rewrite equation (1) by replacing the osmotic p
ressure term
with a term for concentratiort.-

54
E = RYlnP
...{2)
where C = concentration of the metal long in solution.
Since the electrode potential developed by a metal strip is dependent upon the c
oncentration
of the metal long, is it not possible to measure the concentration of metal long
in any given
solution by measuring the potential developed by the electrode? Theoretically th
e answer is in
affirmative. However. practically it is impossible to measure the potential Of a
half cell (one
electrode dipping in a solution). This problem can be circumvented ff we provide
for a second
electrode of the same metal dipping into a solution of a different concentration
. The two electrodes
will now develop different potentials. If the two electrodes and the two solutio
ns are suitably
connected, an electric current will pass from one electrode to another, the elec
tromotive force of
which will be equal to the difference between the two electrode potentials. Thus
,
E =E1-E2
RT In C2 ( l and P2 cancel out since they 1
= nF C kate identical for the same metal]
If we can flx:the potential of the second electrode then the potential of other
half-cells can
be determined relative to it. In other words, the problem of measuring the poten
tial of a half-cell
in the presence of another half-cell is solved by referring all potentials to a
standard reference
potential. The electrode(s) which is used as a standard is known as the referenc
e e/ectrode. We
will consider some of the reference electrodes used in electrometric determinati
on of pH in the
following pages.
1. REFERENCE ELECTRODES
The basic function of a reference electrode is to maintain a constant electrical
potential
against which deviations may be measured, The. desirable characteristics that a
reference

electrode should possess are (0 it should be easy to construct, and (//) it shou
ld develop potentials
which are reproducible even if small currents are passed.
Various reference electrodes are in use and some of them are discussed below.
The Hydrogen Electrode
The hydrogen electrode consists of a piece of platinum foil dipped in a 1 M solu
tion of
hydrogen long. The solution used to provide this hydrogen ion activity is 1.18 M
HC1 (the potential
produced is dependent upon the activity and not the concentration: see Box 1.5).
To increase
its surface area, the platinum foil ts coated vth platinum black. Hydrogen gs at
one atmosphere
pressure is bubbled over the platinum foil, The platinum strip saturated with hy
drogen gas.
acts exactly as the metal electrodes described above when dipped in a solution c
ontaining
hydrogen long. This arrangement in toto is known as the standard hydrogen electr
ode
(Figure 1.20) and it is arbitrarily assigned a potential of zero under all condi
tions.

55
lx 96, 500 Ige 2
Acids and Bases
------ Hydrogen gas inlet
Gas outlet
Platinum strip
Hydrochloric acid
(I.SN)
Fgure 1.20. Construction of a Hydrogen Electrode.
We can measure the pH of an unknown solution if two hydrogen electrodes are imme
rsed
into two solutions of differing hydrogen ion concentrations, the [H] being consta
nt in one of
these solutions. The electromotive force developed in such a system will be an i
ndex of the [H+]
in the unknown solution. If we consider the potential of normal hydrogen electro
de as Eho, and
that of the hydrogen electrode in the unknown solution as Eh, the e.m.f, obtaine
d on c,ompleting
the circuit will be
e.m.f. (volts) = Eh- Eho
If we denote the hydrogen ion concentration in two different solutions in which
we have
dipped the hydrogen electrodes by [H+]I and [H+]2, hen by. the Nemst equation th
at we have
discussed previously, the e.m.f, is given by
e.m.f. = Eh- Eho = --log [ H+ ]i
Substituting the values of R, n and F in the above equation we have
But loge = logo x 2.30258. Substituting this value we get,
8.315 x T x 2.30258
=
I x 96,500

Mercury
Calomel
56
Biophysical Chemistry
We have already seen that the H actity around a standard hydrogen electrode is fi
xed at
I. The above equation therefore can be written as
1
e.m.f. = 0.00019837Tlog
I'
but log can be written as log 1 - log [H+]2. Since log 1 = 0, we have - log [H]2.
As we have
seen in the previous chapter, - log [H] = pH. The above equation therefore become
s
e.m.f. -- 0.00019837 T pH.
At 25C e.m.f. = 0.059 pH
or
pH

(3)
0.059

From (3), it is evident that if the potential difference (e.m.f.) between the st
andard hydrogen
electrode and the electrode in the unknown solution is experimentally determined
, the pH of
the unknown solution can be easily calculated.
Although hydrogen electrode is the standard of reference, it is extremely inconv
enient to
construct and maintain it for everyday work. A source of ultrapure hydrogen is r
equired and it
is essential to maintain the correct partial pressure within the cell. Even trac
es of impurities
either in the gas or in the solution are enough to poison the electrode. Moreove
r, the electrode
responds to numerous redox couples and therefore strong oxidants or reductants a
re detrimental
for pH measurement, Due to these disadvantages its use is extremely limited. It
is, however,
used exclusively .for calibrating other reference electrodes. It is also used fo
r measuring hydrogen
ion concentrations of solutions in which no other electrode will operate satisfa
ctorily.
The Calomel Reference Electrode

The design of calomel electrode varies for different applications, but a general
purpose
calomel electrode can be illustrated as in Figure 1.2 I. The electrode consists
of a strip of platinum
sealed into glass and allowed to dip into mercury. A paste of calomel (Hg2CI2, 0
.1 M.) is held
against the mercury by means of a sintered glass plug or cotton wool. The whole
electrode is
filled with saturated KCI. Contact with an outside electrolyte is maintained thr
ough a porous
ceramic plug.
Side arm for
filling KCI
Sintered Glass
KCI crystals
Ceramic plug
Figure 1.21. Structural features of a calomel reference electrode

pH =
E - 0.246
0.059
Acids and Bases
The calomel electrode is a lalf-cell and can be represented as
Pt I Hg] Hg2C12 I KC1 (saturated)
(Every vertical line denotes an interface at which a potential is developed).
If the KC1 solution is kept saturated, all the potentials developed in the calom
el half-cell will be
constant. All calomel electrodes have a side arm to replenish KC1 solution.
The potential in a calomel hf-cell is derived from the primary reaction
H ++
g2 +2e-2Hg
The corresponding Nernst equation for this reaction is
0.059
I
e.m.f. = E
log
Hg
2
connects calomel and hydrogen electrodes to a voltmeter, a potential of 0.246 my
(at 25C). Thus, if we connect a calomel and hydrogen electrode together and the
electrode is made to dip in a solution of unknown [H], the potential which will d
evelop
be 0.246 my higher than the situation where both the electrodes used were hydrog
en
The equation of pH, when a calomel electrode is used as reference would therefor
e
Calculation of pH
(I)
From the text we know that at 25C using saturated calomel electrode pH =
(E
-

0.246)/0.059. When the electrodes are dipped in a solution the voltmeter reads
0.652. What is the pH of the solution ?
0.652 - 0.246
A$.
pH
=
0.059
= 6.9
pH of the given solution is 6.9
(2) Calculate the potential developed if the pH of a given solution is 2.6.
Ans. 0.4
Calomel electrode is extensively used as a reference electrode for pH measuremen
t. It is easy to
prepare, cheap and quite easy to maintain. The potential developed by this elect
rode is
reproducible and constant.
The Silver/Silver Chloride Electrode
Basic design of this electrode is provided by Figure 1.22. It consists of a meta
llic silver wire
coated with silver chloride and immersed in a saturated potassium chloride solut
ion. The potential
of this system is derived from the primary reaction

Biophysical Chemistry
The potential developed may be calculated from the Nernst equation
e.m.f. = E,--lg[Cl= 0.222-0.0509 log [CI-] (at 25C1
From the above equation it bec.omes clear that the potential of the reference el
ectrode is a
function of the chloride ion concentration (activity). In order that the chlorid
e ion concentration
remains constant at all conditions of humidity, a saturated potassium chloride s
olution is
used. Electrical contact between the reference electrode and the solution being
tested is
maintained through a potassium chloride salt bridge. This Junction is made throu
gh a porous
ceramic membrane embedded in the bottom of the reference electrode.
Silver/silver chloride electrode is extremely easy to maintain and it develops a
reproducible
potential. It is being used as a reference electrode in most of the current Inst
ruments.
Side arm for
filg KCI
Saturated KCI -..
electrode
KCI crystals -Ceramic plug
Figure 1.22. Structural features of an Ag : AgCl reference electrode.
2. THE GLASS ELECTRODE
The measurement of pH by glass electrode involves the use of two reference elect
rodes,
separated by a glass membrane whose function is to establish an electrical poten
tial depending
upon the hydrogen ion activity of the solution being tested. The design of glass
electrodes is
enormously variable, but a basic construction is shown in Figure 1.23. It consis
ts of a high
resistance glass tube with a thin. low resistance glass bulb fused at the bottom
. The bulb is
- responsible for the pH sensitivity; the rest of the electrode is insensitive t
o [H+]. The tube is filled

up with 0. I N solution of HCI. Dipping in this solution is a silver/sflverchlor

ide electrode. The
other reference electrode might be a calomel electrode, but in most current inst
ruments this
electrode also is an Ag/AgCl electrode. When both electrodes are dipped Into a s
ample, the
resulting e.m.f, gives the pH of the solution.

and Bases
Glass stem
: AgCl
eiectrode J
0. IN HCI -. -- pH sensitive
glass membrane
Figure 1.23 Diagram of a glass electrode.
It is essential to understand the nature of the pH sensitive glass membrane to u
nderstand
electrode works. X-ray diffraction studies have revealed that the glass membrane
consists
ia network of silicate and aluminate long (Figure 1.24). The holes in this latti
ce-like structure
be occupied by cations of varying size. These holes, however, cannot accommodate
anions
because of the strong repulsion of the oxygen containing long. By careful manipu
lations during
it is possible to obtain a membrane whose holes can accommodate only H long.
membranes will then be sensitive to hydrogen ion activity.
) gen atoms

con atoms
Catlons
Figure 1.24. Structure of a pH-sensitive glass membrane as revealed by X-ray dif
fraction studies.
How does a glass membrane respond to differences in hy4rogen ion activity?. The
belief is
that the glass electrode works by an ion exchange process. It is believed that t
he membrane
consists of three layers, a dry glass layer sandwiched betweentwo hydrated layer
s. Let us now
see what events can occdr when this glass electrode is placed in aqueous solutio
ns of neutral,
basic, and acidic reactions (Figure 1.25). We begin with the basic assumption th
at in the holes
of the lattice-like network of the two hydrated layers, hydrogen long are presen
t.
(i) When the electrode is placed in a neutral solution : In this situation hydro
gen long from

the concentrated HCI in the inside of the glass electrode become bound to the in
ner hydrated
surface. This results in the release of an equal number of protons from the 'hol
es' of the outer
hydrated surface and the neutrality of the membrane is maintained (Figure I..25
A).

6O
H+C"

NEUTRAL
BASIC
CF ' H
CF H
H+CI"
CI'
H
H+Cl"
e
cl"
H Tj
H+Cl"
H+
u H+
H
Cl" H ."
Cl" H

CI" H
H+CI-Internal
solution
Biophysical Chemistry
ACIDIC
-.
H+ + OH'Na+----H20
:

H+ + OH" Na+-) H20

Cl" H :---b H+ + OH'Na+ H20
H+CI"
.- H+ + OH" Na+.b, H20
CI-H
=--b H+ + OH" Na+---- H20
HCl"
H + + OH'Na +-- H20
H
H

H+Cl"

Internal External
External
internal External
solution solution
solution
solution Solution
(A)
(e)
(C)
Figure 1.25. The glass electrode works by ion exchange mechanism. A schematic di
agram showing events at different
pHs.
go When the electrode is placed in a basic solution : Consider that the basic so
lution is
that of NaOH which dissociates fully to give Na and OH-ions. Since a high number
of OH- long are present outside, the H long from the outer hydrated layer leave t
heir 'holes' and go
into the sample solution where they combine with OH- long to give H20. The Na lon
g remain
unquenched and therefore positive charge at the outside of the glass membrane in
creases. Let
us see what is happening at the inside of the membrane. Since the outside 'holes
' are becoming
vacant, more and more protons from the HCI solution inside, become bound to the
inner hydrated
surface. This leaves the Cl-ions unquenched and consequently the inner side of t
he glass
membrane develops an excessive negative charge. The separation of charge on two
sides of the
glass membrane gives rise to the electrode potential which is an index of the pH
of the solution
(Figure 1.25 B).

(iii) When the electrode is placed in an acidic solution : In this situation, th

e concentration
of H+ long at the outside of the membrane is high. Consequently, the 'holes' in
the outer hydrated
surface are occupied by H long. Now, to maintain the electroneutrality of the mem
brane, H
long from the 'holes' of the inner hydrated layer are released. These H+ long de
crease the excess
of negative charge inside due to CI- long. Thus, while the inside becomes less n
egative, the
outside is becoming less positive. Again, this separation of charges is the sour
ce of the electrode
potential (Figure 1.25 C).
The overall potential of the glass electrode is a contribution of several potent
ials: (/) the
potential of the internal silver/silver chloride electrode; (//) the potential d
eveloped at the inner
glass surface; (No the potential developed due to imperfections in the glass sur
face; and (iv) the
po.t.ential developed at the outer glass surface. However, potentials (/), gO, a
nd (///) are all constant

ELECTRODE
Reference
electrode
(Ag]AgCl,or
Hg/Hg C12)
REFERENCE
ELECTRODE
Internal
referenc
electrode
Ag/AgCl
GLASS
Acids and Bases
61
for a given electrode and it is only (iv) which changes with the pH of the test
solution. For a given
glass membrane, then, all these constant potential may be collected into a gener
al constant EG,
and an equation may be written.
2.303 RT
e.m.f. = Eo +
(pH outside)
F'
We know that E,., R, and F are donstant. Thus, e.m.f, depends only on the pH and
the
temperature. This equation therefore tells us how important it is to maintain th
e temperature
of the buffer used to standardize the pH meter and the temperature of the test s
olution at the
same level for the pH reading to be correct.
When a pH sensitive glass electrode and a reference electrode are dipped in a so
lution, a
galvanic cell is set up. The cell can be written as follows:
GLASS CERAMIC
MEMBRANE
MEMBRANE
electrolyte
solution

(Sat.)
(0.1 N HCI)
Each vertical line in the above representation denotes an interface and a .poten
tial is
developed at each interface. The potential of the whole galvanic cell is the alg
ebraic sum of the
potentials developed by the glass (indicator) and the reference {Ag/AgCI or Hg/H
ggCI2) electrodes.
e.m.f. (cell) = e.m.f. (re0 3, e.m.f. (glass) -Since e.m.f. (ref) is constant, e.m.f. (ceil) varies only with a variation in th
e pH of test
solution (if the temperature is constant). The modem glass electrodes develop po
tentials which
give a linear relationship with pH .changes.
J
(B)
(C}
Floure 1.26. Various kinds of pH-s.e.nsttive electrodes. {a) general purpose gla
ss and reference electrode: (b) combination electrode for pH measurement of flat
moist surfaces; (c) combination micro-spear electrode
for small sample volumes.

62
Bophysical Chemistry
The glass electrode can be combined with an external reference electrode. In oth
er words,
the reference electrode can be built in the same unit. Such an electrode known a
s a combination
electrode is illustrated in Figure 1.26. Also illustrated in the same figure are
various.kinds of
combination electrodes used for different purposes.
The glass electrode can be dsed to measure pH of virtually all kinds of solution
s, including
those containing strong oxidizing and reducing agents. It may be embedded in sem
isolid materials
such as cheese or butter and give a satisfactory pH value. It is also suited for
pH measurement
of biological fluids. It however, has certain disadvantages {which can be circum
vented}. Firstly,
the electrical .resistance of the glass membrane is high. Use of high impedance
amplifiers is
therefore mandatory for potential measurement. Secondly, at high pHs (higher tha
n 9) the
electrode may start developing potentials due to sodium long rather than due to
H long. This
problem has to be ameliorated by manufacturing a special glass with low sodium c
ontent.
3. THE pH METER
The pH meter is basically an electronic voltmeter (or potentiometer) designed fo
r use with
a glass electrode system. It is composed of(/) a reference electrode, (//) a gla
ss electrode responsive
to the pH of the solution surrounding it, and (rio an electrometer, which is a d
evice capable of
measuring very small differences in electrical potentials in a circuit of extrem
ely high resistance.
The pH meters are usually so constructed that the true zero of the voltmeter is
at or near
pH 7. Before use, the pH meter must be standardized with a pH 7 (or pH near 7) b
uffer,
adjusting the zero calibration to give the correct reading with the temperature
control. Then a
second buffer, whose pH is near to that of the sample is substituted and the met
er is adjusted
to give the correct reading with the temperature control, This procedure helps t
o establish the
correct linear relationship between my and pH. Moreover, this standardization mu
st be done
each time a pH measurement is to be done. This is essential because there are su
btle changes
in various potentials due to the ageing of the electrode. The standardization ju
st before a

measurement tends to nullify these potential changes due to electrode ageing.

To be of use, the meter of pH electrometer must give a reading consonant with th
e pH of
the sample. This needs the signal to be amplified greatly before it is strong en
ough to activate a
standard milliammeter or a millivoltmeter. The following equation states that th
e pH sensitive,
galvanic cell potential (E3 to be amplified is
e.m.f. (cell) -- e.m:f. (rei + e.m.f. (glass)
But

e.m.f. (glass)
Therefore
2.303 RT
= E +pH

2.303 RT
e.m.f. cell
= e.m.f.tref +Eo
+--pH
F
or
e.m.f. {cell)
= E + mpH
where E = e.m.f. {rel) + EG, and m = 2.303 RT/F
The amplified signal is then directed to a meter which produces a deflection rel
ated to the
pH of the solution.

Acids and Bases

63
In general, the determination of pH using a pH meter is quite simple. The soluti
on is
placed in a small container. The glass and the reference electrode (or the combi
nation electrode)
are dipped into this solution so that the glass bulb is dipped completely. The p
otentlometer
circuit is closed and adjusted till the null point is obtained. The pH can then
directly be read off
the display. Two general precautions must be taken; (t) As stated above, the pH
meter must be
standardized before every measurement, and (//) when not in use, the reference a
nd the glass
electrode must be kept immersed in water so that the pH sensitive membrane does
not become
dehydrated. The following points must be kept in mind while measuring the pH.
Sodium Error
General purpose glass electrodes are to a certain extent permeable to sodium lon
g also.
Thus they can produce a potential for Na in the same way that they do for H. There
fore in
solutions where Na is present, the actual pH determined by glass electrodes is l
ower than what
it should be. This is so because the electrode reads a sum of both H and Na long,
i.e., the
recorded pH is -log |H and Na] and not just-log [H]. NaturaLly, as the concentratio
n of Na
increases, the pH decreases. The effect of this problem is not so noticeable whe
n the pH is low.
But when one is working at high pH (due to NaOH), the pH reading could be off by
as much as
two units if I M NaOH is used.
There aretwo ways to correct this problem. The first is to use Na impermeable gla
ss
electrodes. However, if this is not possible, then one should work preferably wi
th KOH when
working at high pH and avoid NaOH if possible. The general purpose glass electro
de is not
permeable to the bigger K long.
Electrode Contamination
It is a common precaution to wash electrodes frequently with acid or With deterg
ents or
sometimes both. The reasons are not fa to see. In our discussion on glass electr
odes we have
seen that these glass membranes have holes for H. Naturally, if these holes are b
locked, the

electrode Will give a faulty reading owing to reduced permeability. For biochemi
sts this
permeability problem is quite common since they work With proteins. Proteins can
form a thin
film on the glass. This impedes permeability. This film can be washed away compl
etely with
either acid or detergents. Thus, it cannot be emphasized enough that if one is w
orking With
proteins, the electrode must be washed very frequently with acid or detergents.
Certain commonly used buffers react With the components of glass electrodes. One
example
is that of Tris[tris-(hydroxymethyl)aminomethane] buffer. This buffer can cause
large changes
in pH readings. One should read the manual supplied along With the electrodes ca
refully. The
- buffers that can cause changes in the electrode areusually listed there. One s
hould then avoid
working with these buffers as far as posslble.
Concentration of Long
Previously we have. discussed that the pH is actually a negative log of H activit
y and not
H+ concentration. At low concentration the activity and concentration are the sa
me, However,
at high concentration the activity actually becomes lower than the concentration
. Thus, one
must avoid measuring pH of very concentrated solution. There is another reason f
or doing so.
Other long can also affect the activity of hydrogen long. Thus the pH of a buffe
r will vary both
with its own concentration and with the concentration of other salts in the solu
tion:
If one is preparing a stock buffer solution which is very concentrated, it is wr
ong to adjust
the pH of the stock so as to give the correct pH upon dilution.. Rather the conc
entration should
be so prepared that upon dilution it gives the correct pH; the pH of the diluted
buffer should be
measured.

Biophysical Chemistry
pH papers are for rough use. The commercially available pH papers claim that
measurements can be very sensitive - difference of 0.2 pH units can be detected.
However,
there are numerous pitfalls involved here. For biochemists, the pitfalls are hig
her, and, again
these pitfalls result from having to work with proteins. Many proteins are known
to cause
errors of several pH units when working with pH papers. High salt concentration
can also
cause large errors.
Additionally, some components of the solution may react with the pigments on the
paper
and cause color changes not related to pH at all.
Suggestions For Further Reading
1, Henderson, L.J., The Fitness of the Environment, Beacon Press, Boston, 1958.
2. Edsall, J.T. and Wyman, J., Biophysical Chemistry, Academic, New York, 1958,
Vol. I.
3.
West, E.S., Todd, W.R., Mason, H.S. and Van Bruggen, J.T., Textbook ofBi
ochemlstry, 4th
ed., Macmillan, 1966.
4. Segel, I.H., B/ochem/ca/Ca/cu/at/ons, 2nd ed., Wiley, New York, 1976.
5. Bates, R.G., Determination ofpH ; Theory and Practice, 2nd ed., Wiley, New Yo
rk, 1973.
6.
Wfllard, H.H., Merritt, L.L., Dean, J.A. and Settle, F.A., Instrumental
Methods of Analysis,
6th ed., CBS, New Delhi, 1986.
EXERCISE
1.
Carbonic acld has two pKa. pK = 6.35 and pK2 = 10.3. (a) Draw a titratio
n curve indicating
the p, attern of ionization of carbonic acid as a strong base is added. (b) Iden
tify the pH ranges
where buffering occurs. (c) Identify the predominant chemical species at each of
the following
pH values : 4,5,6,7,8,9, I0, I 1 and 12. (d) Identify the pH at which HCO specie
s would exist in a 50-50 equilibrium with its conjugate base.
You are given two buffers : (a) 0.1 M phosphate buffer of pH 7.7 and (b} 0.1 M p
hosphate
buffer at pH 6.71. If acid is to be added to these buffers, which of them, do yo
u think, will
resist the pH changes better?

When the ratio of salt to acid concentration is unity, the buffer has maximum ef
ficiency.
Prove this statement mathematically taking an example of acetate buffer, pK of a
cetic acid is
4.76.
4.
What is the major ionic species present at pH 7.5 in 0.15 Msolution of (
a) leucine, (b) glutamic
acid, and (c) arginine.
5.
The living cell contains several phosphoester compounds. 3-phosphoglycer
aldehyde, whose
structure is given below, is Just an example of such compounds.
CHO
HCOH
CH2OPOI2 -,
The hydrogen at position 3 are dissociable. The first pKa is 2.1 and the second
is 6.8. Can
you now draw the structure of the species of the compound that would predominate
at
physiological conditions7
6.
How is it that buffers of different compositions can have the same pH? F
or example it is
possible to prepare 0.01 M phosphate buffer of pH 7.0 and 0.1 M phosphate buffer
of pH 7.0.

Acids and Bases

65
7.
/k man suffering from untreated diabetes mellitus is admitted to a hospi
tal. Doctors fear that
his blood pH may have dropped because of ketoacidosis. Analysis of his blood rev
eals that
[HCO ] = 16 mM and Pco 2 = 30. If pK of HCO is 6. I., determine whether the pati
ent runs a risk of acidotic coma. (Note. In plasma under physiologic conditions,
concentration of CO2
and Pco2 are related by the solubility constant for CO2 in plasma which is 0.03
mM/mm Hg,
8. After a course of insulin the man is feeling good and his blood pH has become
7.4. [HCO ]
has increased to 21 mM. Cm you calculate the concentration of CO2 in the blood p
lasma of
the patient at this point? Can you also calculate the Pco2?

2
ION SPECIFIC ELECTRODES
It is a well known fact that metal long have a profound effect on cellular proce
sses. The
importance of the role that long play in cellular activity can be gauged by the
fact that most cells
maintain a very critical Na and K balance between the extracellular and the intrac
ellular
spaces. Any disturbance in this critical balance is detrimental to the cellular
metabolism through
a drastic change in the osmotic pressure resulting in cellular swelling. Another
metal ion, Caz,
is known to act as a minatory intracellular messenger stimulating such diverse p
rocesses as
insulin secretion, chemotaxis, endocytosis, and even cellular proliferation. The
se examples are
sufficient to underscore the importance of studies evaluating the activity of me
tal Long in various
metabolic states and processes.
The realisation of the importance and increased number of such studies involving
long
prompted a swift development of glass electrodes so useful for measuring ion act
ivity in biological
samples.
Ion Selective Electrodes Measure the Activity of Metal Long
Biological fluids contain many different types of Long such as Ca2+, Na+, IC, CI
-, HPO4-,
HCO-s, etc. The ensuing electrical interactions between these long will assure t
hat every ion,
while repelling like charged long, will surround itself with oppositely charged
long. Any given
ion can therefore exist in two forms in a biological system; (0 it can exist in
its free ionized form,
or (//1 it can exist complexed with an oppositely charged ion(s), where the net
charge of the
complex is zero. To understand the point better, let us take an example of calci
um. Calcium
can exist in its free ionized form. Ca2+, or as a chargeless specie(s} like CaCO
3, CaCI2, etc. It is,
however, well known that the effects of calcium, such as nerve conduction, muscu
lar contraction,
cellular proliferation, etc., all depend upon the free ionlzed calcium, Ca2. It i
s therefore much
better ff the study conducted and the instrument employed measures the concentra
tion of free
ionized calcium (or the activity of calcium long) rather than measuring the tota
l calcium
concentration. The same logic applies to the study of other long too. In the lat
er pages of this

text book, we will discuss about a technique known as flame photometry (emission
and absorption
flame photometry} which has been employed as an useful tool to measure mineral c
ontents in
biological samples. This technique, however, measures the total quantity of the
mineral present
(domplexed or free ionized form}. On the other hand, ion selective electrodes me
asure the
concentration of the active species of the metal long.
Basic Principles
An ion selective electrode operates on exactly the same principles as a pH elect
rode (see
Chapter 1). In fact, a pH electr0de is a type of ion selective electrode sensiti
ve to hydrogen long;
Just like a pH electrode, the electrode body contains a reference solution and a
n internal
reference electrode. On to this electrode body is sealed an ion selective membra
ne which acts as

Ion Specific Electrodes

67
the ion sensor. Four different types of ion selective membranes are in use. They
are (i) specially
formulated glass, (//) an ion exchanger dispersed inan inert matrix, (/tO a crys
tal, and (/v) a
liquid ion exchanger. The external reference electrode is either a calomel or a
silver/silver
chloride electrode. The potential developed across the ion selective electrode c
an be measured
on a millivolt scale available in a pH meter. This is proportional to the activi
ty of ion in the
sample. More sophisticated instruments employ specific ion meters (high impedanc
e millivoltmeters) which have readout scales directly calibrated in concentration.
The presently available ion selective electrodes may be divided into four catego
ries depending
upon the ion selective membranes that they employ. The four types are discussed
in brief in the
following pages.
Glass Membrane Electrodes
The selectivity of a glass electrode is a function of the composition of the gla
ss. Three
subtypes of glass electrodes and their selectivity characteristics are presented
below.:
Type : pH, order of selectivity : H >> Na > K, Rb, Cs .

>> Ca2

Type : cation sensitive; order of selectivity : H > K > Na > NH, Li >> Ca2
Type : sodium sensitive, order of selectivity : Ag+ > H > Na >> K. Li .... >
> Ca2
The second two subtypes are in general responsive to monovalent cations and are
more or
less unresponsive to anions.
Appropriate adjustments ofglass composition Change the degree of electrode selec
tivity
and also the selectivity order depicted above. Thus, glass can be made more resp
onsive to
cations by adding to it elements which have coordination numbers greater than th
eir oxidation
numbers to alkali metal-silicate glasses (20% Na20 - 10% CaO - 70% SiO2). This t
reatment
excessive negative change to the glass making it suitable to attract cations
having a proper charge-size ratio. Glasses with a composition of 27% NaO - 5% Al
2Oo - 68%
, show a general cation response. If the above composition is modifiea to 11% Na

2( 18%
7 lO./o SiO, the glass becomes highly sodium selective as compared to other alkali metal
. elecfrodes are very sensitive to silver long also, but this does not pose a pr
oblem in
their biological applications.
Electrode stem
- Internal reference
electrode
ljure 2.1. Construction of a typkal glass electrode
Glass electrodes are preferred where the studies involve measurement of sodium,
lithium,
or silver long because of their high specificity for these long. Other desirable
feature of the glass
is that due to its relative inertriess it can be used in non-aqueous media, orga
nic solvents, and

Ag/AgCl
electrode
also in the presence of lipid soluble or surface active molecules. They also sho
w an indifference
to anions present in the sample unless the anions chemically attack the glass.
The glass electrode consists of a stemof non-cation responsive, high resistance
glass on
which is fused a thin walled bulb of cation responsive glass. Figure 2.1 depicts
typical glass
electrode. For further details about the working of a glass electrode the reader
is advised to
refer to the chapter on acids and bases.
Solid-State .Ion Exchanger Electrodes
In these electrodes, the glass membrane is replaced with a solid-state ionically
conducting
membrane. The ion responsive material is an insoluble or sparingly soluble salt
dispersed in an
inert matrix. Often used inert matrices include silicone rubber, polyvinyl chlor
ide, and other
polymeric materials. In order to prepare the membrane, the ion exchange material
is dispersed
through the inert matrix. Such membranes have good mechanical properties and giv
e
reproducible potentials. The membrane so prepared is then cemented to a glass or
epoxy resin
electrode body. The body holds an internal-reference solution and a reference el
ectrode.
Sometimes, the back Of an ion responsive membrane is coated with mercury, and a
platinum
wire is connected to it which works as the reference electrode.
These electrodes are mainly anion responsive and several varieties are available
for
measuring the halide ion concentrations. The ion responsive material for such ha
lide response
electrodes is usually a silver halide.

Reference
halide solution
Membrane
(Silicone
rubber wiib
silver halide)

Figure 2.2. Diagrammatic representation of a solid

/on exchanger electrode.

Solid-state ion exchanger electrodes have

two drawbacks. The first drawback is that they
have a relatively short working life. This problem
has been circumvented by building the sensor
membrane into a removable cap which can be
replaced as required. The second drawback
concerns itself with the extremely high resistance
of silicone rubber and other matrices. In order to
tackle this problem it is necessary that the'
embedded ion exchange material should provide
enough electrical conductivity across the
membrane. This is achieved by careful dispersion
of the ion exchanger so that each exchanger
particle is in contact with each other within the
matrix. Figure 2.2 is a diagrammatic
representation of a solid-state ion exchanger
electrode.

Solid-State Crystal Electrodes

A crystal or pressed pellets of an insoluble salt can act as ion sensiti
ve elements operating
in much the same way as the salt dispersed in an inert matrix. The cryst
al is precision ground
into a disc shape and fixed onto an electrode body. The manufacturing pr
ocess is closely controlled
to avoid the crystal developing an internal crack or leak. The crystal s
hould also not have high
resistance. Examples can be cited of the lanthanum fluoride electrode (m
easure fluoride) and
silver chloride electrode (measures chloride).
A single crystal of LaF3 acts as the sensing membrane in a fluoride elec
trode. However,
LaF3 has a very high electrical resistance. To cancel this detrimental p
roperty LaF- crystal is
'
3
usudly doped .with europium (II) which lowers the crystal resistance and facilit
ates ionic charge
transport. The LaF3 crystal, sealed into the end of a rigid plastic tube, is in
contact with the
internal solution arid the external solution. The internal solution is 0.1 M wit
h respect to NaF

Ion Specific Electrodes

69
and NaCI. The fluoride ion activity controls the potential of the inner surface
of the.lanthanum
fluoride membrane. NaCI is present in the internal solution so that the chloride
ion can fix the
potential of the internal Ag/AgCI reference electrode. This fluoride electrode c
an measure the
fluoride ion activity as less as 10- M. The fluoride ion may also rspond to hydr
oxide ion concentration. However, this does not pose a problem as hydroxide ion
concentration is kept
constant with a buffer.
In a similar manner, as described for fluoride electrode above, polycrystalline
Ag2S
membrane gives a good sulphide ion electrode. Mixed crystals of AgX-Ag2S compose
the anion
selective electrodes for chloride, bromide, iodide, and thiocyanate.

Reference
electrode
Internal
filling
solution
Crystal
Figure 2.3. Cross sectional view of a
crystal electrode.
Conductance in the crystal is a function of lattice
defect. A mobile ion adjacent to the vacancy defect moves
into the vacancy. Only the mobile ion may move into this
vacancy since this vacancy is tailor made for the charge,
size and shape of the particular ion. Thus only the mobile
ion takes part in conductance, all other long being rejected
by th crystal membrane. For example, in a fluoride
electrode only fluoride long can move into the vacancy
defect thereby taking part in conductance. The electrode
thus responds only to fluoride long.
Solid-state crystal electrodes have a life of about 1-2
years. However, if used at high temperatures, their life
gets shortened considerably (1-3 months). Figure 2.3 is a
diagrammatic representation of the crystal electrode.

Liquid-Membrane Electrodes

The sensing element of these electrodes is a layer of organic solvent in which a

n ion
exchanger is dissolved. Most available electrodes use a porous diaphragm (glass
or ceramic
disc) which separates the inner ion exchanger solution from the test solution. T
he ion exchange
solution keeps the disc always saturated (see Figure 2.4). This type of electrod
es are used for
the measurement of calcium, nitrate, perchlorate and other long.
Reference electrode
Ion-exchange reservoir
Internal filling solution
(Reference solution)
Porous membrane
Figure 2.4. Cross section of a liquid ion ehange electrode.
The design of such electrodes is discussed briefly. They have a typically double
concentric
tube arrangement in which the inner tube contains the aqueous reference solution
and the
internal refence electrode. The outer tube contains the ion exchange solution wh
ich saturates
the pores of a hydrophobic porous disc. The pores of the disc are roughly 100 rn
in diameter.
The porous disc, also known as the membrane, is replaceable. Thus, the ion excha
nge solution
can be changed after removing the disc. This process allows change in the ion se
lectivity of the
electrode, selectivity being a function of the ion exchange solution. The liquid
ion exchanger
used for calcium sensitivity is the calcium salt of his (2-ethylhexyl) phosphori
c acid (d2EHP)
dissolved in various straight ch.ain alcohols. Another type of liquid ion exchan
ger used for

70
BoplujscoJ Cherrtstrtj
calcium is didecylphosphorie acid dissolved In di-n-octylphenyl phosphonate. Thi
s calcium
electrode can be changed Into a nitrate electrode ff the calcium sensitive Ion e
xchanger In the
electrode Is replaced by a substituted nickel (II}-1, 10-phenanthrolIne Ion exch
anger which ts
sensitive to nitrate.
Due to the loss of ton exchanger with each measurement, life of such electrodes
Is extremely
short (1-3 ,months). At the end of such periods, the ion exchanger Is refilled a
nd the porous
membrane replaced with a new one. The electrode Is sensitive enough to measure f
airly low
concentration of tons.
Interferences
Two main types of interferences are encountered by ion selective electrodes. The
y. are
(0 method interference, and (tO electrode Interference.
Method Interference can occur when either
c strength
or temperature of the sample beIng tested
asuring fluoride
long, a low pH might give very low values
so sInce fluoride long
form complexes with the hydrogen long and
ivity. The
method Interference can therefore be done
nditions
diligently.

or all of such factors as the pH, ioni

are not properly fixed. Thus, while me
for the activity of this ion. This is
consequently lose their measurable act
away with by fixing the measurement co

Electrode interference.s are mostly due to the passage of an Interfering ion Int
o the membrane
In lieu of the Ion being measured. Thus, high barium ion concentration In a samp
le beIng
measured for calcium can be detrimental to the experiment as barium long can com
pete with
calcium long for passage Into the membrane. Another type of electrode Interferen
ce occurs
chiefly with crystal electrodes. Surface reactions between long present In the s
ample with one of
the components of the crystal can lead to formation of a second Insoluble comple
x on the
crystal. The crystal electrode consequently loses its sensitivity to the desired
ion. Example may
be cited of a bromide electrode (AgBr crystal) which forms a complex with thiocy
anate ion
(AgSCN) altering its sensitivity to bromide long. Sometimes one of the component
s of a crystal
may form a stable cbmplex with anion In the sample being measured. For example,

citrate long
form a very stable complex with lanthanum long. This results In Increased solubi
lity of the
membrane thereby IncreasIng the lower limit of detection of the fluoride ion.
Applications
(0
Activity measurements are valuable because the activities of Long determ
ine rates of
reactions and chemical equilibria. Thus, ion electrodes have been used for predi
cting
corrosion rates, extent of precipitation, degree of acidity, formation of comple
xes,
solution conductivities, and effectiveness of electroplating bath solutions. The
se are
some of the Industrial uses of ion selective electrodes.
(/0
Ion selective electrodes can have many diagnostic applications In biolog
y. For example,
the chloride electrode is beIng used to assay chloride ion activity In the sweat
of babies
as a diagnostic test for cystic fibrosis. Another application with a potential f
or direct
health .use falls In the area of dental research. Calcium and fluoride electrode
s are
beIng used to study the relationship between tooth decay and saliva ion composit
ion.
(/to
Ion selective electrodes are used to study the control of ion transport
b-y tissues and
cells. Example can be cited of very recent studies by Prentll eLa/where Ca2+ home
ostasis
of human neutrophlls was studied under various stimulating and resting condition
s.
Such studies have provided a deep understanding of the role of long In triggerin
g
neutrophil response to an InvadIng antigen. Transport of Na and C1- long have al
so
been studied In various ceils usIng the correspondIng electrodes.

"'X I I Ion-selective
f" ------ "M .----'-',- -" membrane t--.--.--.----.. Re fe r en c e solution
Ge, i .ob ed
subtrate or enzyme
100
100
10-2
100
- 10-I
- 10-2 - 100 - 100 - I0
Chloride
Cupric
Cyanide
Iodide
Nitrate
Nitrite
Sodium
Ion SpecIj Electrodes
71
(/v) Another very stimulating biological application of ion selective electrodes
is that they
can be converted into enzyme electrodes. These electrodes can then measure the a
ctivity
of an enzyme in a given sample [the only action required
on the part of the investigator is to dip the electrode in
the desired sample). For example, the ammonium ion
electrode can be converted into an enzyme electrode
' measuring the activity of urease. Urea is fixed in a gel
membrane which is fastened onto the bulb of ammonium
electrode. The enzyme urease (kO present in the sample
will then act upon urea in the membrane to give rise to
ammonium long which can diffuse through the
membrane to be sensed by the electrode. The ammonium
electrode thus, is converted into a urease sensitive
electrode and can measure the activity of urease in
various samples. There are thousands of other enzyme

substrate combinations that yield products measurable

Figure 2.5. Construction of an enzyme With ion selective electrodes. The design
of an enzyme
e/ectrode,
electrode is provided in Figure 2.5.
As is obvious from some of the applications listed
above, ion selective electrodes can provide much useful information about cellul
ar processes.
to this tremendous utility potential, a lot of ion selective electrodes having s
ensitivity for
long have been developed in recent years. A list of such electrodes along With t
heir
and the long which can interfere in their functions is provided in Table 2.1.
2. I Particulars of Some lon Selective electrodes

Material Detected
Concentration
Ammonium
10-6100
Bromide
10.5 - 100
Calcium
I0-5- I0
i0-s10-7 10-6 -10-7 lO-5
lO-6
lO-6
Potassium
10-5
Lead
10-7
Range (M} Interfering Long and Compounds
CO2, volatile amines
S2-, I-

Zn2+, Fe2+, pb2+, Mg2+

I-. Br-, NO, SO-, F-, HCO;
S2-, Hg2+, Ag2+
S2-, V
S2NO, Br-, ICO
K, Li
H, Cs, NH
Ag. Cu .

GAS-SENSING ELECTRODES
Two gas electrodes are Widely used in biology; the oxygen electrode and the carb
on
dioxide electrode. Whereas the oxygen electrode is used in many diverse branches
of biology,
the carbon dioxide electrode is chiefly used to measure carbon dioxide in the bl
ood. Both of
these electrodes are described b.riefly in the following pages.

72
Biophysical Chemistnj
The Oxygen Electrode
Although a variety of different anode-cathode combinations for oxygen electrode
are
available, the platinum with silver/silver chloride is the most used cathode-ano
de combination. The often found arrangement of these electrodes is annular with
the tubular silver/silver chloride
anode enclosing the platinum cathode. The electrodes dip into an electrolyte sol
ution (usually a
buffered pgtassium chloride solution) which is held inside an electrode by an ox
ygen permeable
membrane. The membrane might be a very thin polypropylene. Polarization of elect
rodes at 0.6
V is achieved with the help of a mercury cell.
The gas electrodes, including the oxygen electrodes measure the partial pressure
of a gas
in solution rather than its concentration. The term partial pressure is often ab
breviated to pX,
where X stands for the gas whose partial pressure is being indicated. Thus, the
partial pressure
of oxygen is indicated as pO2, and that of carbon dioxide as pCO2.
When the oxygen electrode is dipped into a solut/on containing oxygen, the follo
wing
reactions happen :
(i)
The oxygen molecules from the sample diffuse through the membrane into t
he electrolyte
so that within a short time the electrolyte and the sample come to equilibrium w
ith
respect to the pO2.
(//)
The outermost valency shell of each oxygen atom has a vacancy for two el
ectrons upon
acceptance of which it can be turned into an oxygen ion. These electrons are sup
plied
by the platinum cathode. The reaction at the cathode is :
4e-+ 02 + 2H20 = 4OH(//0
The hydroxyl long so produced at the cathode then react with potassium c
hloride in
the electrolyte solution. The reaction in the electrolyte is :
OH- + KCI -- KOH + CI(iv)
The negatively charged chloride ion produced in the electrolyte solution
are attracted
to the positive anode and donate their electrons. The reaction at the anode is :

CI- = C1 + eAg + Cl = AgCl

There is thus a deposition of silver chloride on to the anode.
The overall result of the above reactions is a transfer of electrons from the ca
thode to
anode. This transfer represents a current flow which can be measured and is prop
ortional to
pO2 of the sample.
Calibration of the oxygen electrode is done with the help of an oxygen free samp
le (5 mg
sodium sulphite in 5 ml 0.01 M borax, or water through which nitrogen gas has be
en bubbled)
and another sample with a known pO2 (equilibrating a liquid with air with a know
n oxygen
content). The response of the calibrated-electrode is exactly linear to the part
ial pressure in the
ample.
The current produced by an oxygen electrode is affected by variation in temperat
ure. If the
oxygen concentration remains constant, a temperature variation of IC would induc
e a 5%
variation in the current. It is thus imperative that the sample measurement shou
ld be done at
the same temperature at which the calibration was performed. If this can not be
done, then
temperature correction must be applied before one reaches to a conclusion about
pO2, of the
sample.

73
Ion Specific Electrodes
A diagram of a typical oxygen electrode is provided in Figure 2.6.
Electrode body
Platinum cathode Ag/AgCI anode
Membrane Electrolyte
Figure 2.6. Construction of an oxygen electrode.
The Carbon Dioxide Electrode
The constituents of a carbon dioxide electrode are (/) a conventional glass pH e
lectrode
with a calomel reference electrode, (/0 a thin plastic or teflon membrane which
is permeable to
carbon dioxide and not to other long, and (///) a standard bicarbonate solution,
usually 0.005 M
NaHCO3 between the membrane and the glass electrode.
When the electrode is dipped into a sample containing dissolved carbon dioxide 0
the
carbon dioxide is allowed to diffuse into the bicarbonate solution by the permea
ble membrane.
The pH of the bicarbonate solution changes, and this change is read by the glass
electrode. This
pH change is reflected by the pH meter which is directly calibrated for pCO2. Th
e response time
of a carbon dioxide electrode is higher because the standard bicarbonate solutio
n has to come
Into equilibrium with the sample. The same temperature variation relationship di
scussed with
respect to the oxygen electrode applies here too.
A diagram of the conventional carbon dioxide electrode is provided in Figure 2.7
.
Combined pH and calomel
reference electrode
Standard bicarbonate solution
Membrane
F/gure 2.7. D/agram of/t carbon d/ox/de e/ectrode.

Applications
The oXygen electrode is being widely used in many different biological experimen
ts wherever
there is a need of measuring oxygen. The carbon dioxide electrode on the other h
and is mainly
used for clinical purposes, oft.e.n for measuring the carbon dioxide dissolved i
n blood or plasma.

74
Biophysical Chemistry
Suggestions For Further Reading
I. Lakshmlnarayanaiah, N.. Membrane Electrodes. Academic, New York, 1976.
2.
Koryta, J., lon Selecave Electrodes, Cambridge monographs in Physical Ch
emistry, No. 2.
Cambridge University, New York, 1975.
3.
Willard, H.H., Merrltt, LL. Dean, J.A. and Settle, F.A., Instrumental Me
thods of Analysis,
6th ed., CBS, New Delhi, 1986.

3
THE COLLOIDAL PHENOMENA
Until the middle of the nineteenth century colloidal systems were regarded as be
ing outside
the realm of well behaved chemical systems because they did not behave in a mann
er expected
of an aqueous solution. Such physico-chemical properties of colloidal solutions
as the exhibition
of osmotic, pressure, electrolytic conductance, lowering of vapour pressure, ele
vation of boiling
point, depression of freezing point etc. were different. However, colloids must
constitute extremely
well behaved systems because life is a manifestation of various colloidal states
. All protoplasm
i8 in colloidal form. Most of the biological fluids, notably blood, lymph, milk,
bile, and digestive
secretions are colloidal solutions. Moreover, the biomembranes may themselves be
considered
to be a manifestation of the colloidal state.
Historical Perspective
The study of colloids as a system began with Selml in 1843 when he prepared coll
oidal
suspensions of Prussian blue, sulphur, and casein. He observed that the preparat
ions were not
true solutions but suspensions of a finely divided state of matter in water.
Michael Faraday, a British scientist, prepared aqueous suspensions of gold in 18
57 and
studied the optical properties of this preparation. On passing a narrow beam of
light through
this preparation he observed the path marked out by a cloudy haze. This could no
t be observed
in a true solution. The above phenomenon, which is due to diffraction or scatter
ing of light by
colloidal particles was further studied in 1969 by Tyndall and is today known as
the Tyndall
effect.
The breakthrough in colloidal chemistry came with the work of Thomas Graham, an
Englishman who performed certain fundamental experiments to prove the existence
of colloidal
systems. He observed that solutions of certain substances diffused at a slower r
ate and were
unable to pass through a parchment membrane. Because of their sticky nature he s
upposed
them to be noncrystallizable and called them co//o/ds (Kolla in Greek means glue
). Substances
belonging to the other class which were readily diffusible through membranes and
also could
be crystallized were termed crystalloids. This primitive system of classificatio
n was not fortunate
as it was seen later that many substances which formed glue-like solutions could

be crystallized.
On the other hand from almost all the crystalloids colloidal solutions could be
obtained. It was
soon recognized by Von Weiman that any substance can under suitable conditions,
be brought
into a state of subdivision to be appropriately termed colloidal. Wolfganf Ostwa
ld in 1907 pointed
out that any matter in finely divided dispersed condition implies the presence o
f another phase,
the dispersion medium. Only when these two phases i.e., the dispersed phase and
the dispersion
medium exist as separate phases, the system can be said to be colloidal in natur
e. Thus a
colloidal system is a state of matter and not a type of matter.
With the invention of the ultramicroscope in 1903 the study of the properties of
colloidal
sols became possible. The appearance of a colloidal solution under an ultramicro
scope is like a
milky haze of counfless points too minute to be separately observed. Based on th
e various size
-of partlcles which could be distinguished, a rough classification was suggested
by Siedentopf &
Zsigmondy. According to them, the particles which were distinct in the ordinary
microscope

Brilliant
red-orange
Spinal fluid dilutions with 0.4% NaCI 76 Boptu3sc Chemlstrg
and appeared as well focussed images in We u]tramJcroscope were called mcrons. T
hose wltch
could be observed only in the ultramicroscope were the submcrons and the others
which were
invisible both in ordinary and ultramicroscope were the am/crons.
The stability of colloidal systems was studied at great length. The importance o
f valency
and its influence on stability of sols was studied by Schultz. Hardy added to th
is information by
looking at it from a quantitative angle. Their findings, formulated as "the Schu
ltz-Hardy rule"
are still in practical use today, one of them being clarification of drinking wa
ter by adding small
amounts of calcium salts. Zsigmondy introduced the term gold number while explai
ning the
protective action of one colloid over another. Later, the gold number of cerebro
spinal fluids.was
introduced into medicine as a diagnostic aid to obtain information about certain
types of
diseases, such as meningitis, neurosyphilis etc. (see Figure 3.1).
Co|omNumber
of
sol
5 Colourless
4 Pale blue
3 Blue
2 /Lilac/purple
Red-blue
0
A = Neurosyphilis
B = Meningitis
to

Normal spinal fluid

lgure 3. I, Gold sol curves for cerebrospinal Jluld give djnostic information
Outstanding contributions in the field of colloidal chemistry have been made by
many
other scientists, notably Freundlich {phenomenon of adsorption), Donnan (Donnan
equilibrium)
Svedberg (sedimentation) and Einstein.
What are Colloids?
As stated earlier, colloids represent a state of subdivision of matter. The matt
er, finely
divided, is uniformly distributed in a continuous medium. However, the dispersed
particles are
neither so large that they separate on standing, nor so small that they can be s
aid to be in
solution. This means that the colloidal state is an intermediate state between a
suspension and
a true solution.
A colloidal system is characterized by particles ranging in size from 1 ml to 0.
1 in diameter. They can be formed either by aggregation of small molecules like
sodium chloride or
by disintegration of large polymers. The particles are non-filterable and can be
observed under
the ultramicroscope as illuminated discs engaged in a kind of random zig-zag mot
ion. This type
of movement is called Brownlan movement after the name of its discoverer Robert
Brown. The
smaller the particles, the more rapid is their movement. This unpredictable chao
tic movement

The Colloidal Phenomena

77
of molecules was explained by Albert Einstein in 1905. The surrounding molecules
of the
dispersion medium continuously collide with the colloidal particles. These colli
sions impart
sufficient kinetic energy to the colloidal particles so as to enunciat a Brownia
n movement.
This movement is, however considerably slower than that of the molecules of the
medium and
can therefore be observed under an ultramicroscope.
If the particles cross the upper size limit they will form suspensions. In that
case, the two
phases separate on standing and the particles can be filtered off easily. An ord
inary microscope
is enough to observe the participants of the dispersed phase in a suspension. In
contrast are
the true solutions whose individual members are invisible even in the ultramicro
scope. Only an
electron microscope can be used for this purpose as the size of the particles in
such a system is
always less than I m.
Another characteristic property exhibited by colloidal system is the Tyndall eff
ect. If a
narrowly defined concentrated beam of light is allowed to pass through a colloid
al system it
appears as a white path. The same is not observed in a true solution (see Figure
3.2). The above
effect can be illustrated as follows. Consider a room with a small shutter on on
e of its walls. As
long as the room is uniformly illuminated the air inside the room seems perfectl
y clear and
transparent. But ff the room is now darkened and a concentrated beam of light al
lowed to enter
shutter, we can see the dust particles in the path of the light clearly. This is
due to
', of light by dust particles. Minute dust particles affect the visible light wa
ves to produce
the phenomenon called diffraction. The same principle is followed by colloidal p
articles. True
solutions do not respond to such a phenomenon because its particles are too smal
l to scatter
(A) Copper sulphate solution
":
(B) Colloidal solution of blue ink n water.

Fre 3.2. Tyndall effect. (A) No licjht scattering by a concentrated solution of

copper sulphate; (B) lJht beam is
scattered and appears as a white path through a colloidal solution of blue ink i
n water

The "I,ndall effect is utilized in the ultramicroscope. In this instrument an in

tense light
.beam from an arc lamp is focussed by the lenses of a compound microscope on the
stage of
another high-power microscope with its optical axis at right angles to the first
one. Other light
is excluded from the field. The individual sol particles scatter light and appea
r as bright discs
gainst a dark background (see Figure 3.3). Modern methods have made the use of u
ltraviolet
light to extend the range of microscopic work. Its. short wavelengths permit for
mation of focussed
images of objects as small as I0 mm in diameter. The images though not visible c
an be recorded
photographically. Figure 3.4 shows a simplified version of an ultramicroscope.
View under ultmmlcroscope. (A) Colloidal panicles appear as illuminated discs of
light (B) Particles of
true solution an: not visible " "

78
Solid
Liquid
Sol
Bophysal Chemtry
Hlgh-power
microscope
"
'i Scattered light
Source o
O
microscope
Colloidal solution
O
O

Figure 3.4. Tyndallization and observation in an ultramicroscope

CLASSIFICATION OF COLLOIDS
As mentioned earlier, colloidal systems are composed of two phase of matter. The
dispersed
phase, also called the discontinuous or internal phase is made up of colloidal p
articles while the
dispersion medium, also called the continuous or extema/phase is made up of the
solvent in
which the dispersion takes place. Numerous types of combinations of these two ph
ases are
possible (Table 3.1) and it is difficult to classify colloids strictly. However,
various attempts have
been made and the classification of colloidal systems as two distinct types the lyophilic colloids
and the lyophobic colloids, based upon the interaction of phases has fdund wide
acceptance.
Table :S.1 Typee of Colloidal 8ytem
DiIrd Diponslon Medium Type
Solid
Solid

Solid sol
Solid
Liquid
Liquid
Gas
Solid
Liquid
Solid aerosols
Solid emulsion
Emulsion

Liquid
Gas
Gas
Gas
Gas
Solid
Liquid
Gas
Liquid aerosol
Solid foam
Liquid foam

Alloys, glasses coloured with.

dispersed metals, e.g., ruby
glass, gemsl pearls,
Gold sol, ferrichydroxide
sol, starch in water.
Smoke, dust in air, volcanic dust.
Butter, cheese,
Milk, water in oil, off in water,
cream.
Fogs, mist, clouds.
Pumice stone.
Whipped cream, froth on beer.
Not known (this type is not seen
because of the high rate of
diffusion of gases).

The Colloidal Phenomena

79
Lyophilic Colloids
The word lyophilic stems from the Greek word philos meaning loving (lyo means so
lvent).
Needless to say that the these are colloidal systems where there is strong attra
ction between
the solvent and the particles. Numerous substances of biological interest such a
s proteins,
nucleic acids and the starches fall in this category. Other significant compound
s are the cholic
acids, soaps, synthetic detergents, and the emulsifying agents. Lyophilic colloi
dal solutions
differ from true solutions only with rspect to the size of the particles which l
eads to a change
both in the properties as well as techniques of study of these systems,
A lyophilic colloidal system can take up different names depending on the nature
of the
dispersion medium. When it has water as the continuous phase it is called a hydr
osol, while
With alcohol as its dispersion medium it is called an. alcosol. But the general
term for this type
of system is the emulsoid.
The emulsoids are easy to prepare and though they resemble the true solutiohs in
many
respects, the great disparity in size of the solute particles confer upon them s
ome distinct
physical properties. Additional properties are conferred upon them because of th
eir charged
polyelectrolyte nature. One unique property of colloidal solution is the Donnan
effect. It is
unequal distribution of diffusible long across a semipermeable membrane due to t
he
concentration of colloidal particles on one side of the membrane.
Due to the high degree of solvation of the particles in an emulsoid, the precipi
tation or
flocculatlon of these systems is difficult..They are stable and therefore are pr
ecipitated only by
high concentration of electrolytes. Also, they exist in a state of true reversib
le equilibrium. To
explain this further, the dispersed phase of lyophilic colloids when separated b
y evaporation or
precipitation clump together in loosely packed aggregates. Theseflocs can be rec
onstituted into
the colloidal form easily by removing the electrolyte from the medium and by mix
ing:
The hydrophillc colloids show a higher viscosity than that of the medium. It has
also been
found that the viscosity ihcreases with"concentration. Two reasons are attribute
d to these
observations.

(/)
The hydrophflic colloids have strong attraction for water. This extensiv
e solvation of
the particle immobilizes the bound water and resists its freedom of movement thu
s
. increasing viscosity.
(//)
With higher concentration of solute, there is more trapping of solvent.
Also the colloidal
particles try to orient themselves in a lattice like structure. This systematic
arrangement
affects the flow of the solvent as the solvent now has to find gaps in this stru
cture to
escape or it has to break down this structflre to enable its flow.
, The optical properties of lyophilic systems are also different. Tyndall effect
for these systems
is minimum. Besides the size of the particles, the difference in refractive indi
ces between the
dispersed phase and the dispersion medium also affect tla In emuloid this differ
ence is not appreciable because of the high solvation character of this system.
Therefore
the Tyndall effect is not s.o prominent.
The lyophobic colloids derive their name from the Greek word phobe (meaning fear
ing or
hating) and thus mean 'solvent-hating'. But we carmot call these ystems 'solvent
hating' in the
strict sense because if it was so, no dispersion would be formed. If the solute
molecule completely
repelled the dispersion medium it would have remained dry. No wetting and theref
ore no
dispersion was poible. However, lyophobic systems do form dispersed systems in w
hich the
molecules remain detached from the solvent though not in complete isolation. Thi
s is one of the
reasons of.their instability. -.-

80
Biophysical Chemistry
The lyophobic systems are also called suspensoids. They easily precipitate out f
orming
irreversible flocculates. But if they are left Undisturbed, despite their intrin
sic instability they
remain unchanged for long periods (see Box 3.1). Research has shown that it is t
he electrical
potential difference between the surface and the solution far away from the part
icles that
determirtes the diuturnity (stability) of the lyophobic system. This electrical
potential difference
arises due to the charge carried by the particles of a sol, and the charge carri
ed by the long in
the surrounding medium.
The optical properties of lyophobic systems are worth mentioning. These systems
are
characterized by solute molecules which stay apart from each other. This allows
increased
scattering of light as the difference in refractive indices of the solvent and s
olute particles is
quite high. The.-ndall effect is distinct and therefore the particles of a suspe
nsoid are more
clearly visible in an ultramicroscope. The viscosity, though, is not so affected
as the solvent flow
is not hindered.
BASIC THERMODYNAM/CS OF COLLOIDAL SYSTEMS
In the early 1900's when the physico-chemical behaviour of solutions was being s
tudied
there was a misconception that the basic principles of physical chemistry were n
ot applicable
to colloidal solutions. Since diffusion was very slow, viscosity was high and fr
eezing-polnt
depression was not so remarkable in these solutions, workers were doubtful wheth
er the laws
of thermodynamics would be obeyed. We now know that colloids do conform to certa
in
fundamental physico-chemical laws as systems containing only. small molecules or
long, only
the scale is different. To understand this, let us first look into the factors w
hich are the sources
of non-ideality of colloidal solutions.
The two factors which determine the solution behavi0ur of biopolymers are (1) th
e great
difference in size of the molecules of the solute with the solvent. (ii) the bon
ding interactions
and frequently charged nature of the solute.
( Size: A feature of solutions of macromolecules is that the solute molecule is
many times

larger in size than the solvent molecule i.e,, the solutions are more concentrat
ed in terms of
weight or volume percent while their molarity is still very low,
One of the determinants of the equilibrium state of a system of many particles i
s the
randomness or disorder of the system. According to the second law of thermodynam
ics, the
term entropy is used to denote such a randomness in a system. In an ideal soluti
on it is assumed
that the solute molecules are of the same order of size as the solvent molecules
so that the
distribution of solute molecules in the solvent is entirely random as is seen in
the case of true
solutions. But this cannot hold true for macromolecular solutions because of the
high solute to
solvent size ratio. In these solutions the centre of each molecule is excluded f
rom a volume of
solution because of the high solute to solvent size ratio. In these solutions th
e centre of each
molecule is excluded from a volume of solution because it is temporarily occupie
d by some part
of another macromolecule. The departure from ideality thus depends on this molec
ular xcluded

... (I)
... (2)
... (3)
... (4)
...
The Colloidal Phenomena 1
volume (6). The quantity B, the second virial coefficient, which serves as a con
venient measure
of solution non-ideality is related to '#' by the equation.
NO
B- 2M2
where N is the Avagadro's number, and M is the mo!ecular weight.
The excluded volume is determined.by the actual molecular dimensions.
8Mv
For spheres, N
4v
so that
B M
2LMv
and for rods, 6 Nd
Lv
so that
B dM

v is the specific volume,

L is the rod length,
d is the diameter of the rod.
The globular macromolecules contribute less to non-idcality of a solution than t
he
asymmetric rods or random coils because the permutations and combination of arra
ngements
possible in a solutiol for the latter are always less in number than the former.
(ii) The bonding interactions : When a colloidal dispersion is prepared, the sol
ute-solute
i Interactions and the solvent-solvent interactions are broken with utilization
of energy and new
bonding nteractions, the solute-solvent interactions are formed with release of
energy. This
liberation of energy is the driving force for mixing. If we do not consider the
'entropy' term
described earlier, dissolution of a solute in a given solvent will be favoured o
nly if the solventsolute interactions are great enough to overcome the solute-solute interactions
and the solventsolvent interactions.
The description of thestate of a solution needs to specify the amount of compone
nts
present and the temperature and pressure. If the temperature and pressure are as
sumed to be
constant, in an ideal solution,
(A)
The internal energy = sum of internal energies of its components in thei
r standard
states, i.e. E or energy change between system and surroundings is zero, and
(B)
The volume = sum of the volume of the components is their standard state
s, i.e. AV
or volume change for mixing is zero. When the volume change is nil at constant p
ressure.
the change in enthalpy (heat content) is also zero.
(C) On mixing, there is a great disorder so that AS or change in entropy is posi
tive.
The combination of the statement of the first and second laws of thermodynamics
gives
us the equation,
.F= &H -T&S
... (6)

82
Biophysical Chemistry
It follows from the above equation that if AS. for mixing is positive, &
F for mixing will be
negative. Only under these conditions, the reaction will proceed spontaneously t
o form an idea]
solutlon.
Since it is the free energy of a solution which is a property of interest, we mu
st know the
contribution of each component of a system to the total free energy. The partial
molar energy is
the amount of free energy contributed by each component of the system. It is an
intensive
variable and is often called the chemical potential, symbolized by ''.
The total free energy Of the solution is then
F = Z nl]i
... (7)
i=]
where ni is the number of moles of component L The chemical potential is the int
ensity factor of
a chemical reaction and may be regarded as its driving force. It also determines
the direction in
which the net reaction will proceed. When the value of AF = 0, the system is sai
d to have
attained equilibrium. In that state, the chemical potential of any com .ponent m
ust be constant.
It also follows that tl must be identical at all places in the system, Otherwise
there will be
further reaction or migration of matter from a region of higher chemical potenti
al to a region of
low chemical potential.
For solutions of macromolecules, the chemical potential of the solvent is import
ant as
many macromolecules interact strongly with the solvent or with one another. In '
poor' solvents,
the solute-solute interactions are preferred. This increases the solvent chemica
l potential and
enthalpy change then is not equal to zero (AH 0). The virial coefficient win be
negative. On the other hand, in 'good' solvents, there is preferential attractio
n between the solvent and solute
molecules so that the solute molecule tends to extend itself and the entropy inc
reases. The
excluded volume is larger in such solvents and the virial coefficient is positiv
e because there is
a great decrease in solvent chemical potential. Both the above cases are deviati
ons from ideallty.

There. are of course, other sources of nonideality in a macromolecular solution

in addition
to the excluded volume. To make a solution behave as an ideal one, a temperature
can be found
(0 temperature or Flory temperature) at which the above enthalpic and entropic t
erms cancel
each other. The second virial coefficient, B, then becomes nll and the solution
behaves ideally.
The lyophilic colloids mostly behave as true solutions, form spontaneously and a
re stable
thermodynamically. On the other hand, the lyophobic colloids are always unstable
thermodynamically. Despite inherent instability, many of the lyophobic colloidal
systems
persist indefinitely. This is attributed to the electrical charge carried by the
colloidal
system. A detailed discussion of the electrical properties of colloids will be d
ealt with
elsewhere in this chapter.
When a warm concentrated solution of starch is cooled, it gradually sets into a
more or
less rigid mass. This product is called a gel and the process by which it is for
med is termed
gelation. Gelation is a property possessed by lyophflic systems. Many factors li
ke pH, temperature
and, concentration of solute as well as electrolytes in the medium have profound
influences on
this process. Change in H concentration can bring about a change in the molecular
constitution
of a colloidal particle thus altering its power to gelate. At higher temperature
s gels generally
liquefy, while cooling favours gelation. With increasing concentration of the co
lloidal particle,
gelatlon is favoured. Also, the gel structure is more firm with higher concentra
tion of solute.
The gIs are thought to possess a definite structure. Nageli described a mesh lik
e structure
for gels. The gels are so firmly set that it becomes very difficult to squeeze o
ut any solvent which

The Colloidal Phenomena

83
is trapped or absorbed within the network of the interlacing fibrils. On standin
g for a sufficient
period of time, most gels gradually contract, extruding a portion of the dispers
ion medium, the
process being known as syneresis. Avery good example of a colloidal system trans
iting into the
gel form is blood clotting. Blood exuding from the wound gets more and rore visc
ous to form
the clot eventually. The clot when examined under the microscope appears as a cl
ose network
of fibrils (fibrin) in which are trapped the blood cells and other liquid compon
ents. The clot is a
rigid structure but on standing it shrinks, exuding a transparent fluid, the ser
um.
Gel formation is not always an irreversible process. Though coagulation of prote
in .and
clotting of blood axe irreversible, solid agar or gelatin formed by the cooling
process can be
brought back to their original lyophilic colloidal solution forms by gentle warm
ing. Gels may
thus be classified into two types, elastic and non-elastic. The elastic gels eve
n after complete
dehydration can be brought back to their original state by addition of water. So
me examples of
this class are agar agar, gelatin etc. The non-elastic ones, like silica gel are
irreversible
An entirely different process may also form gels. Lyophilic colloids, in their s
olvent free
forms, spontaneously imbibe considerable amounts of liquid solvent or solvent va
pour, increasing
in volume and setting into a more or less rigid mass. This process is known as s
welling. The
amount of solvent imbibed may be very large and it is held firmly within the hon
eycomb-like
structure of the gel. If this process is not checked, swelling may continue inde
finitely, leading to
a lyophflic dispersion which gets diluted more and more. But as observed, swelli
ng does have a
limit. It continues as long as the free energy of the system decreases. Once the
macromolecular
chains have become fully extended, swelling will virtually stop. This can be exp
lained in terms
of entropy. As the chains extend to their full, they become more and more ordere
d. This decreases
the entropy of the system with a concomitant increase in free energy. A balance
which results
between the decrease in free energy, (due to solvation of the macromolecule by t
he solvent) and
increase in the free energy brings the whole swelling process to a standstill.
Presence of long in the medium affects swelling. This is due to the high solvati
on
characteristics of the individual long. Some long increase the limit of swelling

while some decrease

it. Viscosity of the gel decreases as swelling continues.
Some gels which are in a semi-solid state can revert back to liquid sol on agita
tion. On
standing the sol reverts back into a gel. This sol-gel transformation without ch
ange of temperature
is referred to as thttropy and it has great biological significance. It can also
be brought about
by a change in hydrogen ion activity or by changing electrolyte concentration. P
rotoplasm is
said to have thlxotropic properties. In fact, the complete mechanism of muscle c
ontraction
involves an elaborate gel-sol transition process controlled by the activity of c
alcium long.
Association Colloids
We have a little earlier discussed the nature of lyophobic systems which are inh
erently
ffnstable and the lyophilic systems which ae thermodynamically stable and are ma
de up of
macromolecules having great solvation power. There exists another class of collo
idal systems
which, though thermodynamically stable, are made up ofmicelles. A micelle is a s
pontaneously
formed aggregate of micromolecules. This aggregate achieves colloidal dimensions
and is a
thermodynamically stable structure. The dispersion finally formed is called the
association
colloidal system.
A striking feature of the association colloids is that the molecules formlng it
are amph/path/c
in nature i.e., the molecules possess both lyophilic and lyophobic groups in the
ir structure. The
lyophobic components form the core of the micelle (aggregate) while the lyophili
c components
are on the surface extending their polar groups into the aqueous solution.

84
Biophysical Chemistry
Because of tiffs dual character of its molecules, association colloids are of gr
eat practical
utility. Most of them are surface-active substances. Examples of this class are
the soaps and
detergents like sodium dodecyl sulphate. While some are excellent solubflizers o
f various types
of organic compounds in water, others are good dispersion stabilizers.

85
The Colloidal Phenomena
Colloids
There are many insoluble constituents in the biological fluids like urine and bi
le. But,
they do not-precipitate out normally. Only under abnormal conditions they
the form of gallstones and kidney stones. How is a normal person safeguarded
pathologies ? The answer lies with protective colloids. Protective colloids or p
eptlzing
as they are often called, do not form a separate type or class of colloidal syst
ems..They
nothing but lyophilic colloidal systems which when added in sufficient quantitie
s to a
sol hinder its precipitation by electrolytes (see Figure 3.5). In other words, t
he
is protected by the emulsoid. The emulsoid therefore, acts in this,case as a pro
tective
An opposite phenomenon occurs if the recipe is altered, the ingredients remainin
g the
called sensit/zat/on. In this, if a small amount of the lyophflic system is adde
d to the

86
Biophysical Chemistry
lyophoblc system, precipitation is facilitated. The suspensold gets sensitized a
nd is precipitated
out by a very small amount of electrolyte. This is because the lyophflic colloid
is now enveloped
by the lyophobic system facilitating flocculation (see Figure 3.6).
Aggregates form
(1 ml, 10%) Na. , --Na
The particle is charged Na
due to adsorption
of anions
. Albumin or
Gelatin
Charge on the
particle is reduced
to zero by the
electrolyte added
Neutralization
of charges lead
to aggregation
and precipitation

,,.- -.-,.=. -: - -'

-- J"-- ,
Precipitatton
t- - .--.,,
.- L' .-.a' --'..--" --'.?
-'-"
-'"-t
"-',..o"
Water film
|
a,----*'on of water
narge on me
t

fails

- o de
thou reduced
further proection)
is not effective to
cause precipitation
Protective Gelation Albumin film
Fjure 3.5. Protective colloidal action on a hydrophobic so/.
&
7 Lyophobic colloidal particle
& Lyophllic colloidal particle
Sensitization
Protection
Figure 3.6. llustratlon of sensltlzation and protection by a lyophoblc colloidal
solution.
The protective action of various lyohflic substances was studied by Zsigmondy. H
e advised
a quantitative measurement called the Go/d Number in order to measure the relati
ve protective
powers of hydrophflic colloids. The Gold Number is the weight in mgs of protecti
ve colloids that
when added to 10 ml ofa Zsigmondy gold sol (contains negatively charged hydropho
bic colloidal
particles), just fails to prevent the colour change from red to blue on the addi
tion of I ml of a
10% solution of sodium chloride. The smaller the gold number, the greater is the
protective
action of a particular lyophilic substance. With the unprotected gold sol, the a
ddition of sodium
chloride brings about charge neutralization, aggregation of the sol particles, a
nd finally,
precipitation.
The .gold number of various fluids of our body presents a picture of our health
profile and
thus c.ap be used for diagnostic purposes by a clinical chemist.
Examples of protective colloids are gelatin, gum arabic, albumin etc.

Th Co//o/da/Phenomena
87
" PROPETIF. OF COLLOIDS
We have earlier discussed the kinetic properties (Brownian movement) and the opt
ical
properties (Tyndall effect) of colloids. The behaviour of colloidal systems can
be understood
properly only when we consider the electrical properties of its constituents as
well as the system
-as a whole.
Electrical Properties of Colloids
The colloidal systems we frequently ncounter are the solutions of biological mac
romolecules
or biopolymers as they are often called. Majority of these biopolymers are elect
rically charged.
The charges may be 'strong' or Xveak' depending on the ionization constants of t
he acidic or
basic groups: The distribution of the charges on the macromolecules may be symme
tric or
asymmetric, and this is independent of the net charge carried by the macromolecu
le. The origin
of these charges may be either extema/or/nternaL When the charges are acquired b
y adsorption
of positive or negative long from the medium, it is called external. It may also
happen that the
charges arise due to active acidic or basic groups present in the inherent struc
ture of the
macromolecule which remain exposed on the surface. In such a case the origin is
said to be
internal.
Consider a spherical colloidal particle witha negatively charged surface. This p
olyelectrolyte
will tend to attract the positive long from the dispersion medium around itself.
As a result, the
macromolecule will soon be surrounded by an/on atmosphere, a region in which the
re will be a
statistical preference for long of the opposite sign. Helmholtz likened this sit
uation to the charge
distribution in a parallel plate condenser. The condenser consists of two concen
tric spheres of
opposite signs, and the two plates of the condenser constitute the so called r/g
/d doub/e/ayer
(see Figure 3.7).
Q --Charge due to adsorbed layer of long
Q = particleCharge carried by the spherical colloidal

Outer sphere of oppositely charged long

Figure 3.7. Helmlwltz "Double layer" model.
With more contributions from Gouy, Debye and Huckel0 a modified theory was propo
sed
to explain the charge distribution around a macromolecule. According to this ele
ctrical double
layer .theory, the long of charge opposite to that of the surface (called gegen/
ons, from the
German gegen meaning against) concentrate and tend to orient as an immobile laye
r on the
surface. As the distance from the surface increases, mobility of the long increa
ses and the ion
layers become more and more diffused (see Figure 3.8}.
If +Q is the charge on the spherical macromolecule, r is the radius of the spher
e, -Q is the
charge on the adsorbed ion layer, and d is the thickness of the double layer, th
e potential at any
point on the surface of the macromolecule will be given by +Q/Dr where D is the
dielectric
constant of the medium. The outer layer of oppositely charged long reduce the po
tential on the
spherical colloidal particle by -Q/D (r+d).

88
Biophysical Chemistry
.- -[ , Compact layer (surface of shear)
- I---.-.--" 4. ,. ---\
"
-- - --" "----',- - ....
r" "" -.parttc-t-e- q+ -#- \- -r---- Mobile long in liquid around X.-- .- -- /" -- -v --- -" the particle {
Diffuse layer)
-[L-r.- Positively charged colloidal particle
I
t
, Immobile layer of negatively charged
long adsorbed upon the surface
from the liquid
Fire 3.8. The electrical double layer around a spherical colloquial particle whi
ch is posvebj charged
The net potential between the immobile layer of long and the diffused mobile lay
er of long
is called the e/ectrok/twttc or zeta potential and is represented by 4.
= Dr D(r+
= (r +d}-r@
Dr (r+d)
" .
Qd
Dr(r +d)
...
The electrical double layer in the phase boundaries produces the -potential as a
result of
lectrostatlc and adsorptive interactions. The zeta potential has a very close re
lationship to the

stability of a sol. The zeta potential of a sol can be very effectively reduced
by addition of
elee'rolytes. The electrolytes decrease the zeta potential to a critical value,
after which
neutralization of the charges takes place resulting in the collapse of the doubl
e layer. When this
happens, flocculation of the colloid takes place. Various other factors which af
fect zeta potential
are surface charge density, dielectric constant of the medium and thickness of t
he double layer.
Great slgrtiflcance has been attached to zeta potential in understanding various
precipitation
reactions of our body, one of them being blood coagulation. During this process,
zeta potential
is reduced below the critical value (mainly due to the increase in calcium long
in the medium)
enabling the dispersed particles to form aggregates. This occurs because the dou
ble layer breaks
down at the 'critical potential' facilitating coalescence of the particles. A fl
occulate in the form of
a clot appears subsequently to stop blood flow.
Existence of charged layers on and around a colloidal particle gives rise to man
y other
potentials besides the zeta potential. The layer of oppositely charged long whic
h is in immediate
contact with the colloidal particle, i.e., the immobile layer, is also called th
e Stem layer. The
potential between this layer and the particle surface is called the Stern potent
/aL The potential
difference between the surface of the particle and all the ionic layers in the s
olution surrounding
the particle is called the electro chemical potential and is represented by E.

Colloidal Phenomena
89
The three potentials mentioned above are related to each other by the fo
rmula
Stem Potential = E ... (9)
(Electro chemical - Zeta Potential)
The above relationship shows that zeta potential is a little lower than the Stem
potential
I this is because it is located further out from the surface of the macromolecul
e. The zeta
of any colloidal solution can be calculated by electrokine*ic measurements like
electro-osmosis and streaming potential. Though the methods are different, all
the same calculated value of the zeta potential fcr any particular system. All t
he methods
the relative motion of the two surfaces in contact.
For the quantitative treatment of electrokinetic phenomena, various assu
mptions have to
First, the double layer is likened to a parallel plate condenser where the plate
s are 'd'
apart and each carry a charge 'q' per sq.cm. The potential difference created be
tween the
plates may be assumed to be equal to zeta potential which now can be written in
form,
4ndq
C ... {lO)
D
D represents the dielectric constant of the medium. With the above equation the
deduction of the zeta potential by the different types of electrokinetic phenome
na
(i) By electrOlahore$i: Movement of macromolectles in an electric field may be o
bserved
measured with a microscopic equipment known as the microelectrophoretic cell. Th
e design
[such an apparatus can be seen in Figure 12.3.
If oppositely charged electrodes are dipped in a solution of macromolecules, the
particles
to migrate in the electric field which is thus created. The velocity of movement
given by

law would be :
EQ
V = -... (11)
E = electric field in volts per cm felt by the particle
Q = moving charge
= viscosity of the medium.
There will also be a frictional force operating which will tend to drag the solv
ent in the
direction resulting in retardation of the velocity of movement of the particle.
When the
force and the frictional force balance each other the particles move with a less
er
velocity given by
DE
4q
... (12}
or
= --

... (13)
DE

Anode
Cathode
Solvent
90
Boptujsc Chemtrv
us, measurement of ze pote revolves the measemet of e ve]ocl of e
cogod pc]es der pressed e]ecc e]d as e vue of oer components of e equaon (13) be
o eas.
iO e: In 1808 Reuss t obseed e flow of water mu a membe of powdered qu when a po
ten derence was applied. Based on is ely eeent,
eoreflc studies elecoecs were made d sever tecques have been ds to study effecve
ly e behaour of macromolecules soluon.
In o preous scuion we studied bef e movement of e cooid pcles under e uence of e
lecc fld. If e pcles e now forcibly stopd from mog, we see at e dispersion meum
now ss mog. is movement of fld elecc field is ced ecss or ecoss.
To delve equation reg e -poten to e eleco-osmoc flow, appatus c set up as foows.
: A U-tube is dided to o m compen bya blk of gel ed side e tube (s gure 3.9). e
whole to is now a solvent.e gel, wch is fact a ckened fo of a lyopc cooid cot mo
ve but it c ow e Ivent molecules to
svee ou it. en elecc coecon is made beeen e o s of e U-tu ou a p of elodes. e so
lvent is seen to se one ofe . Ife solvent ter, e ter flow u e gel tod e negave p
ole because e ter acqs a sive chge (it t be mfloned here at e pous gel membe beh
aves as enoous set of cap robes). Dung e flow of ter ou e gel, e is pferen
adsoon of OH- long. e s of e 'pes' lea e H long to chge e war molecules posively
'.
Original - " Flow of : :
Blek of gel
Figure 3.9. Illustration of electro-osmosls
If W" is the volume of liquid flowing through the capillary per second, then
V = -r2ED
41I
4Vql
{ = r2ED

The Co//o/da/Phenomena
where r = radius of the capillary,
E = potential applied in volts,
= Zeta potential,
91
length of the capillary,
dielectric constant of the medium,
viscosity of the medium,

For a parchment membrane or a colloidal gel 'r" and T are very difficult to dete
rmine. To
clrctunvent, these factors, a different term 'K' was evolved. 'K' is the specifi
c conductance of the
liquid in the capillary, and is equal to
I = strength of current,
a .= cross-sectional area of the capillary,
E = potential gradient in volts cm-:
The relation between the ,potential and the rate of electro-osmotic flow through
a
then stands as
4 K rIV
=

... (15}
DI

The above equation can be used to determine -potetial. The equation states that
at a
electr/c current I, the volume of liquid 'V" flowing through a membrane is direc
tly
to the zeta potential and the dielectric constant of the medium and inversely
to the viscosity '' and the specific conductivity 'K' of the I/quid.
{i//) By streamh potent/a/: A third type of electrokinetic phenomenon observed i
s the
streaming or flow potential when a liquid like water is forced to flow through
A potential develops on either side of the capillary at its two ends (see figure
Glass
capillary
wall

OH"
OH: OH- OH- OH- OH- OH- OH- OH- OH'
H" H*
H
H
H
H
H
H
H"
H* H*
OH" OHOHOH"
OH"
OHOH"
OHOHOH"
Glass
capillary
wall
()
The nature of the potential depends upon the pressure applied as well as nature
of the
It has been suggested that the streaming potential results due to forcible flow
of liquid
tends to separate the oppositely charged layers of the electric double layer. St
reaming
is related to zeta potential '' by the equation.
= .4 KS
PD
... (16}

92
where
Biophysical Chemistry

S
= streaming potential
P
= pressure applied to the streaming liquid
D
= dielectric constant of the medium
K
= specific electrical conductivity of the liquid, and
3 = viscosity of the medium.
Another method for study of electrokinetics is the determination of sedimentatio
n potential.
The phenomenon is known as Dom effect after the name of its discoverer. It measu
res the
electric potential established by the movement of the colloidal particles with r
espect to a Sationary
dispersion medium. The particles move under the impact of a mechanical force, th
e gravitational
force. Normally the potential derived by this method is very negligible, but it
assumes great
significance during centrifugation. When a colloidal solution is allowed to sedi
ment in an
ultracentrifuge under the centrifugal force, a potential difference of measurabl
e dimensions
develops across the ends of the solution.
Precipitation of Colloids
The stability of the two general classes of colloids, the lyophllic and lyophobl
c systems,
differs mainly due to two factors.
(0 charge on the colloidal particle, and
(/0 the degree of solvation.
The hydrophobic colloids (suspensoids) can be flocculated easily by addition of
small
quantities of electrolytes. When a dilute electrolyte solution is added dropwise
to a colloidal
solution and mixed thoroughly after each addition, it is observed that a point c

omes when after

addition of a single drop, rapid aggregation takes place leading to the formatio
n of a coarse
flocculate. The volume of the added electrolyte at this point gives the.floccula
tion value of the
colloidal system.
The flocculation values of lyophobic colloidal systems are lower than those of l
yophilic
colloidal systems. This in turn means that the lyophillc systems are more stable
. Why? Two
movements take place continuously in a colloidal dispersion. One is the Brownian
motion and
the other is the convective movements of the dispersion medium. Both of these wo
uld normally
bring the particles close to each other continuously. Though there are ample cha
nces of contact
of particles, it is seen that coalescence does not occur. This is due to the fac
t that the colloidal
particles are charged. The hydrophobic colloidal particles are electrically char
ged alike and
repel each other. The electrolyte long gets adsorbed upon some of the colloidal
particles,
neutralizing or reversing the charges of the latter. These colloidal particles n
ow serve as nuclei
which grow rapidly by aggregation of other particles finally leading to precipit
ation. If the
electrolyte solution is added rapidly and in excess, with vigorous stirring, pre
cipitation does not
take place. The reason is that charges on the colloidal particles get reversed s
imultaneously so
that the particles continue to repel each other and remain separated.
The effectiveness of a-salt solution to precipitate a colloidal solution also de
pends on the
valency of the oppositely charged ion. Higher the valence, greater is the effect
iveness.
The stability of lyophilic systems (emuIsoids) lies in the fact that the particl
es have a very
strong attraction for the solvent in addition to the electrical charge which con
fers stability to a
sol. As long as the particles remain solvated, neutralization of the charges alo
ne will not bring
about precipitation. Similarly, dehydration alone will only convert the emulsoid
to a suspensoid
but not precipitate it. This property of lyophilic colloids has great biological
significance as most

Colloidal Phenomena
93
physiological colloidal dispersions are of the lyophilic type. Living matter the
refore can
slight changes in its content of inorganic long without disaster. Effective prec
ipitation
lyophilic colloid requires the addition of a large quantity of soluble salt. Thi
s is known as
process, thepower of which depends upon the nature of both the cation and the
anion of the salt. Depending on their ability of dehydration of other particles,
the long are in series called the /yotrop/c or the Hofmeister series. This seri
es determines the
i out power of the different electrolytes (see Box 3.4). Thus addition of large
amounts of
dehydrates as well as neutralizes the charges on the colloidal particles simulta
neously
md causes precipitation. The reversibility of the precipitate of an emulsoid to
the dispersion
forri is also vez difficult to achieve and for this reason they are also known a
s irreversible

94
Biophysical Chemistry

Colloidal Phenomena
95
Besides electrolytes, H and OH- also affect the stability of colloids. While OHLong
the negative zeta potential H long have an opposite effect. Thus by varying the v
alue of
potential to a 'critical potential', precipitation is facilitated. Various other
physical and
heat, UV radiation, acids and alkalies,
mechanical disturbances like ultrasonic vibrations, high speed shaking or stirri
ng can
the conformation of the colloidal particle and thus precipitate it.
Another effective method of bringing about precipitation .is by mutual cancellat
ion of charges.
can be done by adding a negative suspensoid to a positive suspensoid or vice-ver
sa. This is
as mutual precipitation of colloids.
DONNAN EQUILIBRIUM
In a
dispersion, if the particle is a macromolecule o polyelectrolyte nature,
the genel:al physicochemical propexies may arise. The British physicist
in 1911, showed that when two solutions of electrolytes are separated by
a
membrane, potentials arise at the junction. This happens when movement o
f
of the long through the semipermeable membrane is hindered. The hindranc
e may
to the colloidal nature of the ion or the electrolyte may be chemically bondedto
an
like an ion-exchange resin on one side. In addition,
Losmotic pressure difference between the two compartments is observed at equilib
rium. The
for these apparent anomalies was provided by Donnan and therefore the
Donnan membrane equilibrium bears his name to this day.
Let us consider a system to be divided into two compartments by a semipermeable
water to pass through but not the large long of colloidal
In compartment A is present a solution of salt NaR of concentration 'a' where Ris
anion of polyelectrolyte nature. Compartment B is occupied by a solution of NaCl
of
'b'. NaCl will now diffuse from (B) to (A) and some of it may reversely diffuse

back
(B). This continues till the system attains equilibrium.
rlf 'x' represents the net concentration of NaCI that has diffused from (B) to (
A), then at
the situation in the compartments will be depicted as in Figure 3.11 (a and b).
(A)
(B)
Na
(a mols)
Na
(b tools)
()
CI(b mols)
{a
mols)
Figure 3. I i. (a) Distribution of ions.across a semipermeable membrane at zero
time.
(A) (B}

Na
(a + x mols}
(a mols)
Cl{x tools}
Na
(b x mols}
CI(b - x mols}

Figure 3.1 I. (b) Distribution of long across a semipermeable membrane at equili

brium.

96
Biophysical Chemlstr
The rate of diffusion of NaCI from (B) to(A) is proportional to the product of t
he concentration
of Na and C1- in (B) and the rate of diffusion of NaCI from (A) to (B) is proport
ional to the
product of the concentrations of Na and CI- in (A). It might be mentioned here th
at in very
dilute solutions, activity terms can be replaced by concentrations. The situatio
n is represented
as below :
[Na]B [CI-]s = [Na]A [CI-]^
to-x) to-x} =(a+x} (x}
The above equation shows that while in compartment B the concentration of Na and
CI- is the same, in compartment A the concentration of CI- is less than the conc
entration of
But as electroneutrallty requires the presence of equal numbers of cations and a
nions in each
compartment, it follows that
[Nals = [CI-] and
iNal^ = [R'l^ + |el-|^
where [R-I^ is the concentration of the restricted ion in compartment A. The abo
ve equation
points out the fact that the concentration of the diffusible anion is less in th
e compartment
containing the non-diffusible ion than in the other compartment. The restriction
of the nondiffusible anion at the membrane while the cation is tending to diffuse through
it, sets up.a
potential difference between the two solutions. At equilibrium, the solution in
the compartment
A acquires a positive potential with respect to the other because of the slight
excess of Na long.
Thus we have seen that the presence of a non-diffusible ion on one side of a dia
lyzing membrane
causes unequal distribution of every diffuslble ion on the two. sides of the mem
brane. While the
positive diffusible long concentrate in the compartment containing the negative
non-diffusible
ion, the negative diffusible long are more to be seen on the opposite side. The
relative
concentrations of the non-diffusible and the diffusible long is important in a s
ystem in Donnan
equilibrium. If the concentration of the non-diffusible ion is increased, a grea
ter inequality in
ion distribution is observed. This leads to a further increase in membrane poten
tial. On the
other hand, the greater the concentration of diffusible long, smaller is the mem
brane potential

due to the masking of the effect of non-diffusible long. Therefore, the Donnan e
ffect can be
decreased considerably at high electrolyte concentrations.
The osmotic pressure difference observed at equilibrium due to inequalities of d
istribution
of diffusible long in the two compartments can be minimized by dissolving the po
lyelectrolyte in
a salt solution of high molarity (0.1 M or higher) and then using the same salt
solution in the
other compartment. Another modification which can be adopted for protein solutio
ns is to
select a pH very near to the isoelectrlc pH of the protein so that the net prote
in charge is
reduced to a minimum value. Under these conditions, the flow of the oppositely c
harged diffusible
ion across the membrane and its concentration in the compartment containing the
protein
molecules will be decreased. The osmotic pressure in a system exhibith-lg Donnan
effect can be
calculated by the formula
P--cRT
where c = concentration of the long
R ffi gas constant T = temperature
At 27C, the osmotic pressure of a system of the following type can be calculated
thus :

97
5.10 mols

The Colloidal Phenomena

Na (a)
R{B}
Na {b}
Cl- {b}

(at zero time)

Semipermeable membrane
Let a = 3 mols and b = 4 mols, then amount of NaCI diffusing from (B) to (A), re
presented
by 'x' will be,
X
a +2b
This is so because at equilibrium
,
[Na+]s [CI-]s = [Na+]A [CI-]A
or
or
or
or

(b-x}(b-x} = {a+x}(x}
b2-2bx + x2 = ax + x=
b== ax + 2bx
b= = x {a + 2b}
b=
X
a+ 2b

Putting the value of a and b n the above equation, we get

(4)2
16
16
= 1.45approx.
x - (3 + 2 x 4) - 3+8
11
..
Now, calculating the concentration of each of the long in the two compartments w
e

Na
(a.+x) = 4.45
{B)
Na
(b-x) = 2.55

CI(x) = 1.45
R(a} = 3.0
8.90 tools
Cl(b-x) = 2.55

Since the osmotic pressure is given by the equation,

P =.cRT

98
Biophysical Chemistry
by substituting all the values we get,
P = (8.9 - 5. I) x 0.082 x 300 (as 27C = 300K)
= 3.8 x 0.082 x 300
= 93.48 atmospheres
Biological Significance of Donnan Membrane Phenomenon
The Donnan effect plays a significant role in our body because of two basic stru
ctural
organizaUons.
(0
Most of our body membranes are dialyzing membranes, i.e., they allow the
movement
of water, gases like O and COa, long and small organic molecules across them whi
le
disallowing colloidal particles, and
(//)
The non-diffusible protein anions are present in large quantities in cel
ls and in plasma
but not in interstiUal fluid.
Both the above factors promote unequal distribution of diffusible long on either
side of
biological membranes which in turn would result in establishment of a pH differe
nce and.an
osmotic difference between the compartments. Minor pH changes are tolerable for
most of the
tissues but drastic pH changes may pose problems. The body saves itself from the
h .armful
influence of the latter with the help of its buffer systems. But sometimes maint
enance of a
drastic pH difference is a necessity, as is in the case of stomach. Gastric dige
stion cannot
proceed ff the pH is not maintained at a very low level. In this case, the gastr
ic mucosal membrane
gears up to permit the hydrolysis reaction
PCI + HOH POH + HCI
where P represents the non-diffusible protein long. The H long, with associated C
I- long (to
maintain electroneutrality) will move from gastric mucosa to the gastric lumen b
ecause of the
non-diffusible P* long in the protoplasm. This leads to a greater concentration
of H long in he
lumen and subsequently the pH of the gastric secretion fails to very low values.

The Donnan effect applies to all kinds of diffusible long. The red blood ceils p
rovide another
case where Donnan effect determines the distribution of long between the content
s of the cell
and the surrounding plasma. The erythrocyte membrane is freely permeable to HCO
and Cl-anions but is impermeable to the protein haemoglobin which exists in the
anionic form. When
blood flows through capillaries, HCO diffuses into the plasma by passive diffusi
on. About 70% of the HCO formed in the red cells enters the plasma. Na md K are n
ot available for free diffusion due to the operation of sodium-potassium pump. T
o maintain electroneutrality, CIfrom the plasma enters the erythrocytes. But at equilibrium the Cl- ion concentr
ation in RBC
and plasma is not found to be equal. Only 70% of the CI- long present in the blo
od plasma enter
the cells. The unequal distribution of this diffusible ion is mainly due to the
Donnan effect. As
the RBCs of venous blood has more of riCO long, there is a greater concentration
of Cl- long in these RBCs during this exchange phenomenon.
The above examples have made us understand the usefulness of Donnan membrane
equilibrium in biology. However, there are cases where the same effect becomes a
bane to the
system. An example that can be cited is that of oedema in ttssues. When the plas
ma .albumin
content falls below the normal value, salt and water retention tares place in th
e tissues. The
movement of the concerned electrolytes in and out of the ceil membranes occurs m
ainly due to the Donnan effect. Other developments like increased secretion of a
ldosterone and vasopressin
fotlow soon to result in a full-fledged oedema state.

ne CoY.ol Phenomeng
99
The Donnan equilibrium has played a very important role in interpretation of oth
er
physiological phenomena like absorption of glucose by the intestine, the distrib
ution of diffusible
long between blood and the cerebrospinal fluid and in urine formation in the kid
ney.
Besides its role in physiology, it is also being exploited for the benefit of ma
nkind. Kidneys,
apart from being one of the main organs involved in acid-base regulation, also e
xcrete many
toxic substances through urine formation. Naturally, both these functions are al
tered in kidney
pathology with life-threatening repercussions. For such patients, artificial kid
neys are used.
These artificial kidneys have dialyz'ng membranes which form minute channels for
blood on
one side and a dialyzing fluid on the other side. The thin dialyzing cellulose m
embrane allows
all constituents of blood to .diffuse freely in both directions with the excepti
ons of plasma proteins
and cellular components. In this way, the toxic substances accumulated in the bl
ood can
diffuse through the cellulose membrane into the dialyzing fluid. This diffusion
can be tailored
as per the needs of the patients by altering the concentrations and compositions
of the non diffusible long. It is obvious that Donnan effect plays a major role
in the,functioning of artificial kidneys.
Suggestions for Further Read/ng
I.
Edsa]l, J.T., The S/ze, Shape and Hydration of Protein Molecules in The
Proteins, Vol. I,
Part B (H. Neurath and K. Bailey, eds.), Academic, New York, 1953.
2.
Haschemeyer, R.H., and Haschemeyer, A.E.V., Proter: A Guide to Study by
Physfcal and
ChernfcaIMethods, John Wiley and Sons, Inc. 1973.
3.
Ward, A.G., Co[[ofds, Their Propertfes andApplfcatfons, Blackle and Sons
Ltd., London, 1948.
4.
Me Ba/n, J:W., Co//ok/Science, Reinhold Pub]/shlng, CoG., New York, 1950
.
5.
Kruty, H.R., Co//ofd Sdence, Elsevier Publishing Co., New York, 1952.

6.
Adamson, A.W., Physical Chemistry of Surfaces, Intersc/ence Pub]/shers I
nc., New York, 1960.

4
DIFFUSION AND OSMOSIS
Matter tends to move spontaneously from a region of high concentration to a regi
on of
lower concentration until the composition is homogeneous throughout. This moveme
nt of
molecules without any energy expended is called passive transport or diffusion.
The molecules in a solution are in a state of continuous motion as a result of b
ombardment
by solvent and other solute molecules if present. The direction of movement is c
ontinually
changing in a zig-zag fashion. The velocity of this movement, also called Browni
an movement
(see Chapter 3) is given by the equation,
M
... (1)
where C is the root mean square velocity in cm sec-,
'R' is the gas constant having a value of 8.314 10 ergs/degree/mole,
T is the absolute temperature in Kelvins, and
M is the molecular weight of the moving particle.
Due to Brownian movement, the displacement of the molecules per unit time is not
in a
straight line. The small molecules and long (crystalloids) diffuse rapidly while
the macromolecules
owing to their large size and shape have a sluggish motion. Another factor affec
ting the movement
of molecules is the concentration difference. Greater the concentration gradient
, more rapid
will be the diffusion.
Biomembranes are selectively permeable in nature. Diffusion of solutes across th
ese
membranes is mainly of two kinds :
(/) Simple diffusion,
(//) Facilitated diffusion.
Simple diffusion may take place directly through a lipid bilayer, through "holes
" created in
the membrane by membrane proteins or by discontinuities in the lipid bilayer. Bu
t there is no

active participation of any membrane protein in this type of solute movement.

On the other hand, facilitated diffusion compulsorily occurs with the participat
ion of a
carrier molecule, usually a membrane protein, It may perform its task in any of
the three ways
mentioned below :
((}
it may move across the membrane with the substance to release it on the
other side
and then return to its original position to continue the cycle, or

Diffusion and Osmosis

I01
(//)
a conformational change may take place in the transport protein on bindi
ng to the
solute which may help in the translocation of the latter to the opposite side, o
r
(///)
the solute may be shuttled from one membrane protein to another till it
reaches its
destination.
A MOLECULAR - KIITIC APPROACH TO DIFFUSION
Diffusion is a process of equilibration thatis directed along a concentration gr
adient from
a region of higher concentration to a region of lower concentration. At equilibr
ium the molecules
will be uniformly randomized throughout the system leading to an increase in ent
ropy. It is
therefore a spontaneous process, according to the second law of thermodynamics.
Due to the random walk of individual molecules owing to vibrational and thermal
motion,
it is difficult to measure the actual displacement per unit time for each molecu
le. However, a
statistical consideration of all the diffusing molecules as a whole shows that a
t each point
time a net flow exists, which is proportional to and follows the concentration g
radient. This rate
of net movement of molecules can be described by Fick's laws of diffusion.
Fick's Laws of Diffusion
Let us first assume that the solute molecules are moving in one dimension only.
A molecule
situated at the origin initially is capable of moving either to the left or to t
he right in distances
of average length W. We shall call these small displacements as 'steps'. The net
movement of a
molecule can now be given by the excess steps taken to the right over the left o
r vice versa. The
probability of the steps taken in either direction remains the same. If 'n' is t
he total number of
steps covered by the molecule in unit time, the total distance travelled will be
equal to 'r', 'n' is
constituted of the steps taken by the molecule to the right and to the left indi
cated by symbols
'mr', and 'm,' respectively. As already stated that the chances of a leftward or
rightward step are
equal, the most probable end point of any particular molecule will be at the ori
gin itself, i.e., rn
is equal to mr, therefore rn-mr is equal to zero. However, the system contains a
large number of

molecules and some of these molecules might move excessively to the left or to t
he right. As the
time increases, the total number of steps 'n' also increases and with it increas
es the possibility
of more and more molecules moving away from origin by purely random motions lead
ing to
diffusion.
Rather than one, let us now consider a model having two origins separated by a p
lane.
From both the origins molecules radiate out into the solution across the plane l
ying between
the two origins. Naturally such a variation would change the concentration profi
les of not only
the two origins but of the plane also. From the above paragraph we know that the
change in the
concentration profile depends upon the total number of steps taken by the molecu
les (n), distance
covered (.) and on the initial concentration. Therefore, the net displacement pe
r unit time
across the plane in any one direction will be determined by the difference in th
e initial
oncentration of the two origins and by 'n' and W. This is the molecular basis of
lck'sfirst law
of diffusion which is in effect described by rate equation. It states that the a
mount of solute 'da'
diffusing across the areaA (1 sq. cm) in a length of time 'dt' is proportional t
o the concentration
gradient dc/dx at that point, or
' ...
where D is a constant of proportlonallty, also called d{1sion lent. It is indepe
ndent of the
concentration at 'x' and x + dx, and related to 'n' and ',' mentioned above. The
negative sign
arises since diffusion takes place in the direction of decreasing concentration
gradient.

... (3)
where
102
Biophysical Chemistry
While formulating the first law, Fick assumed that the laws of diffusion were an
alogous to
the news governing the transfer of heat derived by Fourier earlier.
METHODS OF DETERMINATION OF DIFFUSION COEFFICIENT (D)
One of the methods to determine 'D' is to measure da/dt, also called the flux, a
nd then
find dc/dx frbm it. From this value we can obtain D as the flux obeys Fick's fir
st law. Due to
diffusion the composition and therefore the refractive index of thesolution chan
ges. Most of the
methods for measuring rate of diffusion involve measurement of such changes in t
he refractive
indices with the help of such techniques as light absorption, fluorescence and T
yndall effect,
More recenfly the interferometric methods of studying the boundary changes have
been preferred.
Experimentally, the measurement of diffusion constant is done by two methods :
(/) the porous disc method and
(//) the free diffusion method.
(0 In the porous disc method the particles are allowed to pass through a porous
disc made
of sintered glass or alundum. A concentration gradient is thus created across th
e porous disc.
A solute is now chosen whose diffusion constant is known and its rate of diffusi
on is measured
by this method. The molecule whose diffusion constant has to be evaluated is now
treated in
identical manner and its rate of diffusion through the porous disc is measured.
From the
obtained data, the diffusion constant of the unknown solute molecule is calculat
ed. The value
of 'D' thus obtained is of a relative nature.
For absolute measurements of'D' by the free diffusion method, Fick's second law
of diffusion
has to be applied. It is a partial differential equation derived from Fick's fir
st law of diffusion.
The simplest assumption during appication of this second law is that the diffusi
ng particles do
not participate in any chemical reaction within the diffusing space. This law ca

n be expressed
by the equation,
de
where - = rate of change of concentration with time
d2c
and- = first derivative of concentration gradient with respect to distance.
For three dimensions, the equation becomes
/
x, y and z are the space variables. The above equation has ALSO been expressed a
s
de B2
--- DVc
... (5)
dt

Diffusion and Osmosis

103
-2 is the Laplace operator, in this case operating on 'c'. To use equation (5),
it has to be
where We
solved first. The solution takes into consideration the boundary conditions and
initial conditions
operative in the system under investigation. These conditions determine which of
the multitude
of possible solutions of a differential equation is applicable to the system. To
simplify the
mathematical dealing, one may employ a specially designed diffusion apparatus wh
ich is
geometrically simple or use an analytical centrifuge The spread of an initially
sharp boundry
between the solute and solvent is observed as the solute diffuses into the solve
nt layer.
Diffusion in biological system is, however, mostly associated with chemical reac
tions. In
such situations the. simplest assumption that can be made regarding the diffusin
g system is
that within the diffusing space the rate of utfllzation of the diffusing solute
is constant, i.e.
independent of time and place. Equation [5) in such conditions will take the for
m
-- = DVc - A
... (6)
dt
where 'A'. represents the rate of consumption. Another frequent observation in b
iological system
is that due to stationary boundry conditions the concentration is independent of
tirne at all
positions of the diffusing.space. Under this condition, dc/dt = 0 and equation (
6) becomes,
D.Vc = A, assuming again the constant consumption of the diffusing solute.
SIGNIFICANCE OF DIFFUSION COEFFICIENT
The speed of movement of a particle is characterized by its diffusion coefficien
t which is a
function of the size and shape of the molecule. Knowledge of the diffusion coeff
icient is necessary
for ultracentrifuge studies as it provides us with information regarding the sha
pe of the particles
in solution. Perhaps it is of more importance during the determination of moecul
ar weight of

macromolecules as there is a distinct relationship between the molecular weight

of a substance
and its diffusion coefficient. Before we proceed to the mathematical treatment f
or obtaining the
relationship between molecular weight and 'D', we shall discuss the molecular na
ture of a
liquid film.
The /atttce theory explains beautifully the
molecular nature of a liquid film. According to this
theory the liquid film appears as a network of
molecules with empty spaces in between (see Figure
4. I). These empty spaces or 'holes' as they are called
are the regions where molecules are trapped during
their translational movement. Molecules thus move
from hole to hole, creating in the process new holes
to trap other molecules. This movment of molecules
from one hole to another is called diffusion.
However, since the molecules of a liquid are
attracted by other molecules adjacent to them, they
are unable to move swiftly in the holes created for
them. Therefore, in order to move a molecule into
a hole energy has to be expended. IfaE is the total
energy required to break the bonds a molecule
makes with its nelghbours to flow into an adjacent hole, will represent the dist
ance travelled by a molecule to fall into a hole. If the initial concentration o
f the sample at the origin is 'c', on

104
Figure 4.2
'dc. . +dx
Distance travelled (.x)
Diffusion of molecules across an energy
barrier. , is the dstance between
successive equtltbrrn posons, and aE
Is the total energy required to enable a
molecule to free from its neghbours and
j'Iow into an acent hole
Bophsca Chemstr
diffusion of the molecules through a distance dx
the concentration at the new equilibrium position
dc
would change to c + - (see Figure 4.2).
Therefore, in order to move into an adjacent hole
the molecules should possess an energy equivalent
to or greater than AE.
Not all molecules possess AE. The fraction
which does possess AE can be represented by the
number e-/R, according to the Maxwell-Boltzmann distribution law.
As time 'dr' lapses, some of these molecules
will acquire the most favourable orientation to move
faster into the hole, so that the total number of
molecules per unit volume diffusing from left to
right will be depicted as

N = cQe-/T
where Q = the number of molecules possessing the correct orientation.
Similarly, the ones crossing over from right to left will be given by
The net movement from left to right direction would now be

Nm = N- Nr
or
Nm = -X --.Q.e-E/T per unit volume
... (7)
dx
If the diffusion has occurred across a plane of area 'A', the net total number o
f particles
that have corssed over from leil to right along the concentration gradient per u
nit time would be
given by
Nm ..2A.Q.eA/RTdc
-... (8)
dt
dx
Comparing eqn. (8) with eqn. (3), we see that
D = ,2.Q.e-Z/RT "
Thus, if the values X, Q and E are known, D can be easily obtained.

4n r3N
3M
... (13)
Di.Jiusion and Osmosis
105
The hydrodynamic properties of a molecule are strongly related to its size and s
hape.
Diffusion is of two main types -- translational and rotational. Out of these, th
e latter is
characteristic of the type of molecule. A large spherical molecule will have a s
ingle rotary diffusion
coefficient. An ellipsoid of revolution is characterised by two coefficients as
it possesses two
axes while a general ellipsoid molecule will have three coefficients, one for ea
ch of the axes.
These rotary diffusion coefficients of macromolecules of various shapes can be d
etermined by
flow birefringence (see Chapter 9) and by electrical methods if the molecules ar
e dipolar in
nature.
Einstein in 1905 gave a relationship between diffusion coefficient and radius of
a
macromolecule on the basis of Stokes' law of diffusion. The assumptions for this
relationship
are that the size of the diffusing molecule is many times greater than that of t
he solvent molecule
and secondly, the solvent is a continuous medium.
Einstein provided the relationship between diffusion coefficient (D) and frictio
nal coefficient
(f} as
D =-... (10)
f
where
k = 1.38 x I0-=3 JK- (Boltzmann's constant) and
T = Temperature in Kelvin.
The coefficient T depends on the shape and size of the macromolecule, but not on
its
mass. For spherical particles, Stokes provided the relationship
f=6lr

... (11)
where r = radius of the particle, and
-- viscosity of the solvent.
Combining eqns. (10) and (11) we get the Einstein-Stokes' equation as
D = (12)
6xrlr
...
4
Since the volume of a sphere of radius 'r' is -- ra, and the volume of a spheric
al molecule is
M;
3
-- where ; is the partial specific volume, M being the molecular weight and N th
e Avagadro's
N
number, we can write

106
...
.., (16)
RT
Do = -- (as 'K' .the Boltzmann's constant is
Nfo
equivalent to the gas constant per
molecule, it Is replaced by R/N)
where fo is the frictional coefficient of a spherical particle.
Or,
Do =
As the macromolecules being studied are of various shapes, the experimentally de
termined
values of'D' are usually smaller than those calculated theoretically on the assu
mption that the
molecules arespherical in shape. The ratio f/fo i.e., the ratio of the frictiona
l coefficient for nonspherical particles (f) to the frictional coefficient of spherical particles (fo
) is termed asfr/ct/ona/
rat/o or dtssymetry constant. The value off/fo for spherical particles is 'one'
and as the deviation
from spherical shape increases for macromolecules, the value of f/fo becomes pro
gressively
greater.
DIFFUSION OF ELECTROLY'rEs
Earlier we have discussed the zlg-zag movement of molecules in solution on accou
nt of
Brownian motion. However, the same theory does not apply to solutions of electro
lytes. The
long here are influenced by the electric field and the net movement is according
to the sign of
their charge. One ion attracts around itself other long of opposite sign and ten
ds to drag them
along with itself. As a result, long of opposite charge do not move singly but r
ather in the form
of pairs. A faster ion is thus slowed down by its 'sluggish' partner while the l
atter is speeded up
by its association with the former. Thus it is difficult to obtain the diffusion
coefficient of any

single ion. What we find actually is the diffusion coefficient of the ion pair,
If an electric connection
is made after the solution of electrolytes has come to diffusion equilibrium, a
flow of current will
be observed. This is due to the dissociation of the ion pairs which under the in
fluence of the
extemally applied electric field now move independently to their respective oppo
sitely charged
electrodes. It is now possible to measure the ionic mobility of individual Long.
Each ion will have a different ionic mobility and the mobility is a measure of t
he speed of
the long as it moves through the solution. Therefore, there is a distinct relati
onship between the
mobility of an ion and its diffusion coefficient. It is given by
)
RT (v, + v2)
D = "
... (171
N Vla2 + v2D
where D is the diffusion coefficient of the electrolyte at infinite dilution, N i
s the Avagadro's
number, R is the gas constant equivalent to 8.314 x 107 ergs/degree/mole, T is t
he temperature
in Kelvins, bt and 12 are the absolute mobility of the two oppositely charged lo
ng and v and v2
represent the number of positively, and negatively charged long respectively obt
ained on
dissociation of the electrolyte (for e.g., ff one molecule of NaC1 dissociates i
t will give rise to one
positively charged ion, Na and one negatively charged ion, CI'. Similarly CaCI2 w
ill give one
positive ion Ca2 and two negative long of CI'}.
The value of 'D' obtained by equation (17) is of limiting nature as it is obtain
ed on the
assumption that the electrolytic solution is infinitely diluted. However, at low
concentrations,

Dtffuslon and Osmosis

107
the actual value of D can be found by taking into consideration the activity of
the solution. The
equation now takes the form : ,
D=DO +(l+dln+ /
)
... (18)
where 7 represents the "mean ionic actlty" coefficient and 'c' the concentration
of the solution.
At higher concentrations the above equation has to be modified further to take a
ccount of
the solvation of long which we shall,'discuss subsequently.
A stable solution is one in which the solute-solvent bonds are of the same order
of magnitude
In the same manner an aqueous solution will be stable only ff the
is capable of forming hydrogen bonds with water in a manner similar to the hydro
gen between the water molecules. When the solute has to diffuse, i.e., move from
hole to
In the lattice, its hydrogen bonds haveto be broken. The energy required to brea
k one
bond is approximately 4.2 kcal at 27"C. Now if the molecule has to move in water
, all
hydrogen bonds that it makes with water need not be broken at the same time. But
ff it
In a lipid layer, all the hydrogen bonds have to be broken together which will r
equire a
mergy. Therefore, the diffusion coefficient will be reduced in lipid solvent
solutes. Some solutes form many such hydrogen bonds with water molecules. If all
these bonds have to be broken when the solute moves, then diffusion will be slow
.
Having gained knowledge about the various types of interactions in a solution wh
ich affect
and on the basis of lattice theory we can now. give an explanation on diffusion
of
:solutes across bio-membranes. It will be appropriate to mention here some struc
tural
characteristics of living membranes as per.the model provided by Davson and Dani
elli. Biological
membranes have a lipid bflayer sandwiched between two layers0f proteins, the lat
ter providing
integrity to the membrane. The lipid layer acts as an effective diffusion barrie
r.
Several reasons have been attributed to this behaviour of the lipid layer. We sh
all discuss them
one.

(0
A molecule has to break all its hydrogen bonds with water if it has to p
enetrate the lipid
In an aqueous environment this would not have been difficult as the H-bonds coul
d be
one at a time and similarly reformed one at a time. Therefore, the diffusion rat
e of a
is lowered when passing through a lipid medium.
(10 The lipid bilayer is a closely knit structure with intermolecular hydrophobi
c bonds
acent side chains of the fatty acids making it even more difficult for a molecul
e to
it. To create even a single hole for a moving solute molecule, a large amount of
energy
to be expended in breaking the hydrophobic bonds.
For a lipid membrane 50A thick, the resistance becomes too much and diffusion is
reduced
extent,
(li0 For large polyelectrolytes, diffusion is slowed down because larger holes h
ave to be
by displacement of a large nnmber of solvent molecules. Larger the molecule, les
ser is
value of 'D' as can be seen in equation (12).
(iv) A hydrated molecule has to shed its bound water molecules to penetrate into
the lipid
again requires a considerable energy. Diffusion thus is retarded. Greater the de
gree
of a molecule, lesser is the diffusion. The values of spherical molecules are gr
eatly
affected by hydration. Cations are slow in diffusion than the anions because of
their bulk, the

... (19)
Ceb
108
Biophysical Chemistry
bulk being chiefly contributed by the water molecules around them. Hydration cau
ses a swelling
of the molecule by binding water, and thisincreases the frictional effect. For m
acromolecules ff
the frictional ratio is more than 2.5, the effect of hydration becomes negligibl
e.
(v) Lastly, there are several sophisticated membrane processes affecting diffusi
on of solute
molecules in and out of the cells.
The anions and cations of an electrolyte solution move at different rates. As a
result a
potential is set up across the membranes through which these long diffuse. It ca
n happen when
two solutions of the same electrolyte at different concentrations are separated
by a membrane
and also when two different electrolytes of the same concentration are kept sepa
rate due to the
membrane. These potentials are called diffusion poten'.ti, and are a characterist
ic of the
membrane as well as the long. If the membrane has the pores of a size that would
not allow
entry of one particular ion through it, the diffusion potential will be high.
Two equations have been provided to calculate the diffusion potentials of membra
nes.
They are :
Case a
E = 0.058 log laA + lab
c + ga
when there are two different salts AB- and CB- of a single concentration separated
by a membrane, and
E = 0.058 laA+ ]an logt
... (20)
1C + laB C2
when there are two different concentrations C and C= of the same salt across a m
embrane. ^,

B, c are the ionic mobilities of the long A, B and C respectively.

The above equation tells us that the relative mobilities of the diffusing long a
nd their
concentrations are the factors determining the magnitude of the diffusion potent
ial developed
across a membrane. However, these potentials are unstable as concentration diffe
rences are
bound to occur on further diffusion.

109
Due to
and Osmosis
always foundto be less than the expect value.This is due to the physiologicaldea
d space. The
anatomical dead
cpacity. The value of K for oxygenin human beings,can be determined from measure
ments of
diffusio.,capacity for oxygen, llng area and the istance through which diffusion
occurs. The
rate of diffu.
lungs, by the
DIFFUSION OF WATER ACROSS MEMBRANE : OSMOSIS
So far we have discussed the free diffusion of molecules and diffusion of solute
s across
process which is complimentary to the ones described above and obeying
mechanism is osmosis. Osmosis is one of the major exchange processes which is
to the living body and therefore has to be regulated carefully. It is not only t
he
of the various long in the body fluids but also their content which is important
for
functioning individual. Osmosis takes care of both of these. By definition it s
theflow
a dilute to a concentrated solution, across a semipermeable membrane till the
equilibrium. The semipermeable nature varies from membrane to membrane. For
it is semipermeable to small long and molecules but not big colloidal particles,
for others
be differentially permeable to certain long, excluding others, while some may be
totally
Biological membranes, though, are mostly specific. They have a vast range of
systems embedded within them which will carefully 'sniff the metabolite to be
while it waits outside and only allow one kind to enter, that which is needed.
the one to go out also has to face scrutiny. This flux of metabolites is associa
ted with of solvent. Some of these movements require energy and thus are termed
active
others just move on across the gradient and therefore called passive. Osmosis be
longs to
It is concerned with the spontaneous flow of solvent only. We can demonstrate
A glass tube open at both ends is chosen and at one end is tied an
membrane in the form of a sack. Now the tube is half-filled with concentrated su
gar

in a beaker of water (see Figure 4.3). After some time we observe that
in the tube has risen. The level of the liquid rises until the hydrostatic press
ure so
sufficient to stop the flow of solvent, or osmosis, into the tube. This hydrosta
tlc
developed as a result of osmosis is called the osmotic pressure of the solution
and is
as the excess pressure that must be applied to the solution to prevent the passa
ge of the
t into it thorugh the semipermeable membrane separating the two solutions.

110
Hydros
pressu
Btophysicat Chemistr

Final level
h,--- ---- Initial level
-----* Sucrose solution
I---- Water
i" -- = -- Animal membrane
---4 (semipermeable)
Figure 4.3 Osmosis through animal membrane
The osmotic behaviour of molecules was first reported by Abbe NoIlet in 1748.
on, the search began for an ideal semipermeable membrane. M. Traube in 1867
membrane of a gelationous precipitate of copper ferrocyanide, Cu2Fe(CN)6 for low
weight solutes in water. For high molecular weight solutes in organic solvents,
made of cellulose, cellulose nitrate or animal membrane have been found suitable
.
Why Exactly Does Osmosis Take Place ?
The answer becomes very simple if we look at it thermodynamically. When a solute
dissolved in a solvent, the energy of the solvent molecules is reduced considera
bly
the solute-solvent interactions. When this solution is separated from the pure s
olvent by',
semipermeable membrane rapid movement of the solvent molecules across the barrie
r
in the direction of the solution. The hydrostatic pressure so developed in the
containing the solution is enough, to increase the free energy of the solvent mo
lecules in
solution. When the free energy of the solvent molecules in the solution is resto
red to
equal to that of the pure solvent, an equilibrium is achieved and osmosis stops.
More
number of solute particles in the solution, greater is the osmotic pressure deve
loped.
osmotic pressure is one of the four. colligative properties that a solution poss

esses
pressure, rise in boiling point, depression of freezing point, being the others)
. Under
conditions it is just like gas pressure, directly proportional to the absolute t
emperature and
concentration, and is independent of the chemical nature of the dissolved materi
al.
When working with a colloidal solution, mention has to be made about the nature
semipermeable membrane. Protein solutions, in general, are prepared in dilute sa
lt
If the membrane is impermeable to both salt and colloidal particles, we obtain t
ota/
pressure. With a membrane impermeable to protein but permeable to
osmotic pressure. The latter is useful while deducing the molar mass of the coll
oidal particles
MEASUREMENT OF OSMOTIC PRESSURE
The first determination of osmotic pressure of macromolecular solutions began wi
th
in 1899 He filtered blood serum through a semipermeable membrane made of
successfully freed the serum from its crystalloid constituents. Osmotic pressure
of the filtrate devoid of protein constituents and the raw (unfiltered) serum sh
owed that
proteins gave osmotic pressures of 30-40 mm Hg. This experiment provided an insi
ght
osmotic behaviour of cells in various physiological processes, namely, the renal
function
the secretory processes. Since then, various attempts were made to design the os
motic
measurement apparatus to a type where the osmotic pressures of the biological
not be affected adversely by extraneous factors.

and Osmosis
111
Sorensen in 1917 first published the data of his measurements of osmotic pressur
e of
solutions under carefully controlled conditions. His results and those of Starli
ng's on
which was reproduced using collodion membranes and better designed modem
showed that a strict control of pH and salt concentration, and knowledge of the
of protein are mandatory while determining the osmotic pressures of protein
The osmometers which were. used by Sorensen and others in the first half of 20th
though efficient, took several days for standardization. The osmotic event was r
apid
:the rise due to capillarity in themanometer tube required several days to reach
an equilibrium
osmometers designed later were easy to handle and more accurate. The manometers
these latter type of instruments contained organic liquids, paraffin, toluene or
alcohols with
could be attained in a very short time. The sample requirement also was low.
instruments required a rigorous thermostatic control within 0.004o. Later, this
problem
s circumvented by the invention of an electronic osmometer by Rowe andAbrams in
1957.
could be operated without thermostatic control and the sample for determination
was
small volume (only 0.5 rnl). A detalled discussion of the apparatus is given in
pressure (P)
Transducer
Electrical cireuit
Galvanometer
An osmometer adaPted for rap dmeasurements of,osmotic pressure using
ample was designed as above by,Rowe and Abrams in 1957. The U;tube has
platinum foil which isconnected

o = 1
A SOLUTE
Biophysical Chemistry:
OSMOTIC BEHAVIOUR OF CELLS
We rarely come across biological membranes which are perfectly selectlvely
the solvent and exclude solute molecules completely. However, due to small pore
size
membranes, though the movement of the solute molecules will be impeded, there
a very slow diffusion of low molecular weight solutes. Let us consider a vessel
fillet
and divided into two compartments (A and B) by a porous membrane. The membrane
impermeable to solute molecules. If a solute like sugar is now added to compartm
ent B,
will flow from A to B because the osmotic pressure of the solution in B is great
er than in
There will also be a diffusion of sugar molecules from B to A at a very slow rat
e. Due to this,
effective osmotic pressure as judged by the movement of solvent from A to B will
be
the osmotic pressure calculated from the solute concentration alone.
The effective osmotic pressure of a solution can be calculated by including a te
rm
Staverman's reflection coefficient, symbolized by o, in the osmotic pressure equ
ation, so that
neff =
The reflection coefficient has an inverse relationship with permeability coeffic
ient. It depen&
on the nature of the selectivly permeable membrane and the nature of the solute.
If AsoLv
represents the effective fraction of the area on the membrane available for diff
usion
particles and ASOLVET, the effective fraction of the area on the membrane availa
ble for
diffusion, then
If the pores in the membrane are of size which would not allow the solute molecu
les to
diffuse, the second half of the equation, i.e.,
ASOLUTE approaches zero and o
approaches I.
ASOLVENT
The significance of o is better understood when we study-the osmotic behaviour o

f living
cells. Living cells have selectively permeable membranes which are very permeabl
e to water and
some low molecular weight solutes, but much less permeable to other substances.
This leads to
the inflow of some solute molecules along with the solvent while others are reta
ined. Under
these conditions, the solution exhibits only that fraction of its total osmotic
pressure that is due
to the solutes which are retained. This fraction of the total osmotic pressure o
f a solution is
termed as its ton/city. If the cells are placed in a medium whose osmotic pressu
re matches that
of the cell contents inside, there is no unidirectional flow of solvent and the
cell size remains the

lon and OsrnosLs

113
same. The medium then is said to be isotonic with the cell. If the medium is con
centrated, its
osmotic pressure is greater than that of the cell interior. Water then will be a
bstracted from the
cell resulting a shrinkage of the cell. The solution is said to be hypertonic. O
n the other hand, a
cell will swell if it is placed in a dilute medium because the osmotic pressure
of the cell Contents
being higher will cause the cell to absorb water from outside and increasein siz
e. The medium
then is said to be hypotonic, The extent of swelling depends on the osmotic pres
sure differences
between cell contents and the surrounding fluid. Tonicity of a solution is there
fore expressed
always in terms of response of cells immersed in solutions. It is a function of
the cell membrane.
In contrast to tonicity, which refers to increase or decrease in size of ceils,
osrno/adty
refers to the concentration of solutes in the solution. In osmometry, concentrat
ions should
always be expressed as molal concentratlons Molal concentrations are expressed i
n terms of
weight of the solvent, and this is unaffected by temperature. Molar concentratio
ns, i.e., grams
per Iitre though are preferable because of the inconvenience of weighing liquids
. This however,
has one important disadvantage in being temperature dependent. Ideal solutions o
f equal molarity
exert same osmotic pressure and therefore they are called/sosmotic. In exerting
a. greater osmotic
than another, a solution is said to be hyperosmotic and hyposmot/c if it exerts
a lower
The osmolarity of a solution is best expressed as osmoles. In quantifying the os
motic
of body fluids, it is convenient to use a smaller unit, the milliosmol (abbrevia
ted
, which equals 1/1000 of an osmoL The milliosmol is equivalent to a solution of
I millimole
a non-electrolyte per litre of water. If a substance iordzes, each ion contribut
es the same
molecule. Thus for example, one mol of glucose, a non-lonizable
has an osmolar value of 1; one mol of sodium chloride (NaCI) will produce 2 osmo
les
long while one mol of calcium chloride (CaCI) will PrOduce 3 osmoles
s it dissociates into one cation and two anions, the total being 3 long.
The osmolarity of the erythrocytes is approximately 280 mflliosmoles. When eryth
rocytes
mM mannitol no change in the size of the ceils is observed. The reflection
for mannitol is approximately equal to 1, and therefore mannitol at 280 mM
is isotonic for red blood cells. If the same erythrocytes are suspended in 280 m
m

the cells will swell, i.e. 280 mM glycerol is hypotonic to the same cells. To ma
ke glycerol
a concentration of 318 mM is necessary to give an effective osmolarity
!280 milllosmoles. The reason for this is the value of G, which is 0.88for glyce
rol. Similarly
solution has to be prepared at a concentration of 337 mM to be isotonic for red
equal to 0.83 for malonamide. Thus knowledge of reflection coeiTcient can serve
as
during preparation of isotonic solutions for living cells.
The above observations make it clear to us that/sosmotic is not synonymous to so
tonic.
hyperosmotic and hyp0smotic are not synonymous to hypertonic and hypotonic.
' of a solution cannot be predicted solely from its known composition. A solutio
n isotonic
can be slightly hyperosmotic to it and vice versa. Consider, for example a tank
divided
which is permeable to solvent and the solute
being completely impermeable to sucrose (see Figure 4.4 case 1). Chamber 1 is
with an squeous solution containing 1.0 M sucrose and 1.0 M glycine while chambe
r 2
a 2.0 M aqueous solution of sucrose. Chamber I is thus isoosmotic with chamber 2
.
the tonicity of the solution in chamber 1 is less than that of the solution in c
hamber
of this hypotonicity of the mixed solution, water escapes to the 2.0 M solution
of
by osmosis. If we empty out the tank now and fill chamber 1 again with the same
mixed
of sucrose and glycine while chamber 2 now is filled with 1.0 M sucrose solution
only
;Figure 4.4 case 2) we observe no net transfer of water from any of the chambers
. This is, in
; of the fact that the solution in chamber I was initially hyperosmotic to that
in chamber 2.
reason for this is the isotonicity of the two solutions. Thus the pattern of sel
ectivity of the
plays a great role i.n the osmotic behaviour of living cells.

114
2.0 M
Sucrose
1.0 M
Sucrose
1.0 M
Glycine
1.0 M
Sucrose
or
n
I
CASE 2 ]
(1) Chamber 1
is hyperosmotic to chamber 2,
(II) Chamber 1 and chamber 2 are isotonic to
each other.
Chamber 1
1.0 M
Sucrose
1.0M
lycine
Biophysical Chemistry
Chamber 2
Chamber 1
Chamber 2
Selectively permeable membrane :

permeable to glycine
CASE i I but not to sucrose

(I) Chamber 1 is isosmotic with chamber 2.

(II) Chamber 1 is hypotonic to chamber 2.
.'. direction of osmosis =====
.'. no osmosis.
Figure 4.4 Osmotic behaviour of cells
MOLECULAR WEIGHT DETERMINATION FROM OSMOTIC PURE
MEASUREMENTS
Osmotic pressure measurements for molecular weight determination is seldom used
today
because of several disadvantages. Firstly, since osmotic pressure is a colligati
ve property and
therefore depends on the number of solute molecules, an impermeant molecule of s
mall mass
has the same effect as a molecule of large mass. Secondly, in practice, since th
e van't Hoffs
equation is followed for osmotic pressure measurements, the osmotic pressure-mol
ecular weight
relationship holds only for very dilute solutions. However, this method has impo
rtant theoretical
advantages. For example, it is indifferent to the size, shape, charge and so for
th of the solute
particles, at least in the dilute solution limit where interaction effects are a
voided. Therefore
this method can be put to use in determining molecular weight of such high molec
ular weight
compounds as polymers, proteins, plastics, etc.
In determining the molecular weight of a protein, measurement of the osmotic pre
ssure of
solutions having different small concentrations of protein are made. The van't H
offs equation is
used for the purpose in the following manner.
l-IV = nRT
As
n = C, i.e., concentration of solute in moles/litre,
V
But as 'C' is equal to c/M

cRT
MI-[

and Osmosis
115
".oncentration is gms/lltre, R is the gas constant, T is the
temperature In Kelvins and H, the osmotic pressure In atmospheres. A graph ofl'[
/c is
against c. The linear curve obtained is then extrapolated to zero concentration
which
the value of the Intercept as equ. to RT/M. From this the molecular weight of th
e proteIn
(see Figure 4.5).
to i
0 &l O 03 0.4 05 (6 0.70
Concentration in gm protein/litre solution
The osmotic pressure measurements give a value of M., the average weight of a mo
lecule.
iis equal to the weight of the sample divided by the total number of moles In th
e sample. Thus,
Weight of the sample (w
w
M. =
or number of moles Number of moles (n)
M
n
Suppose n' of the sample is made up of several fractions, each of a different mo
lecular
weight, i.e., the sample contains nI moles of molecular weight Mi,- n moles of m
olecular weight
M, us moles of molecular Ms and so on. Then,
n]M] + n2M2 + n3M3 +... niMi
Run ffi
n +n. +na .,.nl
Is the number of moles In the l fraction of molecular weight Ml

or
Run - '-niMi
En
Such an average molecular weight Is known as a number average mo/eadar weight.
Measurement of any of the colligative properties of a sample will lead to the ca
lculation of
number average molecular weight as they are proportional only to the number of u
nits In
solution.
The weight average molecular weight gives representation, to various molecular s
pecies In
proportion to their given weights In the given sample. The 'number' term therefo
re is replaced
by 'Ight' of that species In the formula represented as :
My = wlM +W2M +wsMs +"" wIMi

116
or
ffi hiMI,
and so on.
No.
2.
3.
4.
5.
6.
7.
8.
9.
10.
12.
13.
14.
12.0
Table 4.1 enlists the molecular weight of certain proteins as deduced from measu
rements
of their osmotic pressure.

Table 4. I Molecular weights of certain proteins from masuremnts of their omotio

pr.
Name of Protein

Insulin (in acid medium)

Lysozyme {egg white)
Trypsin inhibitor (Soyabean)
Prolactln
Trypsin
Pepsin
Chymotrypsinogen
Chymotrypsin
Ovalbumin
Haemoglobin
Bovine Serum Albumin
Human Serum Albumin
Flbrinogen. Huma plasma
L-Myosin
17.5
24.0
26.5
36.5
36.0
36.0
41.0
44.9- 45.1
67.0
69.0
69.0
580.0
840.0

BIGNIFICANC OF OBMO818 IN BIOLOGY

Most of us believe that life began millions of years ago in the form of minute s
imple
organisms dwelling in an aquatic environment. These primitive living things live
d in an
environment which was more or less constant with respect to the composit/on of v

ar/ous
substances. As time passed, the organisms gradually evolved into more complex an
d highly
organ/zed individuals. This process of evolution finally gifted the with theh- o
wn 'personal' internal environment while the world surrounding them formed the e
xternal
environment (see Box 4.3)

117
Diffusion and Osmosis

118
BiophyslcaI Chemistry
Gradually the constancy of internal.environment became vital for existence of li
fe. It had
to be maintained at all costs. Many physlological processes and most of the orga
n systems
therefore got involved in maintenance of this constancy of composition of the in
ternal
environment, which is also known as homeostasis, by an incessant replacement and
exchange
phenomenon. It is at this point of evolution that the different exchange mechani
sms for the to
and fro passage of molecules in and out of cells got an upper hand. The body, di
vided into many
many compartments by an effective network of membranes worked in a perfectly coordinated
manner. Fluids containing dissolved matter were exchanged continuously between t
he "internal"
and 'external' compartments. In other words, a dynamic equilibrium was achieved.
Osmotic pressure plays a major role in the maintenance of this dynamic equilibri
um
within an individual. The factors responsible for compartmentalization of body f
luids appear to
be largely osmotic and related to electrolyte concentration. This is further mod
ified by 'the
selective nature of the biomembranes. The body fluids distribute themselves acro
ss membrane
structures such a way as to equalize osmotic pressure. This state is one of osmo
tic equilibrium.

/Mffuslon and Osmosis

119
Greater the concentration of non-diffuslble particles on one side of a membrane
as compared to
the other side, more Is movement of water towards It, i.e., towards high solute
concentration.
The organs responsible for malntaining homeostasis in an individual are many. Bu
t the
homeostatic mechanisms or their end organs mainly lle in the intestine for absor
ption and for
exction in the kidney, skin, lungs, liver, pancreas and agaln intestine. These m
echanisms are
well adapted for exchange of water, minerals and metatlotds extstLng as cations
(see Box 4.4).

Biophjsical Chemistry
te/eost, probably due to the similar nature of their functions, r
The plasma proteins, being macromolecular polyelectrolytes, are large bulky mole
cules
which cannot normally diffuse through the blood capillary membranes into the rel
atively protein
free tissue fluids. They thus exert an inward force, the co//o/da/osmotic pressu
re (also called
ondotic pressure) which has a magnitude.of approximately 25 mm Hg. This force is
enough to
hold a certain volume of water within the blood. Though its value is quite less
than the total
osmotic pressure of blood,, it exerts a great influence in maintaining fluid bal
ance across the
capillaries, The colloidal osmotic pressure of the plasma proteins is opposed by
the cap///ary
pressure which tends to direct the fluids out of the capillary into the tissue.
This filtering force
differs in its magnitude at either ends of a capillary. At the venule end it is
as low as 9.0 mm Hg
whereas at the arteriole end it rises to 25.0 mm Hg. At the arteriole end additi
onal outward
forces like the interstitial.fluid pressure (approx. 7.0 mm Hg) also exert their
effect with the
result that there is net fluid shift at this end from the capillary to interstit
ial space.
The opposite occurs at the venule end of the capillary with fluid flowing in fro
m the tissues
into the blood. However, if this balance between the two opposing forces is upse
t under certain
abnormal physiological conditions, fluid accumulates in the interstitial space r
esulting in a
condition known as edema. In this diseased state, a normally negative interstiti
al fluid pressure
becomes positive. This may happen due to cumulative or singular effect of severa
l factors like
high blood pressure, increased capillary porosity and/or low plasma protein cont
ent.
Albumin contributes to around 80/0 of the total osmoUc pressure of the plasma pro
teins.
Because of its considerably low molecular weight compared to globulin, it is a m
ore effective
species osmotically. Therefore, a loss or deficiency of plasma albumin has a mor
e negative
effect on fluidbalance in the body. Lowered plasma albumin leads to diminished b
lood volume
which precipitates into a state of haemorrhagic shock.
- Besides the plasma proteins, an appreciable variation in the concentraUon of c
ertain
electrolytes retained in body fluids can drastically affect a large number of ph
ysiological functions.
The total osmotic pressure of a body fluid is equal to the sum of the osmotic ef

fectiveness of all
the long present. The osmolar concentration of plasma is approximately 0.33 (i.e
., blood plasma
is 0.33 Min its total concentration of dissolved p -rarUcles inclusive of all lo
ng andhon-electrolytes). The cationic components of blood require precise regula
Uon in order to matntaln the osmoUc
balance of blood. The three important cations which require special mention are
sodium,
potasslumand calcium. A major loss of sodium from the body leads to asignificant
lowering of
osmotic pressure of the body fluids which may be enough to cause dehydration whi
le increased
serum sodium leads to fluid retention. An increase in potassium concentration in
blood mainly
affects funcUoning of the heart and also causes dilatation of blood vessels. The
vascular system
thus loses ittonicity. Decreased serum calcium may lead to a generalized muscle
spasm called
tetany.

Diffusion .and Osmosis 121

Osmotic pressure has a key role to play in the functioning of normal healthy pla
nts. The
constant osmosis of water into the cell creates a surplus hydrostatic pressure w
hich keeps the
cell strong and elastic. In this state, the cells are called 'turgid'. When thes
e healthy cells are
placed in.solutions of low and high osmotic pressure respectively the cells eith
er swell or shrink.
But due to the rigidity of the outer cellulose walls, plasmoptysis, i.e., swelli
ng of the cell in not
observed. However, plasmolysis, i.e., shrinkage of the protoplasm from the walls
is a regular
phenomenon observed due to leakage 0fwater from the interior of the cell through
the membrane
to the exterior when cells are placed in hypertonic medium.
Suggestions for Further Reading
I. Glasstone, S., Textbook of Physical Chem/stry, Van Nostrand, New York, 1975.
2. Crank. J., The Mathemat/cs of D/.rusion, Oxford University Press, 1956.
3.
Stein, W.D., Diffusion and Osmosis in Comprehensive Biochemistry (Florki
n, M. and Stotz,
E.H. eds.), Vol. 2, Elsevier, New York, 1962.
4.
Edsall, J.T.. The Size, Shape and Hydration of Proteins in The Proteins,
Vol. 1B (H. Neurath
and K.,Bailey, eds.), Academic, New York, 1953.
5. Robinson, R.A., and Stokes, R.H., E,ctrolyteSolutions. 2nd ed.. Butterworths.
London, 1959.

5
VISCOSITY
If equal volumes of water and castor .oil are allowed to flow out through I0 ml
glass
pipettes under identical conditions, we observe a time difference between the tw
o liquids in the
emptying out process. This means that one liquid flows faster than the other. Th
is difference in
the rate of flow of liquids is attributed to the phenomenon of viscosity. In the
following text we
shall elaborate this term to understand its nature and impact on the living syst
em.
Stationary plane
Force

Moving plane

Stationary plane
Figure 5. I Velocity gradient in a fluld flowlng through a glass tube
In a liquid sample, a layer of liquid experiences resistance when it flows over
another
layer. If a shearing forceI is applied to overcome the attractive forces between
the molecules of
the adJacenta,, layers, Each layer will move with a different velocity (see Figu
re 5.1). The velocity
gradient,
, so formed can be related to the shearing force applied per unit area b
y the equation.
F dv
n = q
{I)
A dx
"'"
F
where = shearing force per unit area (also called shearing stress),
viscosity of the liquid,
dv
=dx difference in velocity between two layers separated by a distance dx.

S Force : If a piece of metal is subjected to a force at its upper surface while

being held fixed at
its lower surface, it will be deformed. The deformation is proportional to the a
pplied force. Such an
apl]led force is called a shearing force.

123
C=O
The coefficient of viscosity, l, can be defined from the above equation as the f
orce per unit
required to maintain unit difference of velocity between two parallel liquid sur
faces which
unit distance apart. The units of viscosity are dynes sec- cm-2. More common uni
ts are the poise) and millipoise (10- poise), after the name of poiseuflle who p
ioneered r of viscosity. Therefore, a liquid is said to have a coefficient of vi
scosity as one poise
a force of one dyne maintains a difference of velocity of one cm sec- between tw
o parallel one cm apart and have an area of contact equal to one sq.cm.
The reciprocal of the coefficient of viscosity is called the fluidity and is giv
en by the symbol
1
=-

... {2)

The term fluidity expresses the tendency of a liquid to flow

s a measure
!the resistance a liquid offers to this flow. Liquids having
viscosity are
'mobile' due to their ease in flowing. On the other hand the
e high
of viscosity and do not flow easily. Most of our body fluids
the

whereas viscosity i
low coefficients of
iscous' liquids hav
are viscous due to

of particles of macromolecular size. Therefore, our main concern in this chapter

will
r viscosity with an eye on the solutions of macromolecules.
When we define viscosity, we assume that the fluid concerned is undergoing a lam
inar
the flow lines of the solvent will cause a change in the viscosity of
the solvent. Now, let us consider the viscosity of a solvent lo. On addition of
macromolecular
particles to this solvent, the viscosity of the solvent increases to a new value
q. The ratio of
solution to solvent viscosity (q/qo) is the relative viscosity. The ratio of the
change in viscosity to
the original viscosity of the solvent is called the specific viscosity and is de
noted by qsp. As the
concentration of the macromolecules in the solution increases, the specific visc
osity also
increases. The concentration 'C' is expressed in grams/ml or grams/100 ml. Many
empirical
equations have been proposed to describe the change of specific viscosity with c
oncentration
but the equation proposed by Huggins has found wide acceptance. It is expressed
as

A plot of q,p/C verses C is shown in Figure 5.2. Such plots are normally linear
for dilute
solutions. On extrapolating the curve to zero concentration, we get an intercept
on the Y-axls.
The value oflp/C at this dilution gives us the b-ttrtns/c viscosity of a solutio
n, depicted by [q]. We
also call it the//m/ring vlscos/ty number. The Huggins constant 'k' in the equat
ion (3) is obtained
from the slope of the curve. The quantity ,plC is sometimes called the reduced v
iscosity.
Concentration
lure 5.2 Plot of reduced viscosty versus concentmt

124
... (4)
... (9)
For moderately concentrated solutions, another equation proposed by Solomon and
Cluta
has been used extensively. The value of [B] determined by this method is only mo
derately
accurate because it requires the measurement of.p at a single concentration only
. Nevertheless,
It is necessary for determination of viscosity for those solutions whose protein
components
denature easily when a very dilute solution of the protein is allowed to flow th
rough a capillary.
The equation is stated as
where In I = In (1+ l)
When macromolecular particles are suspended in a solvent they assume different
conformations. The increase in viscosity then depends primarily on the effective
volume occupied
by the macromolecules. Scientists have made several attempts to quantitatively r
elate viscosity
to the axial ratio of t-he macromolecules as
.p= v$ ... (5)
where v = the viscosity increment which increases with increasing axial ratio p
ffi l/d (the ratio
of length to diameter) of the macromolecule and $ = fraction of the volume of th
e solution
occupied by the particles.
[i)
Sphe
Einste/n gave an equation relating spec/flc viscosity of a di/ute soluti
on of macromolecules
to the volume fraction occupied by the particles. The equation
1sp-- 2.5 $
... (6)
is based on the following three assumptions :
(a) the solute particles have a Very large size ratio to the solvent molecules a

nd are rigid,
(b) the flow rates are very low, and
(c)
the capillary through which the liquid flows must have a large radius co
mpared to
the radius of the spherical particle.
When the solution is moderately concentrated, there is interaction between the f
low lines
of the solute too and to quantify it; another term is addedto equation (7), so t
hat the. equation
now takes the form,
1sp = 2.5 $ 12.6
As the volume fraction $ VeC
=

... {8)

M
where We is the effective hydrodynamic volume of one mole of macromolecular solu
te particles
of molecular weight 'M' and concentration 'C' gm/ml, equation (8} gives
"q, = 2.5V= +
-- -- 12.6// C
C M
As the concentration reaches Lnflnite dilution-, the equation becomes,
|'q) = 2.5 V/M

... (12)
a<<b
factor {V}.
Prolate
{Approx. 165 %}
125
Ellipsoids
Simha provided an equation for the viscosities of ellipsoids of,revolution. The
prolate
e//Ipsolds of revolution are clgar-shaped while the oblate ellipsoids of revolut
ion are dlsc-shaped
(see Figure 5.3). According to derivations by Einstein and later by R. Slmha,
AS O,

sp
p2
2
or v
p
=

14

+ (11)

15(In2p- 1:5)
5(ln2p-0.5)' 15
"'"
The above equation is applicable to prolate ellipsoids. For oblate ellipsoids, t
he equation takes
the form,
(p)
15 tanp
O
Prolate
O Oblate
spheroidal
spheroidal
(Approx. 108 %)

(Approx. 94.%)
a>> b
{a}
0
Prolate
Oblate
{d)
Figure 5.3 Ellpsolds of revolutlon
Oblate
(Approx. 63 %)

Figure 5.4 shows a graph of the axial ratio for ellipsoids of revolution against
Simha's
15 Oblate
"o
lC Prolate
1
25 5 I0 15 20 25
Simha's factor
Figure 5.4 Plot of Simha" s viscosity increment v as a functWn of axlal ratio (a
/ b) for prolate and oblate ellpsokls of
revolution
(til) Linear Random Coils
No particular three dimensional structure can be allotted to a linear random col
l. This is
because the molecule exhibits a .great deal of rotation about the various segmen
ts in the

126
Bfphysfcal Chemistry
macromolecular chain. Hence, a large number of conformations are possible. The r
andomly
cotled macromolecules occupy a very large effective volume in solution. For the
same reason,
the intrnsic vscosltles of solutions of such polymers are often found to be vast
ly larger tha
those for spheres or ellipsoids. The intrinsic viscosity for such soluUons becom
es a measure of
the particle volume. As the conformation of the molecule continuously changes by
the npacts
of the surrounding solvent molecules, only average dmenslons of the molecule can
be deted.
Table 5.1 Itsts the values of intrinsic viscosities, vlscosRy increments and axi
al ratio for a
number of proteins,
Table 5.1 Intrlas/e viseoIt/es, viscosity increments, and calculated axial ratio
s for various
St. No. Name of Prote/n
I00 []
2.
3.
4.
5.
6.
9.
10.
II.
Myoglobin
Tropomyosin (a)"
Serum albumin
[horse)
Serum albumin
0roman}
y-Globulin (human)
Flbrlnogen (human}
Thyroglobulin

Tooacco mosaic virus

3.1
3.9
4.5
4.3
.52
6.2
4.2
6.0
25
7.1
28
5.7
74
8.2
5.6
8.1
35
9.9
39
Axial Ratio Prolate Axial Ratio Oblate
w = 20.1
w = 0.3
3.0
2.1
4.0
3.0
4.6
3.6
4.4
3.3
28
24
6.0
5.0

4.2
3.3
6.0
5.0
17.5
15
7.1
5.9
18
16
w=0.1
w=0.3
3.5
2.3
4.9
3.5
5.6
4.3
5.5
4.0
94
76
8.8
6.3
5.4
4.0
8.3
6.8
44
26
11.1
8.4
56
4O
The values of tropomyosin vary with different conditions of the experiment. The
values expres%-d

here are for acid Solutions at pH 2. I.

intrinsic viscosity,
v' = viscosity increment
w =
degree of hydration, i.e., if hydration is to the extent of I0%, w = O.
I,
:
if 30% then w = 0.3 and so on.
The radius of gyratOrS, Rg, gives a rough measure of the effective radius of a m
olecule (see
Box 5.1). When the molecule is a linear chain, the radius of gyration will be a
function of the
chain length of the polymer and hence of the molecular weight.
2.
Rod/J ofGyratlon : It is the square root of the mean square distance of
all individual electrons from
the centre of gravity of a particle. It is a measure of the three dimensional ex
tension of a particle.

127

Biophysical Chemistry
(ii) For rods,
where Mo isthe
Now, the intrinsic viscosity of a solution of macromolecuIes is related to the m
olecular
weight by an equation stated by Mark Houwink as
(I) = KILL .... (13)
where K and a are constants for a particular solute-solvent system and temperatu
re, and M is
the molecular weight, a is also known as an expansion factor. It depends on the
number of
bonds in a chain and takes account of the expansion of the flexible coil due to
the continuous
bombardment of the solvent molecules on it. If the polymer is dissolved in a 0-s
olvent, (see
Chapter 3) the intrinsic viscosity [I]0 becomes
[1] = KILL'

(14)

as a = I/2 in such a solvent.

If the flexible polymer is placed in a "better" solvent where there iS much inte
raction
between solute and solvent, then the radius of gyration will increase more rapid
ly with M. As a
is dependent on molecular weight, it will also increase. The maximum value Of th
e exponent a
in 'good' solvents is 1.0. The parameters K and a are are determined by measurem
ents of [I] for
each s01ute-solvent system using for calibration samples of known molecular weig
ht.
FACTORS AFFECTING VISCOSITY
Temperature
When a liquid is heated, the molecules in the liquid become more active. This in
creased
molecular activity or molecular motion occurs at the expense of cohesive forces
acting between
the molecules. As a result, the liquid now faces lesser resistance to its flow.
We observe a
decrease in viscosity, and the liquid now flows easily or we ma, say the liquid
has become more
mobile.
The variation of the viscosity of a liquid with temperature is best expressed by
the equation,

I = Aesj ..; (15)

A+E
... {16)
or
log I = T
where A = constant depending.upon the molecular weight and molar volume of the l
iquid;
T = temperature of the liquid;
and E = the activation energy per mole.

Stage B
129
According to equation (16), the plot of log l against the reciprocal of the abso
lute
i.e., l/T, should yield a straight line. This is found to be true for a large nu
mber
Figure 5.5). The explanation offered for such a plot is that astemperature incre
ases,
number of molecules which acquire sufficient energy (activation energy) to take
part in
[ flow increases. This increase in the number of molecules is in proportion to t
he Boltzmann
e-E1er, and hence viscosity decreases in a reciprocal manner.
C
60 40 20 0
-1.7
-1.9
2.9:3.0:3.1. :3.- :3.:3:3.4 :3.5:3.6 :3.7
103/T
lure 5.5 Temperature de1rtence of vscost plotted as log r vs. T
The opposite is seen in gases, i.e., with an increase in temperature, viscosity
rises.
For liquids having many hydrogen bonds some of the energy obtained will be utili
zed in
the H-bonds. The molecules thus require a higher amount of activation energy to
the resistance and increase flow. As a result, viscosity of such solutions is al
ways
temperature.
57C 60oc
Temp.erature
Flgue 5.6 Plot of log of tlme of JIow through
vlscometer of an albumin solution
as a function of temperature,
The temperature effects on viscosity of

lyophilic colloidal solutions are very queer. The

viscosity of albumin solution with changes in
temperature have been studied in detail .by
Ostwald (see Figure 5.6). As the temperature is
raised, the viscosity of the solution decreases.
upto a certain stage (stage A) after which there
is a sudden shoot up and an equally sudden fall
in viscosity of the albumin solution (stage B)..
After crossing this fluctuating stage the viscosity
curve appears even (stage C) and as if in
continuation to stage A. It seems as though just
before coagulation, albumin increases its degree
of hydration and as coagulation is completed it
is left bereft of water. The coagulated albumin is
hydrophobic in nature. The other lyophilic

Blophysa Chemistry
systems like those of starch and gelatin also show such anomalous behaviour thou
gh they may
be quite different from that shown by albumin.
The effect of pH on viscosity of colloidal solutions is very distinct. The macro
molecules
contain a nurn'tmr of positlveand/or negative charges on them (therefore called
polyelectrolytes).
The charges produce electrostatic effects which can profoundly influence the sol
ution behaviour
of the macromolecule.
The charges on the molecules induce opposite charges in the surrounding solvent.
The
electrical double layer which is thus formed moves along with the particles resu
lting in an
increase in friction thereby increasing the viscosity. This increase in viscosit
y arising from the
movement of the ionic atmosphere when the charged molecules move is called the e
lectrovscous
effect. It is affected by changes in pH and ionic strength of the solution. Kras
ny-Ergen derived
an equation for the electroviscous effect. It is written as
3 1 D
r)r = I+2.5 1+
22
j j
...
where k = specific conductance of the surrounding medium,
o = viscosity of the medium,
D = dielectric constant of the medium,
r = radius of the spherical macromolecule.
Fi = potential difference between the surface of the particle and a point in the
medium where the charge density is zero.
Fr = potential difference between the surface and centre of the particle.
As per equation (17), (Fi - Fr ) means the potential difference between the inte
rior of the
particle and the interior of the liquid. However, for practical purposes it is a
ssumed that (FI - Fr )
is equal to the potential difference across the double layer and the term (zeta

potential] replaces it.

When working with proteins, the electroviscous effect is minimized by c out visc
osity
measurements in 0.1 to 0:2 M sodium chloride solution. This greatly reduces the
extent of the
electric double layer and hence leads to reduction of the electroviscous effect.
At the isoelectric pH [the pH at which the net charge on the molecule is zero),
the electrostatic
forces of attraction within the molecule cause it to contract resulting in a dec
rease in the
-viscosity of the solution. Above or below this pH, there is either an excess of
negative charges or
positive charges and the molecule expands. This leads to an increase in the visc
osity. But, there
are some exceptions to this rule. Globular proteins, for example ribonuclease, d
o not show a
appreciable increase in viscosity even when they have attained their maximum pos
itive or
negative charge. This is due to the fact that the molecules of this type lack fl
exibility in the
native state.
If the polyelectrolyte expansion, has to be controlled, the dilution of the macr
omolecul
can be done with a solution of the same ionic strength (isoionic dilutionl. This
will keep th
ionic atmosphere around the macromolecule unchanged during the dilution process.

131
Viscosity bears a linear relationship with pressure in the case ofllqulds. This
means that
as pressure increases, viscosity of the liquid also increases.
Chemical Compotion
The viscosity of liquids generally depends upon the size, shape, flexibility and
rotational
diffusion coefficient (see Chapter 9) of the particles. The degree of orientatio
n ofa particle is
dependent on these factoi's. Liquids with large, elongated molecules show a high
viscosity.
When the solute molecules interact strongly with one another, they form
aggregates and the.
viscosity of the solution increases. Large amounts of dissolved Solids also incr
ease viscosity.
Specific Volume
.J.Batschinski in 1913 ave an equation relating viscosity to the specific volume
of" a
liquid as
C
-

... (18)
v-b

where vl = viscosity, v = specific volume of a liquid and b and c are constants.

On rearranging
Eqn (18) we get.
v = b+-...(19)
The equation shows that as specific volume increases, viscosity decreases and vi
ce versa. This
elationship is applicable only over a range of temperature.
The viscosity measurement employs the use of atleast three different instruments
based.
(0 capillary flow,
(i0 rotation of a cylinder immersed in the solution, and
(//0 the rate of fall of a ball through solution.

The time required when a liquid flows through a capillary under specified condit
ions can
be used to determine the viscosity of the liquid. This. method is based on PoLse
ug/e's Law which
embodied in the equation
- 8 IV- ... (20)
where = viscosity of the liquid,
R = radius of the eapillary in cm.
P = pressure applied in dynes per sq. cm.
t = time of flow in seconds,
V = volume of the liquid in milliliters,
I = length of the capillary in cn

..i (21)
... {22)
132
Biophysical Chemistry
The Poiseuille's Law states that the volume of a liquid flowing through a capill
ary is directly
proportional to. the flow time, the pressure under which the liquid flows, and t
he fourth power
of the capillary radius. Also, the volume of liquid that flows is inversely prop
ortional to the
length of the capillary and to the viscosity of the liquid.
The values R, V, I can be made constant by using a capillary of known dimensions
for all
the liquids. A glass capillary viscometer can serve the purpose. If we measure t
he time of flow of
two different liquids through the same viscometer, then according to Poiseuille
equation, the
ratio of the viscosity coefficients of the two liquids is given by
Since the pressure P and P2 are proportional to the densities of the two liquids
p and Q2
we may write
r12 t2P2 tp
Thus, once the densities of the two liquids are known, the viscometer is calibra
ted for the
measurement of the flow time tt and t2 of the two liquids. This permits us to ca
lculate the
viscosity of the unknown liquid i provided of the reference liquid is known. Wat
er is generally used as the reference liquid. The value /p is also known as the
k/nernat/c vlscostty of the liquid.
Thus, by capillary viscometer the viscosity of one liquid can be determined by m
easurement
of its flow relative to another liquid whose viscosity is known.
Design of a Viseometer
The Hess viscometer is often used to measure viscosity of blood. It utilizes hor
izontal
capillaries instead of the vertical ones usually found in other capillary viscom
eters (see Box
5.2).

Wscoslty
133
Torsion
... (23)
Fre 5.7 A Couette rotatlng-cyllnder viscometer
where T =
r, =
h =
Once the values ofT and 0 are known, l can be calculated by equation (23). The c
onstants
of the viscometer appearing in the equation can be determined by accurate measur
ements.
Alternatively, two liquids, whose viscosities are known can be used for calibrat
ion. The type of
flow obtained in this type of coaxial rotating cylinder viscometer is simpler th
an that in a
capillary viscometer. The only difficulty lles in con.trolllng the temperatureof
the system.
Liquids or solutions for which the viscosity is constant and is not affected by
the shearing
stress are called Newtonian liquids. For thesellquids, the thermal motion of the
molecules is
capable enough to counteract any effects due to flow itself and the liquid thus
exhibits Newtonian
ilow. On the other hand, the non-Newtonian liquids-display a change in viscosity
with variation
in the shearing stress. Non-NeWtonian behaviour is shown by molecules which are
very
dI
Rotation of a Cylinder Immersed in,the Solution
When a rotating body is immersed in a liquid it experiences a resistance in its
movement.
This is due to the viscosity effect of the liquid. The amount of retardation dep
ends on two
factors:
(/) the viscosity of the liquid, and

() the speed of rotation of the body.

-- I
Circular
't-- Ptnter
%'nple
The above principle was utilized by Couette
in- 1890 while designing a viscometer to measure
the viscosity of high molecular weight systems. The
apparatus consists of a rotating cylindrical cu.p.
The inner cylinder is held by a torsion wire (see
Figure 5.7). In the Couette rotating cylinder
viscometer, the liquid rotating in the outer cylinder
causes a torque. This torque and the angular
velocity of the rotating cylinder is recorded. The
viscosity of the solution is related to the torque
produced by the equation,

r2 r2

. .

=T o
torque required to maintain a constant angular velocity,
angular velocity of the rotating cylinder,
radius of outer cylinder,
radius of inneT cylinder,
height of cylinder immersed in the liquid.

134
Biophysical Chemistry
asymmetric or extremely flexible or are in the form of stiff coils as in DNA. Th
e capillary
viscometers produce a very high shearing stress and are therefore unsuitable for
viscosity
measurements of DNA solutions. As the Couette viscometer tends to minimize the n
on-Newtonian
behaviour of solutions, it can be efficiently used for viscosity measurements of
DNA solutions
also.
Rate of Fail of a Ball Through a Solution
The falling ball viscometers are designed to measure viscosity of solutions by a
pplying
Stoke's formula. A particle whose volume is much greater than the molecules of t
he solvent falls
through a liquid with a constant velocity. This constant velocity is attained du
e to two opposing
forces acting on the particle at the same time. They are (/) the centrifugal for
ce acting downwards
to accelerate the body, and (//) the frictional force acting in the opposite dir
ection which causes
an upward drag.6 When these two opposing forces just balance each other, as stea
dy state is
reached and the body now continues to fall with a constant velocity.
Let us consider the particle to be a sphere of radius 'r' and density p. The liq
uid through
which it fails has a density PL" The gravitational force acting on the particle
pulling it downwards
is given by
F = 4/3 r3 (p - pL) g

.,. (24)

where g is the acceleration due to gravity. The frictional force acting simultan
eously on the
particle leading to a visco .us drag can be given by
F2 = 6 r l v

... (25)

At equilibrium, F, balances F2 so that,

4/3 r (0 - P) g = 6 r
2r (P-p)g
or
q =
... (26}

9v
Equation (26) is called the Stoke's equation and is applicable to the fall of sp
herical bodies
in all types of liquid media, provided the radius of the falling body 'r" is lar
ge compared to the
size of the solvent molecule. If the difference is not large, the body tends to
"drop" instead of
failing with a constant velocity.
Inlet -- -Steel bali
] : -- -- Sample
A small steel ball of density p is dropped
through the neck of a cylindrical tube (see Figure
5.8) filled with the liquid whose viscosity is to be
determined, and the time of fall of the ball between
two marks is measured. The same procedure is
followed replacing the unknown liquid by a reference
liquid whose density and viscosity are known. The
viscosity of the unknown liquid can now be
determined by the relative time of flow of the two
liquids as given by the equation

Figure 5.8 The falllng-ball vtscometer

... (27)
Viscosity
135
where

-- viscosity of the unknown liquid,

12
-- viscosity of the reference liquid,
p
= density of the steel sphere,
l
= density of the unknown liquid,
I2
= density of the reference liquid.
t
= time of flow of the unknown liquid,
t
= time of flow of the reference liquid.
For viscosity measurements of macromolecular solutions, which generally exhibit
nonNewtonian behaviour, the Couette viscometer is routinely used. In this instrumen
t viscosity
measurements of macromolecular solutions can be attempted over a wide range of s
hearing
stress. Continuous measurements for a length of time under a specified shearing
stress can
also be performed. However, for asymmetric macromolecules with a molecular weigh
t exceeding
400,000 daltons, which show pronounced non-Newtonian behaviour, extrapolation to
zero
shearing stress is necessary to measure the viscosity. The method which is gener
ally followed is
of extrapolation to zero rate of shear, but in this case, the former is to be pr
eferred. The procedure
for such a measurement is as follows :
A concentration is chosen at which the specific viscosity of the macromolecular
solution is
measured at differing known values of shear stresses. This is to be followed by.
plotting a graph
of specific viscosity versus shearing stress. The graph is then extrapolated to
zero shearing
stress. The value of (Isp) = 0 ('T' represents the shearing stress) so obtained
is plotted along with
values obtained similarly by repeating the procedure at sever.al different conce
ntrations, with
concentration denoting the X-axls in a manner similar to the Huggin's plot (see
Figure
5.2). We can thus find the intrinsic viscosity of the macromolecular solution at

zero shearing
stress. The apparatus which can be used for the above experiment is a modified t
ype of capillary
visoometer proposed by Yang. It is a multigradlent capillary viscometer which ca
n accommodate
particular ranges of shearing stress. The equations given below for maximum shea
ring stress
and maximum rate of shear can be used for calculating the value of the various s
hearing
stresses applied which are in fact, necessary for the extrapolation. For the mac
romolecular
solutions whose intrinsic viscosity can be measured using the Couette viscometer
operated at
varying shearing stresses, equation (30) can be applied.
If the maximum shearing stress is denoted by,? and if Rm represents the maximum
rate
of shear,
. =

(28)

2L
"'"
4V
Rm =

(29)

where V is the volume of the liquid passing through the. capillary of .radius 'r
' length 'L' in 't'
seconds under a pressure amounting to P dynes per square cm.

... (30)
Blphysaxl Cherrdstnj
where 7 is the shearing stress, T is the torque applied to maintain a constant a
ngular velocity,
rd is the difference in radius of the two cylinders of the Couette viscometer an
d h is the height of
the cylinder'immersed in the solution.
APPLICATIONS 0' VIOMETRY
Viscometry has found wide application in the study of macromolecular properties,
and
more so, for proteins. Though among all physical techniques viscosity measuremen
ts are simple,
caution is required in interpreting the obtained data. More often viscometry is
combined with
other physical techniques to obtain fool-proof information about the macromolecu
le being studied.
But in many cases viscosity measurements alone provide definitive information. T
hey are
particularly useful in a showing structural changes in a macr.omolec, e associat
ed with physical
or chemical changes in the environment such as (0 protein denaturation, and/or (
) associationdissociation behaviour of subtmits of macromolecules. In the following text we s
hall discuss in
detail the relationship of some of these properties of proteins with viscometry.
Determation of Aymmetry, Siza md Shal of Maeromollu
Asymmetric particles do not rotate evenly in a velocity gradient and tend to ori
ent themselves
in the direction of the streamline of flow of the liquid. Thus their behaviour i
s non-Newtonlan,
since with increasing stress the degree of orientation of the molecules will inc
rease, The intrinsic
viscosity of such a solution will decrease with increasing shearing stress. Edsa
ll and Mehl
(I 940] working with 0.2% solution of myosin had observed a viscosity ratio of 1
.6 and a viscosity
increment (intrinsic viscosity divided by partial specific volume) of 340 at hig
h shear rates. This
indicated a very . degree of asymmetry of the myosin molecule.
The degree of orientation of a particle depends on its size, shape, flexibility,
and rotational
diffusion coefficient (see Chapter 9]. The combination of viscosity with data ob
tained by other
techniques like viscosity and radius of gyration(obtained from light scattering)
, can give us
Information about the shape of the molecule.

The spherical molecules are compact and therefore give rise to relatively small
viscosity
effects. The rod-llke proteins have a high axial ratio and give rise to very hig
h viscosity increments
because they tend to take up positions with the larger dimension oriented in the
direction of
current. In this position, the degree of orientation increases as the shear stre
ss increases. The
molecules thus lose their randomness.
Staudinger (1932) on the basis of experimental considerations had given an empir
ical
equation relating viscosity to the molecular weight of a macromolecular solute a
s
[l = KILL
Later, Mark and Houwlnk on the basis of theoretical considerations modlfiedthe a
bove
equation and gave a better expression as
[] = KMa
The empirical constant a is dependent on the shape and solvation of the macromol
ecule.
For non-solvated rigid molecules of spherical shape, a = 0. For rigid rods, a 1.
5 whereas for.
semi-rigid rods the value of a should range between 1.0 and 1,5. The flexible ra
ndom coils have
an a value falling in the range 0.5 to 0.8. In the 0 solvent a is equal to 0.5 f
or random coils.
To obtain molecular welghts of macromolecules, the intrinsic vlscosity data may
be combined
with those of diffusion or sedimentatiom The Schachman equations can be appropri
ately used
in such situations which are stated as

... (31)
Viscosity
137
4690 ($2o) [rl]/
(i-%)
6.58xI0-s
and M =
where Szo Is the sedimentation coeffic|ent in Svedberg units at a temperature of
9.0"C. Water is
used as a solvent for this purpose, V is the partial specific volume of the subs
tance (the volume
increment when I gram of dry substance is added to a large volume of solvent) D=
0 is the
diffusion coeiTcient at temperature 20"C.
Protein Denaturation
Denaturation is an alteration of the native structure of a protein. It is genera
lly accompanied
by a drastic change in one or more of the properties of the protein molecule. It
is more often an
unfolding process, for e.g., a globular form might change to a random coil confo
rmation thus
Increasing the disorder of a system. DenaturatiOn can be brought about by a vari
ety of agents
like acids, alkalies, heat, UV irradiation, ultrasonic waves, etc.
The intrinsic viscosity is sensitive to changes in macromolecular structure. The
refore,
viscosity measurements on proteins are useful In indicating changes in molecular
configuration
due to denaturation. When a rigid molecule of low axial ratio is unfolded into f
lexible chains,
there is an observed increase in viscosity, This can be illustrated by the actio
n of heat on the enzyme ribonuclease (see Figure 5.9). At 40"C there is an abrup
t viscosity change indicating a
partial uncoiling of the compact globular form that exists at lower temperature
into the flexible
at higher temperatures. In contrast, myosin when treated with guanidine hydrochl
oride
. [denaturing agent) shows a decrease in viscosity number. It is said that a dec
rease in viscosity
when rigid rods are converted into flexible coils. To obtain a detailed account
of the
denaturation process other techniques have to be combined with viscometry.

3
20oC 30oc 40oc 50oC 60oc 70C
Temperature
Figure 5.9 Vscosity changes by the action of heat on the enzyme ribonuclease
Tanford et.al., after measuring the [rl] of a series of proteins in 6 M guanidin
e hydrochloHde
have shown that an empirical correlation between [r] and the number of amino aci
d residues
written in the form

138
Biophysical Chemistry
[T]] = 0.716 n.ss
... (33)
where 'n' is the number of amino acid residues in the chain. The above equation
is applicable to
protein molecules of all sizes upto 3,000 residues. Thus, viscosity measurements
of solutions
that convert compact proteins to the random cot configuration can be related to
the size of the
polypeptide chain. This can also tell us whether different denaturants acting on
a protein produce
molecules differing in shape or properties.
Chemicai Modification
Most proteins can be modified by drastic treatments. If the modification is of t
he covalent
type where some covalent bonds are broken or formed accompanied by a change in t
he shape
of the protein molecule, the viscosity change is appreciable. Thus measurement o
f intrinsic
viscosity can also help us in detecting chemlcal modification of a protein molec
ule. Splitting of
a peptide bond in a polypeptide chain may result in unfolding of a compact struc
ture resulting
in a considerable increase in intrinsic viscosity, Same can be said about the br
eaking of disulphide
linkages in a flexible coil which may lead to molecular extension.
Association-Dissociation Studies
Association (aggregation) or dissociation of proteins can be studied by viscosit
y
measurements. By convention, if rod like molecules associate end to end, the vis
cosity of the
solution should increase. This is so because such an association is thought to i
ncrease the
asymmetry of the molecule. On the other hand, since side-by-side association dec
reases the
asymmetry, it should also decrease the solution viscosity. These conventions, ho
wever, should
not be taken at face value. To cite an example,, association of myosin increases
the intrinsic
viscosity giving the indication that myosin association occurs end to end. Other
techniques like
sedimentation and light scattering, however, clearly show that myosin associatio
n is side -toside. The 'increase' in viscosity in this experiment can be attributed to an art
ifact whereby.
higher aggregates are produced by the shearing stress in the viscometer. From th
is, it is very
clear that viscosity is not very useful in association-dissociation studies and
is used as an
adjunct technique to absolute methods such as osmometry and light scattering.

Since viscosity is not dependent upon molecular weight of unsolvated spherical p

articles,
it is of limited use in association-dissociation studies of essentially spherica
l particles. However,
if dissociation of globular proteins involves considerable unfolding producing a
random cot, a
considerable increase in the intrinsic viscosity can be observed. Viscosity can
be useful for
understanding such types of dissociations.
Given below are a few examples based on the discussion above.
Studies About Interaction Between Actin and Myosin
Today we understand the mechanism of muscle contraction and the role of actin an
d
myosin in this intriguing phenomenon. However, barely four decades back we were
in dark
about this mechanism. At this time important insight into the relationship betwe
en actln and
myosin and the relationship of ATP with these two proteins was obtained with the
help of
scosity studies.
Albert Szent Gyorgyi and coworkers mixed purified actin and myosin in concentrat
ed salt
soultion. They observed that this mixing resulted in a large increase in the vis
cosity of the
solution (Figure 5. I0). The inference was straight- these two proteins must be
interacting to
generate large polymers. Today we know that this polymer is the actomyosin filam
ent that
primarily constitutes the contractile apparatus of the muscle. They further obse
rved that addition
of ATP to this mixture caused the viscosity to drop to the original level (Figur
e 5.10). The
scientists explained the results in the following way

139
2,0"
0
Actin + Myosin ----> Actomyosin
Actomyosin + ATP ----->Actin + Myosin
0 0.2 0.4 0.6 0.8 1.0 F-actin
1 0.8 0.6 0.4 0.2 0 Myosin
When actin and myosin are mixed the viscosity of the solution rises. Addition of
ATP makes the viscosity
drop. |O)Actin-myosin mixtures; I actin myosin mixtures after addition of A TP.
that Actin from Different Species Acts Sim/larly
In an extremely interesting experiment Oosawa nd his colleagues proved that acti
n from
species indeed can act similarly. All they did was mix actin derived from the sl
ime
(a myxomycete) with myosin from striated rabbit muscle. In purified state the so
lution of
proteins showed low specific viscosity. The moment these two proteins were mixed
,
shot up (Figure 5. I I). Moreover, when ATP was added, the viscosity dropped
of.the previous experiment stated above. This means that actin and myosin from
removed species still interacted to give rise to actomyosin.
.--- 0.5 mM ATP
- - to" 5 mM-A- -TP- # 0.5 mM ATP
0 I0
20 30 40
Time (min)
Figure 5. I I. Effect of A TP on the viscosity of actomyosin in which the actin
was derlved from the $11me mold and the
myosin.from striatedrabbit muscle. Dotted line - pure actin from slime mold; das
hed llne - myosin from
rabbit striated muscle; solid line - mixture of acttn and myosin.

140
Biophysical Chemistry
Idea about the Native Structure of a Protein
Incubation of a protein in 6 M guanidum chloride denatures it. In the denatured
condition
the protein assumes a completely random coil configuration. Thus, if upon incuba
tion i-n 6 M
guanidinlum chloride, one sees an increase in viscosity of the protein solution,
one can safely
infer that the protein was quite compact in its native conformation. An example
is the enzyme
ribonuclease (Figure 5.9). Although in that figure the enzyme was denatured by i
ncreasing the
temperature, the same effect would follow with guanidinium hydrochloride treatme
nt also.
On the contrary, it is observed that with some proteins, the viscosity actually
decreases
when they are treated with 6 M guanidinium chloride. In such cases, one assumpti
on is that f
the protein may be fibrous in nature and that denaturation has given it more fle
xibility as a (
random coil. An example of such a case is afforded by poly(/-benzyl-L-glutamate)
. The viscosity \
of this polypeptide solution actually decreases upon denaturation meaning that t
his polypeptide
exists as a rigid rod in its native condition and the random coil conformation u
pon denaturation
is the more flexible form. The same is true about the protein collagen also.
However, such a conclusion may not be always correct. It could be that the prote
in contains
several polypeptide subunits that have dissociated upon guanidintum chloride tre
atment and
that this is the real cause of decrease in viscosity.
Detection of Intrastrand Disulfide Bonds in Proteins
Assessment of viscosity can easily lead one to detect whether or not a given pro
tein possesses
intrastrand disulfide bonds. When such bonds are present, the polypeptide will n
aturally be
present in a more compact shape owing to the folding that will result through su
ch covalent
bonds. Such proteinh will not give. much change in viscosity if they are treated
with 6 M
guanidinium chloride. However, if such bonds are broken, the same polypeptide wi
ll experience
a decrease in its compactness and in the presence of 6 M guanidinium chloride it
will assume
a random coil formation. The viscosity will increase.
[-mercapt)ethanol can reduce the disulfide bridges into individual sulfhydryl gr
oups, and
if one uses iodoacetate in the reaction mixture one can prevent these bonds from
reforming.
Thus, if consequent to such a chemical treatment, one finds the viscosity of the

protein solution
increasing, it means that the protein, in all probability, has disulfide bridges
. If no change in
viscosity occurs, it may mean the absence of such bonds. Thus calf brain tubulin
has [1] = 36.0
ml/gm when it has not been treated with [-mercaptoethanol. After this treatment
though, [1]
climbs to 44.0 ml/gm. This is a sure indication that disuffide bridges are prese
nt in this protein.
The compactness of a given polypeptide will differ depending upon which cysteins
are
involved in disulfide bond formation. If the cysteins involved in disulfide link
age are situated
wide apart in the polypeptide, the molecule will be more compact as compared to
the molecule
which has disulfide linkage between two cysteins situated very near each other i
n the polypeptide
backbone. Thus, a great change in viscosity upon mercaptoethanol treatment would
mean that
the polypeptide has disulfide bridges formed between distant cysteins, while a s
mall ar
indistinguishable change in viscosity would mean the presence of a disulfide bri
dge between
cysteins that are more or less adjacent on the polypepttde backbone.
Polymerization of DNA
Later, in the chapter on radioactivity, you will come across an example to study
DNA
polymerization using DNA. The example there involves incorporation of radioactiv
ity in the
growing chain of DNA. DNA polymerization can be studied without the use of radio
activity also.
The logic is simple. When polymerization takes place, the chain length of DNA in
creases. When
this occurs, the viscosity of the reaction mixture must increase. Thus, if DNA,
all four 5'triphosphate nucleotides, and Mg2 are incubated with DNA polymerase I, a short wh
ile later,
an increase in viscosity can be demonstrated. This increase will not take place
if one of the

141
not put in the mixture or if Mg2 is absent. This proves that DNA polymerization
all the components in the reaction mixture described above.
of DNA
If a circular. DNA molecule is incubated with a nuclease, what should happen to
the
r of the solution? As long as the nuclease is causing single strand breaks, noth
ing much
happen. This is because the DNA will retain its configuration despite the single
strand
due to the rigidity of base stacking. However, the moment a double strand break
results,
will be rendered linear. This will cause an. increase in the axial ratio and the
r will increase. If the nuclease action is allowed to continue, multiple double
strand
breaks will eventually occur resulting in multiple linear fragments of smaller s
izes. This will
result in a decrease in viscosity. Thus, with circular DNA molecules, the viscos
ity will initially
and then fall (Figure 5.12).
With linear DNA molecules, the viscosity will not increase at all if it is incub
ated with a
Initially, nothing will happen and then the viscosity will start going down.
One can determine whether the genome of an organism is circular or linear using
this
Time
5.12 (A) Circular DNA. Initially the viscosity increases. (B) Linear DNA, Vscosi
ty never Increes. From a
plateau it starts decreasbg with time.
. .
that Certain Dyes Intercalate in DNA
The dye, proflavine binds to double-stranded DNA tightly. When DNA is incubated
with
the viscosity of the complex increases. This can happen only when the length of
DNA
For this, the dye must get inside the DNA structure in a manner that it lengthen
s the
Other techniques can be applied to study the same phenomenon. When DNA is incuba
ted
with proflavine, the sedimentation coefficient of the complex decreases. This ca

n happen if DNA
becomes depolymerlzed due to proflavine. It may also happen if the axial ratio o
f the complex
increases. With the first possibility, a decrease in viscosity must take place.
This is not observed.
With the second possibility, viscosity should increase, and this is observed. Th
us, it seems that
the second possibility is correct. Thus, DNA length indeed increases when it is
treated with this
dye.
Employing fluorescec polarization, one learns that the dye becomes immobilized a
nd
seems to be in the same place as the bases are. This indicates that the dye gets
in between the
bases and pushes them apart thereby lexgthening the DNA chain.
SIGNIFICANCE OF VISCOSITY IN BIOLOGICAL SYSTEMS
The importance of viscosity in human health and disease stems from the fact that
the vital
fluid circulating through each and every part oF the human system, the blood, is
highly viscous
and is affected profoundly by even a small change in the viscosity of the medium
. Generally

142 Biophysical Chemistry

blood flow varies inversely with the viscosity of the blood. Viscosity depends o
n the hematocrit
value, i.e., the percentage of the blood occupied by the erythrocytes, one of th
e major components
of the blood. The large vessels like the aorta and vein have a high hematocrit v
alue whereas the
capillaries and small veins have a very low value due to the difference in the n
ature of flow of
blood through the different vessels (see Box 5.3).
Box5.3
Nature of Blood Flow through Different Vessels
Newtonian fluids (viscosity independent of shearing stress) under conditions of
laminar flow and
constant temperature obey the relationship,
where q = viscosity, y = rate of shear and = shearing stress.
..1161
/ 1494Fm
.$
945um
370,urn
i1111 m115 Prn
0 " i
2O 40 60 8O
Haematocrit (%)
Figure 1 Plot of apparent viscosity of erythrocytes in physiological saline agai
nst hematocrit.
Temperature is regulated at 25C. Figures in rectangles indicate the diameter of t
he
tubes chosen for the experiment
Blood; though inhomogeneous due to the presence of blood cells, behaves as a New
tonian fluid
in blood vessels with internal diameter 1 nm and above, But as the blood vessels
get narrower,
viscosity of blood decreases with increasing shearing stress. This anomalous beh
aviour of blood
is attributed to the erythrocytes as they are the predominant particles of blood
. The apparent

blood viscosity is indicated by the value of hematocrit which is about 45% for m
an (see Fig.l)
The figure indicates the change in apparent viscosity of blood with respect to h
ematocrit values
in vessels of varying internal diameters. At smaller diameters, flattening of th
e curve is observed.
The behaviour can be explained well if we go into the characteristics of the red
blood cells. The
red blood cells are biconcave having extremely flexible membranes. Because of th
is property and
the fluid content of the cells, the R.IB.C.s have a high degree of fluidity. Wit
h increasing shearing
stress, the erythrocytes assume shapes which facilitate their movement. In capil
laries, which
have a diameter smaller than 15 m, even upto 3 m, the red cells undergo extreme
deformation
(they assume an "arrow head" shape). As the shearing stress in these vessels of
narrow bore
increases the.red cells align themselves obliquely along the tube axis. This pro
cess is known as
dal migration. AS a result, the concentration of the R.B.C.s increases in the co
re region and

Vscositj
143
blood gets thinned out near the walls of the vessel.The layers in the peripheral
region now have
a lower viscosity and a high shear rate This lowered viscosity facilitates the m
ovement of the
central column of blood (predominantly R.B.C.) with an increased velocity. Thus,
the average
time spent by an erythrocyte in a narrow vessel like a capillary is shorter than
its counterpart
plasma, the volume element. The hematocrit value is therefore smaller in an acti
ve capillary than
the hematocrit of blood at rest.
Apart from the above parameter, the blbod pressure and the hysteretic (elongatio
n and shortening)
behaviour of the smooth muscles forming the vessel wall are important determinan
ts of the flow
of blood through a blood vessel.
Besides the hematocr/t, the other constituents of blood plasma, mainly the immun
oglobulins
and other plasma proteins also play a significant role in altering the viscosity
of blood leading
to many pathological states. In diseases such as hereditary spherocytosis, the r
ed blood cells
are very rigid and because of this abnormality, viscosity rises.
Variation in the viscosity of blood influences the working of the heart. In seve
re polycythemia,
the increase in viscosity of the blood increases the work of the heart. It was o
bserved by Mackson
in 1936, that the blood of patients suffering from congestive heart failure was
more viscid than
normal, The anaemic person presents a totally different but interesting picture.
In severe anaemia,
peripheral resistance is decreased but the cardiac output increases resulting in
overwork of the
heart. Blood viscosity is increased in several diseased states such as cyanosis,
polycythaemia,
diabetes mellitus, multiple myeloma, icterus and pneumonia.
Amoeboid movement which is a characteristic of all naked protozoa, leukocytes an
d
embryonic nerve cells has very close connection with viscosity changes. During t
hese movements
striking changes in viscosity take place. These changes are of the reversible ty
pe, the jelly like
gel state being converted to the fluid or sol state and vice versa. The changes
in Amoeba proteus
have been well described by Mast (1932). The cytoplasm in these organisms is sur
rounded by a
thin membrane, the plasmalemma. A thick rigid layer of plasmagel lies beneath th

e membrane,
with a thin fluid hyaline layer sandwitched in between. The plasmagel surrounds
a fairly fluid
plasmasol. As locomotion begins the plasmagel at the retreating end is converted
into the
plasmasol. The plasmalemma however, remains attached to the plasmagel and the su
bstratum. The newly formed plasmasol at the retreating end now advances forwards
and is transformed
into the plasmagel at the front. The forward flow of the plasmasol is facilitate
d by the alternate
contraction and relaxation of the plasmagel at the retreating and advancing ends
respectively.
Owing to these differences in the elastic strength of the two regions the cell i
s propelled ahead.
Locomotion is thus affected by the reversible gel-sol transformations as explain
ed above. Similar
gelation changes are observed in many other cells of physiological origin i.e.,
the WBC,
m.acrophage, connective tissue cells and malignant sarcoma cells during cell loc
omotion and
cell division.
Suggestions For Further Study
1.
Edsall, J.T., The Size, Shape and Hydration of Protein Molecules in The
Proteins, Vol. ]
(H. Neurath and K. Bkiley, eds.), Academic, New York, 1953.
2. Tanford, C., Physical Chemistry of Macromolecules, Wiley, New York, 1961.
3.
Yang, J.T., The Viscosity of Macromolecules in Relation to Macromolecule
s, Adv. Protein Chem,
16, 323 (1961).
4. Scheraga, H.A., Protein Structure, Academic, New York, 196 I.
5.
Haschemeyer, R.H. and Haschemeyer, A.V.E., Proteins : A Guide to Study b
y Physical and
Chemical Methods, John Wiley, New York, 1973.

144
Biophysical Chemistry
Problems
I.
If poly 7-benzyl-L-glutamate is denatured with 6 M guanidinium chloride
the viscosit}
decreases. What does this tell you about the structure of the polypeptide?
2.
You are given a plasmid and a linear DNA fragment. Using viscosity measu
rements how can
you prove which is which?
3.
You are given an unknown proteip,When you denatured it with 6 M guanidin
ium chloride,
the viscosity decreased. What possibilities will you consider about this protein
based on thk
observation?
4.
One can manipulate the activity of enzymes by manipulating the pH, ionic
strength, or the
temperature. Suppose hat you have incubated a double stranded linear DNA with a
nuclease
manipulating the conditions so that one single stranded break results every 10 m
inutes. On
an average, it takes about I 0 single stranded breaks before a double stranded b
reak results.
After what time do you think the viscosity of the solution undergo a significant
reduction?
5. A DNA solution has inadvertently been mixed with another unknown chemical and
a-reduction/
in viscosity can be observed. No definite conclusions can be derived from this o
bservation.
However, how many possibilities can you imagine which can give rise to reduction
in vlscosi!
in such a situation?
6.
When one heats DNA, it 'melts', The optical density increases. What shou
ld happen to viscosity
under such a situation? When a salt, say NaCl, is added to the solution and then
the solution
is heated, what should happen? If the salt concentration is increased, what shou
ld happen?
7.

If a polypeptide is incubated with a protease, what should happen to the

viscosity of the
solution?

6
SURFACE TENSION
Dry sand, when sprinkled gently upon water readily floats. The water surface beh
aves as
flit were covered by a fine elastic skin. The same sand particles, if introduced
below the surface
r sink. This anomalous behaviour of the sand particles nearia surface and in the
of the liquid is related to the phenomenon of surface tension. Before we proceed
to
what this phenomenon means, we shall discuss some properties of matter and
matter appears on a molecular scale.
Matter may exist in three different states in nature - solid, liquid, and gas. A
n assembly of
can exist in any of these states provided temperature and pressure are favourabl
e to
equilibrium which is established between the kinetic energy of the molecules and
the
forces between them determines the state of the matter. A solid has molecules co
mpactly
in a regular pattern. All kinetic energy possessed by the molecules is confined
to
energy. A liquid has molecules arranged at an average constant distance but thei
r
do not remain flied due to 'not so strong' attractive forces. Further, the addit
ion of
and translational energy makes them more mobile. As a result, on the macroscopic
the liquid is shapeless but has a definite volume. A gas, however, is not only s
hapeless
has lost its volume definition too. It is because the molecules in the assembly
are only
attracted to each other and the translational energy of the particles has gained
an upper
over the attractive forces. "
In a system where matter is present in more than one state, each state is called
a phase.
or may not be chemically identical with each other. The region of contact of two
is referred to as an interface. Interfaces may be of various types, viz., solid
- liquid, two
luids, gas-liquid or gas-solid. Two solids cannot ordinarily come into close con
tact
trapping any gas or liquid between them. Also, two gases do not form an interfac
e
of their high diffusibility. An interface between a solid and a gas, or between
a liquid
a gas is more commonly termed as a surface. An interface made up of two phases o
f the
compound is highly dynamic in the sense that molecules are continuously leaving
one
entering into another and returning to the original phase. When one moves from t
he

across the interface to the interior of the adjacent phase, one experiences an
m many measurable properties such as density, heat capacity, viscosity, dielectr
ic
electrical conductivity, refractive index, absorption behaviour etc., at the int
erface.
after moving a considerable distance away from the interface, the properties beg
in to
in accordance with the physical state and chemical nature of the material making
up
body of the individual phase.
On examining the arrangement of molecules in a phase, it is observed that in the
body or
phase, take a liquid for example, the molecules attract each other with a cohesi
ve
: which is uniform in all directions because they are surrounded by other molecu
les of the
kind. They are thus free to move in all directions, and free forces of attractio
n are not

146
Biophysical Chemistry
exhibited. At the surface, .on the other hand, the molecules are only partially
surrounded by
other molecules, and as a consequence they
experience an inward pull (see Figure 6.1). The
result of this inward attraction is that the
number of molecules at the surface tends to be
reduced to a minimum and the surface contracts
until its area is the smallest possible for a given
volume of liquid. This behaviour of the surface
is responsible for the nearly spherical shape of
Figure 6. I Cohesive forces acting upon
falling water or oil drops. Drops or bubbles canj
molecules of a liquid so as to
resume a variety of shapes under the Influenc
result in surface tension
of gravity, compression, and stretching but a
soon as they are released, they resume their spherical shape. It !s because a sp
here is a body
having the least area for a given volume.
The special strain that the surface layer experiences as a consequence of the un
balanced
forces is called surface tension. By definition it is the force in dynes acting
along the surface at
right angles [90) to any line one cm in length. The units are dynes/cm. The surfa
ce possesses
a considerable amount of potential energy. It is much higher than the potential
energy carried
by the molecules in the body of the liquid. The surface is eager to accommodate
certain types of
molecules (surface active) always in order to decrease its free surface energy.
If the surface has
to be extended, molecules have to be forced out from the interior to the top and
this requires
.energy. Work has to be done to overcome the attractive forces between the molec
ules and create
the new surface. The work required to increase the surface area by I square cm i
n called the free
surface energy. It is expressed in ergs/cm. Both surface tension and surface eli
iergy have the
same dimension in the CGS system of grams per second2. Numerically they have ide
ntical
values, for e.g., liquid with surface tension of 60 dynes/cm possesses a free su
rface energy
value of 60 ergs/cm2. Therefore, though surface tension is an intrinsic property
of liquids and
surface energy a fundamental property of the surface of the liquid, they are som
etimes used
synonymously for convenience during calculations. The relationship between surfa

ce tension,
surface area and the Surface free energy can be summarized as
Surface Tension x Total Surface Area = Surface Free Energy
Thus, ff the surface tension of liquid is y dynes/cm, the work done in increasin
g the
surface area by 1 sq cm will be ergs, and the surface free energy will be y ergs
/cm2.
The total surface energy per unit area, however, may not be equal to the surface
free
energy alone. As heat must be supplied to the expanding surface to keep the temp
erature in the
surface constant, a heat term has to be added to the surface free energy. The to
tal energy at the
surface per sq cm then will be equal to
dy
p=y-T -...(I)
dT
d
where is the rate of change of surface tension with a change of temperature, and
is the
total energy at the surface. The differential term is always negative as surface
tension always decreases with an increase in temperature.

Box 6.1
Surface Tension
147
The surface tension of a given liquid is a constant value. It depends on the nat
ure of the
liquid. Greater the magnitude of the attractive forces operating between the mol
ecules of a
liquid, higher is the surface tension.
When two immiscible liquids come in contact wit each other, an interracil tensio
n
develops. This is because the dividing surface or interface contracts spontaneou
sly in response
to the attractive forces of the molecules in the interior of each liquid respect
ively. However, this
interracil tension is not an additive property of the surface tension of the two
different liquids.
Instead it is seen that the value is often slightly lower than the larger of the
two surface tensions.
The relation between the interracil tension and the surface tension of the two i
mmiscible
liquids is given by Antonofi's rule which stands as
],2 = ], - 2'
... {2}
where y,2 -- interracil tension between liquids 1 and 2,
/, = surface tension of liquid 1, and
Y2' = surface tension of liquid 2.
The molecules at the interface of two immiscible liquids are more or less satisf
ied and
to this decreased number of unbalanced forces, interracil tension is lowered. It
is for the
same reason that the potential energy at the surface of the molecules of each li
quid is far below
than that seen for molecules in the body of the two liquids individually.
When a drop of liquid is placed gently on a slide, it either spreads out into a
thin fdm or
a drop on the surface depending upon its affinity towards the solid surface. For
this
of solid-liquid interface, interracil tension can be expressed in terms of conta
ct angle. A
angle (e) is the angle between a solid surface and a liquid meniscus (also see B
ox 6.1).
magnitude of this angle is influenced by the forces acting along the surface of
the Solid.,
Useful information regarding the wetting quality of a surface can be obtained by

determining
contact angle and there lies its importance. If the contact angle is zero, the l
iquid wets the
completely. For contact angle values upto 90 the extent of wetting is good but no
t perfect.
For values above 90, wetting is poor or non-existent. (see Box 6.1)
Wetting of Solids and Contact Angle
It haslong been surmised that interracil energies play an important role in phag
ocytosis (engulfing
and killing) of bacteria and other solid bodies by mammalian leukocytes. It migh
t be mentioned
here that the mammalian phagocytic defense consists of the circulating polymorph
onuclear
leukocytes (PMNL) as the first line of defense and the reticuloendothelial syste
m, comprising of
the monocytes and the macrophages, as the second line of defense. For phagocytos
is to occur,
the bacterium and the phagocyte have to be brought into sufficient contact with
each other.
Naturally, the surface energy of the bacter um and the phagocyte are intimately
related to the
degree of phagocytosis. Thus 'a knowledge about the interracil energies of the b
acter um and
the phagocyte can allow us to predict the extent of phagocytosis.
The solid-liquid interracil tensions of solids have been studied using contact a
ngle measurements.
It is one of the oldest techniques devised byYoung in 1805 for studying the natu
re of solid surfaces.
A contact angle e is the angle between a solid surface and a liquid meniscus, as
shown in the
figure below.

p![os
p!nb!'I

149
Surface Tension
Haemophilus influenzae (rough)
18.6
Mycobacterium butyricum
70.0
Streptococcus pyogenes
21 3
Sa/monella typhimurium
20.2
Sa/rnonella arizonae
19.0
Streptococcus pneumon, iae
17.3
Klebsiella pneumoniae and
17.0
Human Neutrophils
18.0 approx.
FACTORS AFFECTING SURFACE TENSION
Temperature
As temperature rises surface tension of a liquid decreases, i.e., surface tensio
n has a
temperature coefficient. The relationship between surface tension and temperatur
e
was first provided by Baron R. von Eotvos in 1886. The relationship may be writt
en as
7(MV)2/3 = K (to - t)
... (3)
where te represents the critical temperature of the liquid, (MV)2/a is proportio
nal to the molecular
surface, and 7 and V are surface tension and specific volume respectively, at an
y temperature t,
expressed in degrees centigrade. K is a constant called the temperature coeffici
ent of molar
surface energy having a value of 2.12 for a number of "normal" unassociated liqu
ids, e.g.,
i carbon tetrachloride, ethyl ether, benzene, etc. For another batch of liquids
like water, alcohols,

md carboxylic acids, the temperature coefficient is not only less than 2.12 but
varies with
This abnormal behaviour is attributed to the associated state of the liquids. In
a state the actual molecular weight of the liquid is not M but xM, where "x" is
the factor of
association, i.e., the average number of single molecules combined to form an ag
gregate. The
above particulars for explaining the nature of the temperature Cdefflcient were
given by Ramsay
: and Shields in 1893, They found that at temperatures not too near the critical
point, the molar
:i surface energy could be expressed by a modified form as
y(MV)2/a = K (t - t - 6) ... (4)
is equal to molecular volume can also be written as M/p where p is equal to the
of the liquid. The Ramsay and Shields equation then takes the form
7(M/P)21 = K(q - t - 6)
... (5)
According to J.D. Van der Waals, the surface tension of a liquid at temperature
't' should
to the critical temperature to by an equation of the form,
7 = to (] - t/t)"
... (6)
a universal constant and to depends on the critical constants of the liquid. 'n'
has a
close to 1.2. Later in 1916, M. Katayama proposed modified form of equation (3)
which is
7----- p;) = K(t -t) = Kt (1- t/to) ...(7)
p" are the densities of the liquid and its vapour respectively at the same tempe
rature.

150
Biophysical Chemistry
The above equation holds quite accurately for normal liquids at temperatures muc
h nearer
to the critical value than does the original form of Eotvos. In equation (4), ac
cording to Ramsay
and Shields, the surface tension of the liquid becomes zero when the temperature
reaches a
value which is lesser than the critical temperature to, by 6C. It follows that at
critical temperature
the surface tension of a normal liquid will become negative. The above predictio
n, however, is
not univerl. Several liquids have shown a value of zero only at the critical tem
perature and
not earlier. For such liquids. Katayama's equation is found to be the most suita
ble. It is expressed
as
y'"/(p- p') = c
... (81
where C is a constant for each substance. For associated liquids the value of C
increases\
slightly as the temperature is raised, but the effect is relatively small.
The above interesting relationship (8) between the surface tension and density o
f a liquid
is also called Macleod's equation (1923) and it holds over a wide range of tempe
ratures. It is
obeyed with considerable accuracy by many organic compounds nearly upto their cr
itical
temperatures. If both the sides of equation (8} are multiplied by the molecular
weight of the
substance M, we get
MY i--= ME = P (constant)
p-p'
The constant 'P' also called the "parachor" by S.Sudgen {1924) is essentially a
molecular
volume modified to eliminate some of the influence of the cohesive forces which
are different for
different liquids. Parachor may be defined as the molar volume of a liquid at a
temperature at
which the surface tension is unity. Two liquids of molecular weight M and M2 hav
ing densities
p and P2' and surface tensions 71 and 72 respectively, will have parachors of th
e value
P] = p] V]7/ and P2 -- 92 V2 Y2]/
where V and V are the molar volumes of the two liquids.

With an increase in temperature, kinetic energy of the molecules of the liquid I

ncreases.
This will weaken the cohesive forces acting between the different molecules. As
a result, the
inward pull exerted by the molecules in the interior of the liquid upon those on
the surface, is
lessened. Hence a decrease in surface tension of a liquid is observed with risin
g temperature.
Effect of Solutes on the ,Surface Tension of a Solvent
Lowering or raising the surface tension of a solvent on addition of a solute dep
ends largely
on the chemical structure of the solute. Substances which decrease the surface t
ension of a
solvent are said to be cap///ary active. Soaps, proteins, organic acids, alcohol
s, esters, ethers,
amines, and ketones belong to this class. Other like inorganic salts, salts of o
rganic acids,
bases of low molar mass, sugar, glycerine, etc., increase the surface tension of
the solvent,
though slightly, and are therefore called cap///ary/nact/ve.
A majority of organic compounds are neither typically polar nor completely non-p
olar.
Consider for example an aqueous solution of fatty acids. The fatty acids possess
a hydrophflic
head (polar) and a hydrocarbon chain (non-polar). The polar part of the molecule
makes it
reasonably soluble. The hydrocarbon residues in the fatty acids are not at ease
in the interior of
the solution and therefore if they are to be brought to the surface, little work
will be required.
Once they accumulate at the surface, surface tension of the solvent will be lowe
red. The capillary

I
Surface Tension
151
activity of these amphipathic molecules thus depends largely on the predominance
of the non
of the molecule. A similar movement towards the surface is seen when non-polar
olutes are dissolved in water. These solutes concentrate preferentiallyat the su
rface and lower
the surface tension of the solvent. They are then said to be positively adsorbed
. Positive adsorption
can be visualized in the form of a surface crust when beaten white of egg is lef
t standing. The
a lbdmin in it accumulates at the surface of each bubble as a result of surface
concentration
md coagulates there. As a result a rigid film is formed at the surface. Saponin,
a plant glucoside,
nd bile salts have remarkable surface tension lowering ability. A very dilute so
lution of saponin
In water exhibits a surface tension of only 20 dynes per cm as compared to 73 dy
nes per cm of
pure water.
On the other hand, inorganic electrolytes in general, raise the surface tension
of water and
are, accordingly, negatively adsorbed at a water surface. In as much as surface
tension is
primarily dependent on mutual attraction of molecules, salts which are ionic and
therefore
polar in nature, by virtue of ion-dipole attractions, tend to pull the water mol
ecules into the
of the solution. In such a situation, if the surface has to be extended, addonal
work
be done to overcome the electrostatic forces. Polar substances are therefore, ca
pillary
above observations are summarized in Figure 6.2. The graph shows three types of
Type I curve corresponds to surface inactive substances. The increase in surface
tension
concentration of these substance is infinite. An opposite effect occurs on addit
ion
surface active substance (type Ill). However, the curve is remarkable in the sen
se that the
in surface tension occurs only upto a particular concentration. Beyond this, any
in concentration will not alter the surface tension of the liquid. Type II curve
the changes in surface tension of a liquid on addition of non-electrolytes or we
ak
The graph depicts a gradual and regular decrease in surface tension of solvent
olute addition.

Concentration
Figure 6.2 Effect of dlssolved substances on the swface tension of the solvent.
I
Surface inactive substances [strong electrolytes {ionic salts} alkali].
II
Non-electrolytes and weak electrolytes.
Ill
Surface active substances {soaps. proteins, etc.}.
The behaviour of amino acids also differ according to their polar nature. Figure
6.3 shows
surface tension of aqueous solutions of amino acids varies with concentration of
the

I
152
Biophysical Chemistry
Solutes which show a decrease of surface tension with increasing concentration a
re said
to exhibit positive surface activity, while those that raise it are said to have
a negative surface
activity; The solutes of the former type arc
Concentration
Figure 6.3 Surface tensions of amino acid solutions at 25C.
I Glyclne
V Lysine
II -alanine
VI y - amino butyric acid
Ill a-alanine
VII y - amino caproic acid
IV -aminobutyric acid
Solutes which show a decrease of surface tension with increasing concentration a
re said
to exhibit positive surface activity, while those that raise it are said to have
a negative surface
activity. The solutes of the former type are also called surfactants. The role o
f surfactants has
been extensively studied in man and it is seen that lung function is intimately
linked with the
amount of surfactant present in the alveoli (see Box 6,2).
Box 6.2 ....
Role of Pulmonary Surfactant: Stablllty of Alveoll
Lungs, the respiratory organs
agraduatedseries oftubes that
medium cimulates through these airways usually
occurs by means of diffusi
and relaxation (inspiration) of the alveoli. The alveoli are
during expiratioD the expiratory muscles

the surface
tension generated bythethin ,aqueous film or
the,pressure suffiCletlyto, eollapsethe alveoli. Because tl
to theradius accord=n.to katlace's lawwhiCh is given as ,
--

end expiration would lead to

as the smller alveoli wou!d empty into the larger alveoli, andso on.
Thisproblem iscircumvented by
mixture oHipidsand proteins, the
interfacewithin'.,the. <

153
Surface Tension
of thelung at the end of each expiration. The collapsed alveoli,, like collapsed
-balloons would
require-greater pressure to inflate them than partially collapsed alveo i and th
is would hamper
smooth:gaseous exchange. ,
Pulmonary surfactant is acomplex mixtureof about 90% lipid 10% protein and small
pementage
lmitoylphosphatidylcho ine
generated by the aqueous
,rmaintaining
t.i,,:
..yelin is,the
;r, but investigations regarding
[
:n=sare inco
studies on surfactant proteins suggesttht these
.
prgteins may play a prominent role in tPie organization of thelipids int
he airway
I'
In man, a direct relationship has been reported between the amou
nt of surface active mater a
11 :
and the pulmonarysurface area. In premature infants, the normal pattern
of ung development s
fii:
= interrupted frequentiyresulting in suffactant insufficiency. A consequ
ence of this is the respiratory
Ji
distresSsyndrome (RDS)wh ch isa eading cause of morb d tyjn these infant
s. An established
[.
treatment of this Complication is introduct on oflhigh concentrations of
oxygen through nspiration
"
accompanied by ventilatory support.This =treatmitis reported to have sho
wn an alteration n the
levels of surfactnt protein, leading to their increased acumu ation!n th

elngs: However, current

..: ..= treatment of the diseae lnvolvetracheal administraton of multipledoseof
mixture of.the
iil : . surfactant materials, namelyphospholipid and thesurfactant"prote us. An
alternate approach for
i... " resolution of acuIeRDS inv es plarmacoiogical internt on but mCich rema u
s to I: elucidated
,.-i regarding the regulation and expiession of the genes itiv01ved in surfactan
t protein synthesisand enzymes involved in surfactant phospho!ipid synthesis bef
ore th s approach can be applied
The tendency of molecules to either concentrate at the surface or remain in the
interior
assumes great significance in the human body. It is this property that is respon
sible to some
extent in forming the limiting membranes (see Box 6.3).
Biomembranes show
proteins

154
Bio1hysicca Chemistry
proteins adhering to lipid-aqueous interfaces because very small amount of prote
ins were effective
in reducing the surface tension of a model ]ilid-water system considerably (see
Figure 1).
Exterior
Protein film
Hyd}ocarbon
chains
Polar head
Lipold film

Protein film
Interior
Figure 1 TheDanielt and Davson model for cell membrane
Untilthe late 1960's bilayer models were often pemeived to be static structures
but studies with a
series of,biophysical techniques involving X-ray diffraction, nuclear magnetic r
esonance (NMR) "
and electron spin resonance (ESR) spectroscopy,-fluorescence microscopy, electro
n microscopy
and others have revealed the dynamic nature of thecell membranes which have been
summarized
in the Singer and Nicolson's fluid mosaic model proposed in, 1972 (see Figure 2)
.
Carbohydrate chains
Proteins
attached to polypeptide
\i 3-'."
backbone
-

:
Llpold film

{ chainSpolar Hydrocarbon ]head ---- . '] ' Protein

s
Figure 2 The Singer and Nicolson model,for cell membrane

Hydrogen-Ion Concentration
Surface tension of protein solutions changes with hydrogen ion concentration. A
minimum
in the surface tension Is observed in the isoelectric zone.
Surface Tension and Vapour Pressure
The surface of a liquid assumes different shapes in different situations. Whenwa
ter is in
a pipette, it has a concave surface. When on a glass slide as a film, it has a p
lane surface, and
in the form of drops it has a convex surface (see Figure 6.4}, When liquid from
a plane surface
forms a drop, there is an increase in surface area. This increase requires more
surface energy.
The molecules of the convex surface are attracted by other liquid molecules to a
less extent now
than they were in a plane surface. The drops also have a higher vapour pressure
than earlier
(plane surface). The opposite is observed for a concave liquid surface. There, t
he surface liquid
molecules are attracted more than they are in a plane surface. The vapour pressu
re here is also
the lowe. st amongst the three surfaces. These observations are compiled in an e
quation given by
Kelvin.

Tension
155
P 2M7
In Po RTpr
... (10)
Po are the vapour pressures of the plane surface of the liquid and convex surfac
e
drop) respectively, M is the molecular weight, y is the surface tension in dynes
cm-, p is
density of the liquid, R is the gas c.onstant expressed in ergs per degree per m
ole (8.31 x
T is the absolute temperature, and r is the radius of the liquid drop or the cap
illary (if it is
oncave surface, r is negative).
The Kelvin equation indicates that liquid drops have a considerably higher vapov
.r pressure
()
@ @ O0
. ()

lgure 6.4 Surface of llquid JIlms.

(AJ Concave surface when in a pipette.
(B) Plane surface when as aJIlm on a glass slide.
{C) Convex surface when as a drop.
MEASUREMENT OF SURFACE TENSION
tension of liquids can be measured by either of the tv, methods: static and dyna
mic. static methods are based on the assumption that the liquid has attained sur
face equilibrium.
uids and solutions of crystalloids the process of attainment, of equilibrium is
very
the static methods are best suitable. But for colloidal solutions a considerable
time is
reach the equflibrium state and therefore the dynamic methods of measuring surfa
ce
preferred. The dynamic methods measure the tension of a liquid before the surfac
e
has had time to form. There are other methods too which fall between the static
and the
methods. Among the static methods, the most commonly used ones are.(/) the capil
lary

(//) the du Nouy ring method, (it/) the Wilhelmy balance method, and (iv) the dr
op-

156
Biophysical Chemistry
Automated Recording Devices
In the middle of the 20th century many automatic recording instruments making us
e of
either the Wilhelmy vertical pull method or the horizontal one were devised. The
se automatic
devices record the pressure exerted by a fflm as a function of the area occupied
. Surface pressure
may be changed by altering the area occupied by a film either by compressing or
expanding the
iflrn. This' force-area relatioriship can be recorded as a curve on a kymograph.
.In another
instrument described by Anderson and Evett a mirror is attached to the horizonta
l float of a
horizontal idm balance. A lens system is placed in such a way that it directs a
beam of light
from a light source on this mirror. Reflected beam of light now illuminates a tw
in phototube.
Any change in the film pressure displaces the light beam and this serves as a si
gnal to the
phototubes to bring about the firing of either of the two thyratron tubes which
in turn activate
a motor. The motor works to restore the float to its original position by means
of a torsion wire.
In doing this, the angular dplacement taking place is tr .ansformed into an elec
trical message
which is captured in a potentiometer recorder.
In 1963, Mendenhall and Mendenhall described an instrument which can carry out t
he
compression and expansion cycles of surface iflms automatically upto a ratio of
70:1. This
phenomenon of alternate compression and expansion is called hysteresis. Study of
hysteresis
loops, i.e., surface tension as a function of area, are of physiological importa
nce. It has been
observed that a surfactant found in the alveoli, responsible for the pressure-vo
lume hysteresis
following contraction and relaxation of the whole lung, exhibits hysteretic beha
viour and thus
prevents the lung from collapsing after a contraction wave (see Box 6.2).
Suggestions For Further Reading
1.
Vold, M.J. and Vold, R.D., Colloid Chemistry, Reinhold Publishing Corpor
ation, New York,
1964.
2..Shaw, D.J Introduction to Colloid and Surface Chemistry, Butterworth, London,
1966.
3. Adamson, A.W., Physical Chemistry of Surfaces, John Wiley & Sons, New York, 1
967.

4.
Weaver, T.E. andWhitsett, J.A., Function and Regulation of Expression of
Pulmonary Surfactant
-- Associated Proteins, Biochemical Journal, 273, 249-264 (1991).

7
ADSORPTION
In the preceding chapter on surface tension we discussed the nature of cohesive
forces
between the molecules of liquids and solids. We observed that surface atoms cann
ot
in the three dimensions as the atoms in the interior do, and thus bonding forces
at the
urfaces are not completely satisfied. In order to satisfy their residual forces,
surfaces of liquids
tend to attract and retain on them gases or dissolved substances with which they
contact. This phenomenon of concentration of molecules or long on the surfaces o
f all
is called adsorption. The substance which is thus adhered to the surface is said
to be the
phase or adsorbate while the substance to which it is attached is the adsorbent
(see
7.1).

"

o
//////////S//////////// AdsorUe.t
Figure 7. I Adsorption : Concentration at tim surface
Adsorption should be carefully distinguished from absorption. In the latter proc
ess a
' retainedon the surface, but penetrates to the interior to become distributed
the phase (see Figure 7.2). However, at times, both adsorption and absorption ta
ke
side by side and it is difficult to distinguish between the two processes experi
mentally.
general term sorption (J.W.McBain, 1909) is implied in such cases.
-- Surface

of light.
158
Biohysico Chem/stry
KINDS OF ADSORPTION INTERACTIONS
The forces operating between the molecules of an adsorbate and the adsorbent are
mostly
short-range forces as suggested by I. Langmuir. These forces may be non-specific
like the
dispersion forces, the orientation forces and the Induction forces. Sometimes ch
emical bonds like
ionic and covalent bonds which are specific in nature may also be involved in th
e adsorption
process. In such a case, when a strong chemical bond appears between a molecule
of the
adsorbate and the surface of the adsorbent, the phenomenon is called chemical ad
sorption or
chemisorption.
The physical forces involved in the adsorption process are non-specific in natur
e like the
dispersion forces or van der Waals forces, These forces arise from attraction be
tween temporary
,r instantaneous dipoles. The origin of an important part of the van der Waals f
orce can be
elaborated thus: owing to the continuing motion of all the electrons in their or
bits within the
atoms of a substance, a constantly changing electromagnetic field is set up outs
ide the substance.
At any gtven instant it may influence upon the electrons in neighbouring matter
in a way that
the electron cloud may be more concentrated at one side of the atom producing fl
uctuating
dipoles. When two dipoles approach each other, their motions are no longer indep
endent. Instead,
two dipoles oriented in the same direction produce an attractive force (see Figu
re 7.3). These
forces are called dlspersionforces because fluctuating dipoles cause the phenome
non of dispersion
11)
12)
13)
iVgure 7.3 Model of dlspersion forces
(I)
Even distribution of electron density of an isolated atom.
(2)
Instantaneous dlpole formatlon due to uneven distrtbutlon of electrona.

(3)
Attraction of two instantaneous dipoles.
Other physical forces of attraction like the orientation forces appear upon adso
rption of
polar molecules carrying constant electrical charges. The electrical charges app
ear on surfaces
either as a result of electrolytic dissociation or preferential attraction of ch
arged long from the
sunndings incompletely satisfied atoms of the surface. For example, charcoal, if
placed in
an aqenvironment is negatively charged because certain valence of the carbon ato
ms
(secondary valence) at the surface which are incompletely satisfied attract hydr
oxyl long from
the medium. These hyflroxyl long holds on to the surface conferring upon it a ne
gative charge.
Polar molecules (these have a net separation of positive and negative charge in
individual
molecules) have weak electrostatic forces between molecules resulting from attra
ction between
opposite ends of individual molecules. Polar molecules tend to orient, themselve
s with their
positive poles toward the negatively charged surface or negative poles toward th
e positively
charged surface (see Figure 7.4). These orientation forces are much weaker than
the electrostatic
forces of attraction between long, but somewhat stronger than the dispersion for
ces between
non-polar molecules.

159
( Polar molecule
circles represent adsorbent
molecules
Surface
,=: Surface
..-----. Iositively
negatively
:
chged
chged
"
(A)
e 7.40ntatn of molecs
ducon forces so play pot role e adsofion process. e elecosc which elecons ier at
tracted to or repelled fro one atom (or groups of atoms)
to oer, e cled inductive effects. adsorbate may not contn dipoles to , but e ele
cc chge on the suace of the adsorbent may duce dipoles in the
molecules ereby attracting them. It may so happen that dipole moments appe
adrbent suace due to inducHon by the dipoles berg adsorbed.
e molecules of e adsorbate approach those of the adsorbent suace, the above
e opposed by forces of repulsion. AdsoHon d desoHon conue till
is reached. A molecule being adsorbed is not associated a sine cene.
centres (see Figure 7.5).

0 0 0 0

I.
o o o o o
I Adsorbate molecules
0-......
Dotted lines represent residual
forces of attraction of surface
molecules

Figure 7.5 Adsorption of adsorbate molecules at multiple sites of the adsorbent

In addition to the non-specific interactions discussed earlier, molecular comple
xes with
bonds are often formed on adsor)ents. Hydrogen bonds are much weaker than normal
bonds but are stronger than thee/dipole-dipole interactions between polar molecu
les.
bonding occurs beositively charged hydrogen atom in one molecule and a
highly electronegtive atom in a second m61ecule (see Figure 7, 6). Further, if t
he adsorbent
of donor-acceptor interactions with the adsorbate, unstable complexes are usuall
y
H -- 0 --H..- ..... H
Figure 7.6 Hydrogen bonding represented by the doffed line, between two water mo
lecules.

160
Biophysical Chemistry
Finally, sometimes the association of the adsorbate and adsorbent may result in
a strong
chemical bond. In such a case a new surface chemical compound is formed which ma
y further
take part in the adsorption phenomena.
ADSORPTION CHARACTERISTICS
As adsorption phenomenon involves concentration of a substance on the surface, t
he
extent of adsorption depends on the surface area. The larger the surface of the
adsorbing agent,
the greater is the adsorption. Therefore, matter in finely divided or colloidal
state exhibits great
powers of adsorption. Finely divided metals like nickel and platinum and porous
substances
like charcoal, fuller's earth, silica gel etc., provide a large surface area and
are best solid
adsorbents.
Physical adsorption is due to the operation of relatively weak electrostatic for
ces of attraction
like the van der Waals forces. These forces are characterized by low heats of ad
sorption (approx.
1 Kl mole-). Therefore, physical adsorption proceeds best at low temperature and
decreases
with increasing temperature ([ Chatelier's principle). The latter occurs due to
the increased
kinetic energy of the adsorbed molecules which causes them to escape more rapidl
y from the
adsorbing surface. Physical adsorption can also be easily reversed by lowering t
he pressure.
In contrast to physical adsorption, chemisorption is associated with high heats
of adsorption
(approx.50-100 kcal mole-). The chemical bonds formed between the adsorbate and
the adsorbent
cannot be broken easily and therefore chemisorption is seldom reversible. Only a
fter heating to
high temperatures, the process is reversed. However, it may happen that the subs
tance desorbed
at higher temperature is not the same as the one originally adsorbed. The heat o
f adsorption
decreases as the amount of substance adsorbed increases, apparently due to progr
essively
decreasing attractive forces as multilayers of molecules are built up on the sur
face of the
adsorbent. Some systems may show physisorption at low temperatures and chemisorp
tion at
some higher temperature.

Adsorption proceeds best from dilute solutions, i.e., it is more nearly complete
when the
ratio of adsorbent to the adsorbate is high.
The surface charge of the adsorbent is an important criterion in adsorption. A n
egatively
charged surface will adhere upon it more of a positively charged adsorbate than
it will do of a
negatively charged one. As for e.g., cellulose strips will adsorb more of the dy
e methylene blue
(positively charged) than of congo red (negatively charged).
The adsorbing powers of many substances can be increased by temperature treatmen
t.
Heating in specified conditions may result in activation of the adsorbent. This
heightening in
adsorbing powers is attributed to an alteration in the nature of the adsorbing s
urfaces. For
example, chcoal gets)activated when heated in streaming steam and then in a clos
ed chamber
at about 800/ .
The molecules adsorbed on a surface show a distinct pattern. They are oriented a
nd
arranged in a definite manner relative to the adsorbing surface as well as to th
emselves. In the
biological systems the patterned and orderly arrangement of the molecules of the
cell membrane
affords useful electrical properties. We shall discuss this aspect of adsorption
in the subsequent
text.
MOLECULAR ORIENTATION
The molecules in the bulk of a liquid are randomly distributed relative to each
other
whereas they are arranged in more or less orderly fashion at surfaces or interfa
ces. This orderly
arrangement of molecules in surface films or at interfaces is called molecular o
rientation. The

161
of molecular orientation was In-st introduced by Hardy and further developed by
Harkins,
Adam and their co-workers.
Molecular orientation results due to the differences between the forces of attra
ction of the
semipolar, and non-polar groups in a substance for another substance which may b
e the
or ariother phase. The polar groups like -OH, -SO3H, -NH2, -COOH, -CN, -CHO,
-NO2, etc., contain atoms which do.not have identical eectronegativities. As a r
esult, a net
of positive and negative chmges occurs. The atoms that possess free pairs of ele
ctrons
a tendency to form co-ordinate linkages. Because of the free pairs of electrons
the
s are surrounded by rather strong forces of attraction. On the other hand, nonlike hydrocarbon radicals have weak forces of attraction around them. This is so
they contain atoms that have nearly equal attraction for the electrons and thus
there is
any difference in the electronegativities. When a molecule containing one of the
polar groups and a non-polar hydrocarbon radical comes in contact with a
it will tend to orient itself in a definite fashion.
Hydrocarbons are characteristically insoluble in water but soluble in organic so
lvents.
if one of the earlier mentioned polar groups are introduced into a hydrocarbon m
olecule,
When such a substituted molecule is added to water it
surface in a characteristic manner. Consider, for example, a solution
acid [CHc(CH)4-COOH] in benzene which is added to water. An emulsion soon
with benzene droplets surrounded by water. The benzene droplets thus form one ph
ase water forms the other. The acid molecules being freely soluble in benzene wh
ile less
: in water are retained in the benzene phase. But at the interface between the t
wo liquids
of caproic acid molecules takes place. The arrangement is interesting. The
,. acid molecules are attracted towards water and therefore they orient towards
water through hydrogen bonds. On the other hand, the C5HI group
acid, a non-polar group, has greater affinity for the organic solvent benzene an
d is
towards it. The acid molecules thUS concentrate at the-interface of the two liqu
ids,
and benzene, with their polar heads toward water and the hydrocarbon tails towar
d
(see Figure 7.7).
Figure 7.7 Orientation of caproic acid molecules across a benzene-water interfac
e
Acids with short hydrocarbon chains like acetic acid (CH3.COOH) and propionic ac
id
are freely soluble in water. Nevertheless, they do orient themselves at the wate
r-

with their polar groups adhering into the water while the hydrocarbon radicals a
re

Surface
162
Biophysical Chemistry
directed above the surface towards the air vertically in a more or less parallel
fashion (see
Figure 7.8).
Solution of propionic
acid CHCOOH in
water
Figure 7.8 Orientation of propionic acid molecules across a water-air-surface
Membranes
The living membranes have various kinds of molecules embedded in them. Some are
truly
polar while some*fire non-polar. There also exist macromolecules like proteins a
nd compound
which are polar at some of their atomic groupings but are non-pglar at o
thers. This
lipids
difference in size, shape and arrangement of atoms in the different molecules in
fluences their
orientation. For example, lecithins and cephalins have been seen to orient thems
elves at a
0
water-lipid interface at an angle of 30 to the water surface, while the similarl
y shaped molecules
of sphingomyetins and glycolipids tend to orient with their long axis perpendicu
lar to the water
surface. Also, the molecular pattern due to orientation is constantly under chan
ge, as influenced
by environmental and intracellular conditions.
The arrangement of macromolecules relative to each other in the protoplasmic mem
branes
is worth mentioning. According to the Singer and Nicolson model proposed in 1972
, the lipid
molecules are arranged in two layers with their hydrophllic heads to the exterio
r while the
hydrophobic hydrocarbon tails face each other. On the hydrophilic heads are adso
rbed the
protein molecules. Protein molecules may also be an integral part of the bilayer
(see Chapter 6
for diagrams). The protein molecules are so arranged because they are more hydro
philic than
the lipids and major part of the water of the membrane tends to be associated wi
th the protein.

The protein molecules are capable of translation or rotation. The long hydrocarb
on chains of
the lipid molecules are bound together at many points along their long axis by h
ydrophobic
bonds. The membrane is dynamic and this poised arrangement of molecules may beco
me
disordered under certain conditions and a transition may occur from the gel-like
ordered phase
of the bilayer to the sol-like disordered phase.
Emulsions
.
Any two immiscible liquids normally forti a two-phase system when mixed
together. But
by vigorous shaking a colloidal dispe,rsion odroplets of one liquid in a
nother may form. This is
an emulsion. As soon as the mechanicai-ffispersive action ceases, the dr
oplets come in contact
with each other and merge to form larger particles in order that the tot
al surface free energy
may be reduced. The coalescence process is rapid and within a few minute
s the droplets merge
to revert to a two-phase system. Usually, one of the liquids is water an
d the other liquid Immiscible
with water can be designated as 'oil' for theoretical purposes. Accordin
gly, we come across

Adsorption
163
emulsions belonging to either of the two classes water-in-oil type (W/O) or oilin-water type
We have just mentioned that emulsions are usually unstable. But there are agents
which
when added to such 'temporary' dispersions convert them into stable ones. Such a
dditional
components are called emulsifiers or stabilizers. Milk is an excellent example o
f an emulsion of
butter fat in water stabilized by casein, a protein. We frequently come across m
any other
stabilizing agents such as gums, soaps, bile salts, and saponins. Most of the su
rface-active
(those possessing surface tension lowering ability) act as good emulsifiers. The
ir
eiliciency lies in their preferential adsorption for one of the two interfaces,
i.e., oil-air or water-This is because the emulsifier is adsorbed on the drops d
uring the formation of the emulsion
reduces their degree of coalescence though it may later on go and lodge itself a
t the oilwater interface. Surface-active substances can be put into two categories depend
ing upon their
number (hydrophile-lipophile balance number). The HLB number is calculated addit
ively
composition of their chemical structure. The ones which show a low HLB number, i
.e.,
to six, lead to 'W/O' emulsions while those with a high HLB number, i.e., betwee
n
lead to 'OfvV' emulsions. Ross and co-workers have shown that the HLB number
to the spreading coefficient and hence in the case of O/W emulsions it is linear
ly
to the tendency of an oil drop to spread over the surface of an aqueous solution
of the
The most important aspect of the emulsifier action appears to be its orientation
at the
between the two liquids. A soa consists of a long hydrophobic hydrocarbon chain
and a hydrophilic polar end (head). In a oil-soap-water system, the polar head b
eing
attracted to water will orient itself towards water and the non-polar tail will
point
the oil. By obeying the principle of minimal surfaces, the liquid which has a hi
gher
tension will tend to draw itself into spheres and will be surrounded by the liqu
id whose
tension was more markedly lowered (see Figure 7.9).
Soap molecules
Figure 7.9 Orientation of molecules in an oil-soap-wter system

/
Thus the emulsifier which is. added tends to collectt the interface between the
phase (oil in this case) and the continuous-plse (water, in this case) to form a
of coating on the surface of the droplet, making it difficult for the surface of
one droplet to
the surface of another, thus preventing coalescence. Harkins pointed out that if
the
of the two parts of the stabilizing soap molecules is such that the polar end is
chain, then a stable emulsion will be obtained in which the area of
of the soap film is greater than that of the oil side. In other words, it will b
e an oill (Figure 7.10). On the other hand, if the polar group of the soap ha a smaller

WATER..
SOAP FILM
SOAP FILM
(1) Abundance ofsodlum soap in
(2)
(3)
a off-soap-water system
resulting in a oil-in-water
emulsion
164
Biophysical Chemistry
cross-section than the hydrocarbon portion, then a water-in-oil emulsion will be
stabilized
{Figure 7.11).

Soap molecules with a large

polar head and a smaller
hydrocarbon tail
Figure 7. I0 An oil-in-water emulsion
Soap molecules with a
small polar head and a
long hydrocarbon tail
F/gure 7. / 1 A water-in-oil emulsion

It was observed that ffan oil was emulsified with water containing a sodium soap
, emulsions
of the oil-in-water type resulted; but when calcium soaps were used, the water-i
n-oil type of
emulsion was formed. Harkins forwarded the theory of oriented molecular wedge to
explain this
phenomenon. According to this, in the case of univalent metal soap, each metal i
on is attached
to only one hydrocarbon chain, but with the soaps of multivalent metals there wi

ll be several
chains for each cation depending on the individual valencies of the metal long.
The hydrocarbon
chains thus have a larger cross-section than the multivalent cation and this acc
ounts for the
stability ofwater-in-tl emulsions. This theory has been tested experimentally an
d proved to be
true with various oleates and stearates by J.H. Hildebrand et. al. However, ther
e are exceptions
to this theory as can be pointed out by the emulsions stabilized by silver soaps
. By this orientation
wedge theory, silver soaps ought to stabilize off-in-water emulsions but these s
oaps have been
seen to actually stabilize the emulsions of the reverse type. Bancraft offered a
different explanation
for the formation of two types of emulsions stabilized by soaps carrying differe
nt cations. As per
his suggestions, whereas a sodium soap is more soluble in water than in oil, cal
cium soaps are
more soluble in oil than in water. Therefore, sodium soaps lower the surface ten
sion of water
sufficiently to enable it to emulsify fat, while calcium soaps decrease the surf
ace tension of oil
and stabilize water-in-oil emulsions (see Figure 7.12).

Critical rail; sin soap

to calcium soap producing no
emulsion
Abundance of calcium s o a p
in a oil-soap-water system
resulting in awater-in-oil
emulsion

Adsorption 16
If in an off-soap-water system, the proportion of calcium soap to sodium soap is
varied,
the emulsifying action of the combination of soaps will depend upon the ratio of
metal long.
i
Clowes found out a critical ratio of calcium ion to sodium ion experimentally wh
ich produced
no emulsion, This critical ratio was Ca : Na = 1 : 4. If calcium is preponderant a
bove this
I
ratio, an emulsion with oil as the continuous
while
phase
results,
any preponderance
of
sodium
I
soap yields emulsions of off-in-water type.
Further work of Clowes on emulsions of both types gave interesting imdings. For
example,
it was observed that an off-in-water type emulsion with a sodium or potassium so
ap as emulsifier
i
may be converted into a water-in-oil system by the addition of salts ofbi-and te
r-valent cations,
e.g., calcium, iron, aluminium, etc. This inversion is due to a phenomenon known
as /on
antagonsrr Long frequently exhibit an antagonistic action. Various long have ant
agonistic
plysiologlca2 actlons upon p'otoplasm. For example, calcium is antagonistic to s
odium,
magnesium to calcium and sodium to potassium. Sodium in hgh concentration s toxi
c to the
living cell, but toxicity is reduced considerably if the increased concentration
is accompanied
by a corresponding increase in the concentration of other long, like potassium I
n thls case.
Magnesium in high concentration in blood leads to p'ofound anaesthesia, but Intr
oduction of

small amounts of calcium may result in prompt awakening and return to sensitivit
y. Calcium
long have a stimulating effect while potassium long have an inhibiting action on
the heart.
Sodium long increase the permeability of cell membranes to water soluble substan
ces while i calcium long decrease permeability. These interactions of Na, K, Ca
and Mg long in living
organisms take an essential part in vital activities. The concentrations of thes
e long and their
relative proportions in tissues and fluids are of such fundamental importance th
at slight
variations in their content or composition produce profound changes in physlolog
lcal behaviour.
Lastly, molecules in an oriented layer at an interface are in a dynamic conditio
n. Molecules
continuously leaving and entering it. An increase in temperature disrupts orient
ation by
increasing the kinetic motion of molecules.
Monolayers
A monomolecular layer, or m0nolayer, can exist only at interfaces. Interfaces be
tween two
hases of the same compound (ice in water, water in contact with water vapour, et
c.) are highly
dynamic and are continuously changing in molecular dimensions. A much less distu
rbed interface
is that between a solid and liquid or between two liquids which are usually inso
luble. However,
most of the data available to date have been recorded either on unimolecular fil
ms spread on
liquid surfaces (liquid-air interfaces) or films transferred on to solid surface
s.
A monomolecular layer can be formed on the surface of water by dropping onto the
clean
surface a solution of the substance in some volatile solvent such as benzene or
petroleum ether. The added solution spreads, the solvent evaporates and the subs
tance is left on the
urface of the liquid (water, in this case} as an insoluble unimolecular film. Ir
ving Langmuir,
the American physical chemist in 1916 introduced new techniques and made some va
luable
and ingenious theoretical and experimental contributions to this field. He evelo
ped a device by
which the two-dimensional pressure due to the spreading tendency of the hsoluble
substance
could be measured. This-device is named after him as the Langmuir trough. It con
sists of a
rectangular shallow trough, one centimeter to a few millimeters deep. The trugh
is coated with
some material (Lucite, Teflon or Nylon)which is not wetted by the solutio/n. A c
oating of Teflon
is preferable as it is very hydrophobic and permits the study of flls under high

pressure
without leaks developing. Quarter troughs may be used for study of protein films
and metallic troughs avoided since small amounts of metallic salts have a stron
g influence on the properties
of films. The trough is filled to the brim with the liquid upon which the films
are going to be

Liquid
166
Biophysical Chemtstnj
formed. The surface of the liquid is cleaned by a movable barrier A, which is al
so non-wettable
(see Figure 7.13).
F/gure 7.13 Schematic view of a simple.film trough
A and B
are movable barriers,
C
is the compression bar,
TI and T2
are barrier ribbons,
W
is the torsion wire.
F
is the.loat.
The barrier is left a few centimeters away from the end of the trough. This oper
ation is
repeated with another barrier B from the other end of the trough. A third barrie
r C, also called
a "compression bar" is then placed in position. It can be moved horizontally to
decrease or
increase the film covered ea {Figure 7.14). The surface of the solution is separ
ated into two
parts by means of a teflon-coated aluminium or mica float, F. A torsion wire W,
is connected to
the float. The float also has two non-wettable ribbons T1 and T connected to the
sides of the
trough.
Movable
barrler
Figure 7.14 Side-view of a Langmuir trough.
The movable barrier can be moved n either direction (indicated by arrows
) to increase or decrease the
.film covered area.
\
j
Once the surface of the liquid is cleaned, a film can be formed on its surface i

n different
ways. The material to be spread in the solid, dissolved, or liquid state, is bro
ught into contact
with the surface and will then spontaneously form a film. Oleic acid, for exampl
e, which is only
slightly soluble in water spreads spontaneously In water. Others, like higher sa
turated fatty
acids have to be first dissolved in organic solvents and then a drop of the solu
tion is placed on

Adsorption
167
water surface to form a film. The choice of solvent is important. One should cho
ose a
solvent which does not dissolve in water, is very volatile and lighter than wate
r.
The spreading of proteins should be carefully attempted. Certain proteins can be
spread
the water surface from the solid state, but, it is preferable to spread them fro
m a dilute
(0.05% or less} with the help of micropipette. The spreading of globulins poses
problems, as in their native state the molecules are water-soluble but on unfold
ing they
' insoluble. This difficulty can be circumvented by adding a small amount of amy
l
the protein solution. This reduces the surface tension of the solution considera
bly
it to spread rapidly on the water.surface.
It is also possible to transfer a monolayer or a system of stacked monolayers fr
om one
another surface. For certain studies, especially the determination of film thick
ness
methods, it is advantageous to transfer the films from the liquid {water) surfac
e to
and has enabled a variety of studies on properties of deposited
polished clean glass slide is first conditioned with a layer of calcium or bariu
m stearate.
then dipped down vertically into a protein film-covered water surface. A protein
monolayer
deposited on the slide. Because the test slide has a hydrophobic surface due to
the
a layer will be deposited during the first downtrip of the dipping process, agai
n
layer will be deposited on the uptrip and so on. In this way a number of protein
stacked one above another.
It is not always necessary for the slide to remain vertical during the transfer
process.
of undenatured protein molecules can be deposited on slides by adsorption. In th
is, a
(coated with calcium stearate/barium stearatel can be stml,] l, tk
i trough containing the protein solution for a minute or so. It is then removed,
washed with
water, and dried. A monolayer ofundenatured protein molecules can be found adher
ing
the slide. The complete process can be followed iran ellipsometer. The ellipsome
ter is a
designed by Rothen which is capable of measuring a film thickness within + 0.3/.
It
measures the change that takes place in ellipticity of reflected light produced
by a deposited
film. The ellipticity and azimuthal position in space of the reflected beam is a
very sensitive and
(over a certain range} function of film thickness.

A monolayer of matter behaves thermodynamically in two dimensions very similar t

o
three dimensional matter. If the surface concentration is small enough, the rela
tionship
film area (surface area} and film pressure {surface pressure} at a given tempera
ture
same dimensions of energy just as does the product of pressure and volume and ca
n be
to the thermal energy term nRT where 'n' is the number of moles, 'R' the gas con
stant
T the absolute temperature. If the area available on the water surface to a cert
ain number
s of the film is reduced, the film pressure increases. Deviations from this law
occur
of both the distribution of the molecules and the orientation of the molecules i
n the
film. Accordingly, experiments on monomolecular layers can yield valuable result
s for
of molecular cross-sections and for the magnitude of molecular interactions,
both with each other and with the molecules of the underlying liquid. The above
dimension to
the use of monolayers was provided by the pioneering experiments of Langmuir in
1916. By
using one of the troughs, described earlier, he prepared a mono!ayer of stearic/
cid
[CH3{CH2}6COOH] on water. He found the tension to increase markedly when ttl.ogl
cules
were pushed together in the stacking position {see Figure 7.15}. The molecules i
n this position
stood upright with their polar heads touching the water surface while the non-po
lar hydrocarbon
chains lay parallel to each other at right angles to the water layer. All the th
ree quantities, /.e.,
the precise number of molecules on the surface, the area of and the forces per u
nit length are
directly measurable. This data is enough to derive the value of the cross-sectio
nal area of the
molecules when held in the upright position.

168
Biophysical Chemistry
Figure 7.15 Monolayer of stearlc acid molecules formed on water n a Langrnulr tr
ough.
A and B are movable bah'lets for cleann9 purpose,
C is the movable barrier used for compression ofthefdm,
F is thefloat.
As for example, stearic acid was found to have a cross-sectional area of 20.5 A
per molecule
by this technique. Knowing the molecular volume of stearic acid (556 A), it was
possible to
calculate the length of stearic acid molecule, i.e.. 556/20.5 = 27.1
The monolayer technique is extremely significant and has numerous applications.
With a
system such as a monolayer, we have the realization of a simple molecular functi
onal unit. In
other words, it is a system whoseroperties are determined by an orderly arrangem
ent of
molecules, i.e., the molecules form a team and the system behaves differently fr
om an independent
molecule. Such functional systems are often encountered in biological structures
, for example,
the proteins interact with lipid, polysaccharides and with other interfacially-b
ound compounds.
In fact, the whole living body is a functional unit where varied molecules inter
act and co
operate with each other.
Many immunological and enzymatic reactions have been studied by surface film tec
hnique.
In addition, various aspects of protein structure can be deduced by investigatio
n of spread
protein films, for example, the study of compressibility of a protein film permi
ts the determination
of the molecular weight of the protein molecules forming the film. Tumit in 1954
has described
a method for obtaining protein diffusion coefficient (D) by monolayer technique
which is less
cumbersome and is speedy too. Lastly, surface films have also been used to study
the effect of
irradiation of proteins and the results supplement the information obtained by m
re conventional
methods.
Adsorption and Surface Tension : The Gibbs' Msorpflon Equation
Surface tension is regarded as one of the different forms of energy acttfig at s
urfaces giving

rise to adsorption. This is more evident at the liquid air interface. Consider f
or example a
solution where the solute components exhibit positive surface activity (lowers s
urface tension
of the solvent). In the course of ordinary thermal agitation, the solute molecul
es are brought to
the surface. As they concentrate in the surface film, they lower the surface ten
sion of the
solvent. In the process, they are restricted in their movement because their esc
ape would cause
an increase in free energy and then work would have to be performed. But the com
plete
replacement of the solvent by solute molecules in the surface will be prevented
by thermal
agitation and forces of molecular attraction. On the other hand, in a solution w
here the solute

... (3)
... (4)
Adsorption
169
component exhibits negative surface activity (increases surface tension
of the solvent), no such
of solute molecules in the surface layer is seen. This is a feature particularly
observed with solutions of inorganic salts. The solute molecules, in thi case, a
re rejected from
the surface and thus have to be content with remaining in the bulk of the soluti
on.
The exact thermodynamic relationship between adsorption and surface tension was
first
derived by J.W. Gibbs in 1872 and later independently by J.J. Thompson in 1888.
It is known
as the Gibbs' adsorption equation which can be derived as .follows.
Let us consider a solution which has attained equilibrium between its bulk phase
and
surface phase. The surface phase consists of those molecules which are close eno
ugh to the
surface to be acted upon fully by the surface forces. We shall designate the the
rmodynamic
properties of the surface phase by a symbol '' and place it as a superscript. A
surface phase
L7
defined in. this manner has an area but no thickness. In other words, it is a st
rictly twodimensional phase. The area of this surface phase will be denoted A. We
by
are
considering
a
L system at constant temperature and pressure and constant composition of the bu
lk phase. The
i., free energy, G, of the surface layer is made up of three factors : (/) the fr
ee energy due to surface
Ltension (?A), (//) the free energy of the solute (n), and (///) the free energy o
f the solvent (2n ). DI and 2 are the chemical potentials of the solute and solv
ent respectively while n and n2
Ddepict the number of moles of solute and solvent respectively. The superscripts
indicate that

W
the functions apply to the surface. Thus, for the surface phase we can write
G o
I
. where S is the surface entropy and T is temperature.
Equation (1) can be obtained from the thermodynamic relationship at cons
tant pressure
,I [)\ ---S, at constant temperature and pressure G = Eiani and the equation dG
= ?dA
i
i! relating change in free energy with the surface tension (y) and change in sur
face area (dA).
r
At constant temperature the equation becomes
G = yA + Bn + B2n
... (2)
If a change in free energy of the surface phase is desired, it can be brought ab
out by
(/) varying the surface area which will cause the change ?dA, (ii) varying the am
ount of solute
which will cause the change Bdn or (//0 varying the amount of solvent, which will
cause the
change B2dn. Thus the differential form of the surface free energy becomes
dG = ydAa + Bdn + l2dn2
But the complete differentil of G is
dG =ydA +A*dy +t,dn' +n'dl +dn;
Comparing Equation (3) and (4) we get

170
... (6)
... (7)
... (9)
... {io)
... (11)
... (13)
Biophysical Chemistry
Now the surface concentration, Ci, in moles per unit area is defined by
Therefore, division of Eqn. (5) by A gives
dy=-CIdBC2d2
The number of moles of solute present in the surface in excess of the number of
moles of
the same solute in the interior is taken to be the molar excess of solute. This
molar excess of
solute is the amount of solute adsorbed at the surface. If the concentration of
the solute is
small, a change in concentration will not affect the free energy of the solvent.
In such a case, the
excess C of the solvent vanishes. Then
dy = - P dt
... (8)
where F is the concentration of the solute in the surface phase. Since the chemi
cal potential is
related to the activity by
.= o + RT In a
where 'a' is the activity of the solute, fin
dt= RT dln a

Substituting equation (10) in equation (8), we have

dy = - F RT din al
For an ideal solution, a = X, the moIe.fractlon, so that
dy = - F RT dln X
1 d7
or r - .
... (12}
RT dlnX
and for dilute solutions where the activity is very nearly equal to the concentr
ation, X becomes I
proportional to C, the molar concentration, and
I dy
Ci .dy
RT dlnC
RT dC
This expression is called the Gibbs' adsorption equation. The equation provides
a
mathematical basis for the generalization that if a substance tends to reduce su
rface tension it
will collect in the surface phase while those that tend to increase surface tens
ion will be retained
in-the bulk phase. We can also see from equation (13) that when the surface tens
ion of a
solution decreases with concentration, dy/dC is negative, I is positive and the
concentration
of the solute in the surface is greater than the bulk phase. The surface active
agents exhibit this
type of behaviour. On the other hand, when the surface tension of a solution inc
reases with
concentration, dy/dC is positive, I is negative and the body of the solution is
richer in the
solute than the surface. This is observed in solutions of many electrolytes. The
above equation
has also been experimentally verified by McBain and Humphrey. They employed an a
pparat-s

Adsorption
171
resembling a microtome to slice off the top layer (0.05 mm approx,) of the surfa
ce from a
was later collected in a sample tube for analysis. The experimental F obtained
satisfactory agreement with that calculated via the Gibbs' adsorption equation.
ADSORPTION FROM SOLUTIONS"
A solution is made up of two basic components : solute, which may be one or more
in
and, solvent. When an adsorbent is dropped into a solution either the solute or
the
may become adsorbed. Adsorpti6n of the solvent is xery rare. When the solute is
taken
from the solution by an adsorbent, it is calledposit/ve adsorpt/or In this case
the concentration the solution decreases after treatment with the adsorbing agen
t. As an example, when a
blue in water is shaken with solid carbon, a part of the dye is removed by
solid carbon and the solution becomes more dilute. Similarly, activated carbon c
an be used
remove acetic acid from a solution of acetic acid in water. Conversely, when the
solvent is preference to the solute, the concentration of the solution actually
increases after
with the adsorbing agent. This phenomenon is known as negative adsorption. When
solvent and solute are adsorbed, the concentration of the solution after the ads
orbent has
will depend on the relative extent of adsorption, whether positive or negative.
Adsorption from solution is very similar to adsorption of gases and is governed
by the
i.e., the mechanism of adsorption is by virtue of electrostatic forces of attrac
tion,
surface tension are the factors resjonsible for an increase or decrease in
The Gibbs - Thompson princle states that "those substances which
urface energy tend to concentrate at a liquid-air interface (in the surface), an
d those
which decrease interracil energy tend to concentrate at a liquid-solid or liquid
interface". It follows from this principle that the lower the surface tension of
the solvdnt,
conditions being uniform, the less will be the amount of adsorption. On the othe
r hand,
is best from solvents of high surface tension, because here the solute causes th
e
decrease in surface energy at the solid-liquid interface. For example, picric ac
id is
adsorbed upon charcoal from aqueous solution than.from an alcoholic one. The
I adsorbed upon charcoal from water may be readily recovered by washing with alc
ohol. The
for this behaviour is that water has a much higher air-liquid surface tension
has alcohol, and so the decrease of the surface tension of the aqueous solution
with
concentration of picric acid is likely to be greater in aqueous than in alcoholi

c solution.
same is observed at the interface of adsorbing solid and liquid solvent.
When various inorganic salts are dissolved in water, the surface tension of the
solution is
r increased. Therefore, solid adsorbents like powdered silica, activated carbon,
or powdered
are raore effective in adsorbing non-eleCtrolytes from a solution than electroly
tes.
The effect of temperature on adsorption equilibrium is worth mentioning. Rise of
of adsorption. This is- in spite of the fact that surface tension
with a rise in temperature. The reason behind this bservation is that molecular
a fundamental cause of adsorption, is disrupted by thermal agitation. For exampl
e,
iodine is added to starch solution, a blue colour appears due to formation of st
arch
However, upon heating, the blue colour disappears due to the failure ofi0dine th
remain
on the starch particles at higher temperatures.
Adsorption from solutions on the surface of solid adsorbents differs from the ad
sorption of
substances like gases, vapours, pure liquids, etc., in that the solution contain
s two
and solvent) forming a closely packed layer on the surface. Upon a change
the concentration, these two components displace each other in the surface layer
, a
observed only during adsorption from solutions. Thus, neither the surface soluti
on
the bulk of the solution has any free sites and only displacement of molecules o
f one
by molecules of the other takes place.

172
Biophysical Chemistry
The amount adsorbed from solutions depends on the properties of the adsorbent, t
hose of
the solution, and of its constituents. Polar molecules that are capable of formi
ng H-bonds with
the hydroxyl groups of the surface of the adsorbent are especially greatly adsor
bed. It is for this
reason that silica which has a hydroxylated surface can smoothly adsorb phenols,
alcohols,
water and amines. The adsorption of organic compounds from solutions of highly p
olar solvents
on the surfaces of polar adsorbents is negligible but adsorption of such substan
ces is strong on
non-polar or weakly polar activated carbons.
An increase in surface area of the adsorbent greatly enhances the adsorption of
substances
from solution. This is clearly evident in the case of porous material like charc
oal, fuller's earth,
and powdered substance like silica.
THE IMPORTANCE OF ADSORPTION PHENOMENA
Adsorption is associated with many of the reactions taking place in the living b
ody. It also
has numerous laboratory applications, industrial applications as well as various
technical
applications. In the following text we shall discuss some of the uses of adsorpt
ion.
Catalysis
The velocity of any chemical reaction depends to a great extent on the active ma
sses of the
reactants, provided other factors are in optimum conditions. Practically, active
mass and
concentration are synonymous and an increase in concentration of the components
of the
reaction facilitates the reaction. In the reactions taking place in the protopla
sm, adsorption
plays a very important role. By this process, substances ordinarily present in l
ow concentration
may have their effective concentrations lncreased tremendously by being accumula
ted at the
interfaces, thus speeding up many chelIfl'cal reactions. Enzyme action also invo
lves adsorption.
The enzymes, being colloidal in nature, have large surfaces and surface adsorpti
on of reactants
is a requisite for the enzymatic reaction to proceed smoothly. The colloidal mol
ecules are
interspersed with a network of membranes which possess enormous surfaces. Adsorp
tion taking

place at these membranes promotes several vital chemical reactions.

Action of Drugs and Poisons
Substances capable of reducing interracial tension tend to accumulate at the int
erfacesI.
To this class of surface-active agents belong varied. "ous drugs andpoisons. The
se substances, as
consequence of reducing interracial tension,' are concentrated at the interfaces
and exert thei
effect from that location. Selective adsorption of drugs may thus be responsible
for specc
action.
Macromolecular Association and Dissociation
Various types of molecular associations seen in the living body are as a result
of adsorption.
These molecular associations between proteins and proteins, proteins and carbohy
drates,
proteins and lipids, and proteins and salts continuously take place in the cll p
rotoplasm and
are vital for the structural integration and function of the living cell. Likewi
se, dissociation of
macromolecules could be dictated by their desorption behaviours.
Histological Studies
Though adsorption need not be followed by absorption, absorption depends upon th
e
concentration of the molecules at the surface or interface. Acidic and basic dye
s which can be
distinguished by their staining power are important tools not only in histologic
al studies, but
also in experimental physiology, where they serve to trace the pathway and distr
ibution of
dissolved substances across cells and tissues. The rate of their entrance into c
ells depends to
some extent on their accumulation on cell surfaces prior to penetration. The cel
l colloids contain

173
their large surface a number of cationic and anionic centers which are the sites
of electrostatic
of the basic and acidic molecules respectively.
in Safety Devices
A bacterial toxin, hormone, or a mineral poison may be inactivated by adsorption
. Ferric
a good adsorbent Is often used as an antidote in cases of poisoning by arsenic.
Gas
are devices containing an adsorbent or a series of adsorbents which purify the a
ir for
r adsorbing the poisonous gases and vapours from the atmosphere.
Applications of adsorption from solution include the clarification of sugar liqu
ors by
removal of Impurities from petroleum oils and motor spirits by colloidal silica
and
of dyes from dilute solutions In a number of solvents,
i
Charcoal can also be used to remove odours from gases. CO, evolved durin
g the
process, often has objectionable odours. These odours can be effectively removed
t adsorption on charcoal. Thus, carbonated beverages can be made fit for consump
tion. Purifiers
used in toilets, refrigerators, etc., are generally adsorbents for gases which e
asily remove the
thus freshening the air.
hromatography
One of the most impoFtant laboratory applications of adsorption is the recovery
and
of vitamins, proteins, and other biological substans by the method of
analysis, the details of which are discussed in one of the subsequent chapters,
title "Adsorption Chromatography".
in Research and Industry
Lastly, adsorbing agents have found wide use in industry and laboratory for puri
fication

Adsorption has been used for purification of enzymes. The impure solution contai
ning
enzyme at a parti..cular pH is brought in contact with an adsorbent such as alum
inimum
After the enzyme has been adsorbed, the adsorbent is washed and then the enzyme
(desorbed) by washing the adsorbent with a solution of another pH. This elution
is also employed in the separation and purification of plant alkaloids, vitamins
and
substances which are difficult to Isolate by ordinary methods. For example, vita
min B
vitamin B extracted from yeast are separated from each other by adsorbing vitami
n B on
gel in acid conditions (pH 3). Under these conditions, While vitamin B is mostly
adsorbed,
is not. Elution of vitamin B from silica is carried out by using an alkaline sol
ution
For adsorption of positive and negative long from solutions, ion exchange resins
have
used in the laboratories and industries. These resins have also found use in med
icine
such purposes as reducing the acidity of gastric juice in cases of peptic ulcer,
reduction of
' and K concentration in body fluids in cases of congestive heart failure and ren
al failure.
also serve to adsorb toxic products of bacterial action in cases of diarrhoea. I
ndustrial
have made good utilization of ion-exchange resins for water softening and
purposes, removal of electrolytes from food material and other products and
purification of metals.

174
Biophysical Chemistry
Suggestions For Further Reading
1. Adamson, A.W., Physical Chemistry fSurfaces, John Wiley and Sons Inc., New Yo
rk, 1967.
2. Garner, W.E., Chemisorptior Butterworths, London, 1957.
3. Sherman, P. (ed.), Emulsion Science, Academic, New York, 1967.
4. Gregg, S.J. and Sing, S.W., Adsorption, Surface Area and Porosity, Academic,
New York, 1967.
5. Shaw, D.J., Introduction to Colloid and Surface Chemistry, Butterworths, Lond
on, 1966.

8
SPECTROPHOT OMETRY
In this chapter an attempt has been made to describe certain relationships among
optical phenomena which produce signals conveying chemical information
either to the concentration of the chemical in a given solution, and/or its stru
cture.
information afforded by different optical signal source is so distinctive and va
luable
each has spawned its own specialized instrumentation, In the following pages the
ory,
and applications of each of the optical signal sources has been discussed
BASIC PRINCIPLES
Light is supposed to have dual characteristics, corpuscular and waveform. Thus,
a
of light may be understood as an electromagnetic wave-form disturbance or photon
propagated at 3.0 108 m/sec, i.e., at the speed of light. The term. electromagne
tic
description of the radiation in that the radiation is made up of an electrical
a magnetic wave which are in phase and perpendicular to each other and to the
of propagation (Figure 8.1A). The magnitude of electrical vector is denoted by
symbol E and that of magnetic vector is denoted by the symbol H. Figure 8-1A den
otes one
of plane polarized light. A beam of light from a bulb consists of many randomly
oriented
polarized components being propagated in the same direction. The distance along
the
of propagation for one complete cycle is known as wavelength, (Figure 8.1A).
may be measured in centimeters (cm), micrometers (m), nanometers (nm), or
units (/), where Inm = 10-3 tm = 10-6 mm = 10- cm = 10-9 m, and 1A = 10-8 cm.
Electric field, E
Direction of
propogation q 1 IIIII III I=:Jllll I I
Magnetic Field,
8. I {A) The electromagnetic wave. The magnetic vector (unshaded) and electric v
ector (shaded) are perpendicular
to each other and to the direction of propagation. Wavelength ()) is the distanc
e between two crests or
troughs.
Sometimes a term, frequency, v, is used rather than wavelength to describe a par
ticular
To understand this term it will be helpful to see Figure 8. IB denoting variatio
n in the
and magnetic field with respect to time at any given location in a plane polariz
ed ray;

number of waves passing through a fixed point on the time axis per second is kno
wn as the

176
Biophysical Chemistry
frequency, v, of the radiation (usually expressed in Hertz or cycles per second)
. Frequency
shares an inverse relationship with the wave, length so that
V = c/X
where c is the velocity of light, 3 x 108 m/sec.
EIectrlc field, E
Magnetic Field, H
Figure 8. I (B) The dependence of electric (shaded) and magnetic (unshaded} vect
ors at a particular location tn a
plane polarized electromagnetic wave.
Sometimes radiat ion, mostly in the infrared region (see later), is characterize
d by another
term known as the w<'e number and denoted by the symbol , Wave number means the
number
of complete cycles occurring per centimeter
I)=11X
The energy E, of a photon can be related to its wavelength and frequency with th
e help of
Planck's constant, h
c
E=h=hm
where c is the velocity of light in vacuum. Remember that wavelength and frequen
cy share an
inverse relationship; this means that as the wavelength increases, the energy of
the radiation
decreases, while the energy increases with the increase in frequency.
A beam of radiation from an electric bulb consists of several wavelengths and is
known as
polychromatic. A beam in which all the rays have the same wavelength is known as
monochromatic.
Electromagnetic radiation is produced by events at the molecular, atomic, or
level. Oscillations of nuclei and electrons in electrical or magnetic fields, mo
lecular bending and
vibration, excitation of orbital electrons, ejection of an inner orbital electro

n and rearrangement
of the other electrons, and nuclear break-up are some of the events which give r
ise to
electromagnetic radiation. Since each of these events differ in terms of the ene
rgy involved, the
radiation they emit will have different wavelengths. Thus, a complete spectrum o
f electromagnetic
radiation will be produced. This spectrum is depicted in Figure 8.2. The events
leading to each
component of this spectrum are also described in Figure 8.2.

Nuclear
transition
X-rays
Microwave
Radio
177
3.125%
Spectrophotometry

Inner
shell
electronic
transition
Valence electron
transition
Molecular
vibration
& rotation
Oscillations of
nuclei and
electrons in
magnetic field

Vacuum I I INear
0.01

0.1
2-180 1400-'780 ] 25,000-125,000 0.005-30 300
180-400
780-25,000
nm
,,
cm

Figure 8.2 The electromagnetic spectrum -- the main regions and their wavelength
s. Physical events involved in
their production are also indicated.

One of the earliest studied characteristics of chemical compounds was theicolor.

That
color intensity should form the basis of most widely used set of chemical assay
procedures is
then but natural. Things are colored because of their ability to absorb certain
components of
the electromagneticietrum with hich they interact. Consider a substance which ap
pears
green in color. The substance in question here is absorbing most other component
s of the
electromagnetic spectrum except green, which is being reflected giving it a gree
n appearance. If
one can calculate the amount of light absorbed by this substance, one can arrive
at a fair
judgement about the concentration of the substance.
THE LAWS OF ABSORPTION
The absorption of light by any absorbing material is governed by two laws. The f
irst of
these laws is known as the Bouger-Lambert law. It states that the amount of ligh
t absorbed is
proportional to the thickness of the absorbing material and is independent of th
e intensity of the
incident light. To understand the above statement let us assume that a thickness
b has the
ability to absorb 50% of the incident intensity of the light passing through it.
If the intensity of
the radiation incident upon such a thickness is assigned a value of 1.0, the out
coming, i.e., he
transmitted beam will have a value of 0.5. If we now place a second equal thickn
ess b, it will
absorb 50% of the transmitted beam, i.e., 50% of 0.5. The second transmitted bea
m will then
have a value of 0.25. This process is illustrated in Figure 8.3 and may well ass
ume the graphical
Figure 8.3 The pattern of light absorption by successive equal path-lengths (b)
of absorbing solution.
form as in Figure 8.4. The successive light intensities are the sequence (0,5)I,
(0.5)2, (0.5)3 etc.
This is clearly an exponential function and may be expressed as
I
e -kb
Io
I = the intensity of the transmitted light,
Io = the intensity of the incident light,

b = the absorbing thickness, better known by the term path-length

178
25.
100% Transmission
50
2
Path length
Figure 8.4 Data of "igure 8.3 expressed graphically by plotting % trmlsslon agal
nt the path lenjth (thickness]
and k = the linear absorption coefficient of the absorbing material. The power t
erm in the
above relationship can be removed by converting to the logarithmic form. Thus,
In I-- = -kb, or In I--?- = kb,
Io
I
changing to common logarithms we get,
Io
2.303 log lo-- = kb
The second law of absorption is known as the Beer's law. This law states that th
e amount
of light absorbed by a material is proportional to the number of absorbing molec
ules i.e., the
concentration of absorbing solution. This can be mathematically expressed in the
form of an
equation similar to the one above.
2.303 log,o -- = k'C
I
where k' = absorptivity constant, and
C = the concentration Of the absorbing material.
We can now combine the two equations for the Bouger-Lambert law and the Beer's l

aw.
Here, k and k' merge to become a single constant a (see Box 8.1). The combined e
quation is
written as
lg,o-Io = abC
I
This equation has been alternately referred to as the Beer-Lambert law, the Boug
er-Beer
/aw, or more simply, Beer's law. This combined law states that the amount of lig
ht absorbed
(absorbance or extinction) is proportional to the concentration of the absorbing
substance and to
the thickness of the absorbing material (path-length). The quantity Io/I is know
n as the absorbance
or the optical density (O.D.). The reverse, I/Io is known as the transmittance,
T (the amount of
llght which escapes absorption and is transmitted).
Absorbance shares a linear relationship with sample concentration. On the other
hand,
the relationship between transmittance and sample concentration is a non-linear
one. It is

Box 8.1
is :the
Mo!ar abso
Spectrophotometj
179
therefore easier to use absorbance as an index of sample concentration. This rel
ationship between
absorbance and transmittance is shown in Figure 8.5.
Percent. transmittmce
- Absorbance
p'igure 8.5 The relationship, between percentage transmission and absorbance.
The two terms are mathematically commutable and so one can be calculated from th
e
other. For this purpose we rewrite the equation so that
A [absorbance) = log Io - log I
but Io is always set at 100% and log 100 = 2, so
A 2-1ogl
In the equation

180
Biophysical Chemistry

182
Biophysical Chemistry
Stating Beer's law a
ional to the
sample concentration
e optical density of
standard solution is
ed from its
optical density. The

bit differently, we can say that optical density is proport

If the path-length is constant. It follows then, that if th
a
known, then an unknown sample concentration can be calculat
formula used for such calculations is

concentration of the unknown

O.D. of the unknown x conc. of a standard
O.D. of the standard

The standard chosen should have an optical density nearer to the O.D. of the unk
nown.
Before we go ahead, it is necessary to instill the concept of what is known as t
he "blank" or
the *reference" solution. To measure the O.D. of any given substance, one dissol
ves it in a
solvent. If the substance does not absorb in the visible region, many times one
adds other
reagents so that the compound becomes colored and absorbs in the visible region.
The solvent
and the other reagents might also absorb in the region in which the absorbance d
ue to the
substance of interest is being measured. The resulting O.D. will then be a sum o
f the individual
absorbances due to the substance and the solvent and the reagents. In order to c
ancel out the
O.D. contributed by the solvent and the reagents, we prepare what is known as th
e blank, which
consists of all reagents used in the solvent but not the substance of interest.
When this solution is
read in a spectrophotometer, a particular reading is obtained on the O.D. scale.
This O,D. is
due to the absorbance due to the solvent and the reagents. This reading is now m
ade zero with
the help of a knob in the instrument. When, subsequently, standards will be read
, the O.D.
observed will be due to the substance only, since the O.D. due to other regents
(which are
added in equal quantities in all the standards and the unknown) has already been
cancelled.
The most accurate way of measuring the concentration in the unknown sample is th
rough
preparation of a calibration curve from a number of standards. The whole process
is described

in Box 8.2.

183
Spectrophotometry
A standard curve should always be prepared to check that Beer's law applies to t
he analysis
performed (in which case the curve would be straight). This is necessary since t
here are
factors that may cause deviations from linearity. The most common factor causing
from linearity is the use of a band of wavelengths to measure absorbance in most
In the strictest sense, laws of absorption apply to monochromatic radiation and
The deviation from linearity in this case, however, is not so severe as
in such experiments. Other factors which cause deviations
are given below.
From Beer's Law
Following factors may cause deviations from Beer's law.
I. Deviations from Beer's law occur usually when hlgh sample concentrations are
being
One effect of high concentration is that the molecules may dimerlze. It is not n
ecessary

184
,
Biophysical Chernistnj
that the absorpUon spectra of the dimers are the same as that of the monomers. I
f the spectra
differ, the absorpUon coefficient will also undergo a change (Figure 8.6) leadin
g to a positive or
negative deviaUon.
The spectral shift is normally due to polymerizaUon. There Is JUSt one wavelengt
h here
where the molar absorpUon coefficient has not changed. This wavelength (2) is .c
alled the
IsobesUc point. At there will be negative deviaUon and at 3 there will be a posI
Uve deviaUon.
High concenaUon may also lead to aggregaUon. Large aggregates then scatter light
. The
intensity of the radiaUon reaching the.detector Is thus lessened. Thus two diffe
rent phenomena
are contributing to the reduction in the transmitted intensity m absorpUon, and
scattering. In
such a case a positive deviation will be seen. A particularly interesting .manif
estaUon of this
phenomenon occurs in the formaUon of micelles (see Chapter 3; Box 3.3, and discu
ssion under
'Asoclatlon Colloids'). Over an extremely small concentration range known as the
critical micelle
concentration (cmc), certain kinds of solute molecules cooperatively associate i
nto large particles,
called micelles, The micelles are huge particles by comparison since each micell
e may co.ntain
more than 100 molecules per particle, The micelles cause appreciable scattering
of radiation in
the X-ray to visible region. Micelles are usually formed by those molecules whic
h have one of
the ends 'phillc' to the solvent while the other is 'phobic.' Examples are fatty
acids, hospholipids,
ionic lipids, etc.
Positive
deviation
--Negative
Concentration
Wavelength
Figure 8.6. (A)A schematlc of absorptWn spectra of the same substance at two dif
ferent
concentrations
Figure 8.6 (B) Schematic representation.of how negative

and positive deviations appear on the chart

Aggregation, on the other hand, can lead to electronic interactions that can eit
her lessen
or enhance the absorption coefficient.
High concentrations can also lead to chemical reactions which will lead to a cha
nge in the
chemical composition of the solution. Naturally, a deviation from linearity will
result.
- Deviations may also occur at low concentrations. The most significant point to
note here is
about proteins. Proteins are known to denature at low concentrations and the den
atured product
has an absorption spectrum that is different from the native protein.
2. Instrumentation limitations may also result in deviations from Beer's law. On
e of the
main causes here is imperfect rnonochromacy. Let us see what this means.
The calibration series for a red solution is measured using monochromatic light
of
wavelength 492 rim, i.e., at the maximum, and of 546 nm, i.e., on the flank. The
calibration
plots obtained are two straight lines having different Slopes. Ife were to make
a third series of
measurements now using a large half-bandwidth filter (20 m) with a maximum of 52
0 nm, the
resulting calibratiu, plot will be a curved line.

Spectrophotometry
..
185
This is only an apparent deviation. The reason is not far to seek. Let us, for t
he sake of
understanding, assume that the filter is passing only two wavelengths. One of th
e wavelengths
is the 'm for the sample and the other wavelength is at the periphery of its abs
orption spectrum
in the red region. Let us say that a given unit of concentration has the. abilit
y to absorb 50% of
the 'm" However, the same concentration absorbs only 25% of the .wavelength at t
he periphery.
From our consideration of the Beer's law in the earlier pages we can say that th
e transmittance
series with every unit rise in concentraon for the .. will be. 1/2, 1/4, 1/8, an
d so on. For the
it will be 3/4, 3/8, 3/16, and so on, It is not difficult to see that if the
absorptions are plotted for both these series against increase in concentration,
result will be a straight llne for each. However, for the whole filter the compo
site transmittance
k i + 2. The series will not rise in an exponential manner as for the previous t
wo individual 2
Thus absorbance plotted for these composite transmittances will give rise to a
(Figure 8.6 (C)). When we think that there are many more wavelengths passing th.
rough
than we have just assumed, it is easy to see why measurement using filters .will
not
in straight lines though there are no other conditions for violation of the Beer
's law.
0.15"
0
2 4 6 8
Concentration
Fgure 8.6 (C) Effect of imperfect monochromacy
imperfect monochr'omacy, other indeterminate instrumentation variations which
include (A) stray radiation reaching the detector, (B) sensitivity changes
, and power fluctuations of the radiation source and detector amplification syst
em.
operation {to be discussed later) tends to cancel out most of the random causes
of
Apart from the factors mentioned above, the following factors can cause deviatio
ns from
i Beer's law.

Effects
Most experimental protocols insist that i heating is requied for color developme
nt, the
be cooled to room temperature before its absorbance is read. This insistence is
not
aqueous as well as other solu/ons expand upon heating. The absorbance in
is always different. Moreover, change in temperature results in a change in the
of solubility, dissociation/association properties of the solute, hydration, and
several
is also reflected in the absorbance. Thus, absorbance measurements always be don
e at a constant temperature.

186
Bophyscal Chemistry
Sample Instability
Experimental protocols for absorbance measurements of a few substances insist th
at the
measurement be done within a very short time of color development. One good exam
ple is the
Fiske Subbarow method of phosphate estimation by the ANSA method. The reason for
this
insistence is simple -- some colored compounds are unstable and undergo changes
within
quite short times. In such cases the color could increase, or decrease. Even mor
e detrimental is
the fact that for some such unstable compountis, even the X, may change.
Fluorescence
Some solutes fluoresce (drugs and drug intermediates often do). For such substan
ces,
deviations occur because apart from the transmitted intensity, fluorescent inten
sity also reaches
the detector.
Turbid solutions always end up giving higher absorbance than what is determined
by the
color.
ABSORPTION SPECTRUM
The pattern of energy absorption by a substance when light of varying wavelength
passes
through it is uniquely characteristic of the substance. This pattern is known as
an absorption
spectrwn.
To establish the absorption spectrum for a given substance, transmittancy or opt
ical density
of a particular concentration of the substance is measured at different waveleng
ths (the path
length is kept constant throughout}. The data so obtained is plotted in the form
of a curve
relating transmittance or optical density (the latter is preferred) to wavelengt
h. An example of a
relatively simple absorption spectrum is shown in Figure 8.7; generally more com
plex curves
are found, Since the absorption spectrum of a substance is usually characteristi
c for it, it may
serve for its identification and may furnish information of analytical value. Th
e application of
absorption spectra is not limited to the visible region of the spectrum but may
be applied
equally wen to characterization of the ultraviolet or infrared absorption of man
y substances.

1.0
Absorbance
0.5
Wawength (nm)
Figure 8.7 A simpl/ged absorption spectnan curve. ), represents a concentration
twice as much as that of x. The
dotted//he cated the wave/ejth of maxm sens/ttvfor spectrophotometrc measurement
s.
Absorption curves of a particular substance vary along the absorbance axis with
variations
in the concentration of the substance; they do not vary along the wavelength axi
s (curve x and
y in Figure 8,7). It becomes clear from Figure 8.7 that although a particular su
bstance absorbs
at numerous wavelengths, there are regions where it absorbs more than in other r
egions. The
wavelength at which the compound absorbs most is known as its absorption maxima,
,.. If
one increases the concentration of a substance, it will be observed that absorba
nce at all

n=l
Energy, J
0
13.6- ground state
187
has increased; however, there is a greater change in absorbance per unit change
.concentration at the wavelength of maximum absorption. This is the reason why o
ne chooses
the absorbance of a compound at its )m, during a quantitative experiment. At thi
s
maximum sensitivity will be obtained in a photometric measurement. Occasionally,
as for example in a procedure where the absorbance curves for reagents and color
ed
overlap considerably, the highest sensitivity may be obtained at a wavelength on
absorption maximum. Thus a knowledge of the absorption spectrum of both, the
compound is essential for the proper selection of an optimum wavelength.
is Absorption Spectrum Specific for a Substance?
How is it that a compound absorbs maximally at one wavelength and does not absor
b at
mother?. Let us assume that a substance S absorbs maximally between 270 and 280
nm
| does not absorb at all at wavelengths beyond 300 nm or below 270 nm. The theor
y of
suggests that absorption o radiation means acceptance of energy embodied in the
to catapult electrons of the absorbing substance to higher energy levels. So why
is it
substance'S accepts energy from radiation of wavelength 270-280 nm to catapult i
ts
levels, but does not accept energy from radiations of other wavelengths?
The answer is provided by the concept of quantized energy. As per quantum theory
, the
of m atom can exist only in certain orbitals which involve different electron en
ergies.
the lone electron of hydrogen can exist in six different energy levels (Figure 8
.8(A)),
lowest level being the ground state. These energy levels are signified by n, whe
re n has
of 1,2,3 etc. Quantum concept forbids intermediate energy levels like 1.2, 1.5 .
.. etc.
to become excited the electron has to Jump from n =1 to n = 2, or n = 3 etc. Qua
ntum
energy of light is not continuous, but is concentrated into packets
energy or quanta. In a low frequency (high wavelength) light each quantum will h
ave a small
T while, in light of higher frequency quanta will have greater energy. Quantum t
heory also
that in order to be excited the electron will accept only that radiation which h
as the

energy to push it into a permitted energy level. In other words it can absorb
wavelength or frequency which provides it with its exact requirement.
to the energy level n = 2 from the level n = 1, hydrogen electron needs a partic
ular
which is provided only by radiation of the wavelength 1,217/i,. Consequently
n=5
n=4
n=3
Energy, eV
0
0.87 x 10-19
- 0.54
1.36 x 10-19
- 0.85
2.42 x I0-19
- 1.51
Excited states

5.43 x 10-19
- 3.40
t
Absorption of radiation of
wavelength 1217 X lifts
electron from n = 1 to n = 2
21.76x10-19
8.8(A)
Ground state and excited state energy levels of hydrogen atom. Associate
d energies for each level are
whiten in Joules and V.

hydrogen absorbs light of this particulkr wavelength. Going by the example of hy

drogen we can
easily explain why the substance S does not absorb at 300 nm but absorbs at 280
nm. Quantum
theory, thus, explains why absorption spectrum is specific for a given substance
. Hydrogen is a
small atom. The very few energy levels available for its lone electron transitio
n give a sharply
defined absorption spectrum (line spectra) for this element. In complex molecule
s, the multlplicity
of energy levels available to electrons belonging to different atoms result in t
he discrete bands
to coalesce and thus, broad bands are observed, which are nevertheless specific
for the given
molecule. Moreover, vibrational and rotational energy levels are also associated
with each energy
level of electronic transition. Excitation of an electron and its promotion to a
higher energy level
is also accompanied by changes in the vibrational and rotational levels. This is
an additional
factor responsible for the broad bands of absorption spectrum of a given compoun
d.
Let us now briefly deal with the types of electronic transitions which t.ake pla
ce when a
given substance absorbs light energy. The following types of orbitals are genera
lly found in the
ground states of organic molecules (see Figure 8.8(B) and Table 8. I),
Bonding morbttals: These are extremely strong and constitute single bonds betwee
n atoms,
The electrons are not at all delocallzed and the distribution of electrons is cy
lindrically symmetrical
about the bond [Table 8.1).
Table 8. I A few examples of some bond types, transitions, and molecular orbital
s
Type of bond
Transition of lowest
elergy
Shapq of molecular orbitals
Ground state
Excited state

g eon system
perpendicular to the
plane of the page.

Note :
Special distribution and sign of wave function of the molecular orbitals
has also
been provided.
Bonding -orbitals: These constitute multiple bonds between atoms and are based o
n a
combination of atomic p-orbitals. The electrons are strongly delocalized and int
eract with the
surrounding environment with relative ease.
n-orbitals : Certain molecules contain heteroatoms (e.g., oxygen, nitrogen). The
occupied
orbitals with highest energy in such molecules are those of lone pairs. These lo
ne pairs are not
involved in bonds (non-bonding) and thus retain their atomic character.
All the above three types are illustrated beautifully by formaldehyde as shown b
elow :
H/ .-------n
According to the molecular orbital theory, when a molecule is excited by the abs
orption of
energy (UV or visible light), its electrons are promgted from a bonding to an an
tibonding orbital
(higher energy sates). The antibonding orbital associated with the bond is calle
d " orbital
and that associated with the g bond is known as the ' orbital. Since non-bonding
electrons are
not decfly involved in bonding, there is no corresponding antibonding orbital fo
r them. The

Spectrophotometry 189
major electronic transitions within the ultraviolet a'Kd visible regions are:
---> o. n ---> a', n ----> =, and n----> x. These transitions are also shown in Fig
ure 8.8(B). The
with these transitions are given below in the decreasing order.
a ----> o > n ---> o* > ---> '> n ---> "
It is clear that the energy required for ----> transition is of a very high orde
r and ' short wavelengths are absorbed for these transitions. The organic compou
nds
which all the valence shell electrons are involved in formation of sigma bond, t
herefore, do
the normal Ultraviolet region (180-400 nm). The usual spectroscopic
ues cannot be used below 200 nm because oxygen absorbs strongly at this range. T
hus,
these transitions, the entire path-length is to be evacuated. It is because of t
his that
below 200 nm is known as the vacuum ultraviolet region.
Energy required for n ----> * transition is lower than that of ----> a transition
. This type
transition usually takes place in saturated compounds containing one heteroatom
with
pair of electrons. Several molecules, including water, ether, saturated alcohols
, and
show absorption attributed to n * transitions.
Even lower energy is required for ----> n" transitions. These transitions take p
lace in the centres of molecules. These transitions are shown by alkenes, alkyne
s, carbonyl
cyanides, and azo compounds etc. Since these transitions are associated with a
energy, they take place at comparatively longer wavelengths easily obtained in a
spectrophotometer, n ----> n transitions usually have very high extinction coeffI
= 104 - 105 M-cm-) ff not forbidden by spin or symmetry selection rules.
n ---> * requires the least amount of energy. Therefore, these transitions frequ
ently appear
shoulders on the long-wavelength side of an absorption spectrum.
All
the above orbitals with ordering of their energies relative to each othe
r along with the
transitions are shown in Figure 8.8(B).
Anti-bonding electrons
Anti-bonding electrons
Non-bonding electrons
Bonding electrons
Bonding electrons
Figure 8.8(B) Schematic rnolecular orbital energy level diagram depicting relati

ve energy levels and allowed

By way of generalizing, it may be said that the absorption bands of almost all o
rganic
normally found in the near ultraviolet and visible regions are due to either n ---> "
n ----> * transitions. One can distinguish between n ----> n* and n ------> n* t
ransitions by
fat the extinction co-efflcients of the peaks at Xm. The n transitions are usual
ly symmetrical considerations and therefore have extinction coefficients of the
order
ust 10, whereas n ---> * transition which are rarely forbidden, have extinction
generally in the magnitude range of 103 - 104.
The transitions just discussed have been summarized in Table 8.2 along with info
rmation
the region of the spectrum in which they absorb.

190
Table 8.2
Types of electronic transitions

Description
Region of electronic spectra

From a bonding orbital in the. ground state

to an antlbonding orbital of higher energy.
(a) o ----> (between orbitals)
(b) . g* (between orhitals)
Vacuum UV (e.g., ethane at 135 nm)
UV (e.g., ethylene at 180 nm, benzene
at 230 nm).

From a nonbonding orbital to an antlbonding

orbital of higher energy
(a) n ----> *
Near UV and visible (e.g., acetaldehyde
at 293 nm. nltrosobutane at 665 nm).
Far UV, or sometlme near UV (e.g.,
methyl alcohol at 174 nm, methyl
iodide at 258 nm).

From an orbital in the ground state to a very

high energy orbital (towards ionization)
Vacuum UV

Lastly, the structural character and the position of absorption maxima depend no
t only
upon the structure of the compound, but also on the nature of the solvent in whi
ch they exist.
The environmental factors that can cause detectable changes in the absorption sp
ectra are pH,
the polarity of the solvent or of neighboring molecules, and the relative orient
ation of ne/ghboring
absorbing groups.

Effects due to pH : It is the pH of the solvent which determines the ionization

state of a
molecule (see Box 1.8 and Tabie 1.7 for clear understanding). With a change in i
onization
0,9"
0.8.
0.7
0.6
0.5.
0.4
0.3
0.2
0.I
0
/ \ A
240 260 280 300
Wavelength (rim)

Figure 8.9. travlolet absorption spectra of 3'-CMP. The nucleotlde was obtained
by enzymatlc degradation of
tRNA. See the difference between the absorption at neutral and acidic pH. Also n
ote the ral
change In the absorption spectra at reglons other than the absorption maxOn

25O
Spectrophotometnj
191
status, the absorption spectra may also change. Given below are the absorption s
pectra (Figure
8.9) of the pyrimidine nucle.otide CMP at neutral and acidic pH. Mark the differ
ence in the
absorption spectra of the same compound in the two states of ionization.
Polarity effects : In general, polar solvents shift the n =" transitions to shor
ter wavelengths and ----> transitions to longer wavelengths as compared to the g
as phase spectra (Figure 8.10). This is true for polar chromophores. For this re
ason polar solvents have
been used to distinguish between n n" and ---> =" transitions. Non-polar solvent
s, on the other hand, produce no such change.
260 270 280 290 300 310
Wavelength (nm)
Fure 8.10. Effect of solvent polarlty on the absorption spectrum of tyroslne. So
lid line represents absorption spectrum
in water while the dashed llne corresponds to 20% ethylene glycol The transition
here is n -- and
therefore the spectrum is shifted towards shorter wavelengths when water, which
is more polar, is used
s a solvenL
Orientation effects: The best example to understand orientation effect is theDNA
. Absorption
coefficient of a single nucleotide is greater than when the nucleotides are arra
nged in a singlestranded polynucleotide. The effect occurs because in a polynucleotide, the base
s are in close
proximity. The absorption coefficient decreases even further with a double-stran
ded
polynucleotide because in this structure the bases are arranged in an even more
ordered manner.
THE CHROMOPHORE CONCEPT
The old definition of chromophore regards it as any system which is responsible
for imparting
color to the compound. Most of the nitro compounds are yellow in color. Clearly,
nitro group is
a chromophore which imparts yellow color. The modem day definition of chromophor
e, however,
akes use of the term in a broader scope. The chromophore is dejlned as any isola
ted covalently
bonded group that shows a characteristic absorption in the ultraviolet or the vl
s.lble region. Some of

the important chromophores are carbonyls, acids, esters, nitrile group of ethyle
nic or acetylenic
groups. Chromophores are known to be of twotypes.
(1) Chromophores containlng electrons and involved in ---> " transitions. For ex
ample, acetylenes and ethylenes (Table 8.3).
(//) Chromophores containing both and n electrons and involved in ---> " and n--> n" transitions. For example, carbonyls, nitrfles and azo compounds (Table 8.3
).

192
Btophystca/Chemfstnj
Auxochrome
Awcochromes are groups which by themselves do not act as chromophores but whose
presence
brings about a shift of the absorption band towards the red end of the spectrum
(longer wavelength).
Auxochrome is thus also known as a color enhancer. Important examples are -OH, OR,
-NH2, -NHR, -NR2, -SH etc. Auxochrome exerts its effects by virtue of its abilit
y to extend the
conjugation of a chromophore by sharing of the non-bonding electrons. This resul
ts in a new
chromophore which has a different absorption maximum and probably an enhancedext
inction
coefficient.
In many instances the absorption and absorbance change either due to interaction
with
an auxochrome or due to change of the solvent. Four uch absorption and intensity
shifts are
known and are detailed below.
Bathochrom/c sh/Jt : This shift is due to the presence of an auxochr0me by virtu
e of which
the absorption maximum shifts towards higher wavelengths. Figure 8. I .YSuch an
absorption
shift is known as the red shift, or the bathochromic shift. Sometimes decreasing
polarity of the
solvent may also cause bathochromic shift
Hyperchromic
shift
Hypsochromic Bathochrom/c
Wavelength {rim)
Figure 8.11 Different types of absorption and intensity shIjs
Hypsochromic shift :This is opposite of the bathochromic shift. This shift is du
e to removal
of conjugation and a change in the polarity of the solvent due to which the abso
rption maximum
is shifted towards shorter wavelengths (b/ue shift). See Figure 8.11.
Hyperchromic effect : This effect signifies an increase in the intensity of the.
absorption
maximum, or a change in the extinction coefficient to a higher value at the same
absorption
maximum. This effect is mostly due to the presence of an auxochrome {Figure 8.1
I}.

Hypochromic effect : This is the opposite of hyperchromic effect and is caused d

ue to
introduction of groups which cause distortion in the geometry of absorbing molec
ules. This
effect signifies that the intensity of the absorption maximum is lowered {Figure
8.1 I}.
INSTRUMENTATION FOR UV-VISmLE AND INFRARED
SPECTROPHOTOMETRY
In order to obtain an absorption spectrum it is necessary to measure the absorba
nce of a
substance at a known series of wavelengths. The instruments that are used to stu
dy the
absdrption or emission of electromagnetic radiation as a function of wavelength
are called

Spectrophotometry
193
or spectrophotometers (colorimeters, if the instrument applies wavelengths only
visible range). More or less similar optical principles are employed in these in
struments.
are, however, some important differences in the specific components used in the
various
spectrum.
The essential components of a spectrophotometer include: (/) a stable and cheap
radiant
source, (tO a monochromator to break the polychromatic radiation into component
or "bands" of wavelengths, (///) transparent vessels {cuvettes) to' hold the sam
ple,
(/v) a photosensitive detector and an associated readout system (meter or record
er).
available commercially involve quite a bit of complex arrangements, but all
represent variations of the block diagram in Figure 8.12.
Sample --- Detectr Amplifier &1
Holder " [ Recorder
Figure 8.12 Block diagram of a spectrometer
,adiant Energy Sources
Materials whlcL can be excited to h/gh energy states by a gh voltage electric di
scharge or
,by electric heating serve as excellent radiant energy sources. As the electrons
of these materials
return to the/r ground state, they em/t radiation of characteristic energies cor
responding to AE,
the energy difference between the excited and the ground energy levels. Some mat
erials have
numerous energy levels very close to each other. Consequently the wavelengths of
radiation
era/tied by these substances take the form of a continuum of radiation over a ve
ry broad region.
These materials would constitute an ideal source for absorption measurements if
the intensity
of all the wavelengths is alike. However, inpractice, this is not so and so we h
ave different
sources for different regions of the spectrum.
Sources of ultraviolet radton : Most commonly used sources of ultraviolet radiat
ion are
the hydrogen lamp and the deuterium lamp. Both the systems. :consist of a pair o
f electrodes
enclosed in a glass tube provided with a qumz window, Te glass tube is tflled wi
th hydrogen
or deuterium gas at low pressure. When a stabilized high voltage is applied they
ernit radiation
which is continuous in the region roughly between 180 and 350 nm. Xenon lamp may
also be
used for ultraviolet radiation, but the radiation produced is not as stable as t

he hydrogen ]amp.
Sources of visible radon :'lngsten filament Imp is the most commonly used source
for
visible radiation. It is inexpensive and emits continuous radiation in the regio
n between
and 2500 nm. Carbon. arc, which prov/des more intense v/sible radiation is used
in a small
number of commercially available instruments.
Sources ofnfrared radton : Nemst Glower and Globar are the most satisfactory sou
rces
o infrared radiation. The Globar consists o a s/I/con carbide rod which when hea
ted to
approxLrnate]y 1200C, emits radiation in the 1-40 region. emst Glower employs a h
ollow rod of zh-'con/um and yttrium. It requires to be heated up to 1500C before
it emits radiation in
the range of 0.4-20 . Globar is more stable than the Nemst Glower.
Wavelength Selectors
All the sources discussed so far em/t continuous radiation over w/de range of wa
velengths.
However, as pe/nted out earl/er in the chapter, the laws of absorption in the st
rictest sense
apply only to monochromatic radiation. Thus, absorption of nrrow band width will
show greater
adherence to Beer's law. Moreover, narrow band radiation will allow the resoluti
on of absorption
bands which are quite close to each other. Therefore, a narrow band width will g
o a long way in
increasing the sensitivity of absorbance measurement. Narrow band widths are mad
e possible
by using wavelength selector(s).

Source
Sample
holder
194
Biophysical Chemistry
'fffYgl'tlg'ffi sel'ecfors are of'two types, filters and mon0chromators.
lailters : Filters operate by absorbing light in all other regions except for on
e, whlch they
reflect. Gelatin filters are made of a layer of gelatin, colored with organic dy
es and sealed
between glass plates. Most modern filter instruments, however, use tinted-glass
filters. Filters
resolve polychromatic light into a relatively wide bandwidth of about 40 nm and
are used only
in coldrimeters. One disadvantage of glass filters is their low transmittance (5
-20%).
Monohromators : As the name suggests, a monochromator resolves polychromatic rad
iation
into Its individual wavelengths and isolates these wavelengths into very narrow
bands. The
essential components of a monochromator are: (i) an entrance slit whlch admits p
olychromatic
light from the source, (tO a collimating device such as a lens or a mirror whlch
collimates the
polychromattc light on to the dispersion device, (No a wavelength resolving devi
ce like a prism or
a grating which breaks the radiation into component wavelengths, (iv) a focussin
g lens or a
mirror, and (v) an exit slit which allows the monochromatic beam to escape. The
entire assembly
is mounted in a light-tight box. For obvious reasons, all the components in a mo
nochromator
assembly must not absorb in the range of wavelengths which are to be studied. A
monochromator
employing a prism for dispersion is shown schematically in Figure 8.13. The effe
ctive band
width of the light emerging from the monochromator depends mostly upon the dispe
rsing element
(prism or diffraction grating) and the slit widths of both the entrance and the
exit slits. Narrow
slit widths isolate narrow bands. However, the slit width also limits the radian
t power which
reaches the detector. Since the effectiveness of the resolving element is of pri
mary importance,
the two kinds most widely used, namely, prlsms and diffraction gratings are cons
idered below.
.... lens
lens
"

Entrance slit
Exit slit
MONOCHROMATOR
l%3ure 8.13 Pasta monochromator
Prisrr" A prism disperses polychromatic light from the source into its constitue
nt wavelengths
by virtue of its ability to refract different wavelengths to a different extent;
, the shorter wavelengths
are diffracted most. The degree of dispersion by the prism depends upon (/) the
apical angle of
the prisln (usually 60), and (it) the material of which It is made. Since it disp
erses the short
wavelengths more and long wavelengths less, the wavelengths at the red end of th
e spectrum
are not fully resolved; they are said to be crowded. This is a major disadvantag
e of a prism.
Two types of prisms, namely 60 Cornu quartz prism and 30 Littrow prism are usually
employed in commercial instruments. The latter is preferred, Simple glass prisms
are used for
visible range. For ultraviolet region silica, fused silica or quartz prism are u
sed. F1ourlte is used
in vacuum ultraviolet range. Ionic crystalline materials are used in the infrare
d region. Some
examples are NaCI, KBr, CsBr, and the mixed crystalline material commonly called
KRS-5,

Spectrophotometry
Figure 8.14 Diffraction grating and the dispersion of polychromatic radiation
Gratings : Gratings (Figure 8.14) are often used in the monochromators of
spectrophotometers operating in ultraviolet, visible, and infrared regions. The
grating possesses
a highly aluminized surface etched with a large number of parallel grooves which
are equally
spaced. These grooves are also known as lines. A grating may have anywhere betwe
en 600 to
2000 lines per mm on the surface depending on the region of the spectrum in whic
h it is
intended to operate it. The principle behind dispersion of radiation by a gratin
g is that it resolves
light into its component wavelength by virtue of constructive reinforcement and
destructive
interference of radiation reflected.
Very often, the monochromator consists of both, a prism and a grating. The prism
, placed
before the grating is known as the foreprism. It preselects a portion of the spe
ctrum which is
then allowed to be diffracted by the grating. This arrangement allows resolution
of a single
wavelength. The major advantage of diffraction grating monochromators is that th
eir resolving
power is far superior to that of prisms. In addition they.yield a linear resolut
ion of spectrum
which is not possible when prisms are used.
Sample Containers
Samples to be studied in the ultraviolet or visible regionare usually gases or s
olutions and
are put in cells known as cuvettes. Spectra of gases are taken using enclosed ce
lls, with an
evacuated cell as a reference. Standard path-length of gas cells is usually 1 mm
but cells with
path-length of 0.1 to 100 mm are available for special cases. Sometimes spectra
of solids may
be taken directly. For this purpose, the solids are generally in the form of pel
lets. The pellets are
kept in pellet holders for absorption measurements.
Most of the spectrophotometric studies are made in solution. The solutions are d
ispensed
in cells known as cuvettes. Cuvettes meant for the visible region are made up of
either ordinary
glass or sometimes quartz. Since glass absorbs in the ultraviolet region, quartz
or fused silica
cells are used in this region. Standard path length of these cuvettes is usually
1 cm. However,
cuvettes of path-length of I mm to 10 cm are available for special purposes. The
surface of the
cuvettes must be kept scrupulously clean; fingerprint smudges and traces ofprevi
ous samples,

by causing interference in the optical path, might cause serious errors in quant
itative measurements. Rinsing with water should normally clean quartz or glass c
uvettes. If, however,
the dirt is abnormally tenacious, sulfonic detergents or nitric acid may be used
. The use of
rectangular cuvettes in spectrophotometers effectively curtails the chances of d
irt being
transferred during handling. The two sides of such ceils through which the light
passes are
precision ground and polished to be optically flat The other two sides are rough
ground glass
and the cell may be handled by these.
Since most of the spectrophotometric studies are made in solutions, the solvents
assume
prime importance. The most important factor in choosing the solvent is that the
solvent should
not absorb (opt/ca//y transparent) in the same region as the solute. However, as
we have discussed
earlier, one should also take into consideration the effect that the solvent may
have on the

196
Bophysical Chemistry
absorption spectrum of the solute. The solvents which can be used in the UV and
visible region
are water, methyl-, ethyl-, isopropyl-alcohols, chloroform, hexane etc. Some oft
en used solvents
with their upper wavelength limit of absorption are given in Table 8.4.
Table 8.4 Upper wavelength absorption limits of some solvents
Solvent
Upper wavelength limit (nm)

Water
Ethanol
Butanol
l-Propanol
Ethyl-ether
Iso-octane
Hexane
Cyclohexane
Acetonitrile
Methanol
Dichloromethane
Chloroform
Carbon tetrachloride
Benzene
Pyridine
Acetone
205
210
210
210
210
210
210
210
210
215

235
245
265
28O
3O5
330

Infrared gas cells are made up of glass. These glass tubes possess NaCl, KBr, or
CaF2
windows for the passage of infrared radiation. The cells have varying path lengt
h; from a few
centimeters to several meters, The latter is achieved by multiple reflections wi
thin the cell.
Liquids are studied as thin films or solutions between NaCI, KBr, or CaF2 plates
. The distance
between the plates (path length) is usually 0.005 to 1 mm. Solids are examined i
n the infrared
as pressed KBr discs or as suspensions in high molecular weight liquids ("mulls"
).
Detection Devices
Most detectors depend on the photoelectric effect, where incident light (photons
) liberates
electrons from a metal or other material surface. Some sort of external circuitr
y collects these
electrons and measures their number as current. The current is then proportional
to the light
intensity and therefore a measure of it. Important requirements for a detector i
nclude (i high
sensitivity to allow the detection of low levels of radiant energy, (ii) short r
esponse time, (iii) long
term stability, and (/v) an electronic signal which is easily amplified for typi
cal readout apparatus.
Change in thermal energy, however, is the basis of detection for infrared radiat
ion,
Consequently detection devices used for this region are different than those ope
rating in the
ultraviolet and visible regions.
Ultraviolet and visible radiation detectors : There are three basic kinds of det
ectors in this
region. Photocells, phototubes, and photomultiplier tubes.
(i) Photovoltaic or barrier layer cells : It employs semiconductor materials.
Semiconductors are crystalline, and the bonding electrons between the crystals o
f some
semiconductors can be knocked out of their positions by incident radiation. Alth
ough a number

of materials are used in photocells (cadmium sulphide, silicon, selenium) seleni

um based
photocells are most common. A typical photocell consists of a thin coating of se
lenium over a

Selenium
Anode
Cathode
Recorder
Spectrophotometry
197
thin transparent silver film on a steel base. This arrangement ensures that elec
trons pass
easily from selenium to silver but not in the reverse direction. Due to the inab
ility of electro ,ns to
move away from the silver film, the silver acts as the collecting electrode for
electrons liberated
from selenium by the incident radiation. The steel plate functions as the other
electrode. The
current flowing between the two electrodes is then measured by a microammeter. A
representative
diagram of the cell is given in Figure 8.15.
Silver
Steel back-plate
Figure 8.15 Selenium based photovoltalc cell
Photocells have a long life and are inexpensive and reliable. They are widely us
ed in
colorimeters but their use in spectrophotometers is becoming limited.
(ll) Phototubes or photoemiasive tubes : The components Of a phototube include(A
} an
evacuated glass envelope (with a quartz window), (B) a semi-eylindrical cathode
whose inner
surface is coated with alkali or alkaline earth oxide, and (C) a centrally locat
ed metal wire
mode. A potential difference of approximately 90 volts is applied across the ele
ctrodes. The
quartz window allows the passage of radiation which strikes the photoemissive su
rface of the
cathode. The energy of the photon is transferred to the loosely bound electrons
of the cathode
surface. The electrons become excited and finally leave the surface and travel t
owards the
anode causing current to flow in the circuit, If the electron collection is 100%
efficient, the
phototube current should be proportional to the light intensity. A schematic dia
gram of a
phototube and the associated circuitry is provided in Figure 8.16.

Evacuated glass
envelope
I Anode
Quartz
Cathode
window
lgure 8.16 Dlram of a photoemislve the. R stands for res/stance
Phototube currents are quite small and require amplification.This is usually acc
omplished
by placing a high resistance (R in Figure 8.16) in the phototube circuit.
(|iI) Pht.multl[r : These detectors are designed to amplify the initial photoele
ctric
effect and are suitable for use at very low light intensities. A photomultiplier
consists of (A) an
evacuated glass tube into whlch are sealed the cathode and the anode, and (B) ad
ditional

Incident
radiation
198
Biophysical Chemtstr
intervening electrodes known as dynodes. The arrangement is shown in Figure 8,17
. The external
circuitry is arranged so that a high voltage (1000 volts) exists between the ano
de and the
cathode. As the radiation strikes the photocathode, electrons are liberated and
the applied
potential difference accelerates the electrons towards the first dynode. Each su
ccessive dynode
is at a higher electrical potential and thus acts as an amplification stage for
the original photon.
The applied voltage causes sufficient electron acceleration to knock out other e
lectrons from
each dynode surface. The liberated electrons are dragged onto the next dynode wh
ere more
electrons are released and this process goes on as a cascade till the last dynod
e. By the time the
electrons arrive at the collecting anode, the initial photoelectric current is a
mplified by a factor
of approximately 106. In practice, photomultiplier tubes are used only for low l
ight intensities.
At higher light intensities, due to their great amplification power, photomultip
liers exhibit great
instability. Inspire of this tendency to be unstable, photomultipliers are the d
etectors of choice
in all modern spectrophotqmeters.
Photocathode
Anode
High voltage
Figure. 8.17 A photomultiplier tube
(iVl Phottditdes : Photodlodes are semiconductors that change their charged volt
age
(usually 5 VI upon being struck by Light. The voltage change is converted to cur
rent and is
measured. A photodiode array is a two-dimensional matrix composed of hundreds of
thin
semiconductors spaced very closely together. Light from the instrument is disper
sed by either
a grating or a prism onto the photodiode array. Each position of diode on the ar
ray is calibrated
to correspond to a specific wavelength. Each diode is scanned, and the resultant
electronic
change is calculated to be proportional to absorption. The entire spectrum is es

sentially recorded
within milliseconds.
Near infrared detectors : These are usually photoconductive ceLls which detect i
nfrared
radiation in the range 0.8- 3.0 . The sensing element is a semiconductor (german
ium,
lead sulphide, or lead teLlurlde). Upon illumination with radiation of appropria
te wavelength,
the electrons of the semiconductor are raised to conduction bands. This causes a
drop in
electrical resistance. Consequently, if a small voltage is applied, a large incr
ease in current can
be noted. The resistance of the system is such that the current may be amplified
and finally
indicated on a meter is recorded.
Middle and far infrared detectors : When middle and far infrared photons are abs
orbed,
their energies are convened to thermal energy leading to a rise in temperature.
Obviously then,
rapid response thermometers such as thermocouples, resistance thermometers (bolo
meters),
and gas thermometers (pneumatic or Golay ceils) are used as detectors in this re
gion.
Thermocouples used in the infrared receivers typically consist of a blackened go
ld lead-teLlurium
metal pin junction which develops a voltage that is temperature dependent. To av
oid heat loss,
the the.rmocoupl is usually enclosed in a shielded evacuated housing. This preven
ts error
causing temperature fluctuations.

Visible
Glass.
Detector
Wave number (cm-1)
Source of radiation
Spectrophotometry
199
Amplification and Readout
Radiation detectors generate electronic signals which are proportional to the tr
ansmitted
light. These signals need to be translated into a form that is easy to interpret
. This is accomplished
by using amplifiers, ammeters, potentiometers, and potentiometric recorders.
Components of UV-visible and i.nfrared spectrophoometers have been summarized in
Tables 8.5 and 8.6 respectively.
Table 8.5 A summary of components of spectrophotometers and colorimeters
Region of electromagnetic spectrum
Ultraviolet

Radiation source
Optical system
Material used in the
Optical system
Hydrogen or deuterium lamp
Prism or diffraction grating, or
a foreprism grating double
monochromator.
Quartz or fused silica,
Tungsten filament lamp,

carbon arc (less used)

Tinted glass filters or interference
filters

Sample holders
Quartz or fused silica, rectangular
cells.
Round glass cells.
Photovoltaic cell.

Detector
Photomultiplier
Table 8.6 A summary of the components of infrared spectrophotometers
Region of electromagnetic spectrum
Near-Infrared
Mid-Infrared
Far-Infrared
12,500,
4000 200 ,

Tungsten filament
lamp
Coil of Nichrome
wire, or Nernst
Glower, or Globar.
High pressure
mercury arc lamp.

Optical system
Quartz prisms or prism
grating double monochromator
Diffraction gratings

-10

with a fore-prism
monochromator.
diffraction gratings.

Optical elements are made up of ionic crystalline materials like NaCI,

KBr, CsBr, or KRS-5.

Lead sulphide, or
lead telluride photoconductive
cells.
Thermocouple, ther- Pneumatic or Golay
mistor, or pyroele- cells, pr bolometers.
ctric.

lflecUng
sector
200
Bphysol Chemtry
Double Beam Operation
Voltage fluctuations inducing fluctuations in the source intensity can cause lar
ge scale
errors in spectrophotometer operation. To obviate this situation, double beam sp
ectrophotometers have been designed (Figure 8.18}. Double beam instruments employ som
e type of
beam splitter prior to the sample containers. One of the split beams passes thro
ugh the "blank"
or reference cell while the other passes through the sample cell. The two transm
itted beams are
then compared either continuously or alternately several times in a second. The
double beam
device, therefore, compensates for fluctuations in the source intensity, the det
ector signal, and
amplifier gain by observing the differences in signal between reference and samp
le at virtually
the same time. The modifications described above make the double beam devices mo
re
sophisticated mechanically and electronicaliy as compared to the single beam dev
ices. Obviously,
the double beam devices are expensive.

Mirror .,

,,. Mirror

Blank
I
I
Miulflplie
ono
Chopper ple
Open sector
Rotating sector
chopper

Figure 8,18 Optical arrangement of a double-beam instrument. A rotaW sector chop

per s also shown.

The beam splitting usually occurs after the monochromator. Rotatingsector mirror
s are
commonly used for splitting or *chopping" the beam (Figure 8,18). The chopped be
ams reach
sample and reference and subsequently to the detector at intervals which depend
upon the
rotational frequency of the chopper. The device then records the ratio of the re
ference and
sample signals.
Dual Wavelength Spectrophotometer
Some metal chelators absorb at One wavelength before chelating the metal ion and
absorb
at a completely different wavelength after the chelation has taken place. An exa
mple is that of
arsenazo III, a calcium chelator. This chelator absorbs at 675 nm before binding
to calcium and
at 685 nm after the binding has taken place. If this chelator is incubated with
a biological
system and the absorbance of the chelator is measured simultaneously at the wave
length pair
675 nm. 685 nm, the ratio of the two absorbances can provide an idea of the calc
ium
concentration in the given biological system. Similarly, in many reaction kineti
c studies it is
necessary to monitor the absorbance changes of two chemical species simultaneous
ly. In many
experiments it is necessary to measure the relative absorbances of proteins {280
nm} and
nucleic acids (254 nm} simultaneously. For all these experiments it is necessary
to use dual
wavelength spectrophotometer.

Z2Cuvette
Spectrophotometry
201
Dual wavelength spectrophotometry refers to the photometric measurement of a mat
erial
by passing radiation of two different wavelengths through the same sample before
reaching the
detector. Light from two different sources is allowed to be resolved into two di
fferent wavelength
with the help of a pair of diffraction gratings. Subsequently the two beams of d
ifferent wavelengths
are made to pass through the same sample by a complex arrangement of a large num
ber of
mirrors (Figure 8.19). Only a single detector, which is always a photomultiplier
tube, is used.
Light source
Mono
chromator

Monochromator
Figure 8.19 Optical arrangement of a dual wavelength spectrophotometer
From the above it is clear that dual wavelength spectrophotometry provides infor
mation
from two wavelengths per unit time. All other factors being equal, the resultant
data should be
more useful than data from a double beam spectrophotometer.
APPLICATIONS OF.UV-VIS SPECTROPHOTOMETRY
Photometry being a very versatile technique, has diverse applications, only a fe
w of which
are summarized below.
Qualitative Analysis
Visible and ultraviolet spectra may be used to identify classes of compounds in
both the
pure state and in biological preparations. This is usually done by plotting abso
rption spectrum
curves. Since these curves are specific for a class of compounds, a knowledge of
the absorption
spectrum can help in identification of a substance in biological milieu. Table 8

.3 provides
information about the absorption ranges of the most commonly occurring functiona
l groups of
biomolecules.
It is quite beyond the scope of this book to deal with the details of identifica
tion of an
unknown compound or the assignment of structure on the basis of its absorption s
pectrum (for
details the student is referred to literature cited at the end of this chapter).
However, a brief
discussion is possible. Before attempting to interpret the absorption spectrum o
f a given
compound, suffioient chemical information about the substance such as the elemen
ts present
should be known. Absorption by a compound in different regions gives some hints
of its structure.
Thus, compounds which do not absorb in 220-280 nm region are usually aliphatic o
r alicyclic
hydrocarbons or their derivatives. Sometimes they might be simple olefinic compo
unds. If the

202
Biophysical Chemistry
compound absorbs between 220-250 nm range, it will usually contain two unsaturat
ed linkages
in conjugation. Absorption in this range could also be due to benzene derivative
s. Presence of
more than two conjugated double bonds usually gives rise to absorption in the ra
nge 250-330
nm. It is a fact that as the number of conjugated double bonds increases, the ab
sorption range
is shifted more and more to higher wavelengths. Thus, -carotene, a precursor of
vitamin A has
eleven double bonds in a conjugated system and appears yellow because the light
in visible
region (450-500 nm) is being absorbed by it. Complex systems will give rise to a
bsorption
curves with several maxima, each of them possessing a characteristic shape and r
ange indicating
the presence of the particular functional group.
As an example to prove how valuable absorption data can be, one can cite the exa
mple of
vitamin K whose structure was determined by the use of its spectral data. The ab
sorption
spectrum of vitamin K has the following absorption maxima: 249 nm, 260 nm, and 3
25 nm.
The maxima around 250-330 nm are characteristic ofa naphthaquinone. Chemical man
ipulation
and comparison of K spectra with several model compounds indicated that the posi
tions of the
absorption maxima were similar to those of 2,3-dialkyl- 1, 4-naphthaquinone. The
structure of
vitamin K determined later is as below:
Also see Box 8.3 for further discussion.
Abetter example, perhaps, is afforded by the four nucleotides (Figure 8.20). If
you dissolve
the four nucleotides GMP, CMP, AMP, and UMP in aqueous solution at neutral pH, t
he absorption
maximum at highest wavelength for GMP occurs at 255 nm, for CMP at 271 nm, for U
MP at
262 nm, and for AMP at 259 nm. With just these absorption maxima, one can tentat
ively
identify GMP from CMP since their absorption maxima are quite removed from each
other.
However, it would be difficult to distinguish between AMP and UMP owing to their
absorption
spectra being quite close. However, one can resort to changes in pH of these sol
utions and the
data so obtained can help us in identification. A look at Figure 8.20 and the te
xt therein will
explain the point.

'0
pounds o
,t,'O
c"0
9"0
L'O
g'O
wv - , .7 6"0

204
Biophysical Chemistry

205
rise?
behavior of
Spectrophotometry
be ,zero
fthe pH is now raisedbeyondt 0.0, what do you
are rosine.
rise in absorption at295 nm will be seen because these tyros nes w I be sh elded
, f only two
tyrosines an; On the surface and two buried in the folds, riein absorpt on at 29
5nm w I only
be hail of what is expected for four tyrosines. Inthe last Case if the pH is rai
sed tosay 13.(,
all tyrosines wi be available for titri0n becausethe protein is now denatured.
largescale unfold ng of the protein
were buried when the protein was in the
surface brings about, a change n the
proteins: Whenever the substrate orthe competitive inhibitor
region or makes certain aminc acid residues
In doing so, it might quite frequ
spectral changes are
obtained with other stud es such as the so vent
quite a bit of information about the structure of the active stem ght be
studies. Solvent
of lysozyme, one observes
maximum of tryptophan is shifted to a longer wavelength.
pectral change corresponds with only one tryptophan residue.. If the
solution of the enzyme with the substrate is again studied by solvent perturbati
on method,
only'three tryptophan residues seem to be on the surface. The conclusion is quit
e Obvious
really. It seems that the e.nzyme has one tryptophan residue in the active site
which might be
involved in the binding. Studies with x-ray diffraction confirm this conclusion.
text, can you answer the followingquestions?

(a)
A protein solution is being heated and its spectral characteristics are
being determined at
250 nm (., for cysteine). At:45oc the extinction of the system suddenly increase
s. This
increase plateaus Out at 55oc. What conclusion should you draw?
(b)
A DNAsolution is being cooled after it was heated..Tle ext nction at 260
nm shows sudden
drop. What happened?

Wavelength
206
Bophystcal Chemistry
Quantitative Analysis
In developing a quantitative method for determining an unknown concentration of
a given
species by absorption spectrometry, the first step is the choice of the absorpti
on band at which
the absorbance measurements are to be made. If the chemical species of interest
has already
been researched upon, its .ultravlolet/vlsible absorption spectrum would be avai
lable in the
literature. If this is not the case, the absorption spectrum of the chemical spe
cie can be dctermcd
experimentally by means of a scanning double beam spectro-photometer. A suitable
absorption
band is then selected from within the absorption spectrum for quantitative measu
rements.
Absorptivity at any given wavelength is constant and is an inherent characterist
ic of the absorbing
substance. Most of the organic compounds of biological interest absorb in the UV
-visible range
of the spectrum. Thus, a number oflmportant classes of biological compounds may
be measured
semi-quantitatively using UV-visible spectro-photometers. Nucleic acids at 254 r
un and prote/n
at 280 nm provide good examples of such use. The absorbance at 280 um by protein
s depends
on their tyrosine and tryptophan content. All proteins will therefore have a dif
ferent absorbance
at 280 nm and may be only accurately assayed if a calibration curve {see Box 8.2
} is plotted for
the pure protein.
What do we mean by selecting a suitable wavelength or alsorption band? There are
certain
rules for the choice. These may be summarized as under (Figure 8.21).
1. Choose an absorption peak with the greatest possible molar absorptivity.
2. Choose a relatively broad peak.
3.
Choose a peak that is as far as possible from the absorption peaks of co
mmonly
interfering chromogens. The same is true for solvents and other reagents used the
absorption band chosen should be as far away as possible from the absorption pea
ks
of the solvent and the reagents.
Figure 8.21. Choice of the absorption band for quantitative analysts.

X has a small absorption coefficient. Quantitative analysis should normally not

be carried
out here. X has the highest absorption coefficient but the peak is too sharp. '3
has a sufficiently
high absorption coefficient (though lower than .) and the peak is broader than .
. k3 then
becomes the best choice. Of course, this must not correspond to absorption peaks
of other
interfering chromogens or the solvent.

207
ay
The quantitative assay of enzyme activity is carried out most quickly and conven
iently
the substrate or the product is colored or absorbs light in the ultraviolet rang
e because
or disappearance of a light absorbing product or substrate can be followed
which gives a continuous record of the progress of the reaction on a
chart recorder. Other light absorbing or light scattering substances must either
be absent
by appropriate blank measurements. Example can be cited of the measurement
of the enzyme lactate dehydrogenase, which is engaged in the transfer of electro
ns lactate to NAD*. The products of the reaction are pyruvate, NADH, and a proto
n.
Lactate + NAD - Pyruvate + NADH + H
One of products, NADH, absorbs radiation in the ultraviolet range at 340 nm whil
e its
NAD, does not. No other component of the reaction, either substrate or
tbsorbs at 340 rim. It is thus very obvious that the progress of the reaction in
the
llrection can be followed by measuring the increment in light absorption of the
system
nm in a spectrophotometer.
simplicity and convenience of optical assays prompts their use in following the
time
of an enzymatic reaction in which neither the substrate(s) nor product(s) have a
ny
absorption maxima. Such type of enzyme reactions are coupled to some other
reaction which has an easily measured optical change. An example of the reaction
hosphoenolpyruvate and ADP yielding pyruvate and ATP catalyzed by pyruvate kinas
e
below.
Phosphoenolpyruvate + ADP v- Pyruvate + ATP
Although neither the substrates nor products of this reaction absorb light in th
e 300-400
the reaction is easily measured if lactate dehydrogenase and NADH are added to t
he
large excess. By manipulating the system in such a manner we obtain the followin
g
I reactions, which have the common intermediate pyruvate:

Phosphoenolpyruvate + ADP Pyruvate + ATP

Pyruvate + NA{)H + H -- Lactate + NAD
Since we have added a large excess of NADH to the system, the system now absorbs
at 340
But from the reactions written above it is clear that for each molecule of pyruv
ate formed
first reaction, a molecule of NADH is oxidized to NAD in the second reaction when
the
converts pyruvate to lactate. Since NAD does not absorb at 340 nm the absorbance
on decreasing with increased pyruvate generation. Such measurements are known as
assays and are fairly routinely used.
Weight Determination
If a compound forms a derivative ith a reagent which has a characteristic absorpt
ion
' at a wavelength where the compound does not absorb, then the extinction
f the derivative is usually the same as that of the reagent. Although the extinc
tion
of the absorption band remains constant in all the derivatives, the optical dens
ity,
is different for compounds of different molecular weights. The molecular weight,
M, of the
be readily calculated on the basis of its absorption data.
M = awb/OD
or M= 10a/E IL

208
Biophysical Chemistry
where w is the weight of the compound in grams per litre, and b is the pathlengt
h.
Molecular weights of amine picrates, sugars, and many aldehyde and ketone compou
nds
have been determined by this method. The method has an accuracy of+ 2%. Molecula
r weights i
of only small molecules may be determined by this method'.
Study of Cis-Trans Isomerism
Sine geometrical isomers differ in spatial arrangement of groups ab.out a plane,
the
absorption spectra of the isomers also differs. The trans-isomer is usually more
elongated than
its cis counterpart. It is usual therefore, for the trans-isomer to have a highe
r wavelength of
maximum absorption and also to have a higher ema' Absorption spectrometry can th
us be
utilized (indeed it has been) to study cis-trans isomerism.
Other Physicochemical Studies
Over the years, spectrophotometry (UV-VIS) has been used to study such physicoch
emical
phenomena as heats of formation of molecular addition compounds and complexes in
solution,.
determination of empirical formulas, formation constants of complexes in solutio
n, hydration
equilibria of carbonyl compounds, association constants of weak acids and bases
in organic
solvents, tautomeric equilibria involving acid base systems, protein-dye interac
tions, chlorophyllprotein complexes, vitamin A aldehyde-protein complex, association of cyanine-dy
es,
determination of reaction rates, determination of labile intermediates, and diss
ociation constants
of acids and bases.
Control of Purification
This is one of the most important uses of UV-VIS spectrophotometry. Impurities i
n a
compound can be detected very easily by spectrophotometric studies by experiment
ally verifying
whether the given compound shows an absorption maxima not characteristic of it.
Thus carbon
disulphide impurity in carbon tetrac, hloride can be detected easily by measurin
g absorbance at
318 nm where carbon disulphide absorbs. Similarly benzene impurity in commercial
absolute
alcohol can be detected by measuring absorbance at 280 nm where alcohol (210 nm)

does not
absorb. A lot many commercial solutions are routinely tested for purity spectrop
hotometrically.
Difference Spectroscopy
Difference spectroscopy provides a sensitive method for detecting small changes
in the
environment of a chromophore. It may also be used to demonstrate ionization of a
chromophore
leading to identification and quantitation of various components in a mixture. D
ifference
spectroscopy involves comparison of absorption spectra of two samples which diff
er only slightly
in their physical states. The common features in the spectra cancel out and the
bands which
are recorded can be interpreted in terms of known differences between the sample
s.
Difference spectroscopy was developed by Chance and Williams in course of their
research
on electron transport chain proteins in the mitochondria. The technique subseque
ntly provided
much needed information about the state and sequence of the electron transport p
roteins.
Difference spectroscopy has also been utilized in toxicology laboratories for an
alysis of many
toxic drugs. Example can be cited of barbiturates which show characteristic chan
ges in absorption
spectra between their keto and enol forms. Moreover, difference spectroscopy is
a necess.ary
tool to study globular protein conformation.
Turbiflimetry and Nepheloraetry
Bacterial Or any other particulate suspension makes the liquid turbid. This is d
ue to
Tyndall effect which addresses itself to light scattering by colloidal particles
(see chapter 3).
While. the liquid in this system might absorb at a particular wavelength, the pa
rticles scatter

Spectrophotometry
209
the incident light. If, then, radiation of a wavelength which is not absorbed by
the liquid is
made to pass through this suspension, the apparent absorption will be solely due
to light scattering
by the particles. The light transmitted by the suspension will have lesser inten
sity than the
light which was incident. Measurement of the intensity of this transmiR, ed ligh
t will allow one to
have an idea of the number of particles in the suspension. Using this technique,
known as
turb/d/metry, one can arrive at a fair approximation of the number of particles
in a given
suspension. This technique is routinely used to measure the number of bacteria i
n a given
suspension. The wavelength used for this purpose is 600 nm. The technique, howev
er, is very
tedious to standardize as the particle size is critical for accuracy (larger par
ticles scatter more
light as compared to smaller particles; thus contamination of small particle sus
pension by a
small number of large particles will give a value far in excess of the number of
small particles
actually present). The principle of turbidimetry is shown diagrammatically in Fi
gure 8.22(A).
Nephelometry is a term many times used synonymously with turbidimetry probably b
ecause
the two techniques are based on a common principle. The major difference between
turbidimetry
and nephelometry is that while the former measures the intensity of transmitted
light coming
out of a suspension, the latter measure the intensity of the light scattered by
the particles in
suspension. The scattered intensity is usually measured at right angles to the d
irection of
incident light. For low concentration this method is more sensitive since zero c
oncentration is
represented by a dark background. On the other hand zero concentration in turbid
imetry means
full illumination. The principle of nephelometry is diagrammatically shown in Fi
gure 8.22(B).
Nephelometry is commonly used for estimating the concentration of microorganisms
. It is also
used for waste-water analysis as well as in the beverages and pharmaceutical ind
ustries to
evaluate the amount of haze present in the preparations.
Sample cell
containing suspension
]
[ (A) f ,Photocell

 o
Light Slit
,
Lens
source
(B)
I
Lens
Photocell detector
Figure 8.22 Principle of turbidimetry and nephelometry.
(A) Light is scattered by particles in the suspension in sample cell. The transm
itted light is therefore of
a weaker intensity. A turbidimeter measures the intensity of this transmitted li
ght and a calibration
cw' beaueen transmitted intensity and particle concentration can be drawn.
(B) Nepheiometry measures the intensity of scattered light rather than transmitt
ed light.
THEORY AND APPLICATIONS OF INFRARED SPECTROSCOPY
As evident from the electromagnetic spectrum diagram, infrared radiation is of m
uch
higher wavelength as compared to the ultraviolet and the visible region. Consequ
ently,
electromagnetic radiation of this region has considerably lower energy. Infrared
radiation is,
therefore, not associated with electronic transitions; rather, it is associated
with vibrational
transitions of molecules as we will see below.

210
In-plane deformations
Scissor and Rock
"
N
F
Symmetrlc
Antlsymmetrlc
DF
IN
Out-of-plane deformations
Twist
and Wag
Figure 8.23 Types of vibrations : (A) Stretching, (B) Bending
All the molecules are continually vibrating. These vibrations are of two types.
The bond
distances between the atoms in a molecule fluctuate to about 0.5A. It should be r
emembered
that while this increase or decrease in bond length is occurring, the atoms rema
in in the same
bond axis. These vibrations are known as stretching vibrations (Figure 8.23{A)).
The other type
of molecular vibration, known as bending vibration (Figure 8.23(B)) involves cha
nges in the
positions of the atoms with respect to the original bond axis. Such variations i
n bond angles
may be about 0.5. Vibrational transitions are low energy transitions and these ene
rgy levels
correspond to the energies of electromagnetic radiation in the infrared region o
f the spectrum.
Consequently, a molecule can absorb infrared radiation of an appropriate frequen
cy, accompanied
by promotion of the molecule to an excited vibrational state. The presentation o
f infrared spectra
differs from UV-visible spectra in that wave number is used in this region rathe
r than wavelength;
infrared spectra are typically presented as percent transmission (transmittance
x 100) versus
wave number.
Cslculatlon of Vibrational Frequencies
The vibrational frequency of a bond can be calculated with the help of Hooke's l

aw
which correlates frequency with bond strength and atomic masses.
Vl
Or
V-,
mass
2n mim2/(m, +rni)
where v is the frequency, k is the force constant of the bond and m and m2 are t
he masses of
the two atoms involved in bond formation. The quantity mrn/(m+ m2) is often expr
essed as m,..
the reduced mass of the system.
Let's try and calculate the approximate frequency of the C--H stretching vibrati
on from
the data given below.
k=5.0 x 10Sgs-2
mass of carbon atom = 20 x 10-24.g

211
Specrophotorrtr
5.0xlO5gs-2
7
2x22

= 9.3x10asDividing the above value with the speed of light we get the value in wave number
which is
3100 cm-L When you experimentally determine the vibrational frequency for C--H s
tretching
you find it ranging from 2800 to 3100 depending on the compound you choose. This
simple
observation tells us that the vibration of any given bond will be affected by th
e other atoms and
their bonds that it coexists with.
If you look at the above equation, it should be easy to surmise that the vibrati
onal frequency
of a bond should increase with the strength of the bond. Also when the reduced m
ass of the
system decreases, the frequency should increase.
The foregoing may make us think that we can predict the frequencies of vibration
for a
given bond. To an extent we can. For example, double bonds are stronger than the
single. So we
can safely say that C--C should have a frequency lower than that of C---C. This
we predicted on
the basis of the bond strength. On the basis of mass we may hazard a guess that
C--H should
absorb at a higher frequency as compared to CmC since the latter has a higher re
duced mass.
However, we are stepping onto treacherous territory if we start predicting frequ
encies on the
basis of masses without knowing the force constant. One example will prove this.
Given below
are a few bonds. Guess which of these bonds will absorb at the highest frequency
solely on the
basis of the reduced masses.
C--H ,. N--H, O--H, and F--H.
Solely on the basis of masses your answer must have been that the absorption fre
quency
falls along the series given. The actual observation is quite to the contrary. T
he frequencies rise
along the series. They rise because all along the series the electronegativity r

ises and with it

rises the force constant.
Thus, although it is possible to predict the frequencies in a general manner tak
ing the
help of Hooke's law, some caution .must be exercised in doing so.
Modes of Vibration
The theory of molecular vibrations predicts that an asymmetrical molecule which
contains
n atoms will have 3n - 6 modes of fundamental vibrations. This means that a mole
cule like CO2
o should possess (3 x 3) - 6, i.e., 3 fundamental modes of vibrations. Likewise,
methane should
have 9 and ethane 18.
Figure 8.23 shows vibrational modes available for AX systems (any atom joined to
two
other atoms, e.g., NH2, NO2, CH2 , etc.). Normally each vibration mode absorbs a
t a different
frequency. Thus a CH group may give rise to two C--H stretch bands, symmetric an
d asymmetric.
However, this is not always true. There will be some vibrations which may absorb
at the same
frequency. Naturally their absorption bands will overlap. Such vibrations are ca
lled degenerate.
Also, there are vibrations whose absorption frequency may lie outside the normal
infrared
region examined.
So far we have been talking about vibrations which are dubbed fundamental. There
are
other frequencies at which bands appear in.an infrared absorption spectrum. Some
of these
bands are called overtone bands. These bands are generated by modulation of fund
amental

212
Biophysical Chemistry
vibrations, Thus, strong absorption at 800 cm-I may give rise to a weaker absorp
tion at 1600
cm-; a strong absorption at 1700 cm- may give rise to a weaker absorption at 340
0 cm-I. Another kind of modulation is when two different frequencies, x and y, i
nteract with each other
(combinations). Such interactions may take place either as x + y, or as x - y. T
he resulting
weaker absorptions are called beats.
You would agree that combination bands are unique to a compound. Since each comp
ound
has a particular arrangement of atoms, a particular kind of combination can only
occur in that
compound and in none other. The combination bands, therefore, assume extreme imp
ortance
because they may be the signature or the fingerprint of a given compound. A larg
e number of
these combination bands fall in the region between 900 cm- and 1400 cm-. For thi
s reason, this region is dubbed the fingerprint region.
In summary we can say as under.
Any given bond in a molecule does not exist in isolation. Thus if we refer to C-H stretch
frequency, we cannot hope that this particular bond is in isolation from the res
t of the molecule.
If this bond exists within a given molecule, the other bonds in that molecule, t
he other atoms
present in that molecule are bound to have an effect on the way this bond behave
s. They are
going to have an effect on the exact frequency it absorbs. Thus a C--H bond in o
ne compound
may vibrate at a slightly different frequency when compared to a C--H bond in a
different
molecule. We may therefore say that the frequencies of molecular vibrations do n
ot depend just
on the masses of the atoms concerned and the strength of their bonds, but also o
n the
arrangement of atoms. The vibrational frequencies therefore reflect the structur
e and
conformation of a molecule. Since the atoms involved and therefore their masses,
the strength
of their bonds, and the arrangement of atoms differ from compound to compound (u
nless the
compounds are enantiomers),it may be said that the infrared spectra of no two co
mpounds are
alike. Conversely, substances that give-the same infrared spectra are identical.
The infrared
spectrum of any molecule is thus absolutely specific and is therefore known as t
he 'fingerprint'
of the compound. -..
The molecules in which biochemists are more interested are the biological macrom
olecules
(proteins, nucleic acids, polysaccharides). Since these molecules have a very la

rge number of
atoms, they will have an enormous number of possible modes of vibration. Conside
r a molecule
which has 100 atoms; it will have (3 x 100) - 6 = 294 fundamental modes of vibra
tion not to say
anything about the bands that will appear out of combinations and other modulati
ons. Biological
macromolecules have much more than 100 atoms. These macromolecules will possess
such an
enormous number of fundamental modes of vibration, that a rigorous explanation o
f their
infrared spectra will be unattainable. And yet the infrared spectra of even thes
e compounds will
provide information about the various functional groups which absorb at characte
ristic infrared
regions regardless of whether they are present in a large or a small molecule (
later we will see
that even conformational studies on macromolecules are done with the help of inf
rared spectra).
The importance of infrared spectroscopy to the study of the functional groups ca
nnot therefore
be overemphasized. It will be apt for us to spend some time discussing the vibra
tions of dlfferent
functional groups of.ubiquitous occurrence.
Infrared Spectra of Common Functional Groups
The regions in which functional groups absorb are summarized in Figure 8.24. The
figure
also illustrates a very simple spectrum belonging to the liquid paraffin Nujol,
a substance often
used for taking infrared spectra of solids.

1028
213
tching I

Spectrophotometry
4000 3500 3000 2500 2000
1500
1000
cra'*

N--H
hing
JL
c--c
I
r
Other
stretching,
1
C "--O
1 | bending and corn-|
N-----oCN
|bination bands.
|
Stetc I'll | The fingerprint
|
Bending [ region
j

Figure 8.24. A summary of the regions of absorptlon correspondgng to the stretch

ing and bending vibrations of the
ma functlonal groups and carbon skeleton bonds. IR spectrum of the Iklukl hydrcx
:oybon, NuJol, is olso

seen.
Obvious from the figure are some rules that must be borne in mind.
1.
Owing to their low mass, the stretching vibrationsf single bonds involvi
ng hydrogen
give rise to absorption at the high frequency end of the spectrum.
2.
The greater the steength of the bond between two similar atoms, the high
er the frequency
of the vibration. Thus, stretching vibrations of triple bonds between carbon ato
ms,
and carbon and nitrogen absorb just below the region where single bond stretchin
g
involving hydrogen absorb.
3.
Below this is the region where stretching vibrations of double bonds bet
ween carboncarbon, carbon-oxygen, carbon-nitrogen, and nitrogen-oxygen absorb. N--H bending
vibrations also absorb in this region.
4.
Bending vibrations are of much lower frequency and usually appear in the
flngerpr/nt
region below 1500 cm-'. N.--H bending is an exception to the rule.
In a book of this size and generality, it is not possible to discuss the vibrati
ons assignable
to each and every functional group. However, a cursory discussion of major funct
ional groups
in structures that may be encountered frequently-in biochemistry follows.
The Carbon Skeleton
Aromatics: C--H str, C=-'C str, C--H def and a group of overtone combination bon
ds are
seen in infrared spectra of aromatics. C--H sir occurs Just above 3000 cm-. Thes
e bands may
be quite weak.
Most aromatic compounds show three of the four possible OC str bands. Of these,
the
band at 1450 cm- may not be seen frequently, and the two bands around 1600 cm-I
may coalesce. Thus, two, three, or four bands may be expected for C'-C sir.
The number of hydrogen atoms on the ring may be characterized by out'of-plane
C--H deformations. Thus these bands are characteristic of the substitution patte
rn on the ring.
The p-substituted benzenes present a strong band above 800 cm

214
Bophyscal Chemistry
Alkanes and Allcyl Groups: Most commonly C--H str absorptions appear Just below
3000
cm-. Out of these .two, the band with the higher frequency is the one assignable
to the
antisymmetric stretch. For the alkyl groups the antisymmetric CH3 def occurs aro
und
1390 cm-.
Alkenes: These skeletons give absorptions quite singular to that of aromatics an
d as such
if an alkene occurs within an aromatic molecule, it is all but impossible to det
ect it.
The most. characteristic feature of alkenes may be the strong overtone bands for
out-ofplane C--H def vibrations. The combination vibration occurs around 1800 - 2000 c
m-, where
few other absorption appear. Trans-alkenes and cis-alkenes are easily detectable
from each
other: the former gives a C--H def band around 970 cm-] while the latter gives i
t around
700 cm-.
Cumulative double bonds give rise to strong absorptions around 2000 cm-. Thus,
C--C-C (allenes) absorb around 1950 cm-I, O-CO absorb at 2350 cm-, C-C---O (kete
nes}
absorb at 2150 crn-, --N--C-O (isocyanates) absorb at 2250 cm-, --N---C-S (isoth
iocyanates}
absorb at 2100 cm-!, and --N3 (azides| absorb at 2140 cm-.
Alkynes: Out of terminal and nonterminal alkynes, the former are easily detectab
le. A
strong C--H str band occurs near 3300 cm-. The weaker CC sir band occurs near 22
00 cm-i.
The nonterminal alkynes have an extremely weak C-=C str and they lack the C--H s
r absorption.
Cas4nyl Group
The stretching absorption of this group has probably been studied more than any
other
group. The CO stretch band is always strong and so the overtone is almost always
visible
around 3400 cm-.
In enols theCO stretch frequency occurs very low at 1580 cm-.
In aldehydes, two C--O stretch bands occur. Just below 1800 and Just above 1800
cm-.
The latter is for antisymmetric stretch.
In saturated esters the C---O stretch band occurs around 1740 cm-. If the carbon

yl group
is in conjugation with a double bond or an aromatic ring, the band occurs lower
around
1720 cm-. Chelation with a nearby OH group weakens the C--O bond even further an
d
consequently the band is lowered even more around 1880 cm-.
If the CO stretch bands occur around 1700 cm- along with a broad band around 300
0 cm- |O--H stretch), the possibility is that we are seeing a carboxylic group.
C-O stretch bands for carboxylic acid anhydrides occur near 1800 cm:.
Hydroxy Compounds
For the primary alcohols C--O sir band is centred around 1300 cm-, and the O--H
defis
between I000 and 1100 cm-.
For secondary alcohols the C--) sir is situated more or less as for primary alco
hols, but
the O--H def is centred around I I00 cm-].
For tertiary alcohols the former occurs between 1300 and 1410 cm- while the latt
er also is sh/fted towards higher frequencies and occurs between I I00 to 1200 c
m-.
For the carbohydrates the detection is made simple by the presence of an extreme
ly intense
OH sir centred on 3300 cm- and a C---O str entred at around 980 cm-.

Spectrophotometry
215
Nitrogen Compounds
Hydrogen bonding in amines modifies both symmetric and antisymmetric N--H str
bands. In dilute solutions free NmH sir can be seen near 3500 cm-. NmH def occur
s around
1600 cm-.
For nitro compounds two NcD str bands occur, one ranging from 1500 to 1600 cm-.
and
the other ranging from 1300 to 1400 cm-.
For nitriles the C eN.str occurs just above 2200 cm- and for isonitriles this ba
nd occurs. just below 2200 cm-.
INFRARED SPECTROPHOTOMI'ER : MODE OF OPERATION
The spectrometer consists of a source of infrared light, emitting radiation thro
ughout the
whole frequency range of the instrument. Light from the source is split into two
beams of equal
intensity. One beam is made to pass through the sample while the other is allowe
d to behave as
the reference beam. The function of such a double beam operation is to measure t
he difference
in intensities between the two beams at each wavelength.
Now the two beams are reflected to a chopper which is rotating at a speed of 10
otations
per second. The chopper makes the reference and the sample beam to fall on the m
onochromator
grating alternately. The grating also rotates, albeit slowly. This rotation send
s individual
frequencies to the detector. It is the function of the detector to convert infra
red thermal energy
to electrical energy.
At the wavelength where the sample has absorbed, the detector will receive a wea
k beam
from the sample while the reference beam will retain full intensity. This will l
ead to a pulsating, or alternating current to flow from the detector to the ampl
ifier. On the other hand,, at the
frequencies where the sample doesn't absorb, both the beams will have equal inte
nsities and
the current flowing from detector to amplifier will be direct,, not alternating.
The amplifier is
designed to amplify only the alternating current. The amplified signal is record
ed by a penrecorder.
This instrument optically balances out the differential between the two beams. T
herefore

this kind of instrument is called optical null recording spectrometer. More soph
isticated
instruments are called ratio-recording instruments. In these instruments the int
ensities of
both sample and reference beams are measured and ratioed.
Samplin Technique
The sampling techniques depend on whether the sample is in vapor phase, a liquid
, or a
solid. In these three phases the intermolecular forces differ significantly and
thus it is imperative.
that the data obtained be specified for the sampling technique used.
Vapor phase : The vapor or gas is introduced into gas ce//s which are either abo
ut I0 cm
long or shorter with a series of internal mirrors (multi-pass cells} which refle
ct the infrared
beam back and forth lengthening the path-length. The gas cell has NaCl windows a
t the ends
(NaCI is transparent to infrared).
Most organic compounds have too low a vapor pressure for this phase to be useful
.
L/quids : Liquids are usually observed as a thin film between two infrared-trans
parent
windows, This is done by squeezing a drop of liquid between NaCl flats.
The flats must be kept cle.an by washing them in toluene or chloroform and they
must be
optically polished using jewellers' rouge. The flats must be.kept dry and they m
ust be handied
by the edges only.

216
Biophysical Chemistry
The thickness of the liquid film varies between 0.01 to 0.1 mm. Pressure is appl
ied to the
flats to achieve the desired thickness.
NaCI flats are used almost throughout the infrared region. However, if the sampl
e contains
water, they become useless. In such cases CaF2 flats are used.
Solids: Solid spectra are recorded in three different ways, mu//s, KBr d
/sks, and deposited
Mulls are prepared by grinding about 1 mg of a solid in a small agate mortar wit
h a liquid
hydrocarbon (Nujol, Kaydol, etc.). Nujol is transparent to infrared over a very
wide range and
for this reason Nujol mulls are very widely used. One disadvantage with Nujol is
that it gives
C--H str and C--H def bands. Thus if C--H vibrations are to be studied, Nujol ma
y not be a
good choice. For these purposes hexachlorobutadiene, or chlorofluorocarbon oils
are used.
Once the mull has been prepared, it is squeezed between flat plates of NaCI and
mounted
for spectroscopy.
To avoid bands due to mulling agents altogether, the sample may be ground with p
ure
KBr (0.1 to 2.0 per cent sample by weight). The KBr must be dry and the grinding
must be
conducted under infrared lamp to not to allow the moisture to condense. With all
these
precautions, however, slight moisture does get in and that results in a band at
3450 cm- due to OH group. Another precaution that should be taken with this meth
od is that the grinding
must be done well. The particle size should be lower than the infrared wavelengt
h, i.e., less
than 2 turn. Poorly ground mixtures tend to scatter more light and lead to erron
eously low
transmissions. Mechanical ball mills are available for the purpose. Once the gri
nding is done
the sample is squeezed, as before, between optical flats.
For solid films, the sample is dissolved in a volatile solvent and then the solu
tion is allowed
to evaporate drop by drop on an NaCI plate. Polymers and fatty materials often g
ive excellent
results as solid films. Solid films are also used for infrared dichroism study.
It must be kept in mind that intermolecular forces are different in solids as co
mpared to
solutions. This is particularly true of those functional groups which take part

in hydrogen
bonding. Thus solid state spectra are different from solution spectra. However,
the number of
resolved lines are far greater in solid state spectra. Many organic compounds ex
ist as polymorphic
variations. These different crystalline forms lead to differences in infrared sp
ectra. It is better to
measure such substances in solution, since all polymorphism is lost in that stat
e.
So/ut/ons : The principal complication here for biochemists is that aqueous solu
tions
cannot be used for infrared spectra. This is because water absorbs infrared powe
rfully owing to
its high absorption coefficient and a high concentration (55 M). This problem is
addressed by
dissolving the sample in DO or D20 -- H20 mixture. Another alternative is to use
chloroform.
This solvent dissolves quite a few polar molecules. However, it suffers from the
disadvantage of
inducing severe conformational changes in macromolecules.
The sample is dissolved in an appropriate solvent to give a 1-5 per cent solutio
n. This
solution is then introduced into a solution cell made of NaCI. The pathlength of
the cell varies
between 0.1 to 1.0 mm. A second cell containing pure solvent is placed in the re
ference beam to
cancel out absorption due to the solvent only. For true compensation, however, t
he pathlength
of the reference cell should be 95 per cent of the pathlength of the sample cell
if the solution
concentration is 5 per cent; it should be 96 per cent if the solution concentrat
ion is 4 per cent
and so on.
Applications of Infrared Spectroscopy
Mainly four types of problems have been addressed by the life scientists employi
ng infrared
spectroscopy. These are : identification of compounds, assaying the rate of reac
tions, studying
the conformation of molecules, and understanding interactions between molecules.

trophotometry
217
Ider.]ation of : Macromoleees ssess a lge number of atoms d as have numerous fde
n braons. s seeg disadvge c be actuy
adve c nse at tt pruces a deed red spec which secs as c et of e concerned mole.
Sups we susct at a ven comund is idcnc
to a o ple, we have to do is to te r sa ofbo csc compounds under flenfl confions
. If e d speca match, e comunds e idenc.
oer approach to idencaflon cs rccose to idencafion of funcon oups.
Here, oaon about vous ncn oups of uo compound is obted nog e ons wch e abon bds
appe. t us usatc s approach the help of example. Consider a substance which has
the elemental foula CHO. See ere c o protons for each cn atom, c compound posses
ses elthcr one
or one double nd. If a double bond volg cn Is present, e icd spectm must show a
sh bd bceen 16 -18 cm-. e elcment foula shows at c
md has a lone ogen atom wch maybe present as a cbonyl oup or as a hydrol
or as cer. If it a hyl oup, e compound must show a sh bd beccn cm. e mpound sho ese bds d us crc Is a double bond n d e lone ogen Is pnt as a hyo
l oup. We c erefore role out
t re ven to e elemen fo. entuy, when e scte lete completely it was fod to be CHa
(CHz)3CH=CH(CHz)7CHzOH; it ssesses go,
e hyl oup d e cbon-cbon double bond as predicted by e red data.
R ofr: Ied speca we wonderful dicaflon for my funcon oups.
eflc acflons volg ese funcon oups eider ese oups e consum or generated e eaflc reacon c be assayed e help of red specoscopy. us, ff e subsate possesses a hydrol oup d
e product doesn't, e rate of e
cflon med by measg e rate of sappece of e H setcg bmflon. Or ff e subsate ds not
possess a cnyl oup d e product does, e rate
ofe rcflon c meased by measg e rate of appece ofe cbonyl setcg bmflon.
e loss or appellee of or en sg of bds has been of oer benefit to e
bihet. My cflon oups bioloc (cro)molecules exchge eff protons e protons of water
. us, when a molecule le prote is dissolved water, e protons
to 0gen, ogen, d su atoms exchge eff protons e protons of ter. enfls we sply ide
n ese oups e help of ed scoscopy. If a prot is sIv DaO, e ae sd protons be exche
d for deuterons.
NH + DzO
ND + HDO
@H + DzO
D .+ HDO
H+DzO
D+HDO
What is happening in the above reactions is that an atom of mass number 1 is bei
ng
for an atom of mass number 2. From our discussion above we know that as the

reduced mass increases the frequency of absorption should decrease. Thus the ban
ds of
for the groups which have exchanged their protons shift to a lower wavenumber. A
shift tells us about the groups that exchange slowly and a fast shift tells us a
bout.
which exchange faster.
For the peptide group, the rate of exchange can be studied by obse -rving the am
ide II band. located at !550 cm- for --CONH-- and at 1450 cm- for COND--.

218
Biophysical Cherntstry
Why should these exchanges be of interest to us? They are of interest because th
ey tell us
much more about the protein than just the exchange. Consider this completely den
atured
proteins and small peptides exchange their protons fast; this is a first order r
eaction with a half
life of about 0.1 to. 1.0 minute; in comparison, proteins in their native state
exchange very
slowly with half lives extending into hours or days for complete exchange. Thus
if we study the
kinetics of such exchanges, it gives us information about the kinetics and therm
odynamics of
the conformational transitions of the proteins concerned.
Study ofconformatlon : Proteins (and for that matter, any macromolecule) contain
a huge
number of atoms. This would mean that their infrared spectra will contain a huge
number of
vibrations, This surely seems like a disadvantage as far as studying conformatio
n of proteins
with the help of infrared spectroscopy is concerned. And yet, infrared spectrosc
opy is of
considerable use in studies of protein conformation. The reason for this is not
far to seek. The
chemical polypeptide repeat unit gives rise to nine characteristic infrared abso
rption bands.
The generally accepted nomenclature, the approximate frequencies, and the approx
imate
description of each mode is given in Table 8.7. Some bands are more useful for c
onformation
studies than the others. The amide A, amide I, and amide II and amide V have bee
n most
frequently used for such investigations.
The amide A and B bands arise from the NH stretching vibration in resonance with
the
first overtone of amide II (essentially NH bending) vibration. These bands are v
ery sensitive to
hydrogen bonding and have been extensively utilized for hydrogen bonding in biol
ogical
macromolecules and pertJnent model compounds. In oriented samples these bands sh
ow marked
dichroism and can be utilized to study the direction of N--H bonds.
Table 8.7 Characteristic Infrared Bands of the Peptide Linkage
Designation
App frequency(cm-}
Description A

B
I
II
III
IV
V
VI
VII
-33O0 }
-3100 }
1600-1690
1480-1575
1229-1301
625-767
640600
537-606
-200
NH stretching in resonance
with NH bending
C-O tretching
C--N stretching, NwH bending
NH bending
OCN bending
out of plane NH bending
out of place C-----O bending
skeletal torsion.

The amide I band is of great value as far as the study of secondary structure of
a given
polypeptide is concerned. The reason for this importance is that the frequency o
f this band
depends upon the environment of the bond. Thus, when the given bond is a part of
the a-helix,
the band appears at 1650 cm-, when it is part ofa [-structure, the band appears
at 1632 and
1685 cm:, and ff the bond happens to be present in a random coil, the band appea
rs at
1658 cm-. In addition, the amide V band appears at 600 for alpha-helix, 700 for
beta structure,
and at 650 cm- for the unordered structure. These bands are all well resolved. A
nd if a protein contains all the three structures, all three bands will appear.
In such a case, measuring the

intensities of these bands, will give an idea about the proportion of amino acid
s which are in a
given configuration. Of course, since D20 is the solvent which is used, one can
never be sure
whether the same proportion of the configurations also occurs naturally in the a
queous medium.

Spectrophotometry
219
There is a totally different mariner in which infrared spectroscopy is applied t
o study
conformations. Here, researchers make use of what is known as infrared
In the next chapter we will be discussing the phenomenon called circular dichrol
sm.
dichroism means the difference in the absorption of right- and left-handed circu
larly
by an optically active compound. The phenomenon used in infrared dichroism is
One can refer to this as linear dichroism. This phenomenon means that
in one direction will absorb plane polarized light differently when the electric
of the light is parallel to the orientation of the molecule and when it is perpe
ndicular to
Whether the compound is optically active or not is of no consequence to this
; even optically inactive compounds will show linear dichroism.
Let's try and understand this application without going into the mathematics of
linear
simple fact is that absorption by an oriented sample will be maximum when the
is parallel to the transition dipole moment of that vibration
is to be excited. Absorption will be zero when the dipole moment of the vibratio
n is
to the electric vector.
If molecules in a sample are randomly oriented, for every molecule which is para
llel to the
a molecule which is perpendicular to it. Also, there will be molecules
all degree of angles with the electric vector. In such a situation there will no
t be any
Thus the trick of studying infrared dichroism is to orient the sample. This can
be
the sample is prepared in the form of a thin film. This film then can be attache
d to
drums which are gently rotated stretching the film slightly. This stretching of
the film
the sample in the direction of the stretching. Such studies can also be done on
liquids
solutions provided the liquid is placed in the space between the two cylinders a
nd the
rotated. The molecules then orient themselves with their long axes in the direct
ion
The importance of infrared dichroism studies can bdst be demonstrated with the h
elp of
following examples.
The conformation of fibrous proteins has been studied by infrared dichroism. Thu
s rat tail
tendons, porcupine quills, silk fibre etc. have been studied. In these proteins
the absorptions at
1640 cm-I and 3300 cm-I are maximum when the electric vector of infrared light i
s perpendicular
to the fibre axis. These two frequencies are for C--O and N--H stretching respec

tively. This
observation can only be interpreted one way - these two bonds in the peptide bac
kbone must be
oriented perpendicular to the fibre axis (Figure 8.25). This inference is backed
up by other
studies on fibrous protein structure as well.
o--c,/
N--H
R---CH
C O
H N/ --T
Fibre axis
-- --R
of the po/ypept/de dm/n 0t st/k (otherflbrous prote/ns may have s/m//ar arrangem
ent).
Note than the C-O and N--H groups of the peptide backbone lie perpendicular to t
he fibre axis.
What is true for these two groups is true for the bases in the DNA structure too
. The bands
1600 and 1750 cm{stretching vibrations for aromatic rings) show greater intensities

220
Biophysical Chemistry
when the electric vector of infrared light is perpendicular to the axis of DNA f
ibre. This again
automatically means that the bases are arranged perpendicular to the axis of the
fibre. This is
borne out by other studies as well is predicted by the Watson-Crick structure.
Iateration between molecules : Polypeptide chains form interchain hydrogen bonds
. So do
the two stands of DNA. Hydrogen bonds have been studied very profitably using.in
frared
spectroscopy.
The reason that hydrogen bonds can be studied with the help of infrared spectros
copy is
that the strength of the bonds of functional groups involved in hydrogen bonding
is altered.
Once the strength, or to put it in more technical terms, the force constant is a
ltered, the-bands
due to the vibrations of these functional groups also shift. This shift gives ev
idence of the
presence of hydrogen bonds and is studied by infrared spectroscopy.
As an example let's consider the hydrogen bond between an NH and a C-O group. Th
e
presence of hydrogen bond here lowers the force constant of both these bonds to
stretching. In
the Hooke's equation, force constant occurs in the numerator. It is obvious that
the frequency
of these stretching vibrations will be reduced.
What about the bending vibrations? Hydrogen bonding places restrictions on the b
ending
of these bonds. Consequently, the bands attributable to bending will occur at hi
gher frequencies.
Apart from hydrogen bonding, other molecular interactions such as the protein- l
igand
binding, enzyme-coenzyme interactions etc. are also studied by infrared spectros
copy.
Disadvantages of Infrared Spectrophotometry
With all the numerous applications listed above, infrared spectrophotometry suff
ers from
two major disadvantages -- the first one of them specifically for the biochemist
s.
The best solvent for many biological works is water. Yet, as we have said earlie
r, water
absorbs intensely in the infrared. Thus, biologists are forced to use other solv
ents such as the
DO, D20--H20 mixtures, chloroform, or even CCI4. The problem is that the data ob
tained
about any molecule or situation in such solvents may not necessarily be true for
aqueous
solutions.

It is very difficult to obtain quantitative data with infrared spectrophotometry

. This is
again a problem related to the solvent. There isn't possibly a solvent that is c
ompletely transparent
to infrared. In order to minimize absorption due to any solvent that is used, sc
ientists use very
small pathlengths. This too is not desirable as far as quantitative results are
concerned because
short pathlength would require higher solute concentration. At high concentratio
n, Beer's law
may not be obeyed.
An alternative is to do away with the solvent and to read the sample in the soli
d state. For
this the sample may be ground with KBr. First of all this method requires very p
ainstaking and
fine grinding because large crystal size would give rise to scattering. Even if
this is done and the
solid is prepared, the problem is of accurately measuring the pathlength of such
a solid sample.
RAMAN SPECTROPHOTOMETRY
When incident light is scattered by intervening sample molecules, the frequency
of light
scattered is the same as that of the frequency of the incident photon. Such osci
llations are said
to be elastic. However, during scattering of monochromatic light by molecules, a
small fraction
of the scattered light is observed to have a different frequency from that of th
e incident photon.
This phenomenon is known as the Raman effect. Since its discovery in 1928 by Sir
C.V. Raman,
the Raman effect has been an indispensable tool for the elucidation of molecular
structure. It
has been of immense use in locating various functional groups or chemical bonds
in molecules
and also for quantitative analysis of complex mixtrares. Only those vibrations w
hich cause a

Spectrophotometry
221
change in dipole moment. Le., a charge displacement, are observed in the infrare
d region. Those
vibrations which do not cause a change in dipole moment and are consequently ina
ctive in the
are observed in Raman spectrn Thus, although the origins of Raman and infrared
are quite different, the two techniques provide complementary informations about
of molecular vibrations and therefore about the structure and conformation of a
Rayleigh scattering results when the .sample molecules scatter light which is of
the same
as that of the incident photon. Naturally we should infer that collision of the
photon
such molecules in not resulting in any energy exchange between them. Some molecu
les (a
less than 1 are capable ofindulglng in energy exchange before scattering
These molecules give rise to Raman effect. A collision between a molecule and a
(in case of light scattering) does not lift the molecule to any quantized level;
rather the
molecule can be thought to be virtually excited (see Figure 8.26). If now, the m
olecule
absorbed the radiation while it was in the lowest vibrational state does not ret
urn to the
level, but to a level which is slightly higher, the emitted or the scattered
will be of a lower frequency as compared to the incident radiation. The differen
ce in
equal to the natural vibration frequency of the molecule's ground electronic sta
te.
such shifted lines, known as Stokes lines are observed in the Raman spectrum and
to a different vibration in the molecule. The Raman spectrum, thus, provides
detailed information about the vibrational spectrum of a given molecules. There
can be other
present in the Raman spectrum. These lines are known as anti-Stokes lines. These
when some molecules absorb incident radiation when they are in a lower state. Th
ese
may subsequently decay to their ground state (which is lower than the state at w
hich
absorbed radiation) and consequently emit radiation which has a higher frequency
than
. anti-Stokes lines may not usually be considered for chemical analysis.
Since most of the molecules indulge in lyleigh scattering, radiation due to it i
s cosiderably
intense than the radiation due to Raman-shifted components. The primary function
of a
spectrometer is to reject totally radiation due to Rayleigh scattering and to de
tect the
Raman-shifted components. The Raman spectrometer should have (i) an intense
light source, (t/) sensitive detection, and (///) high light gathering power. A
laser
unit serves as a good light source and the detection is usually performed by
SPECTROFLUORIMETRY

The phenomenon whereby a molecule, after absorbing radiations, emits radiation o

f a
as fluorescence. Thus a compound which absorbs in the ultraviolet
range. This shift toward a longer wavelength is known
Stoke' s shift [Figure 8.26).
When an atom or molecule absorbs light, the energy of photon absorbed lifts an e
lectron
higher orbital. The excited electron can now return to the ground state in eithe
r of the two
It might do so in one single step in which case it will emit light of the same w
avelength
Fhe electron might, however, return to the ground state in a step-wise manner
energy levels, emitting quanta of radiation corresponding to each energy
. Since each quantum will have a smaller amount of energy, the radiation emitted
will have
the original exciting radiation. It is obvious that the emitted light will
T different wavelengths corresponding to each intermediate energy level the elec
tron
on its Journey to the ground state. Therefore, fluorescence spectra are band spe
ctra.
' are usually independent of the wavelength of the radiation absorbed.

222
Biophysical Chemistry
First excited electronic state

Virtual excited state {Laser frequency)

..... "

I 'l -[ .
' ........
(A)
(B)
1

Excited vibrational
energy levels

Figure 8.26Diagrammatic representation of Rayleigh and Raman scattering. Note th

at no energy exchange takes
place in Raman scattering (B and C).
Fluorescence is an extremely short lived phenomenon (10- seconds or less, see Bo
x 8.3) and therefore can provide information about events which take less than 1
0- seconds to Occur.
Structural Factors Which Give Rise to Fluorescence
Aromatic molecules or the molecules having multiple conjugated double bonds with
a
high degree of resonance stability generally fluoresce. Both classes of substanc
es possess
delocalized n-electrons which can be placed in low-lying excitec single states.
The higher the
number of n-electrons, the higher is the fluorescence. Thus polycyclic aromatic
compounds are
more fluorescent than benzene derivatives. Substituents strongly affect fluoresc
ence. Electron
withdrawing substituents (hCl, --I, .--Br, --NO2, --NHCOCH, hCOOH, etc.) decreas
e
fluorescence. On the other hand substituents which delocalize the n-electron [-NH, OH, --F,
--NHCH, N(CH)2, OCH, etc.] enhance the fluorescence. Fluorescence is often given
by
molecules containing a nonbonding pair of valence electrons. Examples are amines
with a lone
pair on their nitrogen atom.

Molecular rigidity is conducive to fluorescence. Thus among the aromatic compoun

ds,
those that are most planar, rigid and sterically uncrowded are the most fluoresc
ent. Chelation
of aromatic compounds with metal long often promotes rigidity and reduces intern
al vibrations.
Chelation thus promotes fluores, cence (this principle is exploited in measuring
the concentration
of many metal long). Low temperature, glassy state and high viscosity are all pr
omoters of
fluorescence.
Intensity and wavelength of fluorescence of the same compound may vary with the
solvent
used. Solvents exhibiting strong van der Waal's binding forces and solvents poss
essing electron
withdrawing groups diminish fluorescence. "
Drastic changes in pH, especially those changes which affect the charge status o
f the
chromophore influence fluorescence. Thus aniline fluoresces at neutral and alkal
ine pH but
becomes non-fluorescent at acidic pH. Aniso!e which fluoresces at neutral pH is
non-fluorescent
at alkaline pHs.

223
state
Fluorometry : Theory and Instrumentation
Fluorometry is an important analytical tool for the det6rmination of extremely s
mall
concentrations of substances which exhibit fluorescence. Beer-Lambert law, which
we have
considered previously, is also applied to fluorometry in the following form
log ISOL'ElCr -- Ef Cb
ISAMPLE
where ef is the absorptivity of the fluorescent material. C is the concentration
of the substance
and b is the path length. The values of intensities of the incident radiant ener
gy and the
transmitted energy are indicated by ISOLVEr and ISLE respectively. The intensity
of radiation
absorbed is therefore given by ISOLVET -- ISAMPLE. The intensity of fluorescence
is given by
F = k (IsoLV.Z/cr -- ISMPL)
where k is the proportionality constant (< I). Thus,

Slit
recorder
Amplifier
224
F = kIso.VET (I -- I0Cb}
f k[SOLVENTIS written as Fn the relationship becomes
F = Fn (I - 10fcb)
Rearranging
F-Fn = 10fCb
Fn
Therefore,
log = fCb
For Cb < 0.01, F= 2.303 kIso..t eCb. This equation holds good for concentrations
as low as a
few p,p.m.
The instrumentation of a spectrofluorimeter differs from that of the spectrophot
ometer in
two important respects besides other minor variations.
(/)
There are two monochromators instead of one as in a spectrophotometer; o
ne
monochromator is placed before the sample holder and one after it, and
(ii)
As fluorescence is maximum between 25-30C, the sampleholder has a device
to
maintain the temperature.
The main components of a spectrofluorimeter, indicated in Figure 8.27 are :
Sample
Slit
-

- ----- Gt
Source Collimator
or I

..Monochromator 2
Detector

Spectrophotometry
225
a continuous source of radiant energy (mercury lamp or xenon arc);
a monochromator usually a prism (P), to choose the wavelength with which the sam
ple
is to be irradiated;
(//0
a second monochromator (P2) which, placed aider the sample, enables the
determination
of the fluorescent spectrum of the sample;
(/v) a detector, usually a photomultiplier suited for wavelengths greater than 5
00 nm; and
(v) an amplifier.
The fluorescent radiation emitted ly the sample is given off in all directions,
but in most
instruments the sample is viewed at right angles to the incident beam.
Spectrofluorimetry vs. Absorption Spectrophotometry Advantages
(0
Spectrofluorimetry gives extremely accurate results when very low concen
trations are
used. At very low concentrations, absorption spectrophotometry is not at all acc
urate.
To cite an example, sensitivityof absorption spectrophotometry is taxed when I00
mg
of serotonln is to be detected; spectrofluorimetry on the other hand can determi
ne 100
pg of serotcnin with relative ease.
(
The 'Stokes' shift enables use of two monochromators in spectrofluorimet
ry; one selects
the activating wavelength while the other selects the fluorescent wavelength. Th
is
arrangement imparts great spectral selectivity to spectrofluorometers.
(///)
Since there is a direct relationship between sample concentration and th
e intensity of
fluorescence, spectrofluorimeters can make do with quite simple electronics. Thi
s is
generally not possible with absorption spectrophotometers.
Speetrofluorimetry vs. Absorption Spectrophotometry; Disadvantages
(0
A drawback of spectrofluorimetry is a high degree of absorption of fluor

escent radiation
by the emitting sample itself. This is known as quench/ng. Quenching also occurs
due
to impurities. Dissolved oxygen is a very effective quencher. Thus, if precise w
ork is
required, nitrogen is bubbled through the sample to remove oxygen. In old instru
ments
the problem of quenching is obviated by measuring a low concentration of the
sample. The modem instruments use microcells.
(//)
A note of caution : Detergents, filter paper and many laboratory tissues
cause
interference in fluorimetric assays because they can release strong fluorescing
materials.
The cuvettes used for fluorimetry should, therefore, never be washed with a dete
rgent
solution. The cuvettes may also not be dried by wiping them with tissue paper.
The more common applications of spectrofluorimetry include qualitative analysis
spectra and absorption spectra gives a fair idea about the identity
a compound); quantitative analysis (applications include assay of riboflavin, th
iamine,
such as cortisol, oestrogen, serotonin and dopamine, 'organophosphorus pesticide
s,
smoke carcinogens, drugs such as lysergic acid and barbiturates, porphyrins, cho
lesterol,
some metal long); and studies on protein structure (FAD containing proteins). Ta
ble
lists fluorescence maxima of some biologically important compounds. However, mor
e
of spectrofluorimetry are given below.

226
Biophysical Chemistry
Table 8.8 Characteristic fluorescence maxima of some compounds of biological int
erest
Compound
Solvent
Max

Phenylalanine
Phenol
Tyrosine
Tryptophan
Riboflavin
FAD
Chlorophyll a
Chlorophyll b
Fluorescein
Rhodamin B
Indole
BSA
Ovalbumin
Lysozyme
Chymotrypsin
Fibrinogen
Insulin
Water, pH 7.0
Water, pH 7.0
Water, pH 7.0
Water, pH 7.0
Water, pH 7.0
Water, pH 7.0
Hexane
Hexane
0. i N NaOH
0.1 N NaOH
Ethanol
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Water, pH
282
303
303

buffer,
buffer,
buffer,
buffer,
buffer,
7.0

pH
pH
pH
pH
pH

7.0
7.0
7.0
7.0
7.0

348
520
520
665
650
518
630
325
340
332
340
330
332
303

(i) Intracellular free calcium concentration assay : Quin-2 (an EGTA derivative)
, Quin-2 AM,
and Fura-2 are three fluorescent probes which allow us to assay intracellular fr
ee calcium
concentration. These probes are permeable to the plasma membrane and upon enteri
ng the
cytosol combine with calcium (chelation). This chelation gives rise to fluoresce
nce whose
magnitude is directly proportional to the free Ca2 concentration in the cytosol.
These studies
assume importance because of the role of calcium in controlling cellular metabol
ism.
(ii) Fluorescent probes and studies on membrane structure : Fluorescent probes s
uch as
anilinonaphthalene-8-sulphonate (ANS) and N-methyl-2-anilino-6-naphthalene sulph
onate
(MNS), contain both charged and hydrophobic areas and therefore locate at the wa
ter lipid
interface of the membrane. Such compoinds afford important information about thi
s interface.
This is especially so since the fluorescent properties of a molecule, as pointed
out earlier, vary
with its mobility and also with the polarity of the environment. Studies with AN
S have shown
that structural changes occur in mitochondrial membrane during oxidative phospho
rylation.
The probes have also yielded much information about the structural features of t

he plasma
membrane.
(No Assay of membrane potential : Membrane potential of excitable cells is regul
ated within
strict limits. Changes in this potential regulate ion entry into the ceils; Thes
e membrane potential
changes can be monitored by using fluorescent probes such as Di-S-C3-(5),. and m
erocyanine
540 (the latter is not so satisfactory).
(iv) Fluorescent microscopy : Spectrofluorimeter when combined with a microscope
allows
the determination of subcellular location of fluorescent compounds or of materia
ls which bind
fluorescent dyes. This technique has given very important information in the fie
ld of immunology
and pharmacology. Thus, presence of pathological immune complexes may be detecte
d with
the help of FITC conjugates. The presence of an antigen on the cell surface may
be detected by

Spectrophotometry
227
a fluorescence labelled antibody. By using acridine orange, this technique allow
s visualization
of nucleic acids within subcellular organelles. Since this technique alS allows v
isualization of
chromosomes, it is being exploited in genetic counselling specifically to determ
ine whether the
fetus is genetically normal.
(v) Since fluorescent emission is extremely sensitive tolocal environment, it ca
n be used to
mom'tor the kinetics and thermodynamics of the incorporation of a particular sub
unit or substrate
into a macromolecular assembly. It is being used to obtain definitive informatio
n about the
between pairs of loci in a macromolecular assembly. Spectra and the StudF of Protein Structure
As with absorption spectrophotometry, a great deal of information about the stru
cture of
,ven protein can be found out with the help of fluorescence studies. Here too, f
luorescence
changes under different conditions provide the necessary clues about the positio
ns of
amino acids residues in the protein, the composition of the active site, protein
-protein
interaction, denaturation etc.
A few important empirical rules for interpreting fluorescence spectra of protein
s are listed
:
i. The fluorescence of a protein is solely due to the amino acids typtophar., ty
rosine, and
This rule holds true in all cases except where the protein is known to contain
components.
2. Usually it is the tryptophan fluorescence which is studied most often. This i
s so because
weak fluorescence, mostly due to quenching. Such quenching can be due
proximity to tryptophan, a carboxylic group, or an amino group. Quenching also t
akes
if tyrosine is ionized. Phenylalanine has the smallest of quantum potential of t

he three
amino acids.
3. Decrease in polarity of the solvent pushes . max of tryptophan to shorter wav
elengths.
such a situation increases the intensity of the . max.
4. If a shift in . max occurs towards shorter wavelength when the protein has be
en dissolved
solvent, one may conclude that the tryptophan is internal as well as existing in
a nonenvironment.
5. If a shift in . max occurs towards shorter wavelength when the protein has be
en dissolved
non-polar solvent, one may conclude that the tryptophan is on the surface of the
protein
unless the solvent is such that it has brought about large-scale conformational
change in
6. Quenching of fluorescence by known quenchers (iodide, nitrate, cesium ion, ac
rylamide)
provides relevant information. Fluorescence due to whichever of the fluorescing
amino
is quenched, that amino acid should be on the surface.
7.
If a known quencher fails to quench the fluorescence of an amino acid, t
he following
may be considered :
(a) The amino acid is internal.
(b) The amino acid is hidden in a crevice which is too small for the quencher to
enter.
(c)
The surroundings of the amino acid might be highly'charged so that the q
uencher is
being repelled.

228
Biophysical Chemistry
The last case may be verified by trying to quench through a neutral quencher lik
e acrylamide.
If it is able to quench, the existence of the amino acid in a charged environmen
t is confirmed.
Even the type of charge may be determined. If positively charged quencher fails
to quench, the
amino acid is in a positive environment. Ditto with the negatively charged quenc
her.
8. If the substrate quenches the fluorescence due to tryptophan in a given enzym
e, the
tryptophan might be present in the active site, or quite near it.
Extrinsic Fluorescence
There are macromolecules where no fluorescent group is present in the location w
here it is
desired, in such cases biochemists introduce a fluorescent group into the molecu
le. Such a
method is given the name - extrinsic fluorescence. The groups which are introduc
ed in such a
way are catled fluors. To be used as a fluor, the chemical group must saUsfy cer
tain requirements.
(/) It should have the ability to bind tightly at a unique location; (it) its fl
uorescence should be
susceptible to changes in the environmental conditions, and (iti) the group shou
ld itself not
bring eonformational changes in the maeromoleeule in which it is introduced.
The most commonly used fluors for the studies on nucleic acids are ethidium brom
ide,
proflavin, acriflavin and acridine orange. Those preferred for use in protein st
udies are ANS,
1-dimethylaminonaphthalene sulfonate (DNS), 2-p-toluidylnaphthalene-6-sulfonate
(TNS);
fluorescein, rhodamine, and dansyl chloride.
All the above named naphthalene sulfonates (ANS, DNS and TNS) have a peculiar pr
operty
of fluorescing quite weakly In the aqueous medium. But in non-polar surroundings
, the strength
of their fluorescence increases while the florescence maxima shift to shorter wa
velengths. These
compounds are therefore, naturally, used to detect non-polar regions of a given
protein.
To illustrate just how extrinsic fluorescence may be of help in studying maeromo
lecules,

two examples have been cited below.

I. Acridine orange has a property of an increase in fluorescent intensity as wel
l as a shift
in fluorescent maximum to shorter wavelengths when bound to nucleic acids. All t
he more
important is the fact that the fluorescent maxima are different for different nu
cleic acids. Bound
to single stranded nucleic acids (RNA, or single strands of DNA) it gives a red
fluorescence.
Association with double stranded DNA causes it to fluoresce green. Naturally, wi
th the help of
acridine orange you may detect whether the sample contains single or double stra
nded nucleic
aeid, and If the sarple contains both, how much of each type is there.
2. Haemoglobin molecule has two components -- the prosthetic group which is a po
rphyrin
and the protein, known as apohaemoglobin. ANS has a property of giving no fluore
scence when
it is bound to haemoglobin. But an association with apohaemoglobin causes it to
fluoresce.
That it fluoresces when bound to apohaemoglobin indicates that it is binding at
a region which
is .non-polar.
If the porphyrin prosthetic group is now added, the fluorescence vanishes and AN
S is
removed from binding. This indicates that ANS binds at the same site where the p
orphyrin is.
And that the site where the porphyrln binds to apohaemoglobin is highly non-pola
r.
Fluorescence and Energy Transfer
Given below are the excitation and the emission spectra of two different fluors,
x,
and y.

Spectrophotometry
229
I
I
Spectra for (A N. Abson s d by s esn s
d is. N sn s of A os t son s ofB. (C) sn s w n sWn . y fer een 1 2.
Note at e esston sc ofx oveHaps e excitaon spec ofy.
excitation spec we me e rge of wavelens at a fluor must absorb
to fluoresce. f, ou lack of pointed discussion on e pot you e ven to
at e excision specm is sonous e absoon specm for a fluor.
s is deed e case. Howler, somees ngs turn out derenfly,.
Consider a system where go e fluors x, d y e e se soluon. In such a it may we we
be at e luflon is adiated e of e cion of x, e fluorescence due to y be obted. I
n such a case we c say at dion specm for y is derent from its usu abon specm. To
be ct,
was produced by absoflon of much smer wavelens. at happened?
A loc reason for e phenomenon may be as foows: en x s excited, it fluoresce.
e ession specm of x oveHaps e cition specm for y. So, e
, x ab by y, wch en etted oer photon d alonger ve!en espong to its ession specm.
In s laon e ener absorbed by e t
fluor is sfeed.to e.oer raaflon as a mediator. is detely is a possib but s is no
t e reason for e ave phenomenon.
ely as 1928 Wbg d Negele showed at t abrbed by e oflc o ads of a heme prote yeas
t wch were poisoned by cbon monode pcipat sptg off e cbon monode radlc attached
to e heme moie. e efficien of s
press w later sho to 1 %. ere were oer studies about acaflon of ees e help of t.
In ese studies too it could demonsated at ener absorbed at ous ps of e prote mo
lecules could ate to e locaon whe e acflvaon
ced. er oer ents so demonst e fact at abflon of t me comophores cod result fluor
escence etted by oer comophores which we not cit dfly by e t.

230
Biophysical Chemistry
Several mechanisms were proposed for the transfer of energy in protein molecules
. Some
of these were (1) transfer by electron migra.tion in conductivity bands, (2) tra
nsfer by exciton
migration, (3) transfer by proton, hydrogen radical, or hydride ion migration th
rough an ordered
array of water molecules surrounding the macromolecule, or (4) transfer by reson
ance interaction
between chromophores. Since the experimental data fit the last mechanism, this p
henomenon
has come to be known as resonance energy transfer.
Three Conditions must. be Satisfied for resonance energy transfer to occur. Thes
e are
1.
The energy donor must be fluorescent. In fact the efficiency of energy t
ransfer increases
with the fluorescence quantum yield of the donor.
2.
The absorption spectrum of the acceptor must overlap with the emission s
pectrum of
the donor. The more they overlap, the higher is the efficiency of transfer.
3.
The distance between the donor and the acceptor must not exceed 50-100 A
. In fact,
70/ seems to be the upper limit for efficient energy transfer.
It is the last condition that makes this phenomenon of such large interest to a
biochemist.
Let us See why.
If the two molecules engaging in energy-transfer are nearby, the transfer may ta
ke place
at a very high efficiency. However, as the distance between the molecules increa
ses, the efficiency
of energy-transfer may decrease. We may then say that the eJficiency of transfer
is a function of
molecular distances. And this is exactly what makes the phenomenon of interest t
o a biochemist.
Because, then by measuring the efficiency of energy-transfer, a biochemist can p
redict the
distance that may be there between two fluors lying within the same macromolecul
e. Fluorescence
energy-transfer is then some kind of a molecular ruler or a scale.
In 1948 Forster proposed a theory for the resonance energy transfer. He postulat
ed that
the rate of transfer depends on the inverse sixth power of the distance between
the donor and
the acceptor. Thispredicted distance dependence was verified later by experiment
al studies of
fluorescent donor-acceptor pairs separated by a known distance in defined system
s.

The mathematical equation that describes the eciency of transfer as a function o

f molecular
distance is as follows
R6
where E, is the efficiency, R is the distance between the donor-acceptor pair, a
nd R is a constant
related to each donor-acceptor pair.
The above equation may be rewritten for R as follows
= Ro(1-Ej
R
Let us see how E is measured. There are mainly two methods to measure E, Both th
ese
methods utilize three wavelengtths, )h' 12, and )a" The characteristics which th
ese three
wavelengths must possess are as follows : it must be absorbed by the donor effic
iently but not
by the aeeeptor: t2 should be a wavelength emitted by the donor but not by the a
ecelStor; la
should be a wavelength emitted by the aeceptor and not by the donor.

Spectrophotometry
231
It stands to reason that if energy transfer is taking place, the emission ate, t
he wavelength
at which the donor emits, should be quenched. The first method excites the donor
by using ,
and then measures quenching at 2" Let f be the fluorescent intensity at 2 if the
donor was
excited using . Let iv be the fluorescent intensity when the acceptor is absent
and let fv.^ be
the intensity in the presence of the acceptor. The fraction of the donors that r
emain excited is
written as loE. Then
The second method deals with measuring the fluorescent intensity of the acceptor
at 3" The mathematical expression in this case is
(^ V
the fluorescence of the acceptor at 3 when the donor and acceptor both are prese
nt, while f
is the fluorescence of acceptor at X when only the acceptor is present. The exci
tation wavelength
is
From E one can measure R if To is known. To determine To one has to measure (1)
the
fluorescent intensity of the aceptor as a function of , (2) extinction coefficie
nt of the donor,
and (3) the value of Q (photons emitted divided by the photons absorbed) of the
donor when the
acceptor is absent. There is another factor that must be considered but which is
beyond the
scope of this book. This is the orientation factor. A consideration of the orien
tation factor is
important owing to the fact that the fluorescence from the donor is polarized an
d the acceptor
may be at an angle with respect to the. plane of polarization such that maximum
absorption
may not take place. However, the donor and acceptor groups are in rapid motion w
ith respect
to each other and the orientation factor is hardly ever higher than 1.20.
Before energy-transfer experiments are performed, the following conditions must
be met:
(I) if a fluorescent group is to be attached, it should be attached in a manner
that it doesn't
alter the structure of the macromolecule; apart from the manner of attachment, t
he group itself
should be inert so as to cause minimal perturbations in the macromolecular struc
ture; (2)
There should only be one donor and one acceptor; moreover, both should be at kno
wn chemical

.sites;. and (3) The To value for the donor-acceptor pair must be known and shou
ld be in close
agreement with the distance between the donor and acceptor. Once these condition
s have been
considered, the experiment can be carried out.
The first step in identifying a donor-acceptor pair is to ascertain whether the
macmolecule
has an intrinsic chromophore that can serve as a donor or an acceptor. Tryptopha
n residue in
proteins serves as an excellent donor owing to the fact that its emission center
ed at around 330
nm overlaps the absorption spectrum of many potential acceptors. The galactose b
inding
protein from Salmonella possesses Just one tryptophan residue and this is wonder
ful for energy
transfer measurements. Fluorescent coenzymes such as NAD, FMN (oxidized), and pyr
ldoxal
phosphate are dlso attractive potential donors and acceptors. The heme group in
heme proteins
is a very good acceptor since it has an absorption spectrum covering the entire
visible range.
This group is no good as an acceptor since it does not fluoresce.

Ca
232
Biophysical Chemistry
If a good donor or acceptor is not naturally
orescent
analogues are available. Thus benzoadenoslne
adenosine
nucieotides. Protoporphyrin and its zinc and
for heine. Several
fluorescent analogues are now available. The

available in the macromolecule, flu

derivatives are good analogues for
tin derivatives are good analogues
following table lists a few.

Table 8.9 Florescent analogues of a few biomolecules. These analogues can be use
d as
labels or energy transfer experiments
Biomolecule
Fluorescent analogue

ATP, NAD, FAD, CoA, and other

adenine nucleotides
Heme
Thiamine diphosphate
Fatty acids
Phospholipids
ethenoadenosine derivatives
benzoadenosine derivatives
protoporphyrin, zinc and tin porphyrin
thiochrome diphosphate
a- and -parinarlc acid
phospholipids containing parinaric acid,
dialkyl cyanine dyes, derivatives of
phosphatidyl ethanolamtne
Th, Eu

A few examples of the application of the energy-transfer technique to structure

determination
are given below.
Three-Dimensional Structure of tRNA
In the crystalline state the tRNA exists in clover-leaf structure. What is its s
hape when it
is in solution? Is it different or is it the same?
Yang and Soll tackled this problem using energy-transfer studies. They prepared
five species
of tRNA (either fmet or glutamate tRNA from E.co/0 labeled with acridine, dansyl
, anthranilamlde,
or coumarin derivatives at two of the following sites :
(a) the 5'-end,
(b) 4-thlouridine,
(c) dihydrouridine,
(d) 2-thiouridine at the anticodon,
(e) pseudouridine, and
(i) 3"-termlnal adenosine.
- The distances that they calculated from observed transfer efflciencles were as
follows: a
was separated from F by 24, b from fby 38, c from e by 36, d from fby more than
65, and e from
f by 55 amstrong units. The distances that were determined crystallographlcally
are 25, 41,
23, 74, and 53 amstrong units respectively. Thus, except for the distance betwee
n c and e
where the two data do not agree well, all other distances are very close. The co
nclusion that
Yang and Soll reached was that even in solutions tRNA adopts a cloverleaf struct
ure. (see
Figure 8.29).

233
4.
8.29 .Clover leaf structure of tRNA. The diagram shows the. positions at which j
'luorescent labels were
introduced as well as the positions of unusual bases on the basis of which each
arm is given a name.
pyntvate dehydrogenase complex -.
Pyruvate dehydrogenase complex is a multienzyme complex which converts pyruvate
to
It consists of three enzymes : pyruvate dehydrogenase, dihydrolipoyl transacetyl
ase.
dihydrolipoyl dehydrogenase. It has five coenzymes : NAD, CoA, "lipoic acid, thia
mine
and FAD. The enzyme dihydrolipoyl dehydrogenase is centrally placed in the
a lipoic acid moiety attached to a lysine and it is hypothesized that it is this
t moves around the crystal of the enzyme as a swinging arm bringing successive
to the other enzymes in order that the reaction proceeds. Do we have any
support for this hypothesis?
The length of the lipoyllysine arm is known to be 14A. Now if the swinging arm h
ypothesis
the distance between the active sites of any two enzymes within the crystal must
not
28]. This can be more easily understood from the figure given below (Figure
i. However. the energy transfer studies indicate that the distance between the a
ctive sites of
dehydrogenase and dihydrolipoyl dehydrogenase is at least 45A.
The lack of evidence does not mean that the hypothesis is wrong. However, energy
-transfer
have spurred proposition of alternative models. Two of the main alternative mode
ls
are (I) substrate diffusion within the complex, and (2) acyl transfer between ad
jacent

234
CoA
HS
CH3CO 0
14A
{B}
CoA
Pyruvate
dehydrogenase
(E)
Biophysical Chemistry
CH3CO - CoA
Dihydrolipoyl
dehydrogenase
{E3)
Lipolysme arm
O
-< 28A
o
Dihydrolipoyl
14A
transacetylase
(E)

Figure 8.30. (A) The lipoyllystne ann s 14 in length. Thus if the active sites o
f the two other enzymes core placed a maximum of 28 A from each other, the arm c
an reach them. (B) If the distance becomes.greater than

28 , the arm cannot reach the actlve sltes.

Ill +Ix
p - Ill - Ix
Spectrophotometry
235
Changes in the Enzyme Conformation When Substrate Binds
It was Emil Fischer who had, in order to explain the great specificity of an enz
yme for a
group of substrates, proposed the 'lock and key model." The model proposed that
the active
sites of the enzymes are like the key-space of a lock. The substrate behaves lik
e a key. The
correct substrate is like the correct key for a given lock which fits snugly int
o it. The wrong
substrate has the wrong shape and doesn't fit snugly. D. E. Koshland improved up
on this
model when he suggested the 'induced-fit model.' The model suggests that when a
substrate
approaches a given enzyme, both undergo a change in their three-dimensional stru
cture. If the
substrate is true, the change is such that they will fit snugly.
This hypothesis can be tasted by energy-transfer studies. Donor and acceptor gro
ups are
bound to an enzyme and the distance between them is measured when no substrate i
s present.
Then the substrate is added and the distance is measured again. When such studie
s are
performed, a large change in the distance between the donor and the acceptor gro
ups takes
place upon addition of the substrate. This is ample proof for the induced-fit mo
del.
Rhodopsin Serves as a Light-Sensitive Gate
Rhodopsin is the main light absorbing component in the discs of rod cells in the
eye. There
are two parts to it: the protein, opsin and a fluor, cis-retinal. It is the latt
er which absorbs light.
The molecular weight of rhodopsin is 40 KD. A spherical molecule
uld
roughly have a diameter of about 45.. To check whether rhodopsin
nergy-transfer
experiments were performed. The protein already has a good donor
Another fluor
was tied to a sulfhydryl group in opsin. This fluor acted as the

of this size wo
is spherical, e
in cis-retinal.
acceptor.

The efficiency of transfer showed that the two fluors must be about 75A away fro
m each
other. This means that rhodopsin cannot be spherical and, indeed, must possess a
n elongated
protein. In fact, 75,. is a distance big enough to make this protein traverse th

e retinal disc
membrane.
The above data encouraged researchers to hypothesize that rhodopsin may actually
be
serving as a light-sensitive gate controlling the ion flow across retinal disc m
embrane. Subsequent
X-ray-scattering experiments confirmed the dimensions of rhodopsin to be just th
ose found
with energy transfer experiments. And several lines of evidence suggest that rho
dopsin indeed
may be acting as a light-sensitive ion-flow gate.
FLUORESCENCE POLARIZATION
If the orientation of the polarizer is parallel to the plane of polarization of
light, the intensity
o.f the transmitted polarized light will be maximum. This intensity will be zero
if the polarizer
orientation is perpendicular to the plane of polarized light (Figure 8.31). Ther
efore, for a partially
polarized light, the polarization P, may be given by the relationship
Where I and Ii are the intensities observed parallel and perpendicular to an arb
itrary axis.
The value for P varies between +1 and -1. A value of zero means that light is un
polarized.
Other values mean that the light is partially polarized.

236
Polm'lzer
Excitation
Light
source
le holder
Direction of
propagation
Figure 8,31. Production of plane-polarized light. Waves of all orientations fall
on the polarizer, which allows only
those waves to pass whose electrical vector is parallel to the axis of the polar
lzer.
Fluorescence polarization can be determined using the instrument shown in Figure
8.32. The sample is ecited with polarized light. In order to do sol a polarizer
is placed between
the monochromator)and the sample. A second polarizer is placed between the sampl
e and the
detector. The axis 9f this polarizer can be varied from being parallel to the fi
rst polarizer to
being perpendicular. By Varying the axis of the second polarizer, both 11] and Ic
an be determined.
Thus P can be measured. Usually, if the exciting light is polarized, the fluores
cence is found to
be only partially polarized or even unpolarized. The factors leading to this los
s of polarization
are explained below.
monochromator
Polarizer
Emitted
light
m
Emisslon
onochromator

237
A chromophore absorbs polarized light maximally if the direction of the electric
vector of
is parallel to the electric dipole moment of the chromophore. In a solution, the
chromophores
randomly oriented. Only a few of them will satisfy the above condition. Others w
ill have
electric dipole moment at various angles to the plane of polarized light. If thi
s angle is O,
the probability of absorption of polarized light is cos20.
What if the molecule, that is absorbing the plane polarz, ed light a fluor? Will
it emit the
in the same plane as it absorbed? Not necessarily. This is so because the polari
zation
light is not determined by the electric dipole moment, but by the transition dip
ole
The latter is not generally parallel to the former. The probability of emission
of
with the plane of polarization at an angle with respect to the transition dipole
is proportional to sin2.
Note the fact that the transition dipole moment is not generally parallel to the
electric
This is the major factor for the loss of polarization. This non-parallel relatio
nship
the two dipoles ensures that even if the absorbing molecules are perfectly align
ed with
of polarization of the light being absorbed, there would still be loss of polari
zation; P
be less than 1. In-a solution then where the molecules are randomly oriented, th
is
will be even more true. This loss of polarization ( Pvalue being less than 1) is
called
So far we have seen that fluorescence polarization, its value, is a result of th
e fact that the
are not parallel. This value of polarization is called the intrinsic polarizatio
n, Po.
there are other factors that affect polarization and therefore intrinsic polariz
ation is
the value that is observed experimentally.
Consider a molecule. It has absorbed polarized light. In a very short time, it w
ill be emitting
But in the short time between absorption and fluorescence, it moves. This motion
will
transition dipole orientation and will cause loss of polarization. The motion we

are
about here is rotational motion and not translational motion because translation
al
is unable to change the orientation of the dipole. Rotational motion is therefor
e one of
that affect fluorescence polarization. The other factor that affects polarizatio
n
r is the energy transfer that may occur between the molecules. .
In fact both the above factors are complex. The rotational motion of a molecule
is a function
only its own size and shape, but also depends upon the viscosity of the solution
, as well
!the temperature. The larger the molecule, the less will be its rotational mobil
ity, the lesser
be the loss of polarization. Thus macromolecules show substantial polarization o
wing to
really decreased mobility. The higher the viscosity, the lower the rotational mo
tion, the
be the extent of loss of polarization (in fact at very high viscosity, the polar
ization
may be very near that of the intrinsic polarization since the molecule will elic
it very
r rotational motion). The higher the temperature, the more will be the rotationa
l motion, the
will be the loss of polarization.
The second factor is that of energy transfer between identical molecules. Althou
gh resonance
transfer takes place with highest probability between molecules having parallel
dipoles,
take place when the dipoles are not parallel. When this occurs, naturally there
is'a
In the previous pages we have seen that resonance energy transfer is a
of distance between molecules. As the distance increases, the efficiency of tran
sfer "
, energy transfer is also affected by concentration. The more the concentration,
less the distance between the molecules, the more the energy transfer, the more
the loss of
But concentration here is a complex term. To explain with the help of an example
,

238
Biophysical
let's conjure an image of a fluor bound to a macromolecule. Concentration in suc
h
refer to a low concentration of the macromolecule-fluor complex in the solution.
refer to the distance between the fluors on the macromolecule. The higher this d
istance,
lower we will say the concentration is.
" After having considered energy transfer as the second factor affecting
arrive at a good relationship. Even if we completely immobilize a molecule by in
creasin
of the solution to a very high level, loss of polarizition will take place - in
these conditions it
be due to energy transfer. On the other hand, if the viscosity of solution is ve
ry low, the loss
polarization will mainly result from rotational motion of the molecules.
The major conclusion that derives from the above discussion is as
rotational motion, polarization decreases. We need to understand the consequence
s of this
rule as well as understand truly what we mean by rotational motion.
I. Small molecules rotate freely and thus do not give any polarization (P value
is zero)
2.
If these small fluors are attached to another molecule (which may itself
be
polarization will increase owing to the larger combined size and reduced
mobility.
3.
The higher the size of the molecule, the lower will be the rotational mo
bility.
attached fluor, the higher will be the polarization.
4.
A fluor bound to a macromolecule shows good polarization because its mob
ility
greatly reduced by such binding owing to the large size of the macromolecule*.
5.
The nature of binding of the fluor to the larger molecule also affects t
he extent,
polarization. If the binding is rigid (intercalation between bases of DNA) the m
obility
the fluor effectively means the mobility of the whole molecule. In this case
polarization is quite high. On the other hand, if the binding is such that the f

luor move without the whole macromolecule moving ( binding to an amino acid side
which can move with respect to the polypeptide chain) the polarization may not
high as in the first case.
6.
If the macromolecules undergo polymerization, the larger structure will
have
lesser rotational movement. If the fluor is bound to such an associating system,
polarization will increase with association.
7. If the fluor is bound to a macromolecule which changes its conformation, its
will be affected: If the change in conformation leads to the macromolecule assum
ing more ordered structure, it will result in reduced rotational mobility and
higher polarization. On the contrary, if the conformational change leads to diso
rder
the rotational motion is slated to increase and the polarization will decrease.
We have consistently talked about the polarization of a fluor bound to a macromo
lecule. What
the intrinsic fluorescence of the macromolecules and its polarization? For examp
le the
fluorescence of tryptophan in a protein and utilization of its polarization in s
tudying the
There is a problem here. Large proteins move very slowly on a molecular scale. T
hus, to observe
depolarlzation due to motion, the lifetime of the excited state should be suffic
iently long, i.e. , there
should be a good time lag between excitation and emission so that the molecule m
ay
movement in that time and depolarization may occur. For very sma]l proteins, int
rinsic
may be of some use, but for larger proteins, extrinsic fluorescence has to be ma
de use of.

239
Given below a,.e a few examples of use of fluorescence polarization to biochemis
try.
Studies on Proteins
The enzyme lactate dehydrogenase is a tetramer. The association/dissociation pro
perties
been studied using fluorescence polarization. The trick is in tagging dansyl
to the polypeptides. When the monomers associate to form tetramers, the resultin
g
is large. Thus polarization increases as association occurs. When the tetramer
depolarization occurs. It is also seen that depolarization and loss of activity
occur
in hand meaning that the tetramer is the active form while the monomer is inacti
ve.
The effect of various environmental factors, such as the pH, the ionic strength,
and the
the association kinetics has also been studied using the fluorescence
technique.
Tag an extrinsic fluor to an antigen. Incubate the antigen with a mixture of ant
ibodies. If
antibody specific for the antigen is present in the mixture, binding will occur.
This binding
rise to the antigen-antibody complex which will be large. Naturally, the polariz
ation
to the extrinsic fluor should increase owing to lesser rotational motion of the
complex. This
can be used to detect the presence of specific antibodies in a given mixture. No
t just
Even the kinetics of binding can be assayed with the help of polarization.
What is true for the antigen-antibody interaction is true for receptor-ligand in
teraction, as
as for the substrate enzyme interaction. These systems too have been studied usi
ng
polarization.
in Proteins
If one can tag an extrinsic fluor to a protein without changing its conformation
, then
in protein conformation can be studied using fluorescence polarization. Conditio
ns
flexibility of a protein will decrease polarization by giving rise to higher rot

ational
of different proteins and the different conditions that contribute
can be studied.
Presence of disulfide linkages can also be studied with the help of this techniq
ue. If treatment
protein with 2-mercaptoethanol reduces its fluorescence polarization, disulfide
linkages
be present (2-mercaptoethanol destabilizes disulfide linkages and will thus incr
ease the
protein concerned). The method may even be made quantitative for measurement
linkages.

LUCIFERASE
240
Biophysical
LUMINOIVITRY
Luminescence may be described as the emission of light from a chemical
the temperature of incandescence. The phenomenon is usually ascribed to
taking place in solution producing molecules in an excited state. While some
release energy in the form of heat, some others release it in the form of photon
s,.
of luminescent compounds are luciferin, acridinium salts and luminol.
Luminescence has been classified into two categories. Luminescence produced by
chemical means is known as chemiluminescence. Example can be cited of oxidation
of
luminescent compound (e.g., luminol) with hydrogen peroxide in the presence of m
etal
which act as catalyst. Luminescence produced by the intervention of an enzyme is
known
bioluminescence. As compared to chemiluminescence, bioluminescent systems conver
t and
a higher proportion of energy as photons.
Luminescent Measurements
Luminescent measurements have several advantages over spectrophotometry. First o
f they are more sensitive as compared to spectrophotometry. Theoretically as lit
tle as one
(I0-8 mole) of the analyte may be assayed. Although this limit is not achieved i
n
than femtomole quantities are measurable. The second advantage concerns
instrumentation of luminometers. Since luminescent light is virtually monochroma
tic as
result of its emission through a specific reaction, elaborate wavelength selecto
rs, so
for spectrophotometers, are not required in luminometers.
The basic featm-es of luminometers are (i) a light tight chamber in which the
containing the sample can be placed. The chamber is also thermostatically contro
lled,
facility for addition of luminescent reagents in a llght-tight fashion, and (iii
) a detector which
usually a photomultiplier along with suitable amplifier and a recorder. The li
reaction taking place in the cuvette is measured either as a peak value or as th
e rate of chan
in the intensity. While the former method serves to measure the
of interest, the latter is suited for measuring enzyme activities.
Applications
The awareness of the advantages of luminometry have prompted improvement in,
commercial availability of luminometers. Three systems in frequent use are firef
ly,

luminescence and luminol chemiluminescence. The principles and applications of e

ach
systems are described below.
(i) Firefly luminescence and ATP measurement : Luciferase, in the presence of ma
gnesiur
catalyzes the following reaction.
Luciferin + ATP + 02
Oxyluciferin + AMP + CO2 + Photon (562 nm)

The reaction stoichiometry is such that for each molecule of ATP reacting, one p
hoton
of maximal intensity at 562 nm is prodiced. The method, therefore, is the most s
ensitive
method for ATP measurement and much less than one f mol (10-s tool) of ATP can b
e measured.
The system becomes absolutely specific for ATP if all the reagents are purified.
By linkage to
another reaction, this method has been used to assay numerous ATP-specific enzym
es and
their substrates such as creatine kinase, creatine phosphate and triglycerides.

Spectrophotornetry
241
(tl} Bacterial luminescence and measurement of coenzymes : The coenzymes that ca
n be
measured by this system are NADH and NADPH. The system utilizes a purified oxido
reductase
(specific either for NADH or NADPH) obtained from the bacterium Beneckea harveyL
The sequence
of the oxidoreductase reaction coupled to a bacterial luciferase is as follows
NAD{P]H + H+ + FMN
FMNH2 + R.CHO + 02
OXIDOREDUCTASE ) NAD{P)" + FMNH2
FMN + R.COOH + H20+ Photon {495 nm)

A photon of maximal intensity at 495 nm is produced in the reaction in which the

bacterial luciferase catalyzes the oxidation of an aldehyde by oxygen in the pre
sence of
FMNH2. The method allows about 100 fmol of NADH to be assayed. The system can be
utilized to measure numerous oxidoreductases which utilize NADH or NADPH. The
substrates of such enzymes can also be assayed by this system.
(ili) Chemiluminescent assays using luminol : Several compounds have been used f
or
;hemiluminescent systems, but luminol has been the most popular. Luminol is oxid
ized
by hydrogen peroxide at pH 10-11 if copper, chromium, iron or haemin compounds a
re
used as catalysts. Luminol may be oxidized at neutral pH if peroxidases or vario
us other
oxidases are used as catalysts.
O
H
H
CO0+ 2HO + 20H[ "COONH2 O

NH2
+ N2 + 4H20 + Photon
{430 lu)

Photons with maximal intensity at 430 nm are produced. However, the photon
efficiency is only 1% and the method is capable of assaying 1 pg of hydrogen per
oxide or
0.1 pg of peroxidase per ml solution. The system lends itself to measurement of
hydrogen
peroxide produced by specific enzyme methods from such substrates as glucose or
urate.
Additionally, luminol has been much used as a marker in luminescent immunoassays
,
Use of Genetic Engineering in Blosensors
Various laboratories world over are trying to develop biosensors that are based
on the
ability of genetically engineered bacteria to emit visible light in response to
specific
compounds. The ease of measuring light makes this type of biosensor especially a
ttractive.
The following example will make the understanding of the above approach easier.
Vibrio Jischeri, a marine bacterium possesses lux genes which are involved in th
e
emission of light. The lux operon consists of five structural and two regulator)
, genes. Lux
A and the lux B gene code for two subunits of a heterodimer flavin manoo-ygeis-e
luciferase which catalyzes light production by oxidation of a reduced aldehyde.
Aldehyde
production is catalyzed by enzymes encoded by lux C, D, and E genes.
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I

I
I
I
I
I
I
I
I
regulatory C D A B E

242
Biophysical Chemistry
Another bacterium, Serratia marcescens, possesses the mer genes, which are invol
ved in
mercury detoxification. These genes are inducible genes and are transcribed only
when mercury
is present in the medium.
Geiselhardt et. a/. of ClarksonUniversity, New York, have recently fused these t
wo operons
into a plasrnld so that the lux genes are situated downstream of the mer genes a
nd are therefore
placed under the mer promoter. Naturally, whenever mercury is present in the med
ium, not
only are the mer genes, but also the/uxgenes are induced owing to the mer promot
er controlling
the expression of both.
E. coli cells were transformed with this plasmid. These E.colicells, when placed
in a medium
containing mercury, emitted light which was easily measurable. This is then an i
deal biosensor
to measure mercury in any given medium.
Like the mer gene system, genes for detoxification of other elements are also fo
und in
certain bacteria. Bringing the lux genes under the control of the promoters of t
hese gene systems
may give rise to biosensors for other elements as well.
FLAME SPECTROPHOTOMETRY
The flame photometric analysis method is more or less similar to that of spectro
photometry
with the exception that the place of the sample cell is taken by a flame. Conseq
uently, it is the
absorption or emission of specific wavelengths by excited atoms that iS studied
by this technique.
The-optical system and even the photo-detectors used in spectrophotometry and fl
ame
spectrophotometry are identical.
The general method of flame photometry can be applied in two complementary ways:
emission flame photometry and atomic absorption spectrophotometry. Both the vari
ations will
be dealt with simultaneously.
Volatilization of molecules in a flame produces free atoms and then excites them
to higher
energy levels. The characteristic emission spectrum of the element is produced w

hen the excited

atoms return to their ground state. This is the principle of emtssionflame photo
metry. Atomic
absorption spectrophotometry, on the other hand, measures the absorption of a be
am of
monochromatic light by atoms in a flame. Since the transitions available to the
electrons in any
given atom are specified by the available energy levels, atomic spectra are abso
lutely specific for
the element involved. Moreover, the energy absorbed or emitted is proportional t
o the number
of atoms present in the optical path. Thus, apart from giving the identity of th
e element(s)
present in a sample, flame photometry also provides information about the quanti
ty of the
element(s] present.
The amount ofenergy emitted also depends upon the (t) temperature and (ii) compo
sition
of the flame. It is therefore very necessary that the two flame variables must b
e maintained
constant and that standard solutions be used to calibrate the system. The need t
o maintain
flame composition constant also dictates which element should be determined firs
t. Thus, sodium which gives a very high background emission is measured first an
d the quantity of sodium
determined is added to all the standards. Certain elements, such as the alkali m
etals, enhance
the emission of other elements. On the other hand, some substances like aluminat
e and silicate
cause a decrease in emission of other elements. To relieve this deleterious effe
ct certain other
elements known as the releasing agents (strontium or lanthanum) must be added.
Another way to deal with interferences is to measure all interfering components
in a given
sample. After this, standards for each of the components are prepared which cont
ain the
previously determined concentration of interfering components. This is known as
cyclical analysis.

Spectrophotometry 243
About two to three such cycles are necessary before an accurate idea of the quan
tities of all
sample components can be deermed
Organic material in biological samples (which might cause interference) is usual
ly removed
by ashing. To prevent more volatile elements from getting sublimated, the ashing
is usually
carried out under low temperatures in the presence of oxygen. Alternatively, liq
uid ashing, i.e.,
oxidative digestion of the sample in hydrogen peroxide/concentrated sulphuric ac
id solution
may be carried out. A small amount of selenium sulphate, if added, acts as a cat
alyst, while
lithium sulphate is sometimes added to raise the boiling point.
It is advisable to store samples md standards in polythene bottles. This is so s
ince even
the good quality glass containers release metal long.
Flame instability c.an cause large errors in flame photometric analysis. It is,
therefore,
essential that all assays be carried out in triplicate. Calibration cues should
be checked
or reconstructed when the assays are carried out. If high accuracy is desired, a
standard
solution containing more or less the same concentration of the element as the sa
mple
solution, is assayed immediately before and after the sample solution. This meth
od is
known as bracketting. Very frequently, internal standards are used and the choic
e element
for this purpose is usually lithium.
Instrumentation for Emission Flame Photometry
The basic components of a flame emission spectrophotometer are shown in Figure
8.33. The individual components are discussed below.

Nebulizer
Air from -- ....
co
Filter
Sample
Detector Recorder

Figure 8.33 Basic components of an emtssion Jlame photometer

(i) Nebultzers or atomizers : Samples, before they get into the flame, must be c
onverted into
a fine spray, i.e., nebulized. This is necessary because large drops do not rema
in in the hottest
part of the flame for a long time and therefore may not become volatilized and e
xcited.. Because
of this reason, the nebulizer becomes the most critical part of a flame photomet
er so much so
that the efficiency of an analysis depends on the efficiency of the nebulizer.
nebulizers are essentially of the 'scent spray' type whereby a forced stream of
air
at nearly the speed of sound) passes over a capillary tube dipping into the test
solution. This leads to a considerable drop in pressure leading to a suction of
the sample
through the capillary. As the sample emerges from the tip of the capillary it is
broken into a
mist of fine droplets by the flow of air. To eliminate large drops, a cloud cham
ber is placed
between the flame and-the nebulizer where the large drops condense and drain awa
y. The
design of a simple nebulizer is shown in Figure 8.34(A).

.244
Compressed Sample
Drain
Figure 8.34 (A) Design of a simple nebulizer
Since large droplets condense and drain away, there is a large loss of sample (9
0% is lost,
only 10% reaches the flame) and if one has only a small amount of sample to star
t with, this
poses a big problem. To alleviate this problem, an improvement on the basic desi
gn of the
nebIzer has been achieved by placiv an impact bead a few millimeters away from t
he nebulizer
tip. The large droplet, when they emerge from the tip, collide with the bead and
are broken into
smaller droplets. This design lowers the average droplet size and thus improves
the efficiency of
the analysis besides reducing the loss of sample. The design of this nebulizer i
n shown in
Figure 8.34 (B). "
Compressed
Fuel
chamber

Impact bead
Sample
Drain

Figure 8.34 (13) Nebulizer with an b'npact bead

(it) The flame (Also see Box 8.5): Various gas mixtures producing different flam
es
differing in their temperatures are used in flame photometry. Table 8.10 lists s
ome of the
often used combinations.
consuming. Moreover, atleast 2-3 ml of sample are required
a

245
Spectrophotometry
Table 8.10 Fuels. gases and temperatures used in flame spectrophotometry
Fuels
Gas (oxidant}
Temperature (C}
Elements assayed
Acetylene
Acetylene
Acetylene
Propane ,
Natural gas
Air
Nitrous oxide
Oxygen
Air
Air
2400
2800
3140
2000
1500
Mg
Ca
Na, K

(it0 Monochromators: In sophisticated instruments prisms or sometimes even diffr

action
gratings are used. However, for routine analysis of such elements as calcium, so
dium, and
potassium a simple filter might suffice.
(iv) Photocells: These are the usual detectors in a flame photometer. Unfortunat
ely the
flame instability reduces their accuracy. Therefore a multi-channel polychromato

r is used in
some routine procedures to allow measurement of up to six elements simultaneousl
y.
Instrumentation for Atomic Absorption Spectrophotometry
This is practically similar to that of emission flame photometry. An important p
oint of
difference is the need to have a radiation source. It is practically impossible
to isolate a particular
.resonance wavelength from a continuous source by using a prism or a diffraction
grating or
both simultaneously. This problem was solved with the development of hollow-cath
ode discharge
lamps. Such lamps produce monochromatic radiation characteristic of the element
analyzed.
In these lamps the cathode is a hollow tube which is lined by the element in que
stion. The lamp
will thus emit monochromatic radiation characteristic of the emission spectrum o
f the element
involved. Such lamps ha,e now become commercially available for a long range of
elements. In
less sophisticated instruments, a continuous discharge lamp with double monochro
mators is
used.
Instruments with single and double beam optics are available. The double beam op
tical
arrangement is more or less similar to that of double beam apparatus in absorpti
on
spectrophotometry.

Element
246
Bophyscal Cherrtr
Applications
The primary use of flame photometry and atomic absorption spectrophotometry is i
n the
assay of elements in biological samples such as blood, plasma, other body fluids
such as urine,
saliva, cerebrospinal fluid and milk, tissues, cells, organelles, soils, plants
and even in the
macromolecules. Flame photometry is used in routine estimation of sodium, potass
ium, calcium,
magnesium, mhnganese, indium and thallium in a host of biological samples. Flame
photometry
is very sensitive to the estimation of alkali, alkallne-earth and rare earth ele
ments. It also
permits estimation of silver, aluminium, gold, bismuth, cadmium, copper, mercury
, lead,
selenium, iron and zinc. Although most elemental analysis is possible directly,
copper, iron,
lead and mercury need to be 6xtracted from the biological fluids before they can
be assayed.
Atomic absorption spectrophotometry (AAS) is a more sensitive technique and can
detect presence of much less quantities of elements with the exception of alkali
and alkali
earth metals for which flame photometry is preferred. AAS can detect quantities
less than
1 part 10-e of more than twenty elements.
Except for minor differences, the performance of both the techniques is more or
less
comparable. Flame photometry, however, has a very important advantage over AAS i
n
that it allows simultaneous quantitative multielement analysis to be performed.
A list of wavelengths used and detection limits for various elemerts in emission
and
absorption flame spectrophotometry is given in Table 8.11.
Table 8.11 Measurement various elements by emission and absorption flame
spectrophotomet r: Detection limits and wavelengths used
Emission
Absor tion
Wavelength Detection
Wavelength
Detection
(rim)
- limit
(rim)
limit

(parts 10}
(parts 10)
Aluminium
Barium
Cadmium
Calcium
Cobalt
Copper
Iron
Lead
Lithium
Magnesium
Manganese
Nickel
Potassium
Sodium
Zinc
484.2
493.4
326.1
422.7
352.7
324.8
372.0
670.7
285.2
403.3
766.5
589,0
26
0.2
9
0,005
0.5
0. I
0.5
0.001
0.1
0.02

0.001
0.0001
309.3
553.6
228.8
1.0
0.4
0.03

422.7
0.1
240.7
0.2
248.3
0.2
283.3
0.5
670.7
0.02
285.2
0.02
279.5
0.05
341.5
0.1
766.5
0.03
589,0
0,03
213.9
0.02

Spectrophotometry
24 7
Atomic Fluorescence
We have seen the relationship between absorption spectrophotometr" and
spectrofluorometry. A similar relationship exists between atomic absorption
spectrophotometry and atomic fluorescence spectrophotometry. In atomic fluoresce
nce,
the flame retains its role as a source of atoms; these atoms, however, are excit
ed by an
intense source of radiation and their fluorescent emission is assayed at an angl
e of 90
in a manner similar to that of spectrofluorimetry. Lack of sufficiently intense
source for
many elements has been the limitation of this technique, however, with time
instrumental developments are overcoming this problem. High intensity hollow-cat
hode
lamps, or xenon or mercury discharge lamps are used.
Perhaps the greatest advantage of atomic fluorescence is its extreme specificity
and
complete freedom from many forms of interference. Moreover, the sensitivity of t
his
technique is much better than that of the absorption method. We may cite the exa
mple of
zinc and even cadmium where the levels detected are as low as 1 and 2 parts 10-I
respectively.
Developments in the field of instrumentation will surely make available this
technique for use in biology.
NUCLEAR MAGNETIC RESONANCE SPECTROMETRY
The use of nuclear magnetic resonance (NMR) in the determination of molecular
structure has been a major growth point in biochemistry. Until recently technica
l and
theoretical difficulties were major inhibitory factors for the application of NM
R. In recent
times, however, developments in instrumentation and accumulation of basic data h
ave
allowed this technique to be applied to determine macromolecular structure and
interactions.
Many physical methods are comparatively uninformative when applied to aqueous
solutions, and those which can be readily applied often depend on alterations of
molecular
properties and therefore cannot reflect much of the detailed structural informat
ion of
interest. Figure 8.35 provides a comparison of the magnitude of energies and the
frequencies of
transitions over the entire spectroscopic range. It will be observed that a char
acteristic feature

of NMR spectroscopy is the very small value of the energy absorbed in the transi
tion of nuclei to
the excited state. It is natural then, that the appearance of the spectra is dep
endent on slight
variations in electronic configuration. Such spectra can be expected to reflect
the more or less
unaltered structure and conformation of macromolecules in solution.
Frequency 3 x 1015
5 1012
101
I07

4 1014

UV & visible ----Infrared -Microwave - -- Radio---EnergyA , 260

KCal/mole
Electronic &
Transitions------ Vibrational
38
0.56
9.5 xl0-4 9.5 x 10-7
Vibrational
Rotational
ESR
NM R

248
Biophys,.al Chemistry
Most nuclei, the protons, and the electrons possess inherent magnetic fields. Th
e effect of
nuclear fields are, however, too small to be observed in the ambient magnetic fi
eld of the earth.
Artificially created intense magnetic fields can, however, make the nuclei assum
e specific
orientations with corresponding potential energy levels. Nuclear magnetic resona
nce
spectrometry addresses itself towards detecting the minute amount of energy abso
rbed or emitted
as the nuclei jump from one energy level to another.
Magnetic Properties of the Nucleus
In order to explain the magnetic properties of certain nuclei it is necessary to
assume that
the nuclear charge is spinning around an axis. Such a nucleus is then supposed t
o possess an
angular momentum represented by a spin quantum number, I, which is assigned half
integral
values, 0, 1/2, 1, 3/2 ........ 9/2 depending on the particular nucleus. Nuclei
possessing anodd
mass number {either the number of neutrons or the number of protons should be od
d, but
never both odd) are assigned half-integral spin quantum numbers; for example H,
gF, P()
and B(3/2). Figure 8.36 (A) illustrates a nuclei as a spinning sphere. This spin
ning charge
gives rise to a magnetic field. There is an associated nuclear magnetic moment,
, along the
axis of the spin. Nuclei possessing odd numbers of both neutrons and protons hav
e an integral
spin number of I = 1. Thus, the charge on them is distributed non-symmetrically
(e.g., 2H, N).
Nuclei possessing even numbers of both protons and neutrons (2C, O, 3S, etc.) ar
e not measured
in n.m.r, experiments because they do not possess an angular momentum (I = 0) an
d do not
exhibit magnetic properties.
North magnet pole
H
---- Nuclear magnetic
moment
(Magnetic component of I7'

Precessional axis
South magnet pole
/
lgure 8.36 (A) Spinning charge in nuclei generates a magnetlc fleld with a rnagn
etc moment p. When placed in a
powerful unorm magnetlc fleld the spin axes of these nuclei align themselves in
an manner with respect to the fleld and, like the top of a gyroscope, precess ab
out the fleld direction in such a
way that each pole of the nuclear axis sweeps out a c'cular path in the XY-plane
, When a second,
weaker.field of rad frequency (RF} is applted at right angles to the uniform mag
netic fleld, the nuclei
may undergo a transttion to a higher energy level by absorbing the RF energy. On
ly that RF whose
sense of rotatlon is e.mctly equal to the rate of precesslon of nuclear dpole re
quency of RF equal to
precesslon frequency) will be absorbed.

Spectrophotometry
249
Magnetic nuclei assume discrete orientations with corresponding energy levels un
der the
influence of external magnetic fields. The value of I determines the number of q
uantized energy
levels available. This is given by the series :
l,I- 1, I-2

-I

Any given nucleus can have 2I + 1 possible levels or orientationsl Thus

for IH, 15N, gF and
sip, with I = V2, only two orientations are available. These levels are
described as
(0 aligned with the applied field (lower. energy); and (tO antiparallel
to the field (higher
energy). These two levels (Figure 8.36(B)) will have the energy of -Ho a
nd +Ho for low
and high energy respectively, where Ho is the intensity of the applied m
agnetic field and
is the nuclear magnetic moment. The difference in energy, E, is then equ
al to 2Ho.
The general relationship is as follows :
E -- Ho/I
Alignment against
the field
External field, Ho
.
I= +V2
T
No field
<.zE = hv = HoI
I = -V2
Alignment with the field
Figure 8,36 (B) Energy states for nuclei in a magnetic fleld
In order to change from low energy state to high energy state, such nuclei must
absorb the
appropriate quantum of energy. In a magnetic field of several hundred millitesla

(several thousadd
gauss} such nuclei absorb radiation in the radiowave region of the electromagnet
ic spectrum.
This phenomenon is known as nuclear resonance or nuclear magnetic resonance. The
above
equation can, thus, also be written as
AE = hv -- 2Ho (for cases where I =
If we combine the two equations, we get
2rv - 2 ]Ho YHo
hi
here 2 serves to convert linear frequency to angular units of frequency. , which
is obviously
equal to 2r/hI is known as the gyromagnetic ratW and is a characteristic propert
y of the
nucleus. Another mathematical expression for can be derived from the above relat
ionships :
Gyromagnetic ratio =y This relationship is a fundamental equation for n.m.r. The relationship obviousl
y describes
the ratio between the frequency and the field. The ratio is sometimes termed *re
sonance
condition'.

OH
Acetone
Cyclobutane
250
Btophyslcal Chemistry
The frequency of the radiowave absorbed during n.m.r, is dictated by () the isot
ope being
studied, and (tO the intensity of the magnetic field.
Figure 8.37 The n.m.r, spectrum of
methanol (CHaOH)
Obviously all nuclei do not absorb at the
same applied field but the absorption depends
upon the magnetic field which a particular
nucleus, feels. Clearly, the effective field
strength is different for different nuclei as
one nucleus will have slightly different
environment from any other nucleus, Thus,
at a g,ven radio frequency', dlfferent nuclei
will require slightly different magnetic field
strength to give rise to the same effective
magnetic strength which causes absorption.
It is, therefore, a normal practice to measure
the absorbance of a sample at a
monochromatic radiowave frequency while
varying the magnetic field intensity.

n.m.r, spectra are thus a plot of energy absorbed against the magnetic field str
ength, applied.
Such a plot is shown in Figure 8.37. The number of signals obtained on such a pl
ot are indicative
of the different sets of equivalent nuclei or protons. For example, in Figure 8.
37 there are two
signals which are indicative of two sets of protons existing in different enviro
nment, viz., CH3,
and --OH. The area under the peak in such plots is directly proportional to the
number of
nuclei or protons which are responsible for the resonance. For an example one ca
n refer to
Figure 8.37 again; the peak corresponding to CH3 is much higher than the peak co
rresponding
to ---OH.
For a clearer understanding, let us consider the number of signals generated by
some
compounds as examples. Acetone andcyclobutane are considered first. The structur
al formulae
are given below.

Hsc/C ---0
CH----CH
It is easy to see that all six protons of acetone and all eight protons of cyclo
butane exist in
the same environment. Therefore, in an n.m.r, experiment, only one signal will b
e observed for
each of these compounds. The area under the peak of cyclobutane will be more tha
n that under
acetone peak owing to a larger number of protons in the former.
We have already seen how CH3OH gives two n.m.r, signals owing to two different s
ets of
proton equivalents, i.e., sets of protons existing in two different environments
. Another compound
Which gives two n.m.r, signals is 1, l-dibromomethne, CH3CHBr2. Propanal. CH3CH2
CHO, on
the other hand has three sets of equivalent protons existing in three different
environments
(CH3, --CH, and --CHO); this compound gives three n.m.r, signals.
Chemically equivalent protons should also be stereochemically equivalent. Thus,
a given
set of protons is chemically equivalent only if all the protons exist in identic
al environment even
when stereochemical formula of the molecule is written. This principle can be gr
asped better
with the help of an example. The compound 1,2-dichloropropane is expected to giv
e three
n,m,r, signals in the following manner:

251
Heft : Ho - oH
3 2 i
CHz--CH(CI)--CH2CI
In an n.m.r, experiment, this compound gives four signals. To understand this le
t us take
a look at the stereochemlcal formula of the compound.
Cl
1
1,2-Dichloropropane
It is evident from the above formula that the two protons attached to C do not e
xist in
exactly identical environments. Consequently these two protons will give rise to
two different
signals rather than one.
Chemical : Position of Signals
We have seen that the number of signals in an n.m.r, spectrum gives information
about the number of equivalent sets of protons/nuclei that the compound possesse
s. The
position of these signals is indicative of the nature of the environment surroun
ding
the protons/nuclei. For example, the position of the signals can reveal whether
the environment
of proton is aromatic, aliphatic, acetyleni.c, vinylic, or whether the proton ex
ists near an electron
withdrawing group or an electron releasing group. Each o the above named sets of
protons
have electronic environment characteristic of the type: These proton sets theref
ore absorb at
different applied field strengths. It may be said that the electronic environmen
t of the protons
dictates where the proton will absorb in the spectrum.
The electrons involved in covalent bonding are paired and usually do not have a
magnetic field. An applied magnetic field, however creates additional modes of c
irculation of these electrons
thereby generating a small localized magnetic field. This magnetic field is prop
ortional to the
applied magnetic field. Such nuclei, circulation of electrons around which has g
iven rise to a
magnetic field, might not resonate. The phenomenon is known as d/amagnet/c shie
(Figure

8.38), and the nuclei are said to be shie/ded. Such nuclei are not resonating be
cause rather
"than experiencing the fuji magnetic field applied, H0, they are experiencing a
smaller magnetic
field due to the oppeelflon created by the field of electrons.
where is the shielding constant whose Caue depends upon the density of electrons
around a proton or nucleus. It is thus a function of the structure of the compou
nd. For the
above situation, a higher magnetic field will be needed to make the nuclei reson
ate.
Thus, shielding of nucleus proton shifts the position of the signal, Le., absorp
tion upfle.kL

252
Induced
magnetic
field
Ho -.-..-: :..
""" ?...F:.'.i
Electron
circulation
SnPulcenlulslg

Figure 8.38 Diamagnetic shielding. Local magneticJlelds opposing Ho generated by

uuced electronic drculatlon
Magnitude of shielding decreases with increasing electron withdrawing power of t
he nearby
substituents (strongly electronegative substituents such as oxygen). Sometimes t
he induced
field augments the applied field. The nucleus is then said to be deshlelded and
a smaller applied
field is required to make such nuclei resonate. Thus. deshielding shifts the abs
orption downfleld.
The above discussion can be understood better with the help of an example. Let u
s again
consider the n.m.r, spectrum of CH30H given in Figure 8.37. One can see that the
absorption
due to --OH is occurring at an applied field lesser than that required for --CH3
. This iS because
in the former, the proton is adjacent to the strongly electron withdrawing oxyge
n atom. The
proton is therefore deshielded and requires lesser magnetic field to resonate; P
rotons in CH3 are
more shielded and therefore absorb at a higher magnetic field. The signal due to
CH is therefore
shifted upfield.
It is usual, just as we didin case of absorption spectrophotometry, to compare t
he positions
of n.m.r, absorptions arising out of shielding and deshielding with a standard r
eference.
The shifts in positions of n.m.r, absorptions as compared to the standard refere
nce are
known as chem/ca/sh/fls. For proton spectra in nonaqueous media, the reference m
aterial
is tetramethyl sflane (CH3)4Si, abbreviated as TMS. TMS contains 12 protons and
all these
protons are chemically equivalent. Therefore, TMS gives a single sharp signal in

n.m.r, experiment.
Silicon has a very low electronegativity and therefore equivalent protons of tet
ramethyl sflane
are deshielded very little; it may be said that shielding of equivalent protons
of TMS is greater
than most of the organic compounds, Therefore, n.m.r, signal for tetramethylsfla
ne is taken as
a reference and chemical shifts for different sets of protons is determined rela
tive to it. In
comparison to this reference TMS signal, signals for different types of protons
will appear at
different field strengths. This difference in the absorption posttlon of tte pro
ton with respect to
TMS signal is called chemical shift. The magnitude of the chemical shift Is expr
essed as 8-value
which is calculated as follows:
8 = HSAMPUS --HTMS X 10e
where HSAMPLE and HTMs are ,tl e positions of the absorption lines for the sampl
e and
reference respectlvcly, expressed in frequency units {hertz}, and i is the opera
ting
frequency of the spectrometer. is usually expressed in parts per million {ppm}.
Most chemical shifts have values between 0 to 10.

Spectrophotometrtj
253
Another scale used for measurement of chemical shift is known as the x (tau) sca
le. It is
related to scale by the following s.imple relationship:
= 10- "
It is usual to plot n.m.r, signals with magnetic field strength increasing to th
e right. TMS
is assigned a value of 0 ppm and its signal appears at the extreme right (Figure
8.39}. Protons
which experience greater deshielding have larger values. This is so with most of
the organic compounds. A list of values for chemical shifts for protons in diff
erent environments is provided
in Table 8.12.

TMS
Highly shielded signals
]
I0
9
8
7
6r
5
4
3
2
I
0
of protons
Primal>
Hydroxyl
Esters
Acids
Acetylinic
Chlorides
Ethers
Alcohols
Fluorides
Vinylic
Aromatic
Aldehydic
Carboxylic
Phenolic
Enollc
R-CH3

Chemical shift in ppm

R-OH
H-C-COOR
H-C-COOH
CmC-H
H-C-CI
H-C-OR
H-C-OH
H-C-F
C--C-H
Ar-H
RCHO
RCOOH
At-OH
C=C-OH
0.9
1.05.5
2.02.2
2.02.5
2.03.5
3.04.0
3.34.0
3.44.0
4.O4.5
4.65.8
6.09.0
9.0 10.0
9.1
9.0
8.0
8.0

8.0
7.0
6.7
6.6
6.0
5.4
4.0
1.0
4.5
7.8
7.5
6.5
6.0
6.0
6.0
5.5
4.2
1.0
0.0
to -0.5
to +6.0
to -5.O
10.5
4.0
15.0
- 12,0
- 12.0
- 17.0
-2,0

-2.0
-7.0

External
field
254
Biophysical Chemistry
S Uttlng ,
We have dy seen how elg d deselg ect posion of n.m.r. specWa. e n.m.r, speca bec
ome fuer compcated what Is o as spin-sp coup. We m dersd s phenomenon fly e foHo
phs.
Consider eyl brode, CHCHBr: from what we have considered so f, we ow at
s molece cons o ds of protons denot as a d b low.
a
b
CH
CH Br
It Is erefore et at e n.m,r, s of s md Ist of is, We so eect t e o sis be smets,
i,e., e sis each have one peak. e n.m.r, spectm of this compound, however, show
s two sisals
compos of mple, i.e., a cluster of pes. (re 8.). For a up of ptons we
obsee a slgn consisting of a cluster of three pes (plet) whereas e s for b
oup of protons consis of a cluster of fo ps (quiet). y have e ss spt? e molece o
f eyl brode, e sp of (HO c couple e ney meylene oup ee disct ways reon to e e fi
eld. ese ee ways of coup e
8.0 7.0 6,0 5.0 4.0 3.0. 2,0 1.0.
''"-

(A}

(Relnforcmg)

Signal from -

{B) (Not effectlng)

'a' protons
-

(C}

.(Opposing)

Spin influence of
protons 'b'
This is the reason why a triplet signal is given by --CHs.
Let us now consider the reason for a quartet signal for the-CH2 group. The three
protons of the methyl group couple with the adjacent methylene group in four di
fferent,ways in relation
to the external field as outlined below.

i Spectrophotometry
255
/(A) ,
(ReinforcLng : Strong)
,s,,

(B} l (Reinforcing : Weak) External

Slgnal from
(C} 6' 4% (Opposing : Weakl field
'b' protons
(D)
(Opposing : Strong)
Spin influence of protons 'a'
From the above, it is clear that the slittLng of the signals into multlplets has
been because
'the different environments of two sets of protons with respect to the nearby pr
otons. It is
that this mutual magnetic influence between the two sets of protons is not trans
mltted
but through the electrons of the chemlcal bond between the two groups. This
which involves interaction between like and different spins of nearby nuclel is
Analysis of the n,m.r, spectrum of a compound, only the empirical formula for wh
ich is
beyond the scope of thls book. We are, however, at a stage where me e.aa y to
a given compound gives a partlcular n.m.r, spectrum. We again take up the
the first example. From its formula we can deduce that the compound
two signals. It does; but both the signals are multiplets. One signal is a quart
et
We have already seen the particular spin-spin coupling by which we
that the quartet belongs to the methylene group while the triplet belongs
the methyl group. Since the methylene group is nearer to the bromine atom, we ex
pect its to be deshielded more than that of the methyl group. Consequently we ex
pect the
due to the methylene group at a lower applied field as compared to the methyl gr
oup.
figure it can be seen that the signal for methylene group occurs at 6.6 as compa
red | that for the methyl group which occurs at 8,35 .
b a
As a second example let us consider Lelchloroethane, CI2CHCH2CI.As indlctted, tw
o sets
present
and we might expect two signals. Figure 8.41 shows two signals indeed,
a triplet while the other is a doublet. Which of the slgnal belongs to aand
&O
?.0
e.O
.0

4.0
:3.0
2.1)
1.0
0 ppm (6)
:.0
3.0
4.0
U
.0
7.0
8.0
9.0
Hgu 8.41 N'MR specan of bdoroethane. See text for descr/pKon (taken from cata/og
ue).
to b? The proton set b has two chlorine atoms (electron withdrawing) near it
proton set ahas only one such atom near to it. We, therefore, rnlght expect prot
on set
more d6shielded as compared to o. If this is so, the triplet which is present at
a lower
should belong to group b and the doublet should be due to a. Let us also confirm
this by

Bophus.co Chrrtrv
consideng the spin-spin coupling. Splitting of signals from proton set aby the p
roton b should
as follows
(A) ' (Reinforcing)
T External
(B) (Opposing) I field
Thus coupling of proton set a with a single proton b will give rise to a doublet
. On the other
hand signal from proton b will be split by proton set into a triplet in the foll
owing manner.
(A)
(Reinforcing)
(B)
,
(Non effecting)
[ External
(C]
(Opposing)
| field
Thus from spin-spin coupling also we can see that the triplet belongs to b while
the doublet
belongs to o.
Due to all the complications which split and shift the peaks, it is a difficult
Job to
interpret n.m.r, spectra. Usually services of an expert are required. Sensitivit
y of the old
generation n.m.r. Instruments was much less as compared to other optical techniq
ues
and such other techniques as gas chromatography, and mass spectrometry. The mode
m
day n.m.r, instruments, called the Fourier transform n.m.r, are butt around a sm
all,
high speed digital computer. These instruments have revolutionized the practice
of n.m.r.
in organic chemistry.
Instrtunentation
Figure 8.42 shows the basic components of an n.m.r, spectrometer. It would not b
e
wrong to say that the magnets are the heart of an n.m.r, spectrometer. In compar
atively
old instruments extremely heavy electromagnets (103 104 kg) produced fields ranging
from 1-10 tesla. The newer gendration spectrometers, however, use superconductin
g
solenoids which generate fields above 3.5 teslas. The magnetic field can be vari
ed by

about 10-2 tesla by using an auxiliary sweep generator. Since the nuclei absorb
in the
radiowave region, the source of radiation is a radio frequency transmitter. The
sample
must be dissolved to a relatively high concentration in a solvent which is proto
n-poor
or CDCI3). Sample is usually placed n a high precision diameter tube (this minim
izes the
variations in the magnetic field) and is rotated at high speed by an air turbine
. The
absorption signal is then detected by a radio receiver..The signal is amplified
and recorded.
Sample
Radio
transmitter
/
. ,/ Radio receiver
Amplifier
Recorder
/weep coils
Sweep
Magnet
generator
Figure 8.42 Main components of a nuclear magnetic resonance spectrometer
Appl/eations
"(0 Structural diagnosis: The n.m.r, spectroscopy is mainly applied to study the
structure
of small organic molecules and small globular proteins. Thus, structural informa
tion which

Spectrophotometry
25 7
relates to the biological functions of the antibiotics such as valinomycin and g
ramicidin
have been obtained from n.m.r, studies. Structural information about small prote
ins such
as some neurotoxins, various cytochromes, hen egg-white lysozyme, and calcium bi
nding
proteins have been obtained using n.m.r, spectroscopy. For protein studies, howe
ver, the
technique is combined with X-ray tudy for better information.
(ii) Study of dynamic-characteristics of protein structure: Processes which modu
late
intemuclear distances, e.g., .the internal motions of macromolecules or chemical
exchange,
affect the intrinsic properties of n.m.r, resonances and make it possible to pro
be the
dynamic aspect of molecular structure by this technique. Thus, internal motions
of
proteins such as the opening of secondary structure, aromatic side chain rotatio
n,
segmental motion of the main chain, and overall tumbling of protein have been st
udied
using n.m.r, spectroscopy. The proteins which have been studied for their dynami
c
characteristics include histones, cytochrome b5, prothrombin, plasminogen and
chromogranin A.
(iii) Studies on complex formation : Using n.m.r., it is possible to detect very
small
conformati0nal changes. Thus, n.m.r, has been used to study complex formation su
ch as
the binding of a ligand to an enzyme, a drug to DNA, an agonist to the receptor,
or an
antigen to an antibody.
(iv) Biological structures and compartments : Compartmentation is an essential p
art of
the organization that characterizes living systems. In the recent past n.m.r, ha
s been
increasingly used to study this biological structure. Extensive use of n.m.r, ha
s been
made to study the conformation of the lipid headgroups of the biological membran
es and
their interaction with integral proteins of the membrane, n.m.r, has also been u
sed to
measure intracellular pH, membrane transport phenomena, and metabolite
concentrations, interconversions and fluxes.
(v) Quant/tat/ve stud/es : Over the years n.m.r, has been used to determine the
concentration
of metabolites. As a direct method n.m.r, has an undoubted advantage over freeze
-clamping
analysis. Measurement of the phosphocreatine concentration in muscle can be cite
d as a
representative example. Another example can be given of the measurement of
hexakisphosphatephytic acid which accumulates in the pith tissue of plants.

(vi) Thermodynamic studies : In the general case of an equilibrium between two s

taes,
n.m.r, can be used to measure the associated thermodynamic quantities, if the n.
m.r.
response of the two states is different, n.m.r, has been used to measure quantit
ies such
as binding constants, heats and entropies of binding, pKa values, and partition
coefficients
for the distribution of a molecule between different compartments. Examples can
be cited
.of studies made on folding and unfolding of proteins and tRNAs, and the interac
tion between
actinomycin D and deoxy-pGpC, pKa of histidine residues in proteins (e.g. haemog
lobin)
has been determined using n.m.r. The techniques has also shed welcome light' on
the
mechanism of action of proteases and on the mechanism of their inhibition by som
e
proteins.
(vii) Membrane transport: Membrane transport either in in vivo systems or using
synthetic membranes has been studied using n.m.r. The technique has been used to
study alanine and lactate transport in the human erythrocyte by exploiting the d
ifference
in the magnetic susceptibility between the inside and outside of the cells; Very
recently
Na transport in human erythrocytes has also been studied using n.m.r.
(viii) Intact organ studies with n.m.r.: In one of the most intriguing new areas
in
biochemical n.m.r., 31p has been applied to the study of such intact biological
specimens
as heart, kidney, and skelet.al muscle. This has been possible largely because o
f the
development of instrumentation capable of resolving 31p resonances of small mole
cules

599,590,668,629258
Bphsat
Chemtr
such as adenosine triphosphate, adenosine diosphe, .eae phosphate, d orgc
phosphate -- e moles most pot ceHul physiolo. e pK's of er sin molecules of phys
ioloc impoce e ound 7. us their 3p chemlc shifts e
sensive to pH d c be used to mease WaceH pH.
() : Bioloc objects such as its, nuts d ce have n ed proton n.m.r, zeuatoaphy. P
resonce stues of ceH metates is now prodg
sit to ceHul compenflon.
EON SP NCE SPERORY
A chec species odd number of elecons ibits chactesc meflc propees much e e nucle
us. e spng acon of unped elecon generates a maec moment, g. If an intense magnet
ic field is applied, the electron assumes
oenmons ower ener, HJ or st (er ener, + HJ e rid& e ener derence, AE, be 2 Ho. In a mer si to e ato
c nucleus, elecon a ec field is able to absorb ener of e proper frequent, = wch
catapult it from e lower to a hier ener level. is phenomenon is o as ecn reso, d
e tecque wch is employed to study s e of behaor is ced eon sp reso
(e.s.r.) s. a mec field of e oer of gauss (i for e.s.r.) e appropte qut of ener
is obned from radiaon e crowave re,on of e elecoec
has en seen respect to n.m.r., It is coon practice to subject e sple to deg male
fic tensies keepg e crowave frequen const. e mec field is
ved e resonce occas. e ea of e.s.r, pe is fly propoon to e numr of ped elecons e
sple vesflgated d us to e concenWaon ofe ple. In qumflve ysis, pe eas of e sple
e comped to e pe ea of a said
wch conts a o quflW of unped elecWons.
Le n.m.r;, e e.s.r, speca do ebit he sptg wch is caused by temons
een e sg elecWons d adjacent spg malefic nuclei. e e.s.r, specWa,
howler, show no phenomenon wch is pel to e chec s seen n.m.r, sc.
from e above it t en at n.m.r. d e.s.r. e complemen tecques.
stentation
ure 8,43 usates e basic components of e.s.r, specometer. elds of 0 llltesla, required for accurate ork, are enerated by elecomaets. A sweep
enerors a capacl of I0-I00 tesla e so prodded. Monocomac owave radiaon t be read
y obted y us a yson oscator.
ple
yon
urce
t
p cs
Oaor Met

259
Saml)les for e.s.r, mxst be soids. Biological samples which cort laxi[e mouv.t o
i water are therefore frozen in liquid nitrogen before e.s.r, experiment.
Appliemtions
Electron spin resonance spectrometry has proven most helpful in studying mechani
sms
of reactions which proceed through free radical intermediates.
Electron spin resonance spectrometry is one of the main methods to study transit
ion
metal containing metalloproteins, i.e., copper (cytochrome oxidase, superoxide d
ismutase),
molybdenum (xanthine oxidase), and iron (cytochromes, ferredoxin, haemoglobin, e
tc.)
Normally, biological macromolecules which lack unpaired electrons can not be stu
died by
e.s.r, because they do not resonate. These macromolecules can however be "spin l
abelled" (spin
labelling involves the attachment of a stable and unreactive free radical to the
macromolecule)
and studied. Thus, lateral diffusion of glycerophosphatides in plasma membranes
has been
studied by labelling glycerophosphaUdes with a stable nitroxide free radical.
While some proteins or enzymes contain intrinsic functional groups with unpaired
electrons,
there are many which do not. Much information can be gamered about these macromo
lecules
by incorporating in them an extrinsic functional group with unpaired electrons.
Spin labelling
refers to the use of stable free radicals as reporter groups or labels. The term
"spin label" was
first coined by McConnell and co-workers in 1965. Spin labels (stable free radic
als) are usually
molecules containing the nitroxlde molety that contains an unpaired electron loc
alized on the
i: nitrogen and oxygen atoms (Figure 8.44). Adjacent methyl radicals (Figure 8.4
4(A)) were developed
between 1960-65. In addition to the nitroxlde moiety, these labels contain a fun
ctional group
useful in covalent attachment of the nitroxide moiety to a larger molecule throu
gh a C--O,
C--S, or C--N bond. Another class of nitroxides developed in 1967 (Figure 8.44(B
)), known as
*doxyl" are usefu. I for studies with steroids, fatty acids and phospholipids.
Figure 8.44 Tgpa:al spia labels

The biochemical methodology behind designing a spin-label experiment is analogou

s to
for any other reporter group study (e.g., affinity label study, substrate-inhibi
tor analog
spin labels carry those functional groups which have been deliberately designed
to
L at specific high affinity site(s) on the macromolecule and/or covalenfly bind
with hyperreactlve
group in the macromolecule structure. The bound labels, ff they bind covalenfly
are
spin/abe/s; other labels, binding noncovalenfly are known as spin probes. For pr
otein
a large number of covalenfly binding labels with specificity for distinct amino
acids
become available lately. These labels might be classified as alkylaing, acylatin
g,
(Figure 8.45 (A)), and phosphonylating (Figure 8.45 (B)).

260
Biophysicat Chemistry
[A)
(B}
llure 8.45 {A) Sulfonylattng spin labels. {B) Phosphonylating spin labels.
A brief list of the kind of information that spin labels can provide in an e.s.r
, experiment is
given below (also see Box 8.6).
(i)
Rates of catalysis: The rate of some step in an enzyme catalyzed reactio
n can be
under circumstances where a change in motional state of the spin label is exhibi
ted
before and after the reaction. This is possible when (i) the conformational
of the spin label on the protein is sensitive to steps in catalytic mechanism, a
nd/or
when the spin label is (one oi) the substrate.
(ii)
Active site geometry: Use of spin labels has provided much information a
bout the
depth or width of the substrate binding region of various enzymes, One of the ve
ry
important applications has involved the use of spin labels to compare the active
site
conformations of families of related enzymes.
(tii)
Denaturation and protein folding: If a spin label is present in an envir
onment which is
sensitive to the degree of folding or unfolding of the overall protein structure
, much
information is reported by it about the denaturation and renaturation process. O
ver
the years these studies have yielded much important information about the struct
ural
features of these conformational states.
(iv)
Enzyme-ligand interactions: Depending on the environment of placement, s
pin labels
have been shown to be excellent reporters of conformational changes, even subtle
ones. ThuS, spin labels bound at the active slte provide much information about
the
enzyme-ligand interactions. In addition, these labels provide information about
the
pH, salt, or temperature-induced structural changes. These strategically placed
labels
also yield information about protein-protein interactions. In fact, e.s.r, spect

rum of a
spin labeled protein easily detects the narrow line spectra exhibited by proteol
ytically
damaged species comprising a few percent of the total protein.
(V)
Symmetry and orientation studies: Studies of proteins by spin labeling m
ay be applied
to crystalline proteins as well. Spin labeled enzyme crystals provide informatio
n about
their symmetry and orientation.
(vt)
Spin labels have provided insight into the kinetics and mechanism of str
uctural changes
of a variety of condensed phases of DNA.
(vii)
Studies of hydrocarbon and phospholipid motion in lipid bflayers have be
en performed
with the aid of spin labels. Such studies are currently in progress and are expe
cted to

(vaO
261
provide much Information about the domains of phosphollplds involved in these
motions.
Myotonic dystrophy: Roses eL a/. have studied erythrocyte membranes from human
patients affected with myotonic dystrophy using fatty acid spin labels as probes
. As
compared to controls, the label was found to reside in a more fluid less polar l
ocus in
patients. It has thus been concluded that myotonic dystrophy is a diffuse membra
ne
disorder.
(Ix)
Other muscle diseases: Work with spin labels is helping in providing ins
ight about the
molecular nature of many muscle diseases. Studies with spin labels, when taken
together with other measurements, suggest abnormal calcium pumping in diseased
membranes and altered interactions with primary membrane proteins such as the
calcium ATPase.
MOSSBAUER SOPHOTOMETRY
The Mossbauer effect is based on the resonant absorption of nuclear gamma radiat
ions
by certain stable nuclear isotopes. Discovered by Rudolph Mossbauer in 1957, the
phenomenon
a technique which has since had numerous applications in biophysics. With the
of
which is about 2.2% in abundance in the natural isotopic mixture of iron
,
isotopes occur in high proportion in biom01ecules. However, some
may be introduced into biological macromolecules by the so called enrich
ment
Mossbauer isotopes most often used for biological applications are 5rFe, and 2I.
It is
present a complete analysis of this phenomenon in this sectlon; a brief explanat
ion
and a brief idea about the applications of this technique is given below.
We have seen that atoms can transit between excited and ground states by absorbi
ng or
emitting electromagnetic radiations in a manner dictated by their electronic str
ucture. Similarly,
nuclei also are capable of transitlons between certain nuclear energy levels as
a consequence of

absorption or emission of electromagnetic radiations such as -rays. In order tha

t momentum
be conserved, whenever a T-ray is emitted form a nucleus, the nucleus recoils. T
he recoil of
nucleus is an energy requiring process and is defined by the following relations
hip
2
RE = E/2Mca
where M is the nuclear mass, c is the velocity of light, E, is the energy of the
emitted photon,
and RE is the recoil energy. Since both E and RE are derived from the energy of
transition, E is
less than the transition energy AE.

262
Biophysical Cherrdstry
If the nucleus had not lost some of the transition energy in the recoil, the emi
tted photon
would have possessed exactly the same quantum of energy wch could be absorbed by
another
nucleus of the same type. However, due to the energy loss, the emitted photon ha
s less energy,
and it can not be further absorbed by another nucleus. Mossbauer discovered exce
ptions to the
above phenomenon when he found that certain low energy y-emisslons took place wi
thout any
recoil. This became possible because the recoil energy in these cases was not su
fficient to make
the nucleus move as a free particle. The momentum of recoil in these cases was t
ransferred to
the entire molecule in which the nucleus was bound, and the large mass of the mo
lecule protected
any energy loss. In such cases, the emitted y-ray will have roughly the same ene
rgy as the.
energy of transition and therefore will be absorbed by another atom of the same
type: Thus,
when y-absorption is measured as a function of energy of the incident radiation,
a very sharp
resonance absorption line is seen at the transition energy of Mossbauer transiti
on. This
phenomenon is known as the Mossbauer effect and the technique based on this is .
called
Mossbauer spectroscopy.
57Fe is the Mossbauer isotope of keen biological interest because of the natural
occurrence
of iron in a number of proteins such as cytochromes, haemoglobin, myoglobin, fer
redoxin etc.
We will therefore discuss Mossbauer spectroscopy with respect to this isotope. S
Co is the parent
nucleus of Fe, and the former is used as a radiation source in studies of latter
. As shown In.
Figure 8.46; 5C0 decays in two steps.
STCo
14.4 KeV EI y-ray
El
---------G 14.4 KeV
--..----.lG
STFe
Fe
Figure 8.46 Schematic representation of EC decay ofTCo toTFe. Probabflttles of t
he steps aregen tnpercentaBes.
G and E are the.ground and first excited nuclear states respectively. E to G tra
nsition results in the
emission of 14.4 KeV y-ray. Absorption of this energy raises another Fe nucleus

from G E state.
Through EC decay (see chapter 13), it gets converted, in the first step, to the
stable daughter
nucleus Fe. However, the EC decay does not lead directly into the ground state o
f SFe; rather
it proceeds through the first excited state of TFe which is located 14.4 KeV abo
ve the ground
state. This state has a mean life of just 140 nanoseconds from which it descends
to the ground
state by recoil-free emission of gamma ray photon of 14.4 KeV. Since this is the
exact quantum
o.f energy that ground state SFe can absorb, Co is used as the radiation source.
The absorption
of gamma radiation produced by cobalt-57 source by Fe in the sample being studie
d is then
measured at various temperatures, and over a small range of energies on either s
ide of the
transition energy. It is interesting to note how the range of energies is produc
ed. The source,
which emits 14.4 KeV energy is moved by an electromagnetic drive system at const
ant velocity,
O, toward and away from the sample (absorber}. This movement results in a Dopple
r effect, the
energy E of the y-ray varying by a small amountAE according to the relation
AE - (0tc] E
where cs the velocity of lght. The velocity, #, is.positive when the source is m
oved toward the
sample and negative when moved away. A typical measurement requires velocities o
f the order

Spectrophotometry
263
of some mm/s. The y-ray intensity transmitted by the sample is recorded in a pro
portional
counter placed behind the sample (Figure 8.47) and this as a function of the Dop
pler velocity/)
is called the Mossbauer spectrum. As an example, Mossbauer spectrum for methemog
lobin is
shown in Figure 8.48. It should be noted that the energy of incident radiation u
sed in a Mossbauer
spectrum is not expressed in terms of wavelength or frequency; rather, Doppler s
hift velocity is
used for this purpose,
Figure 8.47 Principle of Mossbauer spectroscopy. An electromagnetic drive system
(EDS) moves the energy source
(S) towards and away.from the absorber (A) with a constant velocity i. Ttansmltt
ed radiation is measured
by the proponal counter (Z. Lead collimators (C) dej'be beam geometry.
In the discussion above we have said that 'a very sharp resonance line is seen a
t the
transition energy of Mossbauer transition'. From Figure 8.48, however, it become
s clear that
this is not so and in effect quite a few bands are seen. This is so because the
characteristic
features of a Mossbauer spectrum are affected by the local environment in which
the nucleus
giving rise to Mossbauer effect exists. This local environment is responsible fo
r broadening the
absorption line into a band of variable line width. The band may become further
split by what
is known as hyperflne or quadrupole splitting. Moreover, the band may be shifted
slightly in
energy relative to the standard energy source. This is known as isomer shift. Al
l these effects
depend on temperature and just like in NMR (where different kinds of splitting p
rovided
information about the environment) these effects, since they are produced by the
local
environment, provide much information about the local environment itself. For ex
ample, these
effects tell us quite a good deal about the interactions taking place not only w
ithin the nucleus
itself, but also in the lattice around it.
-1.5
-I.0 -0.5 0 0.5
1.0 1.5
Doppler velocity (cm/sec)
Figure 8.48 Mossbauer spectrum of human methemoglobln cyanide

Most of the applications of Mossbauer spectroscopy to biology have been with res
pect to
the iron containing proteins. For instance, such studies have proved very sensit
ive in
distinguishing different Classes of cytochromes. However, the most studied iron
containing
protein has been haemoglobin and some results of such studies are cited here. Nu
merous
publications discuss the mechanism of reversible binding of an O2 molecule to th
e iron in
haemoglobin. Yet, the mechanism is not understood in detail. Mossbauer studies o
n
oxygenated haemoglobin have provided some evidence that duing the binding of oxy
gen to
iron, one electron is transferred to the oxygen molecule. Therefore, the electro
n configuration of
better described as an Fe3+O configuration, rather than as Fe2+O2.

1.2
264
Biophysical Chemistry
Mossbauer studies have also provided some data on the Bohr effect (chapter 1) wh
ich
speaks about the regulation of oxygen affinity of human haemoglobin via pH chang
es in its
surroundings. Although the inference drawn by such studies is negative, it never
theless will be
helpful in the ultimate explanation. What Mossbauer studies tell us is that the
Bohr effect may
not be due to any change in the immediate surroundings of the iron; the change i
n the affinity
of haemoglobin for oxygen consequent to pH changes must be due to changes in the
molecule
occurring at greater distances, from the iron. Such changes are probably alloste
ric in nature.
Mossbauer studies have provided quite a lot of information about the dynamic pro
perties
of biomolecules. Let us again take the example of haemoglobin. Iron, to which ox
ygen binds is
not situated at the surface of haemoglobin; rather, it is situated in a pocket a
nd oxygen reaches
the iron via a channel which leads from the molecular surface to the heme pocket
.
X-ray diffraction studies tell us that this channel is too narrow for the passag
e of oxygen. How
does the oxygen reach iron? Recent Mossbauer experiments give direct evidence th
at the protein
molecule indulges in a sort of breathing motion which opens the channel momentar
ily wide
enough for the oxygen molecule to pass through.
Mossbauer spectroscopy is now being used along with X-ray diffraction for struct
ure
determination of proteins. The details of the process are beyond the scope of th
is book and it
will suffice to say that the technique is used to solve phase problems in X-ray
structure
determination of proteins.
Apart from these studies, Mossbauer spectroscopy has provided a good deal of inf
ormation
about the electron structure of bacterial ferredoxin.
Some Solved Problems
I.
A tube containing NADH in solution was exposed to. oxidizing atmosphere
by mistake. Now
the solution contains.both NAD+ and NADH. This solution gives an O.D. of 0.5 at
340 nm and
1.4 at 260 nm. Can you find out how much oxidized and how much reduced form of t
he
coenzyme is currently present in the tube? Let us tell you that only NADH absorb

s at 340 nm,
but both NAD and NADH absorb at 260 nm. The extinction coefficients of NADH are 1
5,000
at 260 nm and 6220 at 340 nm. The extinction coefficient of NAD at 260 nm is 18,0
00. Also
assume that the cuvette used has a standard path length.
s.
Calculating NADH concentration from O.D. at 340 nm is uncomplicated sinc
e NAD does not
absorb here.
A = abC
0.5 = (6.22 x 103) (1)(C)
5x10-I
C =0.8x10
6.22 x 103
Thus concentration of NADH is 8 10-5 M
The O.D. at 260 nm is due to both NAD and NADH..Let us find out just how much abs
orbance
is due to NADH.
A = ambC
am for NADH at 260 nm is 15 103
A
= [!5x I0){I)[8 10)
= 120 10-2

0.225 =
A4o,m =
(am 450(G} x Co) + (am 450(Z) + C}
0.36 =
(15 x l0s Co)+(7 x 103Cz)
Aoo,m =
{am 600(G) Co) + (am 600 (Z) Cz)
0.225 =
(3 10Co)+(6.5 x 10Cz)
Solving for Co in terms of Cz (You may do the opposite and yet solve the problem
)
0.225 =
15xlO3
Spectrophotometry
265
Thus, out of the total O.D. of 1.4 at 260 nm, 1.2 is due to NADH. O.D. of only 0
.2 is due to
NAD. It is now easier to find out the concentration of NAD.
= am be
0.2 = (18 x 10s)(1){C)
C = 2xI0-1
= 0.11x10-4
18x103
Therefore, concentration of NAD is 1.1 10-s M.
The solution contains two substances G and Z. Both the substances absorb at 450
nm and
600 nm. The absorbance at 450 nm is 0.36 and at 600 nm is 0.225. The molar absor
ption
coefficients of G are 15,000 and 3000 at 450 nm and 600 nm respectively. The mol
ar absorption
coefficients for Z are 7000 and 6500 at the two wavelengths. Can you calculate t
he
concentration of the two substances? Assume that you are using :a standard pathlength
cuvette.
Let us write equations for O.Ds at both the wavelengths first.
0.36 = 15 x I03C+7 x l0sCz

0.36-7 103Cz =
15 I03Ca
0.36- 7 x I03C
Z
15x103
Substituting the above value of CQ in the equation for O.D. at 600 nm
0.225 = (3 x 10CG)+(6.5 x 10sCz)
0,225 -- (3x103) 0"36-7xi03 Cz +6.5x103Cz
15xi03

1.08xI0 -21x108Cz
0,225 -15x103
Multiplying the right hand term with 15 x 103
6.5x103Cz
+

15xlOa
1.08103 - 21x lOaCz + 97.5x 108Cz

266
3.38 x l03= 1.08 x 103-21 x 10sCz + 97.5 x 108Cz
2.3 x103 =76:5x 103Cz
2.3x103
- 3xI0-SM
Cz 76.5x10s
Therefore concentration of Z is 3 x 10-5 M.
We may now substitute the concentration of Z in the equation of O.D.450 or 600 r
im.
0.36 = (15 x 103C) + (7 x 103Cz)
0.36 =
15 x 103 Ca + (7 x 103) (3 x 10-)
0.36 =
15 x 10CG + 0.21
0.15 =
15 X 103 CG
C =
0.15/15 x 103 = 1 x 10-SM
Therefore the concentration of G is 1 x 10-5 M.
To a 4.0 ml solution of glucose was added 2.0 ml of solution containing excess A
TP, NADP,
MgCI2, hexokinase, and glucose-6-phosphate dchydrogenase. The absorbance at 340
nm
due to NADPH was found to be 0.91 after a considerable amount of time had elapse
d. Can
you calculate the concentration of glucose in (i) the final, and (ii) the origin
al solution. Given
that K values for hexokinase and glucose-6-phosphate dehydrogenase lie far to th
e right.
Assume that you are using 1 cm path-length. The molar absorption coefficient of
NADPH is
6220.
Assumption : The two enzymes added to the tube will carry out the ollowing react
ions :
Hexokinase reactions :
. .
Glucose + ATP Glucose-6-phosphate + ATP
Glucose-6-phosphate dehydrogenase reaction :

Glucose-6-phosphate + NADP*
6-phosphogluconic acid--lactone + NADPH + H
The Kq values are far to the right and ATP and NADP* are in exdess. Therefore it
can be safely
assumed that for every mole of glucose consumed, one mole of NADPH will be gener
ated. The
absorption at 340 nm is solely due toNADPH and glucose and other components of t
he reaction
mixture do not absorb at this wavelength. However, since one mole of NADPH is be
ing generated
for one mole of glucose consumed, measuring NADPH will mean measuring glucose.
A
= ambC
0.91
= (6.22 103)(I)(C)
0.91
6.22 103
CNDpH = 1.463 10- M NADPH
Since we are taking NADPH as an index of glucose concentration, the above is als
o the
concentration of glucose in the final solution. For finding out the concentratio
n of glucose in
the original solution, we will have to take into cognizance the dilution factor.
C (original) = C (final) x dilution factor
The dflutlon factor of the solution will be 6/4, or 3/2.

Spectrophotometry
267
Therefore, C (original) = (1.463 x I0-) x 3/2
C (origlnal) = 2.2 10-4 M.
*
Instead of glucose, if one has to measure glucose-6-phosphate, can the s
ame
scheme of things operate? Also, study the metabolic fate of glucosc-lphosphate. What reaction mixture can you prepare for measuring the
concentration of glucose-l-phosphate?
4. Lactate dehydrogenase was assayed by measuring the. generation of NADH in a 1
cm cuvette
spectrophotometrically. The assay mixture of a total volume of 1.0 ml contained
excess lactate,
buffer, 0.1 ml of enzyme preparation. Semicarbazide is also added to the solutio
n with a view
of capturing pyruvate and carrying the reaction to completion. The absorbance at
340 nm
increased at a rate of 0.04/min. The enzyme preparation contained 50 mg protein/
ml. Calculate
(i} the units of the enzyme present in 1 ml enzyme preparation, and (ii) the spe
cific activity of
the enzyme.
Activity = aA/min = 0.04/min
A = (am) (aC} {b}
AA
(am)(b)
= 6.43 I0- M = 6.43 IX moles/litre
Activity = 6.43 IX moles/lit/min = 6.43 10- Ix moles/ml/min. 6.43 10-3 units of
the enzyme are present in 0. I ml of the preparation. Therefore 10 times the amo
unt will be
present in 1 ml of the preparation. Thus 1 ml will contain 6.43x 10- units.
The enzyme preparation contains 50 mg protein/ml. However, only 0.1 ml of enzyme
was
present in I ml of the assay mixture. Thus the effective concentration of protei
n was reduced
to 5 mg/ml.
Therefore the specific activity of the enzyme here was
6,43 I 0-2
S.A. =
5

= 0.013 units/mg protein.

Suggestions For Further Reading
Ultraviolet and Visible Spectrophotometry
I. O]sen, E.D., Modem Optical Methods of Analysls, McGraw-Hill, New York, 1975.
2.
Willard, H.H., Merr/tt, L.L., Dean, J.A. and Settle, F.A., Instrumental
Methods of Analysls,
Wadsworth Publishing Company, USA, 1986.
3. Brown, S.B., An Introduction to Spectroscopy for Biochemists, Academic Press,
London, 1980.
4. Rao, C.N.R., Ultraviolet and Visible Spectroscopy, Butterworths, London, 1975
.

Biophysical Chemistry
Fluorescence
1. Udenfr/en, S., Fluorescence Assay in B{ology and Medicine, Academic Press, Ne
w York, 1969.
2.
Willard, H.H., Merritt, L.L., Dean, J.A. and Settle, F,A., Instrumental
Methods of Analysis,
Wadsworth Publishing Company, USA, 1986.
3.
Konev, S.V.0 Fluorescence and Phosphorescence of Proteins and Nucleic Ac
ids, Plenum Press,
New York, 1967.
Infrared Spectrophotometry
I.
Wood, D.L., Infrared Spectrophotometry in Methods in Enzymology, Vol. IV
, Academic Press,
London, 1957.
2. Rao, "C.N., Chemical Applications oflnfrured Spectroscopy, ,cademic Press, Ne
w York, 1963.
3.
Colthup, N.B., Daly, L.H. and Wiberley, S.E., Introduction to Infrared a
nd Raman Spectroscopy,
Academic Press, New York, 1975.
Flame Emission and Atomic Absorption Spectrophotometry
1. Alkemade, C. and Herrmann, R., Fundamentals of Analytical lame Spectroscopy,
Hflger, Bristol,
1979.
Pinta, M., Atomic Absorption Spectrometry, Hilger, London, 1975.
,2.
1.
2.
3.
4.
ESR
I.

2.
3.
Metcalfe, J.C., Nuclear Magnetic Resonance Spectroscopy in Physical Prtnc/ples a
nd Techniques
of Protein Chemistry, Part B, Academic Press, New York, 1970.
Haschemeyer, R.H. and Haschemeyer, A.E., Prote/ns, John Wiley, New York, 1973.
Opella, S.J., BiologicaI Nuclear Magnetic Resonanc Spectroscopy, Science, 198 :
158-165, 1977.
Moore, G.R., Ratcliffe, R.G. ad Williams, R.J.P., NMR and the Biochemist in Essa
ys in
Biochemistry (P.N, Campbell and R.D. Marshall, Eds.], Academic Press, London, 19
83.

Willard, H.H., Meritt, L.L., Dean, J.A. and Settle, F.A., Instrumental Methods o
f Analysis,
Wadsworth Publishing Company, USA, i986,
Jost, P.C. and GriiTith, O.H., The Spin-Labeling Technique in Methods in Enzyrno
logy, Vol.
XLIX, Academic Press, London, 1978.
Berliner, L.J., Spin Labeling in Enzymology : Spin-Labeled Enzymes and Proteins
in Methods
in Enzymology, Vol. XLIX, Academic Press, London, 1978.
Luminometry
I.
Campbell, A.K., Holt, M.E. and Patel, A. (1985) in Recent Advances in Cl
inical Biochemistry,
Vol. 3, p. 1. Edinburgh : Churchill Livingstone.
2.
Geiselhart, L., Osgood, M. and Holmes, D.S., Construction and Evaluatlon
of aSelf-Luminescent
Biosensor, Ann. N.Y. Acad. Sci., Vol. 646 : 53-59, 1992.
3 King., J.M.H., eL al., Science, Vol. 227 : 778-781, 1990.
4, Rechnitz, G,A,, Chem, Eng. News, Vol. 66 : 24-36, 1988,
Exerclse
I.
Substances A and B absorb at both 475 nm and 500 nm. These substances ar
e given to you
in a single solution which shows an O.D. of 0.62 at 475 nm and 0.54 at 500 nm. T
he a of
" compound A at 475 nm is 12000 and at 500 nm is 4000, Compound B has an a of 50

00 at
475 nm and 11600 at 500 nm. Calculate the concentrations of the two substances i
f the.
cuvette used to take the reading has a path length of I cm.

269
[A] =0.42 x I0-aM,[B] = 0.37 x I0-M
To a 1.5 ml of a solution containing the enzyme glutathione reductase and an unk
nown
concentration of oxidized glutathione, was added an equal volume of a solution w
hich was
2x 10 M with respect to NADPH. The O.D. at 340 nm stabilized at 0.25 in a 1 cm c
uvette.
Given that the glutathione reductase reaction is as under
Oxidized glutathion + NADPH + H reduced glutathione + NADP*
and that the reaction proceeds completely to the right hand side, can you calcul
ate the
concentration of oxidized glutathione In 1.5 ml of the solution? a of NADPH at 3
40 nm
is 6220. (Hint. First calculate the O.D. of NADPH due to the given concentration
)
Concentration of oxidized glutathione = 0.196 x 10- M in the original solution.
3.
The enzymes glutamate-oxaloacetate transminase (GOT) is assayed by a cou
pled system,
having the following reactions.
(a) aspartate + a-ketoglutarate----- glutamate + oloacetate
(b} oxaloacetate + NADH malate + NAD
(the first reaction is catalyzed by GOT and the second by malate dehydrogenase).
Large excess of NADH is added which gives a good O,D. at 340 nm. The second reac
tion
depletes NADH depending upon the concentration of oxaloacetate generated in the
first reaction. Since both the reactions are going on simultaneously, the rate o
f decrease
of O.D. is driven by the rate of appearance of .oxaloacetate and is therefore th
e index of
GOT activity.
On the basis of the above solve the following problem.
A reacUon mixture containing excess asparate, 0.1 ml serum (which contains GOT).
0.3 }
mole of NADH, and an excess of malic dehydrogenase in a total volume of 0,9 ml.
The
reaction is started by adding excess of a-ketoglutarate in 0.1 ml. The absorbanc
e decreases at the rate of 0.04 unit/rain. The cuvette has a path length of 2 cm
. Calculate
the concentration of GOT in the serum as unltsml.
3.21 x 10-2 units/ml
Calculate the O.D. of the following soluUons at 340 nm in a 2 cm cuvette.
(a) 7.3 x 10-MNADH (b) 9 x 10-2MNADPH (c) 2.2 x 10-MNADH
(a) 0.91 (b) 0.11 (c) 0.27
Make a study of glucose metabolism and on the basis of your study describe a pro
bable

method for spectrophotometric measurement of giucoose-l-phosphate based on light

absorption at 340 nm.
6.
You have prepared two concentrated solutions adenine and guanine. Inadve
rtantly. you have
forgotten to label the flasks and now you are not sure which flask contains what
. Can
spectrophotometry resolve the confusion?
7.
You have two samples of DNA derived from different organisms, A and B, D
NA from A shows
an increase in optical density beginning at 70C and a plateau at 80C. DNA from B s
hows a
corresponding increase in O.D. beginning at 65C and plateaus out at 76C. What is y
our
conclusion?
8.
There is Just one phenylalantne residue in a given protein which is abso
rbing maximally at
259 nm with an a, of 206 (both of which are very near the normal values) if the p
rotein has
been dissolved in 100% water. If the protein is now dissolved in 70% water and 3
0% glycerol
the wavelength of maximal absorpUon changes to 265 nm and extlncUon goes up to 2
17. If
the protein is brought b.ack to 100% water, the values also return to the origin
al values. What
is your conclusion?

270
9.
Biolhysica Cherrstnj
A protein is o to possess a se oph d a se phenyle residue, At a
concenton of 55 mg/ml ff eylene #ycol is added to e soluon to a level of 3/0 go
e
mmum absoflon velen d e ecflon increase as eomped to what ey were
in 0. I M NaCI. If the concenaflon ofe protein is cased to 5 mg/ml 0.1 M NaCI, g
o e X d e encflon increase Howler, if eylene glycol Is added now at is hi
ptein concentration, no fuer specM chges e seen. at conclusions may you draw
about e prote ?
I0
at is absoflon mum ? Does e sctur chmcter d the posion of absoflon
mum depends sole upon e scture of e compound ? Discuss.
11.
at mgement may be present some specophotometers If e eors due to voltage
fluctuaons e to avoided togethe Discuss.
12. Discuss factors which give flse to fluorescence. y e fluorescence spec bd sp
ec ?
13.
A given protein is o to con 12 tophs. If cesium long quench 25% of e
fluorescence, is it vd to conclude at ree of e tophan residues e on e surface,
If not. what consideraOons me e conclusion invMid?
14.
A ven protein conns oy one toph which is shong a fluorescence mum as
at for flee toph. Yet, e intensi of fluorescence is inordinately low. at do you
conclude? er, acid flaflon of e protein tes you at e pK of sion is due to a
cbol. so e acid titration er decreases tenai. at do you ow about is
toph no -- 15.
en S Is bound to a ven prote, fluorescence is obned. However, ff e prote
concenaflon is incased fore addg S, fluoence decreases. Your coen? (Note.
More th one elaon is possible).
16.
Descb briefly e eo of n specome. at foaon c be obned from e
n absoflon s?
17.
at is met by e te chec sh? WHte eples, e shielding d deshieldg effects i
nvolved n specome.
18.
at foaflon is prodded by e positions of sign.s specm? How my sign.s e ee
cted. each of e foOt.g:

(a) prope, ) eol, (c) buol d (d} eyl acetate?

9
OTHER OPTICAL TECHNIQUES FOR
MOLECULAR CHARACTERIZATION
CIRCULAR DICHROISM AND OPTICAL ROTATORY DISPERSION
Natural unrefle.cted light from any light source behaves as if it consisted of a
large number
of electromagnetic waves vibrating in all possible orientations around the direc
tion of propagation.
This unpolarized light can be resolved by Nicol prism into two beams in which al
l the rays
vibrate in only one particular plane. These beams are known as plane polarized (
of course,
since a light wave consists of an electric and a magnetic component vibrating at
right angles to
each other, the term 'plane' in not so right, but the ray can be said to be plan
ar if the meaning
is restricted to convey the position of the electrical component only). Each one
of the plane
polarized beams emerging out of the Nicol prism can be further resolved into two
beams of
circularly polarized rays by a device known as Pockels cell. A plane polarized b
eam is, therefore,
considered to be a sum of two circularly polarized components (Figure 9.1), the
right and the
left circularly polarized components.
If we pass these circularly polarized rays of light through an anisotropic mediu
m, it would
be seen that the plane of polarization has been rotated (Figure 9.1). This rotat
ion of the plane of
polarization might be due to either of the two phenomena considered below.
(i)
An anisotropic medium can refract the right (R) and left (L) components
of the plane
polarized light to a different extent so that I'll nR, where n = the refractive
index. This difference between the indices of refraction of the medium for the r
ight-hand and the
left-hand ray results in retardation of one component more than the other. The
observable result of this preferential refraction is a rotation of the plane of
polarization
since the angle a and no longer remain identical. If the medium preferentially r
efracts the left circularly polarized component the resultant would be a plane p
olarized ray
i'otated somewhat to the right from its original position (Figure 9.1), and vice
versa.
This phenomenon is known as optical rotation, The extent and even the sign of op
tical
rotation differs at different wavelengths. This wavelength dependence, of the op
tical
rotation is known as optical rotatory dispersion.
(//)
If the plane polarized ray has the wavelength falling within the range o

f the absorption
maxima of the anisotropic medium, the medium will absorb it. The optically activ
e
medium, however, shows a preference for absorbing either the left or the right c
ircularly
polarized component.

272
b
I
I
I I
(B)
Figure 9.1 (A) Representation of a plane-polar&ed ray as a sum of two clrcularly
polarlzed rays. EL (s the left and
(B)
Result of the passage of plane-polarlzed light through an optically acti
ve meal:lure. The plane of
polarized l@ has been rotated due to retardation of one of the ciradar component
s, a denotes
optical rotation.
In the previous case of optical rotation due to refraction both the refracted co
mponents
had equal intensity. In this case, however, since one of the components is being
preferentially
absorbed, its transmitted portion will have less intensity than the incident ray
. Thus; in this
case, not only will the plane of polarization be rotated, but the two transmitte
d components will
also differ in intensity. The resultant light is said to be e///pt/ca//y po/ar/z
ed (Figure 9.2]. The
dierential absorbance of the circularly polarized light resultlng from a differe
nce in absorpttvtty
of the two rays is called circular dichroism (CD).
Figure 9.2 Elliptlcalpolarlzatn oftlght upon leaving an optically active medium
in the absorption range of an optleally
active troJitWn` Note that E is shorter than E. 8 describes the CD ellpiaj. It i
s coJculated from the
following relatlonshtp:

Other Optical Techniques for Molecular CharacterizatWn

273
We will now discuss the theory behind circular dichrolsm and optical rotatory di
spersion
individually (as we will see in subsequent pages, optical rotatory dispersion an
d circular dichrolsm
are mathematically commutable so that one can be known from the value of other;
it is only for
the sake of understanding that they are being discussed independently).
The circular dlchrolsm spectrmn may be thought of as the difference between two
absorption
spectra, one obtained using the left while the other using the right circularly
polarized component.
If this s so, one can apply Beer's law to circular dichrosm.
Thus
AL = aLCb
and
A = aCb
where the two subscripts L and R indicate the results obtained employing the lef
t and right
circular components, respectively. Taking the difference, we get
AL-A = (aL--aR) Cb,
... a = -aR = -AICb
extinction coeffcients. The usual representation
spectrum is to demonstrate Aa as a function of wavelength. As circular dichroism
depends
only in those spectral regions where absorption occurs
will not be observed in the regions where no absorption occurs. In the latter sp
ectral
aL and a will be zero.
There are two major differences between CD spectra and absorption spectr
a.
(0
Extinction coeffcients are always positive. However, Aa can assume eithe
r a positive
or a negative sign depending on the relative sizes of aL and aR. Thus. optical i
somers of
any given compound at a given wavelength will give rise to CD spectra of opposit
e
signs, that is
aa isomer = - a isomer
(/0

CD spectra and the absorption spectra differ in the relative size of a a

nd the average
extinction coefficient, (aL + a/2. aa for most of the molecules of biol0gical in
terest are
less than I% of (aL +
From the second point above, we can understand that the value of Aa is very smal
l. How
do we measure it to get the CD spectrum? Ideal situation would be to fill a cons
tant pathcell with a solution of particular concentration C and then measure absorbance d
ue to
left and right components (AL and A) individually and then arrive at the differe
nce
one from another. In actual practice a plane polarized beam is passed through
quarter-wave plate which is rotated from +45 to -45 producing first d and then I c
ireularly
measures the periodicvariation in the transmittance
: when the incident radiation experiences periodic changes in the handedness, ff
we use the
relationship between A and.T,
(A = -log T), we get
a = (-log TL + log TICb
= l/Cb log TR = log Is/I
T. Cb
I,./Io
= log Iz
Cb
IL

274
Biophysical Chemistry
where I,. and IR are the intensities transmitted. If the sample does not exhibit
circular dichroism,
TR and TL will be the same and log (TR/TL) will be zero. On the other hand, if t
he sample shows
circular dichroism, the oscillation frequency of the transmittance will be same
as the oscfllaUon
for the handedness of the incident beam. The circular dichroism of the sample is
determined
from the magnitude of the oscillation in the intensity of the transmitted beam s
triking the
detector.
Since the projections of the electric vectors in circular dichroism differ not o
nly in their
angular velocity but also in length (Figure 9.2), the polarization of light leav
ing the medium is
elliptical. Due to this, circular dichroism is also expressed in terms of molar
ellipticity [0]. The
units of [0] are degree-centimeter squared per decimole (deg-cm21dmole). Those o
f ha on the
other hand are centimeter squared per mfllimole (cm21mmole). The following equat
ion relates
and Aa.
[0] -- (3300 deg mmole/dmole) Aa
Optical Rotatory Dispersion
In contrast to circular dichroism, an optically active sample gives rise to opti
cal rotation
even at wavelengths which do not fall within the region(s) in which the sample a
bsorbs. This is
so since refractive indices are never zero even in regions where the sample is t
ransparent (does
not absorb). Thus even those solutions which do not absorb in the ultraviolet re
gion wig glve
optical rotation in these regions.
Optical rotation, a, is given by
b
a = 180 -(I'll - nR)
However, the common form in which optical rotation is reported is either the spe
cific
rotation [a], where [(] = 100 alCb, or the molar rotation (m'), where(m) = [a] M
/100.
The units for the specific rotation are deg-cm/decagram. The light path b is exp
ressed in
decimeters and the concentration C is expressed in grams/100 cm3. If the specifi

c rotation is
multiplied by molecular weight M and divided by 100, it gives rise to molar rota
tion whose units
are deg-cm/decimole. Note that the units for [M'] and [0] are identical. Experim
ental
determination of [M'] at different wavelengths would give rise to optical rotato
ry dispersion
spectrum.
CD and ORD Spectra
Figure 9.3 depicts absorption, circular dichroism and optical rotatory dispersio
n spectra
for a hypothetical optically active substance which has a single absorption band
. The wavelength
at which the maximum absorption takes place, ,=, is denoted by a dashed line. Th
e similarity
between the absorption and CD spectra wig become obvious from the figure. On the
other
hand, the ORD spectnzm shows a more complicated pattern; no optical rotation is
observed at
X,, but maximum optical rotation is obtained at wavelengths slightly away from ,
=. Even
those regions which are far removed from Zm, x exhibit some optical rotation. Th
is property of
opUcal rotatory dispersion is very useful for compounds which absorb maximally a
t vacuum
UV where determination of CD is tedious; for such compounds optical rotatory dis
persion can
be measured at wavelengths far removed from the vacuum UV. If measurements of op
tical
rotation are limited to these regions which are far removed from the absorption
bands, the
molar rotaUon is described by the Drude Equation.

275
O.lm'l
Other Optical Techniques for Molecular Character/zat/on
9.3 Idealized absorption, clrcular dichrolsm and optical rotatory dispersion spe
ctra for a hypothetical compotmd
which s optk:ally active. It s assumed that the compound has only one absorption
band. Absorption
maxlmo are denoted by the dashed vertical lines. (A) and (B) denote spectra of c
ompounds which are
rrdrror brcu3es of each other. Further dLscusskm n the text.
where K and o are constants. Rearranging the above equation we get
.2 [m'] -- . [m'] + K
It would seem from the above equation that a plot oD.:[m']versus[m'] will be a s
traight
llne. In fact the above equation fits optical rotation of transparent aqueous so
lutions of numerous
biological compounds in the visible region of the spectrum. If the equation does
not make a fit,
the inference usually is that the visible optical rotation of the biological sam
ple is due to
contribution from more than one optically active absorption band.
"
Figure 9.3(B) depicts the absorption, circular dichroism and optical rot
atory dispersion
spectra of the mirror image of the hypothetical molecule considered in F
igure 9.3(/I). Note that
while the absorption spectra in both cases are identical, the circular d
ichroism and optical
rotatory dispersion spectra have changed signs If a racemic mixture of t
his hypothetical
compound is subjected to the above experiment, the absorption spectnm wo
uld still be identical
but the circular dichroism and optical rotatory dispersion spectra would
cancel out.
It is interesting to note that the ORD curve resembles the first derivative of t
he CD curve.
ORD and CD are therefore coupled phenomena which in principle are mathematically

commutable. It is possible to calculate CD spectrum of a given compound from its

ORD spectrum
by applying the mathematical relationship known as Kronig-Kramer transform. Inte
rpretation

276
BiophysPzd ChemLstry
of ORD spectrum is made difficult by the fact that it is complicated and extends
to regions far
removed from ,,. This is especially so for those molecules which have multiple o
ptically active
absorption bands. For this reason circular dichroism, which is easier to interpr
et, is preferred.
Applications
(1) CD/ORD of Proteins ,
Optical ativity of a macromolecule is usually a resultant of three factors. In t
he first
place, the constituents of the macromolecule; for example proteins are made up o
f L-amino
acids and these residues would contribute to optical activity. Secondly, these r
esidues might be
arranged in an ordered fashion such as the DNA double helix or the a-helices of
proteins, and
this arrangement is also a basis for optical activity, Thirdly, an asymmetric di
stribution of
charges or dipoles about a chromophore, as prevalent in a rigid tertiary structu
re, will also give
rise to optical activity.
Obviously, if one determines the optical activity of a given macromolecule, an a
nalysis will
provide good information about the general structure of that macromolecule. ORD
and more
preferably in modem days, CD spectra are used to deduce the chain conformation o
f
macromolecules. Although, by modem standards, X-ray diffraction crystallography
is the method
of choice for structure determination of biopolymers, spectropolarimetry continu
es to make
important specific and complementary contributions. X-ray analysis might be virt
ually impossible
if sufficient quantity of the material is not available or if the molecular weig
ht is too high or if
crystallization is a problem. In all these cases spectropolarimetry comes handy.
Besides, this
method consumes very little time as compared to X-ray analysis which is an extre
mely lengthy
procedure.
To understand how measurement of optical activity can provide us an idea about t
he
conformation of a macromolecule, we will consider an example of synthetic polype
ptide poly-Llysine. This synthetic polymer is readily soluble in water at pH 5.7. At this pH
the e-amino
groups are protonated. Consequently the polymer can exist in a variety of confor
mations. The

polymer may then be thought as existing in a random cot state in solution. Obser
ve the CD
(Figure 9.4(B)) and ORD (Figure 9.4(A)) of this random cot (only CD is discussed
; observe,
however, that wherever extinction is maximum for CD, ORD is zero). There is a we
ak positive
circular dichroism at 217 nm and a strong negative circular dichroism at 197 nm.
If one removes the positive charge of z-amino groups by raising the pH of the so
lution to
1 I. 1, the polymer adopts an ordered structure known as the a-helix. Observe th
e drastic change
in the CD and ORD as a consequence to this confo.rmational change. Negative circ
ular dichroism
is now seen at 208 and 222 nm and a strong positive circular dichroism is seen a
t 191 nm. The
synthetic polypeptide can be converted to the -pleat sheet structure by heating
at 52C for 15
minutes and then cooling. Again, the CD and ORD undergo drastic change. A positi
ve circular
dichroism is now seen at 195 nm and a negative band is seen at 217 nm. It may be
mentioned
that while all these drastic changes take place in the optical activity as a con
sequence of changes
in conformation, not much change in the absorption spectrum is observed which ex
hibits only
one absorption maximum for all conformations at i90-200 urn.

277
-20
Other Optical Techniques for Molecular Characterization
[m'] x 10
CURVE
A
I I. I00% a Helix
Rdom coil
190200 210 220 230240250
CURVE'
I. 100% a Helix
2. ioo%
3. 100% Random
coil
19o 200 'i0 220 230 240,250

9.40pti6al rotatory dispersn (A) and circular dichroism (B) of poly-L-lysine in

the a-helicczi random coil, and
-conformatins. Descriptn in text. (Adapted from. Greenfield. N.. and Fasman. G.D
., Biochemistry, 8:4
108, 1969.)
Let us see whether the same pattern of optical activity is found for other prote
ins of
conformation. Figure 9.5 depicts CD spectra of two proteins, sperm whale myoglob
in
phosphate buffer pH 6.86. From X-ray diffraction studies we know that
whale myoglobin exists largely as an a-helix (about 80% of the amino acids are p
resent
eight segments of a-helix).
200
220
WAVELENGTH {nm)
L20

240 .-0 220

240
WAVELENGTH (rm)

9.5
C'radar dhrolsm spectra for (A) sperm whale myoglobln, and (B) eoneanava
lm A,
(A) Sperm whale myoglobin in phosphate buffer, pH 6,86.
(B) Concanavaltn A n phosphate buffer (curve I) and concanavaln A n phosphate bu
ffer + 0.018 M
sodan d.ajl sulphate (coe 2). Descrtn m tex (Adapted from. Maitre, W.L et. oi.,
mochomtry,
15 : 4264, 1976)

278
Bophysk Cherrrlstrg
Observe that its CD spectrum shows the same qualitative features as observed for
a-helical
poly-L-lysine. The CD spectrum for concanavalin A has the same qualitative featu
res as that of
poly-L-lysine -pleat sheet structure (Figure 9.5(B)). X-ray diffraction studies
confirm that there
is no a-helix in concanavalLn A and about 50% of the amino acids adopt the -plea
t sheet
structure. Figure 9.5 also depicts CD spectrum of cocanavalin A upon addition of
sodium
dodecyl sulphate. The spectrum shows the same pattern.as expected of an a-helix.
We can infer
that the detergent sodium dodecyl sulphate has induced formaUon of a-helix in co
ncanavalin
ConformaUonal changes in the enzyme do take place when a substrate interacts wit
h it.
We have already seen that CD and ORD are responsive to conformational changes. T
herefore
CD and ORD measurements provide a sensitive method of studying enzyme substrate
complexes,
A case in point is the study carried out by Teng et.al, on the enzyme p-hydroxyb
enzoate
hydroxylase. The CD of pure enzyme is shown in Figure 9.6. It was known that the
enzyme has
a tightly bound FAD moiety. One of the substrate for it was p-hydroxybenzoate. H
owever, it was
not definite whether NADH, or NADPH was the other coenzyme needed. A CD.spectra
was
obtained for the enzyme in the presence of NADH and NADPH, The results of the ex
periment
are shown in Figure 9.6. From the results it is clear that upon NADH addition th
e CD spectrmn
of the enzyme solution does not change significantly, whereas upon NADPH additio
n the CD
spectrum of the enzyme solution undergoes significant alteration. It can therefo
re be concluded
that the enzyme specifically binds NADPH which must be the coenzyme used in" the
reaction.
i
ENZYME + NADH
0.02
ENZYME + NADPH
|
Figure 9.6 Effect of NADPH on CD spectrum of the enzyme p-hydroxybenzoate tujdro
lase. Description tn text
(Adopted from Teng, N., eL al,, J. BioL Chem.. 246 : 5448-5453, 1971).

Figures 9.4, 9.5 and 9.6 show that CD and ORD are responsive to conformational c
hanges
of proteins. This is the reasonwhy CD and ORD are extensively used to study conf
ormational
changes in proteins and polypeptides. It might, however, be pointed Out that rel
ationship between
ORD, CO, and chain conformation is not as simple as we hope it tobe. Many protei
ns which
have been shown to exist largely as a-helices do not show the qualitative featur
es of a-helix
shown in Figures 9.4 and 9.5. Moreover, changes in optical activity may not be t
otally assignable
to conformational changes; solvent effects might also play a large role. With al
l these ambiguities,
CD and ORD remain important methods to study the structure of proteins.

-7
220 240
260
Other Optical Techniques for Molecular Characterization
279
(2 CDORD of Carbhydrate
CD and ORD are also frequently used in studies on carbohydrates. CD can indicate
the
nature, extent, and position of substitution in carbohydrates. Perhaps the best
example of
such studies is afforded by studies on oligosaccharides from blood-group substan
ces and human
milk.
Chain conformations of a-and -llnked glycan derivatives have been studied throug
h CO.
The studies suggested that x-linked amylose tricarbanilate adopted a hellcal con
formation,
whereas the -linkedcellulose derivative did not. The CD spectra of these derivat
ives is shown
in Figure 9.7. Frequent use of CD has been made to study not only the conformati
on of other
polysaccharides but also their association in certain polysaccharide gels like a
lginate and pectin.
OR
CHOR
OR
IAI 6-1,4
CH2OR
CH20R
OR "
OR
(B) a- 1,4
G.R., in New Techniques n Bphys.s Cell Biology, Vol. I, Wtly, 1973).
An extremely important application of CD is its use as a 'finger-printlng' techn
ique for
qualitative analysis of trace amounts of carbohydrate materials such as mucopoly
saccharides.
9.8 shows the CD spectra of dermatan sulphate (a mucopolysaccharide containing 1
,3sugar) and heparitin sulphate (a mucopolysaccharlde containing 1,4-1inked
sugar). The s ctra for these two mucopolysaccharides differ from each other

I01
----Dermatan Sulphate
8
/!
Heparifln Sulphate
Fre. 9.8CD spectra of two mv.copolyso.ccharldes. (Adapted from Morris, E.R., and
Sanderson, G.R., in New
in Bhyslcs md Cell BWlogy, Vol. I, Wiley, 1973)

4x I0"
2x 10
-4x I0
DenatUred
28o
BphsP.al Chemtry
significantly and this difference lends itself to an important clinical applicat
ion. An accumulation
of these two polysaccharides occurs in the urine of patients with the Hurler syn
drome. The
syndrome has two variants: the Sanfllippo variant where the primary polysaechari
de accumulated
is heparitin sulphate and the Hurler variant where a mixture of dermatan and hep
aritin sulphate
is excreted. Through chemical means it is very difficult to identify the two muc
opolysaccharides
as separate from each other. However, if after some preparation the urine is sub
jected to CD,
an identification is readily made. CD has also been used as a quantitative metho
d for estimating
optically active polysaccharides such as mucopolysaccharides. Here it is extreme
ly useful because
it can measure a very small amount of optically active polysaccharide present in
a large excess
of optically inactive substances (e.g., pectin in the presence of citric acid an
d sucrose).
(3) CD/ORD of Nucleic Acids
Over the years, CD and ORD have also been extensively used in the study of nucle
ic acids.
The ribose phospIiate backbone of nucleic acids does not absorb significantly ab
ove 180
nm. Therefore when we measure CD of nucleic acids, the most important contributo
rs are the
bases : the purines and the pyrimidines. In themselves, both these kinds of base
s are symmetrical
chromophores. However, they become optically active when attached to a ribose su
gar by means
of an N-glycosidic bond. Their optical activity increases further when they assu
me helical
structures.
At 270 nm, the purine bases have. a negative signal whereas the pyrimidine bases
have a
positive signal. Since the bases contribute to the CD signal owing to the chiral
ity induced by
their attachment to ribose, the base composition of a given nucleic acid should

contribute to
the CD spectrum. It does also. However, this contribution is almost insignifican
t when compared
to the contribution made by the orientation of the bases. We know that the bases
are stacked
over each other. It is this stacking that contributes most to the CD spectrum of
the nucleic
acids. CD spectra therefore give us more information about the conformation of n
ucleic acids
rather than their composition.
-2 x I0
..Ac acid
poly (A)

240 260 .280 300

(rim)
Figure 9.9 CD spectrum of adenylc acid, polyadenyl(c add [poly (A)] and denature
d poly (A). Explanation n the text.
CD (and of course ORD) is therefore an extremely sensitive probe of conformation
al changes
in polynucleotides. How sensitive this probe is will become clear ff one compare
s it with simple

I0
5
Other Optical Techniques for Molecular Characterization
281
absorption spectrometric studies. If we study the absorption of light of 258 nm
by adenylic acid
and by polyadcnylic acid, we find that the latter absorbs 26% less than the form
er. The
hypochromicity is due to the formation of an ordered structure by the polymer. N
ow take a look
at the CD spectrum {Figure 9.9} of adenylic acid and polyadcnylic acid. The diff
erence between
the CD spectra of the two is much larger than the 26% decrease in absorption. Al
so shown in
the same figure is the change that occurs in the CD spectrum of polyadenylic aci
d upon
denaturation. Again note the large difference in the native and the denatured sp
ectra. It is
exactly due to such extreme differences that CD is such a sensitive probe of cha
nges in nucleic
acid conformation. Since it is such a fine probe, CD/ORD is used to study (1) si
ngle-doublestrand transition of nucleic acids, (2) denaturation of nucleic acids due to var
ious agents such
as temperature, pH, ionic strength, etc., and (3) binding of proteins, other sma
ll organic
compounds as well as cations.
A few examples of the use of CD/ORD in studying nucleic acids are provided below
.
of B-DNA is D/fferent as Compared to A-DNA
B-DNA is the native form of DNA; what this means is that the DNA in our cells is
in this
Outside the cell, we can obtain B-DNA when the humidity is 92% and the
is an alkali metal such as Na. This is a right handed helix with a diameter o{20A
.
helical twist per base pair is 36 and the pitch is 34. There are I0 bases per turn
of the
spectrum of B-DNA is shown in Figure 9.10. Note the negative band centered aroun
d
nm and a positive band around 275 nm. The zero occurs at about 258 nm.

-I0
-15

210
230
250
270
290
310
Wavelength (nm)

Figure 9. I0 CD of B-DNA. Explanation in the text

this with the CD of A-DNA (not shown in the Figure 9. I0). A-DNA is 26 ,/ in
has 11 bases per turn of helix, has a helical twist of 33 per base pair, and a pi
tch of
This DNA shows an intense positive band at 190 nm, a good negative band at 210 n
m, and
positive band at 260 nm.
Curiously, the samecharacteristlc is given by duplex RNA. It may therefore be as
sumed
duplex RNA assume the A-form.
Base-Stacking
ORD spectrum of polycytidylic acid shows that the [m]292 is 35,160 deg M-Icm-. T
his high
value means that poly(C) is very asymmetric. In other words this means that it i
s helical. The
that suplort this helicity can also be determined easily.. Addition of formaldeh
yde does
change [m]29 significantly. Formaldehyde is a compound that disrupts hydrogen bo
nding.
were a function of hydrogen bonding between bases, formaldehyde addition should

282
Biophysical Chemistry
have reduced [rn]292 considerably. Since this did not happen we believe that hyd
rogen bonding
does not contribute significantly to the helicity of the polynucleotide.
On the other hand, when ORD spectrum is determined when poly(C) is suspended in
90%
ethylene glycol, the [m]29 is found to be 7223 deg M-cm-. This is a huge reducti
on and it
means loss of asymmetry or of helicity. Ethylene glycol disrupts interaction bet
ween hydrophobic
groups. A reduction by such a substance means that the helix was mainly supporte
d by
hydrophobic associations between the bases. ORD spectra therefore provide a very
good support
for base stacking.
DNA has a Different Conformation in Chromatin
Does DNA have a different conformation in chromatin as compared to the conformat
ion in
the free state? It would appear so from the X-ray diffraction studies and the CD
studies. When
bound to histones, the CD band for DNA shows about half the maximum ellipticity
and half the
area of the spectrum for free DNA. From the example about base stacking given ab
ove, we know
that the DNA CD spectrum is mainly due to base stacking. Therefore, the chromati
n spectrum
may be telling us that the bases .are less stacked in chromatin. The point is co
nfirmed by X-ray
diffraction studies which give the same result.
X-ray diffraction is a far better tool to obtain detailed structural information
. However, CD
scores over the former in that it is rapid and can give reliable information abo
ut DNA-histone
interaction as well as about the parameters that affect this interaction. X-ray
diffraction studies
are not such a good idea when we want to study the DNA-histone interaction depen
dence upon
such factors as the pH, ionic strength, and the concentrations of the interactin
g species. As a
result of CD studies we know that DNA interacts with different histones differen
tly and with
different conformational changes in each case.
Instrumentation for Measuring Optical Rotatory Dispersion
Optical rotatory dispersion is measured by a spectropolarimeter. The instrument
possesses
the following basic parts:
(i) A light source

(ii) A monochromator
(///) A polarizer
(iv) An analyzer
(v) Sample tubes
(vt) A photomultiplier
The light source can be a tungsten filament lamp, if a continuous light source i
s desired.
For specific wavelengths xenon-or mercury-vapor lamps are used. If tungsten fila
ment lamp is
used, the monochromator has to be sophisticated. However, with either sodium vap
or or mercury
Vapor lamps, simple filters are required (this is because these two lamps emit l
ight of a limited
number of wavelengths, for example sodium lamp emits only two-589, and 589.6 nm)
.
Polarizer can be any of the three types: a Nicol prism, Glan-Thompson prism, or
a polaroid
filter. The last is the least desirable as it never gives 100% polarization. The
Glan-Thompson
prism is the best and used in most spectropolarimeters.
When the sample is introduced between the polarizer and analyzer prism, optical
rotation
is produced, i.e., the plane polarized light emitted by the polarizer is rotated
. The extent of
rotation can be measured by rotating the analyzer with respect to the polarizer
till the rotation
is fully compensated. In more sophisticated instruments, a quartz-wave compensat
or is
introduced. In these instruments, the polarizer and the analyzer are left perman
ently crossed

Other Optical Technkltes for Molecular Characterization

283
with respect to each other. The optical rotation produced is then compensated by
a piece of
quartz which rotates light in the opposite direction to that of the sample. The
two quartz pieces
are laevo-and dextrorotatory.
Instrumentation for Circular Dichroism
Even an ordinary spectrophotometer can be adapted to measure circular dichroism.
The
only necessity is to provide some means of producing d and I circularly polarize
d radiation. The
usual arrangement for this is to pass a plane polarized beam (obtained through a
ny of the
polarizers listed above for polarimeters) through a quarter wave plate. A rotati
on of this plate
from +45 to -45 produces respectively, d and l circularly polarized radiation. If
a large
wavelength region is to be covered, several such quarter-wavelength plates would
be required,
each catering for a particular band-width. The absorption by the sample of the r
ight and the left
circular components is then alternately measured to arrive at the CD spectrum.
Specialized instruments measuring circular dichroism are also available and have
a
wavelength range of 185-600 nm. Recently composite CD/ORD measuring instruments
have
also become available and offer a wavelength range of 185-800 nm.
Source
Lin(ar
e
polarizer
Light of changing
circular polarization
Photomultiplier
er

Figure 9.11 Depicts the schemat/c diagram of specialized CD instruments

CD measurements would, in principle, require two light sources: one for L and th
e other
R circularly polarized light. Each would have a monochromator for wavelength sel
ection.
commercial instruments have just the once source from which L and R circularly
light is generated. The trick here is to pass the plane-polarized light through
a crystal

is subjected to an alternating electric field. The crystal is called an electroo

ptic modulator
a photoelastic modulator (the older CD instruments have a Pockels cell in place
of this
It has a remarkable property in that the polarity of the field determines which
component will be transmitted. The current is alternating and therefore the L an
d R
are transmitted alternately. The beam is then allowed to pass through the sample
it is transmitted to the photomultiplier. The output of photomultiplier is then
processed to give the final result. CD instruments normally operate in the
range of 185-600 nm. Recently composite CD/ORD measuring instruments have
also become available and offer a wavelength range of 185-800 nm.
ROTATIONAL DIFFUSION
We have seen that molecules in solution show translational movement caused by th
e
of the solvent. In addition to this translational movement, each solute molecule
.
to its centre of mass. This motion is known as rotational diffusion and is descr
ibed
diffusion coefficient O. It has the units of reciprocal seconds and expresses

Molecule
284
Biophysical Chemistry
the average angle of rotation per unit time. Rotational diffusion coefficient, w
hich is a constant
for a given solute, can be expressed in terms of frictional coefficient
e ' KTI frot
Rotational diffusion coefficient is related to the dimensions of the solute part
icle. Consequently
its measurement assumes importance since it can provide an idea about the dimens
ions of the
solute particle. Its measurement is particularly valuable in case of molecules o
f high axial ratio
(length/width; for example long thin rods) where it can provide a precise measur
e of the length
of the solute particle in solution.
Three methods are mainly used to determine rotational diffusion coefficient. The
y are
(1) flow birefringence, (2) electric birefringence, and (3) polarization of fluo
rescence. Of these,
although electric birefringence can yield 0, its main use is in the study of dip
ole moments. All
the three methods of determining 0 are discussed in the following pages.
(1) Flow birefr/ngence
Birefringence means double refract/on. The double refraction can be given rise t
o by the
mIsotropy of molecular arrangement which causes I/ght to travel along one ax/s o
f the crystal
with a different velocity as compared to the velocity at which it t.ravels along
the second s,
Many Crystalline substances are b/refringent since they possess two indices of r
efraction along
different es. Such substances m/ght give rise to certain recognizable optical ch
aracteristics
when studied under polar/zing light microscope. Solution of m/sotropic molecules
, on the other
hand, does not give rise to b/refringence. This is so since, at rest, the molecu
les in e solution are randomly oriented. If however, the molecules are forced to
zltgn themselves in a
uniform way either by imposing an electrical field (electr/cal btrefringence, el
ectr/cal double
refraction) or by means of hydrodynmIc shear (flow b/refrlngence, stremng btrefr
lngence,
double refraction of flow), btrefringence will be observed (F/gure 9.12).
Fure 9.12(AJ Situation at rest. Particles are randomly oriented. At this stage n
o birefringence will occur.
(B}
Hydrodynamic shear applied. This gives rise to a velocity gradient. The
particles become aligned to

the stream line. Birefrlngence may occur in this state.

(C)
Microscpic situatin f a prlate ellipsoid particle in a velcity gradienL
Arrows indicate stream line.
FIw birefringence can be understood properly with the help of a commonplace exam
ple.
A curious thing happens when an aged colloidal dispersion of vanadium pentoxide
is stirred
slowly; the path of the stirring rod lights up since the colloidal particles ori
ent themselves along
the stream lines. This effect is observed because of the difference in the amoun
t of light reflected
by the symmetrically oriented particles as compared to the randomly oriented one
s. In a similar

Rotatin
cylinder
Other Optical Techniques for Molecular Characterization
285
way, when a solution of nonspherical solute particles is subjected to hydrodynam
ic shear, the
fleld of polarizing microscope will light up due to birefringence; the brightnes
s of the field will be
directly proportional to the birefrlngence. "
Theoretical considerations indicate that the double refraction of the system app
ears as a
result of the equilibrium between the orienting effect of the applied field and
the disorienting
effect of the Brownian motion. The intensity of the birefrlngence depends on the
flow rate and
the shape of the particles, but is independent of the birefrigence of the indivi
dual particles. In
fact even Optically isotropic particles will give rise to birefringence provided
that they have an
anisotropy of the system as a whole and not the individual ansotropy
i. tlmt gives rise to flow birefringence.
The direction of the optical axis of the solution which is determined by the ave
rage orientation
macromolecules, as a function of shearing force, gives the rotary diffusion cons
tant of the
from which their size and shape are estimated by applying an appropriate
Hydrodynamic shear : Flow birefringence is usually measured in an apparatus whic
h
concentric cylinders (Figure 9.13). Two types of apparatus are available dependi
ng
whether the inner or the outer cylinder is rotated. For various reasons the oute
r cylinder
------- Light source
\
>Condensing lens
C Y Diaphragm
).t Condensing lens
Po r

Concentric cylinde
Liquid between the
cylinders
( Quarter wave plate
Half-shadow wedge
- Condensing lens

(B)

9.11t AI Iommr representon oft/w.flow birefrtnnee Olm. Inddent lht is conjed by

t.
condenser on to the opening in the dW.phragm. The diaphragm now acts as lht sour
ce. The light
now falls into the lluld between the on,ntc cylinders aJter Imssing through a con
denser and
nlcol polarizer. For measurement of the extinction angle, quarter wave plate and
half-shadow
are removed. They are required only for measuring birefrirjence. Light now passe
s through the
analyzer nlcol and is then focussed by the condenser on to the observirj telesco
pe through which
optical patterns are observed.
(B)
Cross-sectlon of the concentric cylinders, o is the angular velocity of
rotation. Rz and d are radius of
the fixed cylinder, space between the flxed and the rotating cylinders respectWe
ly. R is the sum of
Rz and d. It is on these factors that the shear gradient G is dependent. G is gi
ven by the relatWnshtp.
oR2
Gd

286
Biophysical Chemistj
rotating model is more preferable, although its design is a bit complicated. The
outer cylinder is
connected through a pulley system with a motor, the rotating speed of which can
be changed by
voltage control. The macromolecular solution is placed in the space between the
two concentric
cylinders. As the outer cylinder rotates, while the inner cylinder is stationary
, a velocity gradient
is set up. This velocity gradient produces a torque on the molecules tending to
align their long
axis tangential to the direction of the flow. The higher the axial ratio of the
particle (length/
width), the greater the degree of alignment will be. As the velocity gradient is
increased, the
angle between the long axis of the particle and the direction of flow (stream li
nes) decreases; in
short, the particles align themselves in a manner which offers least resistance
to the flow.
Anyone who has observed a small wooden stick floating on water would be able to
visualize the
alignment of the particles. Wherever the current of water is strong, the stick d
oes not rotate but
aligns its long axis in the direction of flow (the molecules, however, turn very
slowly, depending
on their rotational diffusion coefficient).
The optical system : The alignment of the molecules is determined by passing par
allel
light from a polarizing prism, called the polarizer, through the solution, and t
hen through
another polarizing prism, called the analyzer, placed at right angles to the fir
st. The optical
system for the measurement of birefringence consists of a light source, a collim
ator lens, a
filter, a polarizer, a quarter wavelength plate, and an analyzer, The sample sol
ution enclosed
between the two concentric cylinders is located between the polarizer and the qu
arter wavelength
plate (Figure 9.13). The polarizer and analyzer are usually Nicol prisms or occa
sionally polaroids.
Calculation of Rotational Diffusion Coefficient
When the outer cylinder is not rotating, i,e., when the solution is at rest, it
is isotropic. The
incident light coming through the polarizer, therefore, remains linearly polariz
ed when it comes
out of the solution and is blocked by the analyzer which is placed at right angl
es to the polarizer.
The field appears totally dark. However, when the outer cylinder is rotated, the
particles align
themselves along stream lines and the solution becomes anisotropic and birefring
ent. The
refractive index of this solution is no more the same in all directions. The lig
ht transmitted no

more remains linearly polarized, but becomes elliptically polarized and passes t
hrough the
analyzer. As the orientation of the particles is increased by increasing the spe
ed of the outer
cylinder, more light passes through the analyzer. It would be seen that light pa
sses through
every point except for four positions. These four dark areas, which correspond t
o no birefringence,
form a cross known as the cross ofisocltne (Figure 9.14). This dark cross is see
n to form an
P

i/
\
A
[
.Particle
P
Figure 9.14 The cross of tsocline. The cross forms an angle with the polarizer a
xis (P-P). The same angle Is formed by the cross and the analyzer axis (A-A). Th
s ts the extinction angle 2. The intersection of the long axis of
the particle with the stream line also gives rise to the same angle. However, th
e former relationship utilized to measure .

G
kT
Other Optical Techniques for Molecular Characterization
287
angle with analyzer axis and with the polarizer axis, both the angles being of t
he same extent.
This angle is known as the extinction angle, and is denoted by the symbol ;(i
The same angle, X, is made by the long axis of the particle with the stream line
(Figure
9.14). The extinction angle depends upon the shear gradient G; the angle decreas
es as the
speed of the rotating cylinder increases. I,n fact G depends upon the speed of t
he rotating
cylinder and the distance between the two concentric cylinders.
Rotational diffusion coefficient can be calculated if G and are known. The follo
wing relationship is utilized for the purpose
60
tan 2X G
After some time the rotating system attains equilibrium. At this point the parti
cles for
remain aligned in a preferred orientation. This preferred orientation is denoted
symbol a. a is a function of the dimensions of the solute particle and is descri
bed by the
relationship
It is actually the rotary frictional coefficient from which the shape of the par
ticle in
of its axi.al lengths can be calculated. This rotary frictJ.on coefficient can b
e calculated
the following relationship
where is the rotary frictional coefficient, k, is the Boltzmann constant and T i
s the
once rotary diffusion coefficient has been experimentally determined,
can be calculated from it.
The relationshipbetween the rotary frictional coefficient and the shape of the p
article is

axial length. For a long prolate ellipsoid (Figure 9.15) with semiaxes
iand b where a> 5b
3
16 moa
9.15
The prolate ellipsoid model. The model is obtained by rotation of an ell
ipse of major axis a and minor
ax/s b about the a axis.
o is the viscosity of the solvent. The axial ratio (a/b) of the particle concern
ed can be
by intrinsic viscosity measurements.

288
32 ob3
3
180S
Biophysical Chemistry
For oblate ellipsoids
Here b is longer than

Figure 9.16 A schematle representation of the plot to determine

Q. c is measured for different values of G and
dz
dX I
dG
is related to 0 by -- =
dG 12 @ "
Rotational diffusion
can be measured by another
Here, ; is measured for
of G by varying the
cylinder is rotated. The Values of X
obtained are then plotted against
(Figure 9.16). When a is less than
the relationship is as follows I
G
=
4
120
It is thus possible to relate
initial slope of the plot shown in
9.16 to G by
dz

I
dG
120

(Experimentally, the angle between the polarizer axis and the cross
two times, each time the cylinder.rotating in opposite directions. The differenc
e between
two positions is equal to 2).
Dimensions of several macromolecules have been determined using flow
For example, the length of the major axes of flbrinogen and serum albumin were f
ound to
670/ and 190/ respectively. Both these values are in agreement with values for t
he
macromolecules found by other hydrodynamic methods. Some other example
whose shape has been determined using flow birefringence are myosin, tropomyosin
,
tobacco mosaic virus etc.
Relationship Between Flow Birefringence and Molecular Weight
If is the angle of rotation of the analyzer in degrees, btrefringence of the sol
ution.
be calculated by the equation
where . is the wavelength of the incident light in vacuum and S is the path
solution.
Once the degree of birefringence has been calculated, it can be related to the w
eight
average molecular weight of the macrom, olecules by the following equation
An
= BnM
C

Other Optical Techniques for Molecular Characterization

289
where Bn is a constant characteristic of the solute, C is the concentration of t
he solution and M
is the molecular weight.
Although fl0w birefringence has been used many times for determination of molecu
lar
weight of macromolecules, the method depends upon assumptions regarding the mole
cular
geometry which involves an appreciable degree of uncertainty. Moreover it requir
es evaluation
of empirical constants. These serious drawbacks have limited the application of
this method for
molecular weight determination.
2. Electric birefringence
Another technique to study the dimensions of macromolecules on the basis of thei
r charge
properties is electric birefringence. In flow blrefringence macromolecules were
aligned with the
help of hydrodynamic shear. In electric birefringence an electric field is used
to align the
macromolecules and the alignment is determined by the electrical.properties of t
he molecules.
Macromolecules are forced to align by application of electric field. The fiel is
usually
applied as a short pulse of the order of microseconds. The degree of alignment i
s then determined
i from the birefringence produced. The measurement involves determination of int
ensity of light
passing through crossed polarizing lenses and falling on a photomultiplier tube
where it is
converted to an oscilloscope signal. The optical system is so desigrted that whe
n the molecules
align and the solution becomes birefringent, a tracing immediately appears on th
e oscilloscope
screen.
Inspite of providing a considerable amount of information about macromolecules,
the
method has a serious disadvantage which limits its application; it can be of uti
lity only when
in which the macromolecules are placed is relatively nonconducting.
The rise and decay of birefringence (Figure 9.17)
(2 e
provide two different informations about the
Rir-e "macromolecule. At the onset of the electric pulse, the

shape of the rise of birefringence provides information

cay
about the orientation of the induced and permanent
dipole moments of the molecule. The decay of
, Time ( sec)

birefringence is due to rotational diffusion coefficient

of the macromolecule. The decay, then, provides a

9.17 Oscilloscope tracing showing the
precise measure of the rotational diffusion coefficient,
trans/ent e/ectrlc blrefr/ngence of
flbrtnogen in solution (0. I M
which as we have seen earlier can provide an idea
glyclne, pH 7.9)
about the shape of the macromolecule.
From the dipole moments one can determine the
pattern of distribution of charged groups on the
;. The method is of considerable utility to study reactions in which charge
take place on the protein molecule. For example, activation of a protein in whic
h
are cleaved from the protein can be studied with the help of electric birefringe
nce.
activation (where two peptides with a charge of-4 are cleaved by the action of t
he
thrombin) can be studied with the help of this technique. In a likewise manner,
charge
protein can also be studied. For example, the technique is utilized to study
of a charged hapten to an antibody specific for it.
Polarization of fluorescence
Polarization of fluorescence has been often used to determine rotational diffusi
on coefficients
' macromolecules. Unlike other hydrodynamic methods, this technique is not limit
ed to

290
Biophysical Chemistry
In this technique the sample is irradiated with polarized light of the w
avelength of absorption.
The absorption of this light now depends on the orientation of the electric dipo
le of the solute
molecules. Those molecules whose electric dipoles are in closest alignment with
the field direction
will absorb the most. The lifetime of excited state of a solute molecule after a
bsorption of llght is
usually 'of the order of 10 nanoseconds (nsec). If the alignment of the solute m
olecules does not
change within this time, the resultant fluorescence will show the same extent of
polarization as
that represented by the absorption distribution. In this case a maximum value fo
r the fluorescence
polarization will be obtained. This, however, seldom takes place. In solutions,
Brownian motion
leads to rotational diffusion which randomizes the orientations of chromophores
during this
period (10 nsec). This results in fluorescence depolarization additional to that
caused by initial
random distribution of chromophores. Brownian motion has a large effect on small
molecules
(faster rate of rotational diffusion) whose alignment gets randomized fast and t
heir solution
shows complete depolarization of fluorescence. On the other hand, those chromoph
ores which
are tightly bound to macromolecules have a slower rate of rotational diffusion (
characteristic
times for this process are of the order of 100 to 1000 nsec). Thus with these ch
romophores only
a partial depolariztion can occur during lifetime of the excited state (10 nsec)
. The rotational
diffusion coefficient of these chromophores ( and therefore the macromolecules t
o which they
are bound) can then be calculated by comparison of the resultant polarization wi
th that which
would be obtained if no diffusion took place. In practise this is done by measur
ement of
fluorescence polarization as a function of viscosity and subsequent extrapolatio
n of the data to
infinite viscosity. This is the simplest method by which rotational diffusion co
efficients of
macromolecules can be determined. Moreover, the method is free from those limita
tions suffered
by hydrodynamic methods and is sensitive to very small quantities of sample.
Fluorescence polarization may be characterized by emission anisotropy. When the
exciting
beam used is polarized, the emission anisotropy is given by the following expres
sion
r+ 2I

where Ii and I, are the principal components of the polarized fluorescence vibra
ting in parallel
and perpendicular directions respectively (with respect to the direction of the
exciting beam).
Il and I are in practice determined by measuring fluorescence intensities at righ
t angles
to the exciting beam. This is made possible by using a rotating polarizeralterna
Uvely transmitting
the two directions of vibration (Figure 9.17). Normally the two polarized compon
ents are measured
using the same photomultiplier.
LIGHT SCATTERING
If a parallel beam of white light illuminates a colloidal solution, faint bluish
light can be
observed laterally. This phenomenon, caused by the scattering of light, is commo
nly known as
the Tyndall effect after its discoverer J. Tyndall. Although light scattering is
more pronounced
in colloidal solutions, it is observable in all transparent media like gas, any
pure liquid solution
and even in crystals. Lord Rayleigh formulated the fundamental laws of light sca
ttering in 1871
by calclflating the polarizability of individual gaseous molecules placed in an
oscillating
electromagnetic field of a light beam. As per this theory the incident electroma
gnetic impulse of
a beam of light causes electrons of an isotropic particle to vibrate in unison w
ith it. An oscillating
electric moment is thus induced in the particle. The oscillating electrons now b
ecome sources
of scattered or diffracted light which will mostly have the same frequency as th
at of the incident
beam. The intensity of the transmitted beam is consequently lowered. The intensi
ty of the
transmitted beam, and therefore, the intensity of the scattered light can then b
e expressed as a

where
Other Optical Techniques for Molecular Characterization
291
function of the number of centres of scattering (i.e., particles or molecules) p
er unit volume.
Thus, if a value for Avogadro's number is adopted, quantitative measurement of l
ight scattering
can provide an idea of the molecular weight of the scattering specie. Smimov and
Bazenov of
U.S.S.R. and Putzeys and Borsteaux of France were the first to apply light scatt
ering toward the
calculation of molecular weight in 1935.
P
we
y
"
p
Ill I
Figure 9.18 A simple setup for Jluorescence polarization measurement. S
is the Ikjht source; we is the exciting
frequency; Pz .is the flxd polarizer; vf is the Jluorescence frequency;
P2 is the rotating polarlzer which
alternately transmits Ill and I ; D is the photomultlpler. Monochromator
s and collimators are not shown.
A mathematical relationship describing light scattering phenomenon can be writte
n in
much the same way as the Lambert's law
I
Io = intensity of the incident light,
I
= intensity of the transmitted light,
= turbidity,
and
x
= the path length.
Turbidity,, depends upon the number and size of the scattering particles. For li
ght
scattered at 90 to the incident beam in ideal systems, the turbidity can be expre
ssed by the

following mathematical relationship

32z
3
X4 a
where n = index of refraction of the solution, no = index of refraction of the s
olvent,
, = the wavelength of light in vacuum,
and a = the number of solute particles per ml.
To introduce a term for molecular weight into the above mathematical relationshi
p, a is
replaced by its equivalent CN/M (where C is the concentration of the solute in g
ins per ml, M is

292
Biophysical Chemistry
the molecular weight, and N is Avogadro's number). The mathematical expression c
an now be
written as
where k is a proportionality constant defined by the relationship
The relationship /C = kill is for ideal system. For nonideal systems the relatio
nship takes the
following form ,
kC 1
=m+2RC
M
where R is the interaction constant and is dependent upon the solvent used. This
equation can
be used to calculate molecular weight after obtaining light scattering data.
Molecular weights obtained by light scattering data are weight average molecular
weights
(See Chapter 4). The method has a srious drawback in that it is very sensitive t
o the presence
of aggregates and large scale errors may creep, in molecular weight determinatio
n if formation
of aggregates is not guarded against. As it is, molecular weights obtained by li
ght scattering are
considerably higher than those obtained by other methods.
The apparatus used for measurement of light scattering is called light scatterin
g photometer.
Figure 9.19 is a schematic representatibn of a typical light-scattering experime
nt. The solutions
must essentially be dust free, Fine pore filtration or high speed centrffugation
might be performed
to get rid of the dust particles. Presence of aggregates must also be avoided at
all costs. The
presence of any of the two Impurities stated above will lead to false scattering
values.
[ ser t
OO
Figure 9..19 Schematic of a typical- apparatus for light scattering nts. Monochr
omatic light is obtained from
a laser.source. Lens. L focusses thecollimated beamofwavelength and intensiJloon
to thescattering
cell. D is the detector, usually a photomultiplier which records the scattered r
adiation, D is mounted on

a moveable support so that by chancjing its position around the scattering cell,
scattered intensity at
any angle 8 can be measured.
Although in the present text we have touched only the aspect of molecular weight
determination by light scattering measurement, it should be noted that the pheno
menon is
also useful in providing data on the size and shape of the dissolved macromolecu
les as well as
information on the thermodynamic properties of the system. Moreover, owing to th
e rapidity of
the method, light scattering offers unique possibilities of following the kineti
cs of macromolecular
reactions involving a change in the size or shape of the dissolved particles.

Other Optical Techniques for Molecular Characterization

293
X-RAY DIFFRACTION
It is very instructive to wonder as to where molecular biochemistry would be tod
ay if there
had been no X-ray analytical methods to discover the atomic architecture of biop
olymers such
as proteins and nucleic acids. The importance of this method of s.tructural anal
ysis can be
stated simply by saying that the structure of DNA was discovered largely due to
the availability
of X-ray crystallography data.
The wavelengths of X-rays are of the same order, viz., 10-s cm as the distance b
etween
individual atoms in a crystal. This'similarity led Max yon Laue in 1912 to make
a brilliant
suggestion that crystals could act as natural and very fine three dimensional di
ffraction gratings
for X-rays. W. Friedrich and P. Knipping verified this idea by passing a beam of
X-rays through
a crystal of zinc blende. This resulted in a deflrdte diffraction pattern on the
exposed photographic
plate. A pattern of this type is generally referred to as the Laue pattern.
Previously this method was used to calculate the wavelengths of X-rays, the crys
tal playing
the part of a diffraction grating. W.H. and W.L. Bragg were the first to call at
tention to the fact
that, since a crystal is composed of a series of equally spaced atomic planes, i
t may be employed
not only as a transmission grating, but also as reflection grating. An X-ray bea
m striking these
atomic planes will be diffracted in such a manner as to cause either interferenc
e or reinforcement
of the beam diffracted from the outer plane. The whole beam would behave as if i
t has been
reflected from the outer surface of the crystal.
To understand the theory of this method better, consider Figure 9.20. A wave fro
nt, LP, of
X-rays is approaching a series of identical, parallel and equidistant lattice pl
anes, AA,. BB, CC,
which constitute the atomic planes of the crystal. Part of the beam, LM, will be
reflected at M
along RUN, at the angle of reflection, 9. Similarly, the beam PM'will be reflect
ed partly at M' along
RUN', and partly at Q on the second plane along QMN. To emerge along RUN, the se
cond beam
travels a longer distance than the first, the distance Plt QM as compared to the
distance LM.
If the length of the path LMN differs from that of PM" QMN by a whole number of
wavelengths,
then the two beams will be in phase at-M, will reinforce each other and a maximu
m intensity
beam will result. However, if the two beams are out of phase, interference will

result, and the

intensity of the reflected beam will be less than maximum. It is thus clear that
the condition for
maximum intensity beam is that the distance
PQM-LM = n
where is the wavelength of the X-ray used and n is an integer equal to 1,2,3,4,
etc., and is known as the order of reflection.
N
R
Iure .0 ReJleetlon of X-foxes y l:u"lle[ loes
If we now draw a perpendicular from M to the extension of line PM" Q, and draw a
nother
from M to Rerpendicular to AA, BB and CC, it becomes clear that

294
But, from the figure it is clear that QM = QR. Thus
PQM = PR
But
LM -- PS
Biophysical Chemistry

Therefore, PQM = LM = PR - PS
= SR
Thus,
SR = rv
From the figure it is also clear that angle SMR is (3. Now, since MS was drawn p
erpendicular
toPR,
SR
Sin(3 MR
and
SR = MR sin 0
= 2d sin (3
where d is taken as the distance between any two atomic planes in the crystal.
Therefore
rvX = 2d sin (.
This relationship, known as the Bragg equation, is applicable to reflection from
other
planes parallel to AA and BB. This simple expression directly relates the wavele
ngth and order
of reflection of theX-rays to the interplanar distance d and the angle of maximu
m reflection, 0.
The relationship allows calculation of the ratio )Jd by measuring n, and (3. Mor
eover, if d is
known, the wavelength, Z, of the X-ray Can be calculated. More importantly, if t
he wavelength,
Z, of the X-ray being used is known, the distance between the successive lattice
s of the crystal,
d, can be calculated. X-ray diffraction can thus tell us a great deal about the
structure of the
crystal.

Every atom in a crystal scatters an X-ray beam incident upon it in all direction
s. As all
crystals, even the smallest one, are made up of a very large number of atoms, th
e chance that
these scattered waves would constructively reinforce would be almost zero but fo
r the fact that
the atoms in a crystal are arranged in a regular repetitive manner. The Bragg eq
uation described
above states the condition for diffraction of a beam of X-rays from a crystal. W
hile maximum
contribution to the intensity of the diffracted beam is afforded by the atoms lo
cated exactly on
the crystal planes, maximum destructive interference is meted out by the atoms e
xactly halfway
between the planes. Atoms located at intermediate positions interfere constructi
vely or
destructively depending on their exact location, but with less than maximum effe
ct. The scattering
power of an atom increases as the number of electrons it possesses increases. Th
us, although
.the position of the diffracted beams depend only upon the size and shape of the
repetitive unit
of a crystal and the wavelength of the incident X-ray beam, the intensity of the
diffracted beams
depend also upon the type of atoms in the crystal and the location of the atoms
in the unit cell
(the fundamental repetitive unit). Therefore, if one considers the direction and
the intensity of
the diffracted beams, no two substances can have absolutely identical diffractio
n patterns. The
diffracUon pattern of a crystal can thus be compared to a 'fingerprint' and prov
ides identities of
the components of the crystal.
Reciprocal Lattice Concept
Diffraction phenomenon can be interpreted most conveniently with the aid of reci
procal
lattice concept. A plane may be represented by a line drawn normal to the plane.
The orientation
of the plane would then be described by the spatial orientation of this line. Th
e length of the

Other Optical Techniques for Molecular Characterization

295
normal is usually fixed in an inverse proportion to the interplanar spacing of t
he plane it
represents.
203
003,
'302

"-

/ ' I02
301
(00}
" (0|
Figure 9.21 A profde of several planes in the unit cell of a crystal. Norrnals t
o the planes are also shown.
When normal are drawn to all the planes in a crystal from a common origin, the t
erminal
points of these normals constitute a lattice array. However, since the distance
of each point
from the origin is an inverse or reciprocal of the interplanar spacings, this is
called a reciprocal
/att/ce. Figure 9.21 shows, near the origin, the traces of several planes in a u
nit cell of a crystal,
namely, the (I00}, (001}, (101L and (102} planes. The normals to these planes ar
e called the
reciprocal lattice vectors ha and are defined by
The lattice array is defined by three reciprocal lattice vectors in three dimens
ions. The
magnitudes of these three vectors are given by
b =oo = d
010
The directions of these vectors are defined by three interaxial angles a', [Y, y
'.

296
The Bragg equation now might be rewritten in a form that will relate the glancin
g angle O,
with the other parameters described above.
In the above expression the numerator represents one side of a right triangle wi
th 0 as
another angle and the denominator represents the hypotenuse (Figure 9.22(A)). "l
-le construction
in Figure 9,22(A) can also be interpreted as shown in Figure 9.22(B}. ,e diamete
r of the circle. ABC represents the direction of the incident X-ray beam. The li
ne ,AD. drawn through the
origin of the circle and making an angle 0 with the incident beam defines a crys
tal plane which
satisfies Bragg diffraction condition. The llne BD forming an axgle 20, with the
incident beam
and an angle 0, while the crystal plane represents the diffracted beam's directi
on. Then the

297
Other Optical Techniques .for Molecular Characterization
line CD is the reciprocal lattice vector to the reciprocal lattice point Dh lyin
g on the circumference
of the circle. The vector h originates at the point on the circle where the dire
ct beam leaves the
circle. Now when a reciprocal lattice point lies on the "sphere of reflection" (
a sphere formed by
rotating the circle upon its diameter -'-), the Bragg equation is satisfied. Und
er no other
conditions will Bragg equation be satisfied.
. Diffracted

/ beam
beam
A
Figure 9.22 Diagrammatic representation of the diffraction condition
In a diffraction experiment the crystal can now be pictured to be at the centre
of a sphere
of unit radius. The reciprocal lattice of this crystal will then be centred at a
point where the
direct beam leaves the sphere (Figure 9.23). It thus follows that with rotation
of the crystal, the
reciprocal lattice will also rotate. Whenever a reciprocal lattice point will in
tersect the sphere, a
reflection will emanate from the crystal at the sphere's centre and will pass th
rough the
intersecting reciprocal lattice point.
The Determination of Crystal Structure
"the. Bragg equation makes it clear that if the glancing angles 0 are measured f
or the
various orders of maximum reflection, and if the wavelength of the impinging X-r
ay is known,
the distance d between successive lattice planes of a crystal can be calculated.
Two methods
are most popular. The rotat/ng crystal method and the powder method.
Incident X-ray
beam
Sphere of reflection

ooI
.........

------2
....
|/'-- .,
1

1
'2
'-'
4

Crystal rotation axis

298
Bophgscel ChemLstn3
Rotating crystal method : As the name suggests, the experimental set up is such
that a
monochromatic X-radiation is Incident upon a crystal which is rotated about one
of its axis.
lgure 9.24 Diagrammatic representation of
Bragg X-ray spectrometer.
Description in the text
Thereflected beams lie as spots on the surface of cones
which are coaxial with the rotation axis. In order to
determine the positions of maximum reflection intensity,
use can be made.either of the Bragg X-ray spectrometer
(Figure 9.24) or a photographic film. In the X-ray
spectrometer a beam of X-ray of a definite wavelength
cming from the anticathode of an X-ray tube, X, is
allowed to escape through a slit S. The beam then falls
upon a known face of crystal C, mounted on a rotating
turn table. The position of the crystal at any given time
can be read on the scale L. The rays reflected by the
crystal are allowed to reach an ionizing detector D
through another sllt. The detector is usually a Geiger
cbunter. The apparatus is so designed that the reflected
ray always enters the detector D, and the Intensity of
such a ray is determined. The glancing angles, 8, which
satisfy the Bragg equation, give the strongest reflections.
The procedure is repeated for all the planes of the crystal.
This method necessitates the use of a large crystal
with well defined faces. Moreover, since reflections of

several orders must be examined for a large number of faces, the total labor Inv
olved is enormous.
This, however, is compensated by relatively simple Interpretation of the results
.
The Imwcler mthod : In 1916, P. Debye, P. Scherrer and A.W.Hull devised the powd
er
method of X-ray crystallography which went on to become a widely used method. A
narrow

0
Figure 9.25 Diagrammatic repre
sentation of the principle
of the powder method.
Description in the text.
beam of X-rays falls on the finely powdered substance
P. The powder is usually coated on a hair or inside a

thin walled glass tube. The diffracted rays collide with

the photographic film F which is arranged In a circular
arc with P at its centre. As crystals In the fine powder
are oriented in all directions, a large number will have
their lattice planes In the correct positions for maximum
X-ray reflection to occur. All crystalline particles whose
(100) planes are In proper angle with the Incident X-ray
beam will give rise to first order maxima In directions
lying on a circular cone (Figure 9.25). Other crystals
whose (I 10) planes are in proper angle with the Incident
X-ray beam will produce another circular cone. Circular
cones will be produced In a similar manner for other
planes. The distance between the powdered substances
P, and the spot of Intersection of undiffracted beam with
film, I, is fixed for a given X-ray camera system. Thus,
In order to measure 0, for a particular reflection, the

only necessity is to measure the distances such as/D or IE (Figure 9.25). The st
ructure of the
crystalline material can then be determined.
Chol of X-Radiation
Bragg's equation can be rearranged and written as follows:

Brass
Other Optical Techniques for Molecular Characterization
299
The resultant of the terms in the parenthesis never exceeds unity. Due to this u
se of long
wavelength radiation is not very useful as it limits the number of reflectlons t
hat can be observed.'
On the other hand, in case of a crystal having a very large unit cell, usage of
short wavelength
radiation results in a crowding of individual reflections making the interpretat
ion a tough job.
Clearly then, the choice of radiation has to be a compromise between the two. ex
tremes.
Additionally, the chosen radiation should not fall Within the absorption range o
f the crystal
material. In such a case the crystal absorbs the radiation and emits fluorescent
radiation in all
directions. This radiation darkens the film making it difficult to locate the di
ffraction maxima
sought. Thus, the wavelength of the chosen radiation should be outside the absor
ption range of
the crystal material.
Specimen Preparation
Use of single crystal is preferred for structure determination because the data
so obtained
is easy to interpret. The crystal in this case should be of such a size as is co
mpletely bathed by
the incident radiation. The crystal is generally affixed to a glass capillary wh
ich, in turn, is
fastened to a brass pin (Figure 9.26). For protein crystals, however, it is nece
ssary to mount
Crystal
Shellac
Powdered
sample
Glass capillary
Picein wax
Glue
F@ure 9.26 TWo methods of specnen mounts for X-ray di.'actloru (A) Single crysta
l, and (B) Powdered sample.
the crystals in an enclosed space. This is so since the protein crystals contain

within themselves
a large amount of solvent from which they are grown. About half the volume of pr
otein crystals
is of solvent. If such crystals are exposed to air, liquid evaporates and the cr
ystals shrink
deteriorating the quality of the X-ray pattern obtained,. Therefore, protein cry
stals are placed in
[ thin wall capillary wliich is then sealed at both ends. Sometimes the interior
of the capillary
is coated with a hydrophobic film to make for a cleaner mount.
When a single crystal of sufllcient size is not available, a polycrystalline agg
regate is formed
into a cylinder whose diameter is smaller than the diameter of the incident X-ra
y beam. At
other times the powdered substance might be coated on to a hair and the assembly
is fixed to
a brass pin (Figure 9.26).
X-ray Diffraction and Molecular Weight
A unit cell might contain more than one asymmetric units. Prom the space group a
nd the
unit cell volume, the volume of the symmetric unit can be calculated. If the app
roximate molecular
(M) of the macromolecule is known from other methods, the number of molecules in
the
symmetric unit can be calculated. If we now denote the molecular weight of the a
symmetric

1 V
300
Biophysical Chemistry
macromolecule by W, the volume of the dry unit cell by V, and the number of asym
metric units
per unit ceil by n, the following expression Can be written
where k is a constant.
From the above equation it is obvious that if one knows the number of asymmetric
units
in a unit cell, one can arrive at a fairly accurate value of the molecular weigh
t of the molecule.
There are, in fact, few other methods of molecular weight determination which wi
ll give a value
as accurate as this from a single simple experiment. It should however, be borne
in mind that
the molecular weight as determined from X-ray diffraction data of the crystal mi
ght be a multiple
or submultiple of the true molecular weight and might also differ from the value
of the molecular
weight determined by other methods. The molecular weight as determined by X-ray
data is
therefore termed as "crystal molecular weight".
Suggestions for Further Reading
I.
Imahori, K. and Nicola, N.A., in Physical Prlnctples and Techniques of P
roten Chemistry, (S.J,
Leach, ed.) Part C, Academic Press, New York, 1973, p. 358.
2.
Sears, D.W. and Beychok, S., in Physical PHnciples and Techniques of Pro
teln Chemistry, (S.J.
Leach, ed.) Part C, Academic Press, New York, 1973, p. 446.
3. Adler, A.J., Greenfield, N.J. and Fasman, G.D., Methods in Enzymology, 27 : 6
75, 1973.
4.
Morris, E.R. and Sanderson, G.R., in New Techniques in Biophysics and Ce
ll Biology, Vol 1,
(R.H. Pain, and B.J. Smith, eds.)p.113, John Wiley, London, 1973.
5.
Edsall, J.T., The Sze, Shape and.Hydration of Protein Molecules in the P
roteins, Vol.l, Part B,
(H. Neurath and K. Barley, eds), pp. 549-716, Academic Press, New York, 1953.
6. Beychok,-S., CircularDichroism of Biological Macromolecules, Science, 154 : 1
288 (1966).
7.

Caldwell, D.J. and Eyring, H., The Theory of Optical Activity, Wfley-Int
erscience, New York,
1971.
8.
Feeney, J., Fluorescence Nanosecond Pulse Fluorimetry, in New Techniques
in Biophysics &
Cell Biology, Vol. 2, pp. 233-287, (R.H. Pain and B.J. Smith, eds.), John Wiley,
London, 1975.
9.
Yoshioka, K. and Watanabe, H., Dielectric Properties of Protelns II. Ele
ctric Brefrlngence and
Dichrolsm in Physical Principles and Techniques of Protein Chem/stry, Part A, (S
idney Leach,
ed.] Academic Press, New York, 1969.
I0. Stacey, K.A., L/ght Scatter/ng in Physlca/Chemistry, Academic Press, New Yor
k, 1956.
I I. Tlmasheff, S.N. and Tbwnend, R., /.jht Scatterb in Phy$1ca/Pr/nc/es and Tec
hniques of
Protein Chemistry, Part B, (S.J. Leach, ed.} Academic Press, New York, 1970.
12.
Dickerson, R,E., X-ray Analysis and Protein Structure in The .ProteUs, 2
nd ed., Vol, 2,
(H, Neurath, ed,) Acadera/c Press, New York, 1964.
13.
Willard, H,H,, Meritt, L,L., Dean, J,A. & Settle, F,A, Jr,, Instrumental
Method of AnoB,
8th ed, CB$, New Delhi, 1988,

CENTRIFUGATION
Maeromolecules are almost insensitive tO gravitational settling. This is because
, for small
gravitational force is so minute that the random bombardment of the molecules of
;surrounding medium far outweighs the directing.force of gravity. Thus, only on
standing for
extended period of time will concentration gradients develop in undisturbed solu
tions.
sedimentation would then appear to be completely useless for separation or
of macromolecules in solution. This, however, is not completely true. A
solution to this difficulty is to increase the gravitational potential energy by
. the
in a vessel rotating at high speed. The particles in the solution will t
hen
a centrifugal force is acting upon them in addition to the gravi
tational
Rwas in 1923 that Svedberg and Nicols employed a centrifuge for the first time t
o increase
so as to speed up the rate of sedimentation for the purpose of measuring
sizes. This pioneering work was followed by gradual development of ultracentrifu
gation
is probably the technique most responsible for our current understanding of cell
ular
The applications of this technique range from collection and separation of cells
,
and molecules to the study of molecular weights of macromolecules.

A yplml rotor with holes to accomodate

vessels Jled wh suspension. The vessel
and the rotor Iki are also shown.
The basic components of a centrifuge
are (t) a metal rotor with holes in it to
accommodate a vessel of liquid (Figure
10 I{A} and (tO a motor or alternative means
of spinning the rotor at a selected speed.
All other parts that different centrifuges
consist of are accessories for maintaining
the environment within which the
centrifuge operates or for modifying the
working of the rotor itself during
emergencies,

BASIC PRINCIPI, OF SEDIMENTATION

An object moving in a circle at a steady angular velocity will eperience a force

, F, directed
This is the basis of centrliation. Angular velocity in radians, o), and the radi
us of
r, to cent/meters, collect/rely determine the magnitude of the force F.
F= ' r
.,, (I}
F might be expressed in terms of earth's gravitat/onal force if it is divided by
980. The
referred to as the relative centrifugal force, RCF. RCF is more frequently refer
red to
'number times

303
The speed of a centrifuge rotor might be expressed in terms of rlSm or RCF. Howe
ver,
centrifuges owing to their designs would have different radii (distances between
the
axls and the. middle of the sample tube) which would give rise to different RCFs
at the
is illustrated below. Consider two centrifuges operating at the same rpm, 12000.
two centrifuges, however, have different radii, 4,8 and 8.0 cms. Calculating for
the RCFs
both the centrifuges we get
(/) RCF = (1.119 x 10-5) (12,000)2 zt.8
= 7,734 x g.
(ii) RCF = {I.I19 x 10-5) (12,000) 8.0
= 12,891 x g.
In view of the above, and to avoid ambiguity in scientific work, it is better to
express the
acting upon a particle in terms of RCF rather than the rpm. A nomogram based, on
(4) relating RCF to rotor speed and r is given in Figure 10.2.
Radius
Relative
Rotor speed
(in cm)
centrifugal force
(rev rain-')
20151000
00.. SOoOOO
40.
109"
'00 .:',00
15O0 15,00O

310 "- I000

50, 5000
10.2
Relationship between radius, rotor speed and relative centrifugal force.
By aligninj the values of the
radius and rotor speed, one can.find out the value of the relative centrifugal f
orce. The right side and the
left side of the RCF scale correspond to the right and left side of the rotor sp
eed scale respectively.
Apart from RCF, the rate of sedimentation of a given particle would also depend
upon its
characteristics such as its density, and its radius. The characteristics of the
medium in
the particle is suspended -- its density, viscosity -- will also tend to affect
the rate of
of the given particle.
Consider that centrifugal force is being applied to a particle. As he particle s
ediments, it
have to displace some amount of the medium of suspension. This displacement will
result

...(I0)
Centrlfugatlon
305
4. The higher the viscosity of the medium, the slower will be the particle movem
ent.
A different way to look at the above mathematical expression is to consider the
time
required for a spherical particle to sediment to a given distance in a centrifug
al field as a
function of the variables listed above. An expression for sedimentation time can
be obtained by
integrating equation (8}.
9
t=-- x
Inr--b
where
t = sedimentation time in seconds,
rt = radial distance from the centre of rotation to the liquid meniscus,
and
rb = radial distance from the centre of rotation to the bottom of the tu
be.
The above equation makes it clear that a mixture of heterogeneous particles ( fo
r example,
broken cells) can be separated by centrifugation on the basis of their densities
and/or size, This
can be achieved by two methods,
Letting the particles (different organdies in case of broken ceils) sediment to
the bottom
of the tube, 0rganelles differ in their size as well as their densities. Natural
ly, they will sediment
at different rates and thus some will reach the bottom before the others. Once a
particle reaches
the bottom and packs there (such a packed deposit under sedimentation is called
a pellet), we
can removethe supernatant (the suspension above the pellet) and resuspend the pe
lleted particles
in a suitable medium. We can repeat the procedure with the supernatant and pelle
t different
organelles differently. This is the principle of the so called dtfferent' centrl
fugatton and we will
discuss it later.
Letting the particle be sedimented for a fixed time at a fixed sedimentation vel
ocity.

Since each particle sediments at a different rate, all the particles will be fou
nd at different zones
within the tube ailer sufficient time has been allowed to pass. This principle i
s exploited in
density gradient centrlfugation which, again will be discussed later.
We defer the theoretical discussion here to certain other sections where it will
be more
useful.
INSTRUMENTATION
Many centrifuges of different designs are available in the market. However, all
centrifuges
can be roughly categorized into three different types on the basis of their oper
ating speed.
"Desk Top Centrifuges
These are very simple and small (can be placed atop a desk and hence the name) a
nd are
the least expensive. They are normally used to collect rapidly sedimentlng subst
ances such as
red blood cells, yeast cells or bulky precipitates of chemical reactions. These
are also known as
.clinical centrifuges since most of the clinical work is done by these models, T
heir maximum
speed is usually 3000 rpm and they do not have any temperature regulatory system
.
The main difference between the centrifuges of this category is the maximum carr
ying
capacity. Separation can be carried out in 10, 50 or 100 cm tubes. Larger capaci
ty (4-6 dm)
centrifuges are also available and there are centrifuges whose rotors can accomm
odate bottles
of fairly large capacity. In all these centrifuges the rotors are mounted on a r
igid shaft. It is
therefore very important that the contents of the centrifuge tubes are balanced
accurately and
that they are never loaded with an odd number of tubes. Moreover, if the rotor i
s only partially
loaded, the tubes have to be placed diametrically opposite to each other to disp
erse the load
evenly. These precautions are true for all types of centrifuges discussed here.

306
Biophysical Chemistry
High Speed Centrifuges
High speed centrifuges can operate with maximum speed of up to 25,000 rpm provid
ing
about 90,000 g centrifugal force in the process. They are usually equipped with
refrigeration
equipment to remove heat generated due to friction between the air and the spinn
ing rotor. The
temperature can easily be maintained in the range 0-4C by means of a thermocouple
. The
highest carrying capacity may be 1.5 dm3. These instruments are routinely used t
ocollect
microorganisms, cell debris, cells,, large cellular organelles, precipitates of
chemical reactions
and immunoprecipitates. Although these centrifuges are useful in isolating sub-c
ellular organelles
such as the nuclei, mitochondria, lysosomes etc. they are of little use in isola
ting smaller
organelles such as the ribosomes, microsomes, etc. Neither are they useful in se
dimenting
individual molecules for want of sufficient centrifugal force.
The Ultracentrifuge
The ultracentrifuge can operate at speeds up to 75,000 rpm providing centrifugal
force in
excess of 500,000 g. At such speed the friction between air and the spinning rot
or generates
significant amount of heat. To eliminate this source of heating, the rotor chamb
er is sealed and
evacuated by two pumps working in tandem making it possible to attain and hold v
acuums of
1 to 2 . Apart from this the ultracentrifuge has a refrigeration system which ca
n maintain the
temperature of the rotor between 0 and 4C. The drive shaft on which the rotor is m
ounted is
merely 1/16 inches in diameter. The small diameter allows the shaft to flex duri
ng rotation
accommodating a certain degree of rotor imbalance without spindle damage, The sh
aft is made
up of aluminium or titanium alloy of high tensile strength to withstand the grea
t forces generated
during centrifugation.
To prevent the rotor from operating at speeds which exceed its maximum rated spe
ed, all
centrifuges possess an overspeed device. Operation of rotor at excessive speeds
can result in an
explosion with the rotor being torn apart. To contain such explosions the rotor
chamber is
always enclosed in a heavy armor plate.
The resolution power of an ultracentrifuge can be gauged from the fact that it c
an resolve
two types of DNA molecules differing only in the fact that one of the two types
contains SN (the

heavier isotope) in place of the naturally occurring 4N isotope of nitrogen. Wit

h this backdrop it
is easier to understand how the ultracentrifuge has helped biochemists in partic
ular and the
biologists in general to not only isolate, but to study the structure-function r
elationship of
those subcellular organelles which could previously be only observed under an el
ectron
microscope. Over the years the ultra has been used to isolate viruses in pure fo
rm. This has
allowed careful analysis of their composition. Moreover, the ultra has also been
of much use in
such analytical applications as the characterization of macromolecules (proteins
, RNA, DNA)
not only with respect to their molecular weights, but to some extent their confo
rmations also.
Ultracentrffugation can be carried out either with a desire to obtain certain bi
ological
material in isolation from the components that it associates with (e.g., isolati
ng a cell type away
from all other cell types, obtaining a sub-cellular organelle without any contam
ination from
other organelles or fractionation of a macromolecule) or with a purpose to chara
cterize a
macromolecule or sub-cellular particle with respect to its molecular weight or s
edimentation
coefficient. Centrifugation for isolation and purification of components is know
n as preparatory
centrifugation, while that carried out with a desire for characterization is kno
wn as ana/yt/cal
centrifugation (we will discuss both these types in the subsequent pages).
The ultracentrifuges also are of two types -- the preparatory ultracentrifuge, a
nd the
analytical ultracentrifuge. All the description given above was that for prepara
tory ultracentrifuge.
Given below is a description of the analytical ultracentrifuge.
Analytical Ultracentrifuge
The analytical ultracentrifuge is more or less like the preparative ultracentrif
uge described
above in that it operates at almost the same maximum speed providing the same RC
F; it also Is

Motor
Centrifugatlon
307
refrigerated and has an evacuated chamber; the alloy used in the rotor and the s
haft are the
same. It, however, differs from the preparative ultracentrifuge in having differ
ent type of rotor
and in possessing a specialized optical system to monitor the progress of centrf
fugation. The
rotor of an ultracentrlge is elliptical (Figure 10.3 A) and has two holes for ho
lding two centrifuge
cells. One cell is known as the analytical cell while the other is known as the
counterbalance or
the counterpoise cell. The rotor holds these cells vertically whether it is at r
est or rotating. The
main function of the counterpoise is to counterbalance the analytical cell. It i
s a precisionmachined block of metal with two holes drilled at calibrateddistances from the c
entre of rotation.
These holes serve as calibrations for measuring the distances in the analytical
cell. The analytical
cell is sector shaped and can hold a liquid column about 14 mm high. It has a ca
pacity to
accommodate about 1 cma of sample. The upper and lower planes of the analytical
cell are
transparent having quartz or synthetic sapphire windows. The windows are provide
d for the
passage of light to monitor the progress of centrifugation. The optics used in a
n analytical
centrifuge are either Schlieren optics or Rayleigh interference optics. The Rayl
eigh interference
operates on the basis that the region of the solution in analytical cell harbour
ing macromolecule
will have a refractive index higher than the rest of the solution. At the beginn
ing of sedimentation
this peak of refraction will be at the meniscus. With the progress of sedimentat
ion, however, as
the macromolecules move down the cell the )eak also shifts giving direct informa
tion about the
To vacuum
Counterpoise
Lens Photographic plate
Mirror
Lens
DriVe shaft
Analytical cell

Rotor chamber

Lens
Monochromator
Slit
Light source

14 40
3o8
Bphysat Cherrs
sedimentation characteristics of the macromolecules. The whole optical informati
on
continuously photographed. Figure I(.3 (B) provides diagrammatic representation
of
analytical ultracentrifuge system.
Analytical ultracentrlfugation has made it quite easy to measure the
coefficients and molecular weights of macromolecules. Molecular weights
species, even when they are in a gross mixture, have been measured by this techn
ique.
measuring molecular weights, it has several other applications discussed elsewhe
re in
chapter.
One point must be made clear here. The name 'preparative ultracentrifuge' is a b
it of.
misnomer. This is so because apart from being used for preparative purposes, it
is used quite
frequently for analytical purposes too. This will become clearer when we
applications of this type of ultracentrifuge.
Roto
Basically, rotors come in four varieties : fixed-angle rotors, vertical tube rot
ors, swinging.
bucket rotors, and zonal rotors. The first three rotors are discussed below. Zon
al rotors will be l
discussed in a later section.
Fted-ang/e rotors: These rotors have holes within their body and one can slide t
he centrifuge
tubes within these holes. Since the holes are at an angle (between 14 and 40) to t
he vertical,
the tubes and the solution within also take the same angle (Figure 10.4). Under
centrifugal
Centrifugal
field

Tube angle
Axis of rotation
Centrifugal
field

[D)
(E)
Figure 10.4. The flxed-angle rotor. (A) CroSS section of a typical flxed-angle r
otor. (B] If the centrifuge tube {s fllled
with a gradient and placed in the rotor: no centrlfugal force ts applied. (C) Ap
plication of centrlfal fore
leads to reorientation of the sample and the gradient, (D) The sample components
separate. (E) Gradient
reorients as the rotor stops.

Centrlfugatlon
309
field, the particles move radially outwards, travel only a short distance before
they strike the
wail. The particles then slide down the wall and the pellet is formed at the out
ermost point of
the tube. This sliding down makes the sedimentation quicker (wall-effect). Howev
er, there is a
severe disadvantage - particles differing highly in their sedimentation characte
ristics can only
be resolved in these rotors; particles which do not differ much in their sedimen
tation behavior
are not resolved. (For more, see discussion under wall-effects).
Vertlcal-tube rotors: These rotors too have holes within their body in which one
can slide
the centrifuge tubes. However, these holes lle parallel to the rotor shaft and n
ot at an angle. As
the rotor accelerates and centrifugal field is applied, the solution within the
tube reorients
through 90. This reorientation makes it lie perpendicular to the axis of rotatio
n. As the rotor
decelerates, the solution orients back to the original position (Figure 10.5).
(A)
Centrifugal field
J
Axis ofrotation
(B)
(c)
(El
(F)
Centrifugal field

Figure 10.5. The vertical tube rotor. (A) Cross-sectlon of a typical vertical tu
be rotor, (B) Tube.filled with gradient and
sample; no centrifugal force applied. (C) Application of centrifugal jeld leads
to reorientation of the
gradient and the sample. (D) The orientation that the gradient and the sample as
sume under ccmtrlfugal force. (E) The sample components separate. (F) As the rot
or decelerates, the gradient and
sample begin to reorienL (G) When the rotor stops, the original orientation is r
egained.
Sedimentation in these rotors occurs across the diameter of the tube. The partic
les thus
have to traverse the shortest possible distance and sedimentation is quicker tha
n the other
rotors. Moreover, the tubes lie at the edge of the rotor. This makes rm quite la

rge and the RCF,

at its minimum, is more than what is possible in any other rotor. This is anothe
r reason why
separation in these rotors is quicker.
The pellet in such- rotors will be deposited all along the outer wall of the tub
e. This can be
a disadvantage as the pellet may fall back into solution at the end of centrifug
ation. Even if it
doesn't fall back, it may not be easy to reconstitute the whole pellet and some
loss of yield may
be inevitable.
Swfnglng-bucketrotors: As against fixed hole type rotors we have seen above, the
se rotors
have buckets that swing out to a horizontal position when the rotor accelerates.
The solution in
the tube reorients to lie perpendicular to the axis of rotation and parallel to
the applied centrifugal
field. When the rotor decelerates, the tubes fall backto their original position
and the solution
too regains its original orientation (Figure 10.6).

310
(c)
(E)

Centrifugal field
Biophysical Chemistry
Centrifugal field

Tube at res
Tube during centrffugation
Axis of rotation
F3ure 10.6. The swLnfling-bucket rotor. (A) Cross section of a ttjpical swinging
-bucket rotor. (B) Centre tube fllled
with the gradlent and sample; no centrifugal force applied. (C) As the centrifug
al force is applied, the
tube swings out and orients at rjht angles to the axis of rotatlon. (D) The samp
le components separate.
(E) When the rotor stops, the bucket reorients to its original position.
Even in swinging bucket rotors, the particles strike the wails of the tube and s
lide down.
These rotors too suffer from wall-effects like the swinging bucket rotors. But t
hese rotors are
normally used for density-gradient centrffugation and the density gradient can c
ushion and
lessen the wall-effects.
Wall Effects or Trajectory of a Particle Inside the Rotor Tube
The centrifugal force acts on the particle in an outward direction. Thus, when p
erforming
centrifugation in a fixed angle rotor, the particle does not travel in a straigh
t line towards the
bottom of the tube. Rather it moves outward within the tube till it hits its wal
ls and then slides
down this wall to be pelleted at the bottom (Figure 10.7). This results in a rap
id sedimentation
of particles in a fixed angle rotor tube. This is known as the wall effect. This
behaviour, however,
gives rise to strong convection currents due to which it is not possible to reol

ve particles which
vary very little in their sedimentation characteristics. On the other hand, this
rotor design is
Axis of rotation
Supernatant
Cite: tee!e tube of fluid [I
//1
Boundaxy of |
Descending layer ---./
sedimenting
(High eoneentration]/(
particles
id
Thin layer of
Pellet Aseending layer
(Low concentration)
Figure 10.7 Schematic diagram of convectlon currents generated due to wall effec
ts. The diagram shows parles
descending along the wall of the tube (descending layer)

Centrifugation
311
very helpful in separation of particles which vary in their sedimentation rates
by a significant
order of magnitude.
This particle behaviou is true for swinging bucket rotor tubes also, albeit to a
lesser
extent. This is because the particles in a centrifugal field, rather than sedime
nting in parallel
lines, fan out radially from the centre of rotation. Thus, in these cells also t
he particles hit the
wall and then .slide down them to the bottom. Since swinging bucket rotors are g
enerally used
for density gradient centrifugation, the gradient usually decreases these convec
tion currents
due to wall effects.
Attempts have been made to' minimize wall effects by using a sector shaped cell
in the
swinging-bucket rotor. However, the best way to minimize the wall effects is to
use a zonal
rotor. The discussion of zonal rotor is deferred to a later section as an unders
tanding of density
gradient centrifugation is essentiaJ to understand its working.
PREPARATIVE CENTR/FUGATION
Preparative centrifugation is concerned with the actual isolation of biological
material for
subsequent biochemical investigations. Preparative centrifugation methods can be
divided into
two main techniques depending upon the medium of suspension in which the separat
ion is
carried out. Separations carried out in a suspending medium which is homogenous
are known
as differential centrifugation while those carried out in a suspending medium ha
ving density
gradients are known as dens/ty gradient centrifugations.
1. DIFF]INTIAL CENTRIFUGATION
Recall equation (I0) from our previous discussion of basic principles of sedimen
tation.
9
rb
t=--
In2
...

The above equation makes it clear that a mixture of homogeneous partlclcs can be
separated
by centrifugation on the basis of their densities and/or their size. This can be
achieved either by
the time required for their complete sedimentation in a fixed centrifugal field
or on the extent of
their sedimentation after a given time in a fixed centrifugal flcld.
If we now substitute in the above equation the approxlmatc data about the shape
and
densities of various intraccllular particles {we keep the mcdinm and therefore i
ts characteristics,
and the centrifugal field constant} we can arrive at a fair estimate about the t
ime requlrcd for
each of them to sediment completely in a given centrifugal field. In a similar m
anner we can
va the value of the centrifugal field applied for each intracellular organelle a
nd again find out
the time required for each of them to sediment completely under various centrifu
gal fields. If we
assemble all the data so generated we would find that under a flcld of roughly 1
000 g the cell
debris and the nuclei would sediment if the field-is applied for about 15 minute
s. Similarly,
upon subjecting the homogcnate to centrifugatlon for 15 minutes at 10,000 g, the
mitochondria
and lysosomcs would pellet out. Similar data would also be generated for other i
ntracellular
organelles {all the data represented here is approximate and it varies with tiss
ue and species
differences}. The best strategy that we can adapt now to separate the tissue hom
ogenate into
various organellcs is to centrifugally divide it into a number of fractions by i
ncreasing the
applied centrifugal field at each step. We can choose the centrifugal field in s
uch a manner that
a particular organcllc sediments during the already known time of centrifugation
to give a
pellet. The pellet and supernatant arc separated at the end of each step and the
supernatant
reccntrifuged to sediment another lighte" intracellular organelle. This is the e
ssence of differcntlal
centrlfugation.,FIgure I0.8 provides an outllnc of a typical fractionatton proce
dure based on
differential centrifugation.

312
lO00'x g
I0,000 x g
I00,000 x g 100,000 x g
Pt
5-15 rain
15 mln
I-2 hr
of
microsomes
Tissue
Pellet of
of
(.. Pellet of
homogenate
nuclei
mitochondria +
some heavy
lysosomes
solubles
lgure 10.8 Outline of a typlcal fractwnatlon by differentW.l centratlon
Every pellet has to be washed several times. This is so since the pellet obtaine
d is never
pure, i.e,, apart from the desired particles, it consists of contamination from
the other pin.des
l.'.i.. Small sized particles
Large sized particles
(i)
Time of
centrifuUon .

Large sized particles

Medium sized particles
- Small sized particles

Resupenson ofthepellt and reeen yeld apellet which sfcdrly putw, Howwr,
th yidof

Centrifugation
313
,contained in the homogenate. To understand the situation let us take a look at
Figure 10.9.
Before the centrifugation is initiated all the particles of the homogenate are h
omogeneously
distributed throughout the centrifuge tube (Fig. 10.9A, (i)). Centrifugation-res
ults in sedimentation
particles at their respective sedimentation rates till a pellet is formed at the
bottom of the
tube (Figure 10.9A (ii) and (iii)). This pellet, however, is not entirely made u
p of the large
some of the lighter particles, originally suspended near the bottom of the tube
also
and thus contaminated the pellet. Repeated resuspensions and recentrifugations
pellet (Figure 10.9 B and C) however, yield a fairly pure pellet of large partic
les. It
pointed out here that the repeated washings invariably reduce the yield of the f
inal
Inspite of its reduced yield differential centrifugation remains probably the mo
st commonly
method for isolation of intracellular organelles from tissue homogenates because
of its
ease, convenience and time economy. The drawback of this method is ofcourse its
poor
the fact that the preparations obtained are never pure. For experiments which st
rictly
absolute purity of preparations an alternative method known as density gradient
centrifugation (see below) is used.
2. DENSITY GRADIENT CENTRIFUGATION
As opposed to differential centrifugation, where a homogenous medium is used for
gradient centrifugation employs medium which has gradients, The separation
centrifugal field is therefore dependent upon the buoyant densities of the parti
cles.
gradients, apart from exerting their separating effect, eliminate mixing of sepa
rated
due to convection and mechanical vibrations. This method gives a much better
than differential centrffugation. Density gradient centrifugation has two variat
ions,
centrffugation, and isopycnic centrifugation.
Centrifugation
The gradient used here has maximum density below that of least dense sedimenting
particle.
gradient is reasonably shallow, The technique involves careful layering of a sam
ple
on top of a preformed liquid density gradient whose density continuously increas
es

the bottom of the sample tube. Centrifugation is then performed at a comparative

ly
speed for a short time. The sample particles travel through the steep gradient a
nd form
zones depending upon their sedimenting rate. For separation to be achieved b thi
s
it is necessary that centrifugation be terminated before any of the zone reaches
the
centrifuge tube. This method is useful for separating particles which differ in
size
Sample containing
particles of
different sizes
small particles
medium psrtlolss
largo
pilrllolee
A D
;;: Imnll portloloe Medium eled pmrtleloe ..oLarOo pertleloe
| O. I 0 RaW.zonal centratton. (A) Sample layered over a continuous densWj gradi
ent. (B) Posgton of zones
due to dterent sze of parcles at the termlnaton of centrtton. Note that separati
on s due to
d{lerences in sze and shape,

314
Biophysical Chemistry
but not in density, Thus, while this method is extremely useful for separation o
f proteins
possessing nearly identical densities but differing only slightly in their molec
ular weights, it is
not at all useful for separation of organdies such as mitochondria, lysosomes an
d peroxisomes
which have different densities but similar sizes. The method has been very usefu
l for separation
of RNA-DNA hybrids and ribosomal subunits. Figure 10.10 illustrates the salient
features of
rate-zonal centrifugation.
Isopynic Centrlfugation
For better understanding of this technique, it would be good if equation (9) is
recaged
In the above equation if one substitutes identical values for the density of the
particle and
density of the medium, the rate of sedimentation, , will become zero. If a densi
ty gradient is
now prepared in a tube in such a manner that the density goes on increasing towa
rd the
bottom of the tube and a solution of different particles is centrifuged in this
medium, different
particles differing in their buoyant densities will travel different lengths and
become stationary
at a region where the density of the layer below them is greater than their own
buoyant density.
This is the essence of Lopycnlc centrg@atlon also known as the n eqW10rlum
centr0at/o
The method differs from rate-zonal centrifugation in two significant ways. (i) A
s opposed
to rate-zonal centrifugation, where the grad/ents were shallow, isopycnic method
used gradients
which are reasonably steep so that at maximum, the gradient density is greater t
han the most
dense sedimenting species. (il) As opposed to rate-zonal method where centrifuga
tion is carried
out for a limited period of time, isopycnic method allows centrifugation for pro
longed periods at
relatively higher speeds to permit all species to seek their equilibrium densiti
es. Table 10. I
summarizes the differences between the two techniques.
Table 10.1 S--igniflcant differences between rate-zonal and iso.lcnic eenation
Rate-Zonal
Isopyeni
Synonym

s-zonal, sedimentation veloc/ty,

Density equil/bration, sedimentation
equilibrium.
Gradient
Shallow, maximum gradient density
Steep, maximum gradient density
less than that of the least dense
greater than that of the most dense
sedimenting specie, gradient
sedimenting specie, continuous or
continuous,
discontinuous gradients.
Centrifugation Incomplete sedimentation, low
Complete sedimentation till
speed, short time.
equilibrium is acheived, high speed,
long time.
Separations
RNA-DNA hybrids, ribosomal subDNA, plasmallpoproteins, lysosomes,
units etc.
mitochondria, peroxisomes etc.
There are two ways in which isopycnic centrifugation may be carried out. One way
is to
carry out a preliminary centrifugation of the sample at an RCF which will sedime
nt particles
heavier than the one desired. The supernatant is then layered over a medium whic
h has the
same density (isopycnlc) as the desired fraction. If centrifugation is now carri
ed out, the desired
fraction wiB-migrate to the middle of the liquid column. The fraction lighter th
an the desired
one will be at the meniscus, while the one that is heavier will be at the bottom
of the centrifuge
tube.

315
The alternate way is to prepare a continuous density gradient in a tube. The gra
dient
should span the range of particle densities of interest. No pretreatment of the
sample is necessary
and it can directly be layered on top of the densitygradient column. Upon centri
fugation individual
particles will sediment down the tube till they encounter a gradient whose densi
ty is greater
than their own buoyant densiW. The particles will thus become stationary forming
distinct
zones (Figure I0.II).
Srnlde cont|inlng
clMere cleities

Centrifugal force acting

on the particle
Higher density : acts
as a cushion
(A)
The partles have formed d,rent zones n regions of the gradient havbg densities s
bnilar to their
Isopycnic separation depends solely on the buoyant densities of the particles to
be separated
and not on their shaPe or size. Thus, the method is not useful for separation of
proteins many
0fwhich have similar buoyant densities inspire of differences in molecular weigh
ts. It is however,
very useful for separation of such intracellular organelles as mitochondria, lys
osomes, and
perox/somes which do not differ in size but differ in their buoyant densities. T
he method is also
useful for nucleic acid fractionation.
Table 10.2 lists the densities of a few important organelles and other macromole
cules in
sucrose soluUons.
able 10.2 Approximate densitie of particles in sucrose solutions
Particles
Denai (n/cm

Golgl apparatus
1.06 1.10
Plasma Membranes
1.16
Smooth endoplasmlc reticulum
I. 16
Intact oncogenic viruses
I. 16 - I. 18
Mitochondrla
Lysosomes
Peroxlsomes
Plant viruses
Soluble proteins
Rhino- and enterov/ruses
Nucleic acids, ribosomes
Glycogen
1.19
1.21
1.23
1.30- 1.45
1.30
1.30- 1.45
1.60- 1.75
1.70

Materials
316
Biophysical Cherntstnd
Gradient Materials
Pickels (I 943), Brakke (I 95 I) and Kahler and Lloyd { 195 i) were the first fe
w scientists to
use stabilizing gradients. They used sucrose for these gradients. Sucrose still
remains the
materialin most general use.
A gradient material should meet several requirements. There is no ideal all-purp
ose gradient
material. Sucrose is used routinely for rate-zonal centrifugation, while cesium
chloride may be
the usual choice for isopycnic centrifugation. Apart from allowing the desired t
ype of separation,
the gradient material should have the following properties :
(0 it should not affect the biological activity of the sample being Separated,
(iO it should not interfere with the assay technique,
(tit) it should be easily removable from the purified product,
(iv) it should not absorb in the ultraviolet range,
(v) it should be non-corrosive to the rotor,
(vO it should be easily sterilizable,
(vii) it should be cheap and readily available, and
(viii) it should preferably be recoverable for reuse ....
Table 10.3 provides data about most commonly used gradient materials along with
their
maximum densities at 20C.
Table 10.3 Types of Gradient Materials
Maximum density
Sucrose (66%, .5C}
Silica sols
Glycerol
CsCI
Cs acetate

Cs formate
Flcol
Sorbitol
Renografln
Urograffin
Polyvinylpyrrolidone
Diodon
RbBr
RbCI
K formate
Na formate
Metrizamlde
1.33
1.37
1.26
1.91
2.0
2.1
1118
NA
NA
NA
NA
1.37
1.63
1.49
1.57
1.32
1.45

NA = Not Available.

Among the routinely used materials, sucrose interacts with the particles being s
eparated
and can ecert pronounced effects on the results obtained. The interaction varies
from one type

Centrfugatlon
317
of the particle to the other. Non-membraned solid particles are not affected as
much as the
membraned particles containing a large percentage of water. This fact might be u
sed successfully
separate particles of overlapping buoyant densities by deliberately using a medi
um which
interacts more with one kind of particle than with the other, modifying the buoy
ant density of
one with respect to the other. In addition many commercial preparations of sucro
se contain
ribonuclease and absorb UV light. These impurities have to be removed before the
gradient is
prepared,
Other popular materials are Picoll (high molecular weight sucrose polymer and
eplchlorhydrln), Ludox (silica sols) and Metrlzamlde. Metrlzamlde has a disadvan
tage in that it
moderately quenches scintillation cocktail necessitating some correction when se
parated
components are to be assayed by scintillation spectrometry.
Selection of correct gradient material is very important for a satisfactory sepa
ration by
gradient centrlfugatlon. Different particles react differently with different gr
adient materials
and consequently possess widely varying densities in different gradient material
s. A correct
choice of the gradient material would therefore be able to accentuate even small
differences in
densities between two particles leading to their complete purification. On the o
ther hand, an
: incorrect choice of gradient material can lead to a situation where inherently
large differences
between the densities of two particles can be lessened, leading to a less than s
atisfactory
of Density Gradients
Two types of gradients may be prepared; discontinuous or 'step' density gradient
, or
ntinuous density gradient.
Discontinuous density gradient (where the density increases abruptly from one la
yer to
may be prepared very easily by carefully layering solutions of continuously decr
easing
over each other with the highest density solution being placed at the bottom of
the
tube. The sample is then layered at the top layer and the tube is spun under the
experimental conditions. Discontinuous gradients are especially useful for the
of whole cells or sub-cellular organelles from tissue homogenates. They are also
useful for separation of lipoproteins.
Continuous gradients (where the density decreases linearly from the bottom of th

e centrifuge
to the meniscus) may simply be prepared by allowing the discontinuous gradients
to stand
long time. The discontinuous layers slowly merge, through diffusion, to give ris
e to a lindar
gradient. However, this method takes very long time if viscous solutions are use
d in
of gradients since diffusivity decreases with increasing viscosity.
Altematively use of a special device may be made of to prepare density gradients
of a
nature (Figure 10.12}. The device has two chambers. The chamber which drips into
tube contains very dense solution. This chamber is connected to a second chamber
s less dense solution. The first chamber has some stirring device. When the tap
of
first chamber is opened, the dense solution drips into the centrifuge tube. The
level of
the first chamber thus decreases and is compensated by the flow of the less dens
e
the second chamber. The stirring device, which has already been activated, stirs
solution of two different densities and the chamber now contains solution of a d
ensity
'less than it originally had, Thus the solution which drips into the centrifuge
tube is of
decreasing density, In this way a continuous linear gradient may be formed.
gradients are useful in the separation of many proteins and enzymes, ribosomes

318
Stirrer
Biophysical Chemistnj

Dense solution of
gradient material
Less dense solution of
gradient material

Density gradient
Figure lO.12.Dic@ram of a gradient maker. The device has two interconnected cham
bers. The chamber through
which the solution drips in the tube contains very dense gradient material solut
ion. The other chamber
has solution which is less dense.
Sample Application to the Gradient
The volume of the sample that can be applied to the centrifuge tube depends upon
the
cross sectional area of the gradient exposed to the sample. Thus centrifuge tube
s of inner
diameter of 2.5 cm will accommodate only about upto 1 cm3 while tubes with diame
ter of 1.6,
em will accommodate only about 0.5 cm of sample. Large volume of sample, if adde
d, will
result in loss of proper resolution through broadening of the separated zones. L
esser volumes
of sample will not affect resolution but might be difficult to detect after sepa
ration.
The concentration of sample that can be applied is also limited. The general rul
e is to
apply a sample concentration which is about 10 times less than the concentration
of the starting
gradient.
The sample may be
gradient inclined
sample
trickles down the
like DNA, a broad

applied through a syringe which is held about 2-3 mm above

at an angle of 45 touching the wall of the centrifuge tube. The
wall of the centrifuge tube without disturbing the gradient.
mouthed pipette is used in place of a syringe.

Recovery of Samples

Two methods are in vogue. The first involves puncturing of the centrifugation tu
be at the
bottom with the help of a needle and collect the dripping medium in separate tub
es in fractions
of roughly equal volume (Figure 10.13A). A peristaltic pump might be employed to
suck off the
medium from the hole at the bottom.
The second method is known as the displacement technique. A cover with an outlet
is
placed at the top of the gradient. Through another hole in the cover a tube is p
assed to th
bottom of the tube. A very dense.solution (more dense than the highest density i
n the
is pumped through this tube. The dense solution, when it reaches the bottom, dis
places the
gradient layers which start coming out from the outlet provided in the cover (Fi
gure 10.13B)
and can be collected in separate tubes.
The fractions so collected can be analysed for separated components. If the cent
rifugation
has been carried out to obtain sub-cellular organelles, the presence of these ca
n be assayed by
measuring the activities of marker enzyme specific for each organelle.

3i9
Solution
[-. Fraction
density (60%)
[ cllectr1
Least density { 15%)
Sample zone
4----- Intermediate density (30%)
U
1
4----- Sample zone
Highest density (45%)
Solution density (60%)
(A) (B)
I O. 13 Sample recovery. (A) The bottom of a celluloid centrifuge tube i
s pierced with a hypodermic needle. The
fractions dr/p into a series of tubes. (B) Collection of samples separated on a
discontinuous gradient
by displacement technique. The three layers of gradient have been assigned arbit
rary density values.
A higher density solution when ptunlaed into the tube displaces the gradient whi
ch is collected into
of Roto
If the method adopted is rate zonal centrifugatlon, swinging bucket rotors give
better
The reason for their being suited better to this technique is the longer pathlen
gth that
rotor offers. Also, the wall effects are considerably less and the contents do n
ot reorient
acceleration and deceleration. Since rate zonal separations are not based on the
densities
there is a danger that such reorientations may disturb the separated
It is true that longer pathlength of the cell means a longer time for separation
. However,
time required .can be shortened by using half-filled tubes. In this case not onl
y is the

shortened but the centrifugal force at the meniscus also increases. Both these
contribute to limiting the time and as such rate zonal separations can be achiev
ed in
required for a fully filled cell.
Fixed angle and vertical tube rotors separate sample zones better than swinging
bucket
the technique being used is Isopycnic centrffugation. The reason for this is tha
t the
in these rotors is banded over a large surface cross-sectional area. Also, since
the
is on the basis of density and each component becomes immobile at regions equal
to
the reorientation of contents at deceleration is not a great disturbing factor.
As would be clear from the earlier discussion on rotors, fixed angle and vertica
l tube
bring samples to equilibrium in a shorter time as compared to the swinging bucke
t
It should be clear from Figures 10,4 and 10,5 that as the rotor tube angle decre
ases, the
length also decreases. The particles thus have less distance to travel. Naturall
y, the
tube rotors have the shortest pathlength and as such sedimentation is quickest i
n
rotors.
in Zonal Rotors
In a previous section we have already discussed that the centrifugal force acts
in an
making the particles move radially in both the fixed angle type or the swinginggives rise to strong convection currents due to wall effects. If, however,

320
Biophysical Chemistry
centrifugation is carried out in a sector shaped cell the particles will not mak
e a contact
the wall even when they move radially (Figure 10.14A). This is made possible by
what is known as a zonal rotor.
Minir01zing convection currents, however, is not the main reason behind
centrifugation. The main reason behind the use of such rotors is the small amoun
t of
that can'be accommodated in conventional rotors during density gradient
the largest swinging bucket rotors have a combined bucket capacity of only I00 m
l.
it very difficult to prepare sufficient quantities of materials required in many
experiments. limitation can be understood more clearly if we consider the follo
wing example. To purify
enzyme citrate synthetase it is necessary to make a preparation of glyoxysomes
a rich source of the enzyme in question). However, the amount of glyoxysomes req
is tremendous and it would require many days to achieve the quantity by using
swinging-bucket rotors and the density gradient methods described above. This pr
oblem
be solved by using zonal rotor which was first developed by Anderson and his col
leagues.
Initial position
of sample
Positions of
separated
components
Figure I O. 14 (A) Sector shaped cell of a zonal rotor. Wall effects do not pose
a problem. (B) Design c
(B-XV). Note the four veined core. Arrows indicate ports at the base ends of the
veins.
Figure I0.14B shows the design of a zonal rotor. As evident from the figure, the
zonal
is more or less a flattened sphere which has its interior subdivided into four e
qual
(sector shaped) by means of a four- veined core.
Figure 10.15 Cross sectlon of one quadrant of a
zonal rotor showing {i) the special
holes drilled into the sepia for
gradJent loading, and (iO sample port.
G-Channels through which gradient

opposed to conventional methods, the zonal

is rotated while it is still empty, i.e., without a
density gradient. When the speed has been brought
up to 3000-4000 rpm, the gradient is loaded
the rotor (dynamic method). This is possible
because the four veinings of the core are hollow
because of the holes drilled into them. These holes
are continuous with the holes onto the perlmete
of the rotor's interior and with the special movable
seal and bearing assembly. The gradient can thus
be pumped into this special seal and bearing
assembly from where it will reach the sector shaped
quadrants while the rotor is moving (Figure I0.15).
Again unlike conventional methods (in which
the most dense solution is poured first) the gradient:
in a zonal rotor is established by pumping the
material of the lowest density first. In the

Centr/fugat/on 321
subsequent sgcs materials of increasing density arc pumped which displacc the lo
west density
layer toward the centre of rotation. The last to be added is the densest materia
l own as the
fluid cushion. This operation contues l the entire sector shaped cavity is, fu.
There are four
more holes present at the base of each of the four veins. Utilizing these holes
(sample ports) the
sample material can be loaded on to the top of the gradient so that it is very c
lose to the centre
of rotation. Once the sample loading is complete, the movable seal and bearing a
ssembly is
dismantled andthe rotor chamber is closed and evacuated. Subsequently the rotor
speed is
increased to the desired level where the sedimenting species will move radially
till they reach a
density which is similar to their own, When the centrifugation is over a yery de
nse solution is
pumped through the veins to the periphery of the rotor's interior. This operatio
n displaces the
gradient along with the separated sample components out through the central part
s previously
used for sample loading: The effluent can be collected as equal fractions in a s
eries of tubes.
ANALYTICAL CENTRIFUGATION
1. BASIC PRINCIPLF. OF SEDIMENTATION (CONTD.)
Having seen the preparative uses of centrtfugation, let us turn our attention to
the analytical
applications, Let's first deal with 'boundary sedimentation,' a name wh/ch is a
variant for
differential centrlfugation that we have dealt with above. It is called boundary
sedimentation
because in this form of centrtfugatton the initial position is a homogeneous sol
ution, As the
entrifugation starts; particles start to sediment leaving a clear layer of solven
t above.A boundary
is thus formed between clear solvent and the region where the particles are pres
ent, Much
information about the particle is now obtained by following the progress of this
bound.ary
during sedimentation.
There are quite a few factors affecting sedimentation that we are yet to address
. First and
foremost, the equations considered so far do not take into account th design of
the centrifuge,
the concentration of the suspension, and the nature of the medium. All these com
plex factors
do affect the sedimentatlonproperties of a given particle.
Secondly, all the equations we have considered so far are for spherical and unhy

drated
particles. What if the particles are not spherical and/or hydrated? In such case
s, the actual
radius of the particle, that we have been considering in the above equations, ge
ts replaced by
the effective radius or the Stokes radius. The asymmetric particles sediment at
a lower rate
owing to the larger friction they experience. To arrive at a mathematical expres
sion for the
sedimentation rates of such particles, equation (9) is suitably modified to give
dr 2 r {f
wherefis e cflo coecient for e asec or hydrated pcles dfo is e
fcon coeent for e unhydrated, sphec pcle, fifo is e con rao for a ven
molle.
For pcles wch e more or less sphec, for eple e obul proteus, is ratio
is clor to 1( I to 1.4). For fibrous proteus, og to eff long, r-e nature, e rao
be nsiderably g. For DNA too, it berg we asec, e ratio be lge.
e above reflonsp esmbshes a we pot point : ff s s s b ss et, tg w dnt der s. e c
e of s t may not become eately cle. But let's consider a protein its native stat
e d e e prote e denated ste. e confoaons ese o states der dely.

322
Biophysical Chemistnj
The above example is extreme. Let us consider a better example, and a more usefu
l one at
that, An enzyme is supposed to change its conformation subtly when a true substr
ate approaches
it. Thus, an enzyme without substrate, and an enzyme with the substrate may have
sllghtly different conformations. They will then sediment at different rates.
This relationship then becomes the principle on the basis of which one can explo
it
centrifugation for studying the conformation, and, indeed, changes in the confor
mation of a
given macr0molecule.
Yactors Affecting Sedimentation Velocity
We have already discussed the particle related parameters and the environmental
factors
that affect the rate of sedimentation of a given particle. However, there are ot
her factors factors which arise due to complex interaction between the particles themselves,
and between
the particles and the environment --that affect the sedimentation behavlour of t
he particles.
These factors need to be discussed. In the above discussion it has been mentione
d that the
shape of the molecule largely decides the sedimentation rate of that particle. H
owever, the
shape of a given molecule is not a static characteristic; it is largely affected
by the solvent( see
chapter 1, and 8). Also ignored so far is the fact that when a molecule is made
to move under a
brute force, this shape will be further deformed. Another factor is the charge t
hat the molecule
may carry, These charges may interact with each other and modify the sedimentati
on behaviour.
It is also important to consider that the solvent molecules are much smaller com
pared to the
particle. Moreover, with everything else constant, the concentration of the part
icle may itself
affect the sedimentation - at high concentration, the particles may themselves i
mpede their
own migration! A brief discussion of these factors is given below,
F,ct of Concentration: Consider macromolecules which are very large and asymmetr
ic
(extended}, Imagine that they are rotating. Now as these macromolecules rotate,
they will occupy
a very large volume of solution. As concentration of such molecules increases, t
he frequency of
collision also increases. Further imagine that these molecules approach each oth
er. It is very
difficult for the solvent molecules to move in the opposite direction. This rest
riction on the
movement of solvent molecules around the approaching macromolecules means that t
he effective

viscosity is higher around the macromolecules. Naturally the sedimentation rate

of the
macromolecules will be less. Please note that in equations (8), (9), and (I0) th
e term for viscosity
does not include this modified viscosity around the macromolecules.
Now consider two examples. (I) What happens to the sedimentation velocity of a g
lobular
protein when its concentration is increased? (2) What happens to the sedimentati
on velocity of
a large DNA molecule when its concentration is increased?
In both the above cases the chances of collision will increase as the concentrat
ion rises.
However, in the first case the molecules are largely spherical. They will occupy
less solution
volume since they are not extended. The effect on viscosity of the medium around
them will be
quite less. The sedimentation velocity will decrease definitely but to a small e
xtent.
In the second case though, the things will be entirely different. DNA is a very
extended
molecule. Thus, as the concentration of this molecule is increased, the viscosit
y around it will
be largely affected, the chances of collision will be that much more, and the ef
fect of sedimentation
velocity also will be substantial.
Thus, concentration dependence of sedimentation velocity increases with the size
and
asymmetry of the macromolecule.
Then there is the Jhonston-Ogston effect. Consider a situation where you are sed
lmentlng
a mixture of macromolecules, some very large and some small by comparison. In su
ch a mixture,
everything impedes the sedimentation of everything else. The larger molecules im
pede themselves
but they also have to move through a solution of slower-moving molecules. At hig
h concentration

Centrlfugation
323
then the larger molecules are so much impeded that they might move along with th
e smaller
molecules. In extreme cases, the entire mixture may move at the same sedimentati
on velocity
and hide the fact that there are more than one species having different sediment
ation rates. In
view of such anomalies, it is very necessary that sedimentation be done at very
low concentrations,
And even then the experimental results may not be entirely factual.
Fect of Speed: From the equations we discussed above it would seem that the rate
of
sedimentation should be proportional to the centrifugal field. However, this may
not be so
always. At high concentrations such proportionality may not exist for macromolec
ules, Let us
see what may happen here. If the spe,ed is quite high, the macromolecule moving
ahead leaves
a wake behind it. This wake can accelerate the macromoiecule moving behind it so
much that
it can reach the macromolecule moving ahead. This can lead to aggregation and fo
rmation of a
macromolecular cluster that will sediment at a higher rate and pellet quickly.
For very large macromolecules, such aggregation can occur at high speeds even if
one is
working with very low concentrations. This is especially true of DNA owing to it
s large size. This
effect becomes more pronounced as the molecular size increases. Thus, for chromo
somes, this
effect is extremely high. It is therefore absolutely essential that one works at
low speeds if correct
results are to be obtained.
F.ffet of Charge: Biological macromolecules are usually charged, If sedimentation
of
such macromolecules is carried out in solution of low ionic strength (0.01 M), a
peculiar situation
may result. The neutralizing counterions of the solution are very small as compa
red to the
macromolecules. It is obvious that the ionized macromolecules will sediment much
more quickly
than the small counterions. Consider that the macromolecules bear a negative cha
rge and the
counterlons are all positive. A small time after starting sedimentation, a clear
separation of
result - negative charges in front and positive way behind. A potential gradient
would thus get created such that it is against the direction of sedimentation. T
his effect can
slow down the velocity of the macromolecules.
This problem can be done away with by carrying out sedimentation at higher ionic
strength

2. SEDIIENTATION COEFFICIENT
The velocity of a particle per unit centrifugal field is referred to as its sedi
mentation
It is denoted by the symbol 's'. The mathematical expression of s is therefore
V
s =
...(12)
2
0r
Now if you reconsider equations (9) and (11), it will be obvious that if the env
ironmental
are kept constant, the sedimentation rate will become proportional to c02r.
v = s t02 r
...(13)
Look at equation (13). Compare it with equations (9) and (11). It should be fair
ly apparent
In view of the environmental factors being kept constant, s Is a measure solely
of the
particle rethe size, the shape, and the density. It is not surprising then that
velocityexperiments are carried out with a purpose of determining s.
In the above discussion we have taken the composition of the suspending medium t
o be
However, in reality, sedimentation rate studies are performed using a wide varie
ty of
systems. If s is to reflect properties due solely to the particle, then we must
find
way to correct the experimentally determined values to a constant environmental
set. The

62 '
324
Bbphyslcal Chemistry
The experimentally determined value of s is affected by temperature, solution vi
scosity and
density. By convention, the observed value of s is corrected for that value whic
h would be
obtained if water was used as the suspending medium at 20 C. This is done by subs
tituting
the observed value of s into the following formula
2o o (1-v) ...14)
where so is the experimentally obtained value of sedimentation coefficient and s
o.w is
sedimentation coefficient that would be obtained with water as solvent at 20C. S
2o,w is dubbed
as the standard sedimentation coec/enL T is the viscosity of water at temperatur
e T, o the
viscosity of water at 20C, c the viscosity of the solvent at a given temperature
and o the
viscosity of water at that temperature. It is normal to keep the temperature of
the solvent as
close as possible to 20C. P,w is the density of water at 20C, PT is the density
of the solvent at
temperature T, and is the partial specific volume of the particle,
Most values ors for biological particles lle between 10-I" and i0-n seconds. For
convenience
the value of $ is usually reported in svedbergs (S) and 1 svedberg is taken to b
e equal to 10-Is
seconds. Thus when we say that the prokaryotic ribosome is 70S, what we mean is
that it has
a sedimentation coefficient of 70 x I0-s seconds.
Factors Affecting Standard Sedimentation Coefficient
Mo/ecular Shape: Lrge, rigid, exended molecu/es wil/ce more cflon wh/le the sphe
rlcd
molecules face less riction. The higher ction in rod-like molecules is because t
hey cannot
bend. However, if the/r rigidity is destroyed, the/r entation coefftcient /ncrea
e markedly
as the friction decreases. This is best illustrated in the example given below o
f DNA denaturation.
The bases in double stranded DNA exist in a highly stacked manner. Moreover, the
bases of the two strands are in contact with each other through hydrogen bonds.
These two
factors are primarily responsible for the rigidity of DNA.

40 44 48 52 56 60
Temperature (C)
Figure 10.16 A plot of sedimentation coe.ent values of T7 DNA of molecular weigh
t 25 million as a functlon of
temperature. DNA was heated in a solutlon of pH 7.S and O.01M wtth respect to PO
c After heatlng, the
solution was cooled to 20C for centrlfugatlor Formaldehyde was added to prevent r
eformation of
hydrogen bonds when the solutWn was so cooled. Note that the value of s has incr
eased b{1220 at
the h@hest pot.
Imagine a long DNA molecule that is being heated. Heating tends to break hydroge
n bonds.
In the regions where the hydrogen bonds are broken, the bases become less stacke
d. Since

Centrlfugatlon
325
both the rigidity imparting factors are undermined in these regions, the DNA mol
ecule can
bend at these points. Previously the molecule was fully extended. Now, owing to
loss of rigidity
at places, the molecule can bend and its extendedness is reduced. Naturally, it
should feel less
friction and its sedimentation coefficient should increase. As the temperature i
ncreases, more
regions where the DNA can bend are created and the molecule becomes progressivel
y more
flexible. Thus with an increase in temperature, the sedimentation coeffcient of
double stranded
DNA should increase. This is exactly what is observed in an experiment like this
(Figure 10.16).
Molecular weight: Figure 10.16 describes only half the experiment discussed abov
e. If
the temperature of the solution is allowed to increase continuously, all the hyd
rogen bonds
between the two DNA strands are broken and the molecule is rendered single stran
ded. The
moment this happens, the curve that was rising, drops precipitously. This happen
s because
the molecular weight is suddenly halved (Figure 10.17). This is enough to indica
te that s is
dependent upon molecular weight of the molecule.
40,44148 52 56 60
Temperature (C)
Figure 10.17 Continuation of the experiment described by the plot in Flgur 10.3.
The dashed llne represents
sepcwatlon of strands.
Also evident from the above two plots should be the fact that frictional coeffci
ent and
molecular weight are very related functions. Thus any study to find out the rela
tionship between
s and M, in fact, becomes a study to find out the relationship between frictiona
l coefficient and
molecular weight. Such studies become especially important since many of the res
earchers
prefer to characterize a macromolecule by Just finding out its sedimentation coe
ffffcient and not
its molecular weight. This is done because it is simpler to determine the former
. The sedimentation
coefficient is then supposed to reflect the molecular weight of the macromolecul
e.
After several studies the relationship between s and M for DNA was found to be
s2o.w = 2.8 + 0.00834 M'497 ...(15)
This relationship is valid for double stranded NaDNA at neutral pH in 1 M NaCI w

hen the
sedimentation is carried out in a sector shaped cell and if the s value lles bet
ween 10 and 60 S.
Although the relationship exists, it is not very good to calculate the molecular
weight from
sedimentation coefficient values. This is so since a small error in the measurem
ent of
sedimentation coefficient can be amplified and a value of molecular weight so ob
tained will be
oil significantly.
For proteins, however, the situation is qttite different to that of DNA. Here s
varies as
M/. Thus a slight error in calculating sedimentation coefficient is reflected in
a molecular
which is Just slightly off.

...(12)
30.
20.
0.001 0.01
326
Biophysical Chemistry
Sol,t Bind|rig: Binding of small solute molecules to large macromolecules affect
s their
s values. Thus there is a difference in s valu of the same DNA if it is suspende
d in NaCI and if
it is suspended in CsCl. In the latter, it has a higher molecular weight and the
refore a higher s
value.
Much more significant is the change in the shape of macromolecules that can resu
lt asa
consequence of solute binding. Here, such a change is a function of the flexibil
ity of the
macromolecule. For example, if sedimentation coefficient of double stranded DNA
is determined
in a 0.001 M and a 0.05 M solution of NaCl, there is virtually no difference bet
ween the s
values. The double stranded DNA molecule is not only very extended but is very r
igid also. On
the other hand, s values for a single stranded DNA are significantly different f
rom each other
when the ionic strength of the solution differs. DNA bears negative charges due
to the phosphate
groups which are ionized. The molecule thus carries a large negative charge. The
se negative
charges repel each other and keep the molecule extended. In a solution of low io
nic strength
(0.001 M NaCl), these negative charges cannot be overcome and the molecule remai
ns extended.
On the other hand, if the ionic strength is high (0.05 M NaCI), the negative cha
rges due to
phosphate may be neutralized by the counterions in the solution and the DNA may
collapse
into a random coll. The random coil is more compact as compared to the extended
DNA structure
and therefore sediments at a rate faster than the latter (Figure 10.18).
Double-stl'd

0.05
0.I
NaCI (M)
lgure 10.18 Solute effects depend on the.flexibility of the macromolecule. Doubl
e stranded DNA, which is rigid, is
almost unaffected by an increase in NaCl concentration. On the other hand the s
value of single
stranded DNA. which is.flexible, undergoes a large change with increment in NaCl
concentration.
Measurement of Sedimentation Coefficient
Recall equation (12)
v
We have seen earlier that v = dr/dt. Therefore
dr/dt
I
d In r
2
2
...(16)
t0r
0
dt
A plot of log r versus t produces a straight line whose slope will be t02s (Figu
re 10.19). Since
to2 is decided by the investigator, the slope provides a determination of s.
We have seen above the conditions that affect sedimentation coefficient. In addi
tion we
have also seen other conditions which affect rate of sedimentation. These condit
ions also affect

(C}
Centrifugation
327
the sedimentation coefficient. When we measure sedimentation coefficient, theref
ore, we have
to take care to minimize the effect of these factors. Thus to avoid charge relat
ed effects, the ionic
strength of the solution must be between 0.05 to 1.0 M. The pH must be controlle
d within
suitable limits with the help of a buffer. To nullify speed related effects, mea
surements are
performed at several speeds. Concentration affects sedimentation coefficient. We
have seen
that the best results can only be obtained at very low concentrations. Thus, mea
surements are
done at afleast four different concentrations.*
Time (minutes}
lqgure 10.19 Plot of log r vs. time in minutes. The slope is used to arrive at t
he value for s,
Concentration Distribution
Each component sediments at a rate dependent on its sedimentation coefficient. T
hus, in
boundary sedimentation, we should see the following situations as the centrifuga
tion progresses.
Initially, the particles are distributed uniformly throughout the solution. Ther
efore, ffabsorptlon
or refraction of light is measured at this point of time, the whole cell (the di
scussion here
pertains to sector-shaped cells since wall effects are almost non-existent in th
ese ceils) should
give the same data. As the sedimentation starts, the particles start moving towa
rds the bottom
of the cell. Once this happens, clear solvent is left behind at the meniscus. Wh
ether one is
measuring the absorption due to the particles or the refractive index, the param
eter now differs
at places where there is clear solvent and where the particles have migrated. In
the initial
Fure 10.20 Concentration distribution after a given period in a cell fllled with
a homogeneous solutn. (A) Poslt2on
in the cell. (B} A plot of concentration vs. distance: the region where the oonc
entration changes is called
bounda; the upper flat portion is called the plateam (C) Concentration gradient;
plot of derfvata of
concentratgon; this is what is obtained with scldieren optics.

* True sedimentation coefficient can only be obtained at zero concentration (s 2

0. )" This, however, is
o is measured at several concentrations and extrapolated to
an experimental impossibfllty. Here s
knfln/t dflutlons to obta/n a value of so.. at,zero concentration.
Moreover, one should also remember that the sedimentation coefficient that has b
een deterrnined is
of a macromolecule that is bound to counterions and to ligands, ff any. Thus, de
pending on which
counterion has been used and how much it binds to the macromolecule, the sedimen
tation coefficient
will differ.

materials.
M
328
Biophysical Chemistry
conditions when the particles were uniformly dispel;sed throughout the cell, no
boundary could
be seen because the entire solution refracted or absorbed similarly. Now the cle
ar solvent refracts
or absorbs differently as compared to the region where the particles are present
. Thus, at the
interface, there is a boundary now. As the sedimentation progresses, this bounda
ry will move
toward the bottom (Figure I0. 20 ).
Figure 10.20 is for the simplest situation possible : all the particles in the c
ell have a Single
sedimentation coefficient; in other words, all the particles are of the same kin
d. Figure 10.20
(B} is a plot of concentration of the material being sedimented versus distance
travelled, The
region where the concentration changes is the boundary. There are two features t
o this plot: the
distance sedimented and the height of the boundary. The distance sedimented is a
measure of
s and the height gives the concentration of the material concerned. Figure 10.20
(C) is a plot of
the derivative of the concentration curve, or the concentration gradient. This p
lot is what is
obtalnedwlth schlieren optics or other monitoring techniques. The area under the
peak in this
plot gives the concentration of the component.
Let us consider a cell in which two materials with distinctly
ation
coefficients (Figure 10.21) are being sedimented. Since there
, we obtain
two boundaries. The boundary that is more to the bottom has a
ne that is to
the left has a lower s. The heights of the boundaries tell us
ation of the two

different sediment
are two components
higher s and the o
about the concentr

{A)
{B)
Figure 10.21 Same asia. 10-20. But here there are two components n the so/utWn.
The s values of both the
components are quite dljferent from one another.
What if the system contains a mixture of many components which have sedimentatio
n
coefficients differing only slightly from each other? We do see a boundary which
is diffuse

(Figure 10.22).
lure 10.22 Concentran dtrbutlon m a cell contoJnm a heterogeneous solution of se
veral component
slhtly n their s values from one another.

329
Distance
Distance
Distance
Centrifugation
From the above discussion following points emerge:
(ll
(2)
a homogeneous solution gives a very sharp boundary;
if the solution contains two materials, two sharp boundaries will be produced wh
ich
will look like steps;
(3) if the solution has mmly components differing little in their sedimentation
coefficients,
the boundary will have many steps fusing into each other -- the result will be a
sloping
curve
(4) although we have not mentioned it so far, the sharpness of the peak is also
a function
of the diffusion coefficient of the component -- the less the diffusion, the sha
rper will
be the peak.
With these points at the back of our mind let's do a bit of exercise and then se
e the
applications of boundaIT sedimentation.
Look at Figure .10.23. What information do you see in these figures? Don't read
the
in the text given below. First make a mental assessment. Then check with the
explanation.
10.23
Concentration distribution of different kinds of solutions after a given
.period of time. F,xplanation in
the text.
Figure I0.23(A) tells us that there are four components in the material being se
dimented.
the components have sedimentation coefficients quite distinct from each other. A
lso, the

component has a concentration higher than the other three. The other three have
similar
What is not provided in the text and what you have to do yourself is to make a
derivative of the concentration curve.
Figure 10.23(B} is a heterogeneous mixture of many components whose sedimentatio
n
differ only a little from each other. Make a plot of the derivative of the conce
ntration
for this too. From this curve how can you tell whether it is a large diffusion c
oefficient
has broadened the peak or it is due to the heterogeneity of the mixture?
Figure I0.23(C) is quite interesting, It also is a heterogeneous mixture of many
components.
it is different from I0.23(B) in the fact that there is one component in this se
t which
a sedimentation coefficient far removed from the rest of the components. This Co
mponent
faster and the rest of the components differ little in their sedimentation coeff
icients and
behind. The component moving ahead has a sedimentation coefficient higher than t
he rest
Make a plot of the derivative of the concentration curve for this too. In what
would it be different from the others?

330
Biophysical Chemistry
We have already seen that the heights of the boundaries tell us of the concentra
tion of the
components. And since concentrations are additive, the heights of the
tell us
the ratio of the Concentrations of the components. Thus, if in Figure 10.23(C),
is called a and it is a fraction f, of the mass of the total material, then
f = /(a + b). Here b is the faster moving component.
With this in the background let us see some applications of boundary sedimentati
on.
3. APPLICATIONS OF BOUNDARY SEDIMENTATION
Proof That M is ReqIred for Endonuclease Activity
If double stranded DNA is incubated with an endonuclease, strand breakage may oc
cur.
such a DNA solution is sedimented, the data we get can be summarized in Figure 1
0.24.
the endonuclease treatment has not begun, all DNA gives essentially a single
that all DNA present has the same sedlmentatlon coefficient. As nuclease activit
y is started, the
plot changes. There are now two components very clearly visible; one component i
s
moving and the other component is actually a mixture of several species havingve
ry close
sedimentation coefficients. These components are actually the'broken pieces of D
NA created by
the nuclease action. The faster moving component is the intact DNA. As more time
is allowed
for nuclease action, the concentration of the faster moving component decreases
as we can see
in the figure by the diminishing height of the boundary due to it.
If at zero time we put EDTA in the DNA solution and then begin nuclease action,
the whole
scenario changes. No matter how much time we allow nuclease to act, we continue
to observe
essentially a single peak; the one corresponding to the intact DNA. EDTA is a go
od chelator,
divalent cations. This tells us that nucleases require divalent cations for thei
r action.
other studies we confine, that it is actually the magnesium divalent cation that
they
their action.
Figure 10.24 Effect of EDTA on endonuclease activity. (A) No nuclease added. All
DNA has the same

ooe.ienL (B) Endonwlease added. Slow moving component seen which must be a mlxtt
we of many
different sized fragments. (C) Incubation for a longer time with endonuclease re
sults in further:
fragmentation of DNA. (D) Nuclease added after EDTA has been added at zero tine.
Only a sL'ue
species of DNA ts present. EDTA inhibits nuclease action.
Proof for General Theory of Allosterism
In 1963 Monod, Changeux, and Jacob wrote a truly prophetic paper proposing what
we
call today the general theory of allosteric regulation of enzyme action. Making
studies on the
enzyme threonine deaminase their model, they suggested, among other points, that
the active
site is quite different from the regulatory site; that there could be two differ
ent kinds of regulators
- inhibitors and activators, and that the sites for the regulators may be situat
ed at different
places in the enzyme. At the time they proposed the theory, the evidence support
ing them was
fragmentary and inconclusive. Direct confirmation of the model came from the stu
dies by Gerhart

2.8 S
0,0
2 x 10-4M
4x
5.6 S
B x 10"4M
x 10-4M
14x 10-4M
Centrifugatlon
331
and Schachman on the enzyme aspartate transearbamoylase (ATCase). Most of the ev
idence
was obtained from sedimentation studies on this enzyme.
When Gerhart and Schachman treated the enzyme with p-mercuribenzoate (PMB) and
subjected It'to sedimentation, the results depicted in Figure 10.25 were obtaine
d. When the
enzyme was not treated with PMB, only one boundary was seen. However, as the con
centration
of PMB was increased, other boundaries appeared. At PMB concentration of 14 x 10
-4 M, the
boundal3, due to the intact enzyme completely disappeared and in lleu of that tw
o boundaries
appeared. One sedimented at 5.6 S and the other at 2.8 S. This is clearcut evide
nce that the
ee consisted of at least two different subunlts, one larger than the other. The
molecular
weight studies (again with the help of sedimentation) provided that the larger s
ubunit had a
molecular weight of 96000 and the smaller one of 30000.
PMB
PMB
10.25
ZJectofp- onthe0faspartatetmnsautj/aseTCase)atostdmn/ts.
The above sbc flgures pertain to ultn:wentrfge sedlJrwntan velocity runs. Sedb'n
entolWn is from the
meniscus on the left to the bottom of the tube on the right. When no PMB is pres
ent, the enmjme

sediments as a single peak. As the concentration of PMB creases, gradually the e

nzj.rne splits nto
two peaks wn sedlmentation oefficlents of 2,8 and 5,6 S. At 14 I 0 M, the enzyme
seems to have
fu dia to ts subuntts.
Enzyme studtes with the isolated suburdts provided the following data. The large
r suburt
stJll bnd to the substrate and carry out catalytJc activity. However, it could n
o longer
CTP like the tntact enzyme did (CTP is an trblbttor of the ev.zyme). The smaller
suburt

332
Biophysical Chemistry
could still bind CTP but had no catalytic ac.t.ivity. If the two subunits, separ
ated by sedimentation
were mixed again, the molecule which was the same size as native ATCase and whic
h had both
the catalytic and the regulatory functions could be regenerated. This was total
confirmation of
the ideas that Jacob and Monod had mooted.
Proof forKoshland's Induced-Fit Theory
Gerhart and Schachman's studies provided direct proof for another theory of extr
eme.
importance in biochemistry : Koshland's induced-itt theory. For decades scientis
ts had believed
in tlie Fischer's lock and key theory to explain the phenomenon of enzyme-substr
ate specificity,
The theory proposed that the substrate and the enzyme acted like the key and the
lock - the
correct substrate could fit the enzyme active site correctly ad brought about ac
tion; the incorrect
substrate could not fit likea bad key. Koshland changed this 'rigid' theory and
proposed that
the substrate and the enzyme were too flexible for such a theory. He proposed th
at both the
substrate and the enzyme changed their conformation as they approached each othe
r. If the
correct enzyme-substrate approached each other the change was such that both com
plemented
each other; otherwise the change was detrimental for an optimal fit. The direct
proof that
conformational changes did occur in the enzyme were also obtained by an extensio
n of Gerhart
and Schachman's studies on ATCase.
Figure 10.26 depicts the effect of succinate concentration onthe sedimentation c
oefficient
of ATCase. Succinate is a substrate analogue for this enzyme. The figure makes i
t distinctly
clear that as succinate concentration rises beyond a critical point there is a s
teep change in the
sedimentation coefficient of ATCase. In our discussion above we have seen that s
edimentation
coefficient depends very largely on the conformation of the particle. This then
is the direct prod
for a conformational change in the enzyme upon addition of the substrate analogu
e.
4.03.6 -

3.2 =
2.8
2.4
2.0
1.6
1.2
0.8
0.4.

i0-8
lO-S
10-4
10-s
10-i
Succinate Concentrat/on (M)

Figure 10.26 Effect of succlnate (a substrate analogue) on the sedimentation coe

,L'lent of ATCase. See the change
tn the sedimentation coejclent of the enzyme as succate concentration increases.
Since the
sedJmentatWn coefficient s decreasing, the enzjme may become, more asymmetric as
it binds to the
substrate. Mark the antagonistic behaviour of CTP. It tends to increase the sedi
mentation coeffieenL
Its opposite action here is reflected by the fact that it is an inhlbitor of the
ATCase.

333
The above studies were given even sounder footing when the two scientists demons
trated
that the changes in the sedimentation coefficient brought about by rising concen
tration of the
subtrate analogue could be reversed almost completely by adding CTP, which is an
inhibitor
for the enzyme.
Binding of a Cofactor to an Enzyme
Cofactors are tiny molecules as compared to the enzymes. As such, the cofactors
have
very small sedimentation coefficients. However, when they bind to the larger enz
yme, the two
molecules sediment together. This means that the cofactor will suddenly have a l
arger
sedimentation coefficient owing to this binding. The boundaries, before and afte
r binding, can
be observed by absorption optics ff the cofactor absorbs in the UV or the visibl
e range. This is
the theory behind studies of such bindings.
NADH is a cofactor for the enzyme lactate dehydrogenase. NADH absorbs well at 34
0 nm.
NADH alone is sedimented, a boundary which sediments slowly is seen and the
. However, when chicken heart lactate dehydrogenase
to the coenzyme solution a new boundary appears which has a sedimentation coeffi
cient
(Figure 10.27). Now there are two boundaries, both absorbing at 340 nm : one sed
imenting
0.2 S and the other at 7 S. The enzyme does not absorb at 340 nm. Thus ff we obs
erve a
at 7 S, it must be that NADH is binding to the enzyme and settling with it. As t
he
of the enzyme is increased, the boundary at 7 S has more area under its peak
area under 0.2 S peak diminishes. This is even more confirmation that NADH is bi
nding
the enzyme.
This simple assay is one of the best tools to assay the binding of NADH to lacta
te
of course for other cofactors to their enzymes) as a function of such conditions
ionic strength, concentrations of the enzyme and the cofactor themselves, temper
ature,
10.27
Absorption plots of sedimentatlon of NADH (340 nm}. {A] NADH alone sedim
ents at 0.2 S. (B} NADH
bound to Idctate dehydrogenase - sediments at 7 S. M is the meniscus and B signi
fies bottom of the
Subunits of Ribosomes

If pure ribosomal preparation from E. co/t is sedimented at a high Mg concentrati

on, the
giving a single peak sedimenting at 70 S. However, ff Mg concentration
the same preparation sediments giving three peaks: one at 70 S as before, anothe
r
8 and the third at 30 S (Figure 10.28). IfMg concentration is further reduced, t
he 70 S
entirely and the 30 S and the 50 S peaks enclose more area. This tells us that t
he
ribosome is actually formed of two subunits that can associate and dissociate de
pending
concentration.

334
'gure 10.28
Bfophgsfcal Chemistry
Corlc.
of
MgCl
M
30S 50S 70S B
Drawrgs of schl:leren paerns of rlbosome$ and subunlts sedimented at d{ferent co
ncentrations of
MgCl. In the bottom rectangle the concentration of MgCl is ristrj from 0.0002 M.
At 0.0002 M, the
subunits are completely dissociated. However, as the concentration rises, some a
ssociation into 70 $
particle is see. The top rectangle is at 0.01 M MgCI. At this concentration, rib
osomal subunits are fulb3
associated.

4. BAND SEDIMENTATION
In the above section we discussed analytical applications of boundary sedimentat
ion
However. band sedimentation (density gradient centrffugation) differs significan
tly from boundary
sedimentation as we have seen while discussing preparative centrifugation. Since
most of the
principles applying to band sedimentation have been discussed earlier (preparati
ve centrifugation}
we will just try and see the problems and advantages of band sedimentation visa
vis boundary
sedimentation before we discuss the applications of this technique.
Boundary sedimentation is more adapted to analytical centrifugaton. This is beca
use it is
difficult to prepare a density gradient in the small analytical ceil. This probl
em, ironically,
be addressed by not preforming a density gradient at all. What is done is that t
he sample is
initially mixed with the gradient medium to give a solution of uniform density.
The gradient will
then self-form when sedimentation is begun. It is almost mandatory here to use t
he salts of
heavy metals (cesium chloride, CsCI) or to use sucrose/mede. The sample is now m
ixed
in this concentrated heavy metal salt solution. When centrifugation starts, CsCI

molecules
sediment to form a concentration gradient which automatically results in a densi
ty gradient.
Once this happens, sample molecules which were previously uniformly distributed
throughout
the tube. now reorient themselves according to their buoyant densities - they ei
ther rise or sink
to the region where the solution density is equal to their own buoyant density.
This technique
suffers from the disadvantage that very long centrifugation times might be requi
red to achieve
equilibrium. But still it is used frequently as an analytical procedure to deter
mine the buoyant
density of a given particle.
Another problem area in band sedimentation is the determination of sedimentation
coefficient. Since there is a density gradient, the net force acting on the mole
cule is never a
simple function of distance. Secondly, if the determination is being done in a p
reparative

Centrifugation
335
centrifuge, there may be wall effects which would disallow a true value to be as
signed to
sedimentation coefficients. Thirdly, again if a preparative centrifuge is being
used, the movement
of the band may not be visualized and therefore the investigator will have to de
pend only ontwo
valtes - the initial position and the position of the band at termination of exp
eriment. Thus, the
rate of migration of the band will never be known.
The first problem can be solved by the use of isokinetic gradients. Isokinetic g
radients are
those gradients where the viscosity and concentration of the gradient at any poi
nt in the tube is
selected such that the particle migrates at a constant velocity at all distances
from the centre of
rotation. The exact explanation of this is as follows. We know that the velocity
of the particle
increases as it moves farther from the centre of rotation. The concentration and
the viscosity of
the gradient at different points is then to be chosen so that this increment in
the velocity is
exactly nullified and the particle migrates constantly. It is easy to see that t
he concentration
and viscosity of the gradient at any point depends on its distance from the cent
re of rotation if
it is to be isokinetic. Also easy to see is the fact that if for a given centrif
uge tube, 5% to 20%
sucrose gradient is isokinetic, for a longer or shorter centrifuge tube, a diffe
rent composition of
gradient would be isokinetic. This detail can be ignored only at considerable pe
ril to the
experimental determination of sedimentation coefficient. Such indiscriminate det
erminations
can and do quite often result in large scale discrepancies in the values ofs obt
ained by different laboratories for the same particle.
The second and the third problem cannot be addressed unless analytical dntrlfuge
is
used. The way around these problems is to determine relative value of sedimentat
ion coefficient
sedimenting the unknown particle with those particles whose seiimentation value
has been
determined by analytical centrifugation. However, such values may frequently be
far from the
actual values. '
While these problems do exist for band sedimentation, there are its uses which m
ake it an
ensable technique. If the mixture consists of particles with discrete sedimentat
ion
c.oefficients, this technique is the best to either separate them or study their
behaviour. Bourdary

sedimentation may fail here for the simple reason that if there is a minor compo
neit of a high
molecular mass among other components of smaller masses, the former may just bec
ome
indistinguishable. On the other hand, if the mixture is heterogeneous, bound'y s
edimentation
is the best method to use.
Band sedimentation has been used extensively in the study of nucleic acid struct
ure and
function. We give below a few examples.
of Nucleic Acids
Isopycnic ultracentrifugation is one of the most commonly used procedure
s for separating
nucleic acids. For DNA, the gradient is most often constructed with CsCI. The bu
oyant density,
p, of double stranded Cs DNA depends on its base composition:
0 = 1.660 + 0.098Xc
Thus CsC1 gradient separates DNA on the basis of its base composition. The stran
ds
higher GC content sediment faster,
Single stranded DNA is -0.015 g.cmL denser than the corresponding double strande
d
DNA. Thus it is easy to separate these two with the help of density gradient cen
trifugation. RNA
iis quite dense and CsC1 is not a suitable gradient maker for this nucleic acid.
Thus gradients of
Cs2SO
4 or sucrose are to be prepared. RNAs get separated on the basis of thei
r size. On the
hand. the RNA-DNA hybrids can be separated on the CsC1 gradients.

336
Blph.D sicctl. Chmtstvt9
Classical Method of Plasmid Purification
This method, which is less used today, is nevertheless a great example of the us
e
sedimentation technique for the simple reason that it takes advantage of the sha
pe of the
macromolecules in order to separate them. As such it is hugely instructive of th
e range of the
technique and the manipulation that a researcher can resort to in order to explo
it the technique
fully. ALSO notable is the fact that both boundary and band centrifugation are r
esorted to.
The first step in purifying a plasmid is the lysis of the host cell. This is the
trickiest part of
the whole procedure because'both, incomplete lysis and total dissolution of cell
s, will result in
greatly reduced yield of purified plasmid. Ideal lysis is where the cell has bee
n broken just
enough to let the plasmid escape without too much of the chromosomal DNA coming
out. Lysis
is normally done with incubating the cells with lysozyme initially followed by a
NaOH or SDSI
treatment. If lysis progresses well, most of the chromosomal DNA released is of
high molecular
weight and can be pelleted along with other cellular debris with high speed cent
rifugation. This
leaves a supematant that contains the plasmids.
The supernatant obtained above is then mixed in a solution of CsCI and ethidium
bromide
(EtBr). Isopycnic sedimentation is now carried out. As discussed above, CsCI is
a fast
substance and will generate a density gradient automatically. EtBr is an interca
lating agent. It
binds to DNA by intercalating between base pairs. This makes the DNA unwind. A c
ovalently
closed circle such as the plasmid cannot unwind completely. However, the linear
chromosomal
DNA can intercalate a large amount of EtBr and unwind to a great extent. As inte
rcalation
proceeds, the density of DNA goes on reducing. This leads to an interesting situ
ation -- the
density of the linear DNA can reduce much more than the density of the plasmid.
At
concentrations of EtBr, the plasmid has a higher density than the linear DNA. Th
is
in the densities of the two species of DNA leads them to band differently in the
centrifuge
lqading to their ultimate separation.
Role of Rho Factor in Termination of RNA Synthesis

We now know that Rho factor terminates RNA synthesis. This knowledge is based
the experiments conducted by J. W. Roberts in 1969. He isolated the protein fact
or and
that it had a role in synthesis, termination taking recourse to band sedimentati
on. The
behind the experimentation is simple and compulsive. If Rho factor is indeed inv
olved
termination, its presence in the reaction medium would lead to synthesis of smal
ler RNA. overall shape of the RNA molecules is the same, smaller RNA would sedim
ent slower
bigger ones. The experimental setup had two reaction mixtures. In both, the E. c
oli
DNA acted as the template. In both, RNA polymerase along with required buffers a
nd
conditions was present. In one of these, Rho factor was present. In the one wher
e Rho
was not present, [4C] uridine triphosphate was one of the ribonucleotides and in
the other
UTP was present. After incubation, the reaction mixtures from both the tubes wer
e
upon a preformed Isokinetic sucrose density gradient {59/0- 20%} and sedlmented.
sedimentation, the tube was punctured and the fractions collected. Figure 10.29
tells us
results of the sedimentation experiment. The [3H] RNA is collected in a much lat
er fraction
compared to the [4C] RNA. This is clearcut evidence that the [3H] RNA is shorter
.
presence of Rho has resulted in synthesis of shorter RNA meaning that it is inde
ed factor.

337
Centrifugation

,,' ',,/ l,.

,, .- ,,,. [5
10
15
20
Fraction No.
ljure I0,29 Effect of Rho factor on sedlmenmtion rate of the sIntheslzed RNA. Th
e solid line represents RNA
made with [H] UTP. while the dashed line represents RNA made with [C} LrI'P.
Sedimentation in denaturing gradient has been a variation highly used in nucleic
acid
studies. At high pH. DNA denatures and the two strands separate. This seemingly
simple event
induces large scale changes in the sedimentation behaviour of DNA and allows stu
dy of
phenomena which could not be studied in normal density gradients, Denaturing den
sity gradients
mean isokinetic gradients (5% - 20% sucrose) in 0.1 to 0.3 M NaOH. The main uses
of this
variation are given below.
First of all one must consider the fat that if a single strand break occurs in a
double
stranded DNA. the sedimentation pattern of such a DNA will not vary in a normal
density
gradient. The strength of the hydrogen bonds and the hydrophobic associations wo
uld not
allow the two strands to separate. On the contrary, if such a DNA is migrated in
an alkaline
gradient {Figure 10,30). the two strands would separate and the strand in which
the break
exists will migrate as two different fragments. And if there are more breaks tha
n one. thoo,se
many fragments will be created and all will migrate independently if their base
composition is
sufficiently different or if they have sutT1ciently different sizes.
Denaturing
pH
lgure 10,30 Denaturation of DNA ges rise to derent specles of DNA for the intact

and the nP- kc, d double-stranded

DNA. "

338
Bophyscal
This may seem qu/te/nslgniflcant actually. at s at aut dg out
bs DNA? Nog cept at s s-e we se bs wch was lot at DNA reptes a dsconuous mer lea
g to e pucon of what e O fraents (s chapter 13: 'pulse-chase labeg').
D Re.cared onuoy
If one o E. l for y generaons a mium coning
of e ces con e bel at r place. If s DNA
ent, most DNA sedenm a d hang a we s. Hoer, when s ted e adlen, a s con of e DNA
dents we slowly obaon tes of DNA at Is double sd. But one couples e and obaon e
t, e conclusion ached Is at e se stud bs e DNA.
If E. non-radloacve mi e pulse ladled [H]
e DNA sedent e aent, a we low s. ere Is oy one laon for Is. dloac c
repcag poon of DNA. d such corated raoac dents at a low s, repcag DNA must be se
sd as sin fraents which must later be veny
by e ee gase. ese aents e ed Ok frents er e
ese conclusions have been cated d today we ow at DNA ds pte
d/scont/nuous manner.
Native
Denatured

The same logic that has gone into the above study can also let us know about les
s
thLngs such as the rate of depur/nat/on (depurlnSon does not lead to s/ngle stra
nd

Centrlfugation
339
however, the loss of a purine base exposes the phosphodiester bond there to alka
line hydrolysis;
in alkaline gradients therefore, each depurination will lead to single strand br
eak and can be
identified as such), and radiation damage to DNA.
There is another use for denaturing gradient in DNA studies. Linear DNA and cova
lenfly
cosed circular DNA may differ from each other in their s values substantially. B
ut this is not
the case with all the conformations of DNA. For example, a nicked circle and a n
on-supercoiled
covalent circle have the same s values. Moreover, even where there are differenc
es, these may
not be sufficiently large. Thus a linear molecule and a nicked circle do not hav
e widely different
s values. Likewise a nicked circle anda naturally occurring supercoil have close
ly situated s
values. It is naturally difficult to distinguish between these conformations of
DNA by conventional
density gradients, However, denaturing gradients are of considerable value here.
A look at
Figure 10.31 will make evident the effect of denaturation on some of the conform
ations of DNA.
Naturally, with such drastic change upon denaturation, different conformations a
ssume widely
differing s values making the identification of these conformations more easily
possible.
How to Confirm that a Given DNA is a Covalently Closed Circle
If one grows bacteria containing a plasmid in a medium containing [3HI thymidine
, all
DNA is labeled. If these cells are now lysed and the DNA sedimented through an a
lkaline
gradient, two bands are observed - one corresponding to the bacterial chromosome
( whose s
value is considerably smaller owing to the fact the gradient is denaturing and t
he strands
would have separated) , and the other having a large s value which could be due
to a closed
circle. The peak area tells us that the rapidly sedimenting band corresponds to
a very small
fraction of the total DNA. This data merely indicates that the band could be a c
losed circle. How
to confirm that it is indeed a closed circle?
A look at Figure 10.31 points a way out if closed circles are nicked, they give
rise to an
open circle and in a denaturing gradient the strands will separate. This will ma
ke the resulting
structure to sediment slowly and the fast sedimenting band will disappear. The d
isappearanc

is confirmation that the fast sedimenting band was indeed a closed circle.
5. DETERMINATION OF MOLECULAR WEIGHTS
Two approaches are in vogue for determination of molecular weights through centr
lfugation,
the sedimentation velocity method and the sedimentation equilibrium method, the
former being
more popular. A third method, the approach to equilibrium method is also used.
(i) Sedimentation velocity : The method measures the speed of the moving boundar
y
(represented by a peak due to increase in refraction in the region where macromo
lecules are
present)..When the ultracentrifuge is operated at high speeds, the randomly dist
ributed particles
migrate through the solvent radially outwards from the centre of rotation. The i
nformation
hbout formation and velocity of the boundary between the portion of the solvent
that has cleared
of macromolecules and the portion of the solvent still containing the macromolec
ules is provided
by either Schlleren or Rayleigh interference optics. Electrical forces, like dou
ble layer phenomena
(see chapter on colloids) will produce an ion cloud which can impede the movemen
t of large
It is therefore ncessary to use either isoelectric protein or to add a neutral s
alt like
0, I M KCI. The pH or ionic strength is adjusted to make the macromolecules neut
ral.
The molecular weight of macromolecule.or sedlmenting particle can be determined
by the
Swdberg equation
RTS0,w
D(1-p)

340
where
anhydrous molecular weight of the macromolecules,
gas constant,
absolute temperature,
sedimentation coefficient of the molecule (see below),
diffusion coefficient of the molecule,
partial specific volume of the macromolecule,
density of the medium.
Biophysical Chemistry

Centrifugation
341
{i Sedinetttbn et/tilibrtum : This is a method which does not require measuremen
t of
diffusion coefficient of the macrom01ecule whose molecular weight is to be calcu
lated. In this
respect it scores more than the sedimentation velocity method for which diffusio
n coefficient is
a necessity. The method involves centrifuging a macromolecular solution at a spe
ed which will
exactly balance the tendendy of the macromolecule to disperse in the opposite di
rection due to
diffusion. The centrifugation is continued till a balance is established between
the sedimentation
and diffusion of the macromolecule so that no net movement occurs any more. The
concentration
gradient set up in the analytical cell can then be measured and substitution of
the values in the
following equation gives an idea about the molecular weight of the macromolecule
where
R = gas constant,
T = absolute temperature,
o = angular velocity,
p = density of the solvent,
= partial specific volume,
C and C2 = concentrations of solute at distances r and r2.
Obtaining equilibrium used to take a very long time (several days to a week). Ho
wever,
with short analytical cells (I-3 mm) and use of overspeeding techniques the time
has been
shortened to a few hours. ,The technique in its present form is more popular tha
n the
sedimentation velocity technique.
Box 10.2
ght by Sedimentation
umbr|um : Some Problems
subjectedt

342
Biophysicat Chemistry
(ill) Approach to equilibrium: Calculation of molecular weight by sedimentation
equilibrium
method depends upon the fact that when equilibrium is reached the flow of solute
vanishes at
every point in the cell, i.e., there is not change in concentration at any point
. Quite some time
is required for the tube to reach such an equilibrium through continued centrifu
gation. However,
at all time, even without centrffugation, two places exist where flow of solute
is zero. These
places are the meniscus of the solution and the bottom of the cell. This stateme
nt Is based upon
the fact that matter can not be pushed through eithe of the extremities. Thus, f
or all purposes,
the equation for sedimentation equilibrium must apply to these points at any tim
e from the
beginning of the experiment. This particular application of sedimentation equili
brium method
is known as the Archibald method (Archibald pointed out the above possibility).
Initially this method had the advantage of rapidity over the conventional sedime
ntation
equilibrium method. However, since it depends upon the data from the extremities
of the solution
column, precise measurements are not possible and the method is inferior in accu
racy. Moreover,
since the revival of sedimentation equilibrium technique with specially designed
small and
narrow cells and better suited optics (Rayleigh interference), this method has m
ore or less
fallen into disuse,

Centrifugation
343
Exercise
I.
You have been given two DNA solutions and told that they have identical
base pair composition
and the same length. However, upon sedimenting you find that they sediment at di
fferent
" rates. Can you provide an explanation?
2.
A boundary which takes 30 minutes to reach halfway down the cell when ce
ntrifuged at
10,000 rpm would take 15 minutes to reach the same position when the rpm is doub
led to
20,000. Is this statement true or false? Provide math6matical proof for your ans
wer.
3.
A boundary that takes 30 minutes to reach halfway down the cell when cen
trifuged at 10,000
rpm will take 60 minutes to reach the bottom of the cell if the rpm remains cons
tant. State
whether true or false. Give reasons for your answer.
4.
A student prepares an isokinetic gradient for sedimenting DNA. Later in
the day he switches
his mind and decides to sediment a protein solution. He uses the same gradient.
We believe
he is in a spot of trouble. Can you explain why?.
5.
A worker left a DNA solution on his table for a week. He remembers that
the pH of" the
solution was somewhere near 4. In order to detect whether the DNA is still alrig
ht he sediments
a small aliquot on a sucrose gradient and finds that a single band is observable
, However,
when he migrates another aliquot on an alkaline gradient he finds that apart fro
m a major
band, there are several other bands that migrate slower. What may have happened
to the
DNA?
6.
You are given a solution of DNA and told to prove that it contains doubl
e stranded DNA. In
how may ways can you prove this fact?
7.
What kind of sedimentation behaviour is expected of a protein that becom

es more compact
after denaturation?
8.
A protein Solution that has been purified sediments as a single boundary
. After treatment
with PMB, again a single boundary is observed. However, the boundary migrates ve
ry slow
compared to the earlier boundary. How do you explain this observation?
9.
A particular sedimentation run takes 40 minutes to complete. However, yo
u have to leave the
lab after 20/25 minutes. How can you complete the run within that time?
10.
After conducting rate zonal sediment=tion of RNA in a CsCI gradient, the
worker finds out
that none of the regions in the tube absorbs at 260 nm. Why should that be so?
Suggestions For Further Reading
I. Svedberg, T. and Pederson, K.O. (1940), The Ultracentrifuge, Oxford Universit
y Press, New
York.
2. Schachman, H.K. (1959), Ultracentrifugation in Biochemistry, Academic Press,
New York.
3. Bowen, T.J. (i 970), An Introduction to Ultracentrifugation, John-Wiley, Lond
on.
4.
Birnie, G.D. and Rickwood, D. (1978), Centrifugal Separations in Molecul
ar and Cell Biology,
Butterworth, London.

11
CHROMATOGRAPHY
If the individual components of a mixture have widely dissimilar physical and ch
emical
properties, it is very easy to separate one from another. But as the individual
components of a
mixture get more and more similar in physical and chemical properties, it become
s increasingly
difficult to separate them from one another. For example, most amino acids resem
ble one
another rather closely in physico-chemical properties. It is impossible to separ
ate a given amino
acid from a mixture of several by conventional separation methods such as fracti
onal
crystallization. A separation might, however, be achieved readily using chromato
graphy.
The first detailed description of chromatography is generally credited to Michae
l Tswett, a
Russian biochemist, who separated chlorophyll from a mixture of plant pigments i
n 1906.
Because of the nature of the pigments in the sample, each band had a distinctive
color. Thus
the name of the process was coined from the Greek words for color (chromo) and "
to write"
(graphy).
The term chromatography bunches together a family of closely related extremely p
owerful
separation methods. The feature common to them all is that two mutually Immiscib
le phases
are brought into contact with each other. One of these phases is stationary, whi
le the other Is
mobile; the mobile phase either moves oxer the surface or percolates through the
interstices of
the stationary phase. The sample mixture, introduced into the mobile phase under
goes
repeated interactions (partitions) between the stationary and mobile phases .whi
le being carried
through the system by the mobile phase. Different components of the sample mixtu
re interact
with the two phases differentially on the basis of small differences in their ph
ysico-chemical
properties. Since these different rates of interactions govern the migration of
the sample
components through the system, each one of the components migrates at a differen
t rate. The
compound which interacts more with the mobile phase and least with the stationar
y phase
migrates fast. The component showing least interaction with the mobile phase whi
le
interacting strongly with the stationary phase migrates slowly (retarded). This
differential
movement of the components is responsible for their ultimate separation from eac
h other.

Partition Coefficient
Partition coefficient {also known as distribution coefficient) is a definitive t
erm normally
used to describe the way in which a given compound distributes or partitions its
elf between two
immiscible phases, the stationary and the mobile phase. As described above, the
solute upon
entering a chromatographic system, immediately distributes itself between the st
ationary and
the mobile phase. If, at a given time during chromatography, the mobile phase fl
ow is stopped,
the compound will be in equilibrium between the stationary phase and the stopped
mobile
phase. At this stage, the concentration of the compound in each of the phases is
described by
the partition coefficient, K, which is expressed as follows

Chromatography
345
where C and Cm are the concentrations of the compound in the stationary and the
mobile
phases respectively (actually, the correct distribution coefficient will be give
n when one considers
the activities of the compound in the stationary and the mobile phases rather th
an their
Concentrations; but since there is no way of determining the activitie in a chro
matographic
system, concentration is used instead of activity). Thus, if the partition coeff
icient of a substance
between cellulo,se {stationary phase) and carbon tetrachloride (mobile phase) is
0.2, it means
that the concentration of the substance in carbon tetrachloride is five times th
at in cellulose.
This concept of partition coefficient is the basic principle of all chromatograp
hic methods.
All chromatographic systems consist of the two phases named above. The stationar
y phase
may be solid, gel, liquid, or a solid/liquid mixture, which is immobilized. The
mobile phase may
be liquid or gaseous. The choice of the mobile and stationary phase is made in s
uch a way that
components of the sample to be resolved have widely differering partition coeffi
cients.
Types of Interaction: The Nature of Partition Forces
The distribution of a solute between the stationary and the mobile phase is a re
sult of the
between the solute molecules and the molecules of each phase. The partition
therefore, reflects the relative attraction or repulsion that the molecules of t
he two
show for the solute molecules and for themselves. These attractive or repulsive
accompanied by a release or consumption of energy. The amount of interaction
provides a measure of the strength of the interaction and serves as a criterion
to classify
physical or chemical in nature. In chromatography, reversible interactions
importance; most of the interactions that are of interest in chromatography are
physical in nature, although, some chemical reactions can not be excluded from
It is the molecular constitution, which is fixed by the species of atoms present
and by the
of bonds between them (metallic, ionic, covalent, co-ordination), which decides
the
and the intensity of the physical interactions. Dispersion forces and electrosta
tic forces
which contribute most to partitioning of the solute between
Let us first deal with the dispersion (London) interaction. This interaction is
of a non-polar

In a non-polar liquid such as carbon tetrachloride, London's dispersion interact

ion is
only force present between two molecules. These non-polar molecules do not posse
ss any
dipole moment. The interaction is a resultant of instantaneous dipoles formed
the nuclei and electrons at zero-point motion of the molecule. Dispersion forces
are
When two non-polar molecules of the same type approach each other closely enough
for
orbitals to overlap, the weak attraction changes to' repulsion. Thus, non-polar
exist in a state of random distribution to give a disordered array. Another nonpolar
(whether a solute or a solvent) will mix in all.proportions since neither kind o
f the
any attraction between them. From the foregoing, it is easy to understand that a
solute molecule will interact more with the phase which is non-polar; this solut
e
move fast if the non-polar phase is the mobile phase or will be retarded more an
d
if the non-polar phase is the stationary phase.
Let us now deal with the polar interactions. The first kind to be discussed is t
he
interaction. The solvent whose molecules have permanent dipoles exhibits much
intermolecular attraction as compared to the non-polar molecules. Thus only thos
e solute
which exert either a higher or at least equal attraction with the solvent molecu
les as
to the attraction of solvent molecules for each other, will be able to mix with
the
Therefore, a polar sample component will interact more with the phase which
polar and move fast or be retarded more depending on whether the polar phase is
the
or the stationary phase respectively.

346
Biophysical Chemistry
Another type of polar interaction which is very important In chromatography Is
hydrogen bonding. A hydrogen bond is formed between a molecule in which hydrogen
is attached
to strongly electronegative atoms (e.g., oxygen, nitrogen, fluorine) and a molec
ule which possesses
unshared pairs of electrons. The actual strength of hydrogen bonds varies with d
ifferences in
molecular geometry, resonance, acld-base behavior, and the nature of the neighbo
ring atoms.
However, a generalized statement may be made: the interactions arising due to hy
drogen bonding
are relatively strong and in some instances interaction energy may approach that
of a weak
chemical bond. A common example of a solvent which is stabilized by hydrogen bon
ding is
afforded by water. It exists as a highly hydrogen bonded, albeit, relatively ope
n structure
consisting of clusters of 4-5 water molecules. If solute molecules have to inter
act with such a
polar solvent, they need to break this highly ordered, structure. Roughly 4-6 Kc
al is required to
break each hydrogen bond. To do so, the solute should either have the ability to
form hydrogen
bond with water, or it should have the ability to ionJze. Those solute molecules
which have
either of these abilities interact maximally with polar solvent like water. Othe
r non-polar solute
molecules simply do not interact with polar solvents. Hydrogen bonding influence
s partition of
solutes and therefore their chromatographic separation tremendously when one of
the phases
has hydrogen-bonding ability.
In addition to above interactions, which can be grouped as physical in nature, t
here are
other interactions, important in chromatography, that may be grouped as chemical
in nature.
The one interaction of this type which is extremely important for ion-exchange c
hromatography
(see later) is the ion-ion interaction characterized by electrostatic attraction
between mutually
oppositely charged long. Yet another interaction important for a comparatively r
ecenfl developed
chromatographic procedure is the interaction dependent upon biological specifici
ty between a
macromolecule and a ligand (affinity chromatography; see later). These forces do
not fall under
the perview of true partition as in actual terms partition concerns itself with
differential solubility
of a solute into two knuniscible solvents. Yet, as all chromatographic procedure
s involve differential
distribution of solute molecules between two phases, all of the forces involved
may be discussed
as partition forces.

SURVEY OF CHROMATOGRAPHIC PROCEDURES

Each of the chromatographic procedure described below utilizes different types o
f
interactions to achieve resolution of sample components.
The Partition Principle: Partition Chromatography
When a solute is allowed to equilibrate Itself between two equal volumes of two
immiscible
liquids, the ratio of the concentration of the solute in the two phases at equil
ibrium at a given
temperature is called the partition coefficient. A mixture of substances with di
fferent partition
coefficients can be quantitatively separated by a technique known as countercurr
ent distribution,
first developed by L.C. Craig, in which many repetitive partition steps take pla
ce (see Box 11.1).

347
Chromatography

348
Biophysical
Partition chromatography is a logical extension of the countercurrent partition
principle
for achieving chromatographic separation of mixtures. The technique, originallyd
eveloped by
A.J.P. Martin and R.L.M. Synge has since been applied to an enormous nunber of s
eparations.
The separation is achieved in a hge number of partition steps which takes place
on
microscopic granules of a hydrated insoluble inert substance, such as starch or
silica gel packed
in a column or layered on a plate, or the granules of cellulose in paper. The gr
anules, though
nert, are hydrophilic and as such are surrounded by a layer of tightly bound wat
er. This tightly
bound water, since it is immobilized, serves as the stationary phase. On the sur
face of this
stationary phase flows the mobile phase of an immiscible solvent containing the
mixture to be
separated. The solute molecules are subjected to microscopic partition processes
between the
immobilized water layer and the flowing solvent. Since the process takes place o
n the surface of
each granule the number of partition steps is so great that the substances move
along the
column or surface at a different rate as the mobile phase flows through it.
The principle of partition is exploited in gas/liquid chromatography (GLC) techn
ique
also. Separations depend upon the partition of the solute molecules between a li
quid,
supported on a suitable solid, and the gas flowing through the system.
In true partition chromatography, the only factor which Influences the movement
of a
compound as the solvent travels along the stationary phase is the relative solub
ility of that

Chromatography
349
compound in the two phases. Substances more soluble in the mobile phase will mig
rate greater
distances as compared with the substances more soluble in the stationary phase.
Other
compounds of intermediate solubility between the two phases will migrate to inte
rmediate
distances depending upon their partition coefficient.
Adsorption Chromatography
Substances differ in their adsorption-desorption behaviour between a moving solv
ent
(a liquid or a gas) and a stationary solid phase. This behaviour of a substance
can be exploited
to achieve its separation. Adsorption is a surface phenomenon which signifies a
higher
concentration at an interface as compared to that present in the surrounding med
ium (see
chapter 7). Adsorption should not be confused with absorption, which signifies t
he penetration
of one substance into the body of another.
For the purpose of chromatography, the term adsorption has limited meaning; it u
sually
denotes interactions involving hydrogen bonding and weaker electrostatic forces
of the substance
with the adsorbent. The solute molecule which interacts more with the adsorbent,
which is
also the stationary phase, is retarded more while less interacting solute molecu
les are retarded
less. In this way a separation of sample components is achieved.
Ion-Exchange Chromatography
This procedure was first developed by W. Cohn and may be defmed as the reversibl
e
exchange of long in solution with long electrostatically bound to some sort of i
nsoluble support
medium. The ion exchanger consists of an inert support medium coupled covalently
to positive
(anion exchanger) or negative (cation exchanger) functional groups. To these cov
alently bound
functional groups are bound, through electrostatic attraction, oppositely charge
d long which
will be exchanged with like charged long in the sample. Thus, if anion exchange
chromatography
is performed, negatively charged sample components will interact more with the s
tationary
phase and will be exchanged for like charged long already bound to the matrix. T
hese sample
long will be retarded whereas other uncharged or positively charged long will no
t be retarded to
the same degree and will be eluted out fast. The situation will be exactly rever

sed for cation

exchange chromatography.
Molecular Size: Gel-Filtration Chromatography
This technique exploits the molecular size as the basis of separation. The suppo
rt medium,
a gel, consists of porous beads where pore size is strictly controlled. Macromol
ecules smaller
than the pores get entrapped in the pores (and move slowly), while those bigger
than the pores
travel unhindered through the column (and elute out faster than the smaller mole
cules). Thus
the main interaction between the solute and the stationary phase is with respect
to the size and
.. this is ultimately the basis of separation also. This technique is also used
to determine relative
molecular weight of a given macromolecule.
Affinity Chromatography
The technique utilizes the speciicity of an enzyme for its substrate (also recep
tor for its
agonist, antibody for antigen) or substrate analogue for the enzyme's (other pro
teins with biological
specificity) separation. A substrate analogue is coupled to the gel matrix and t
he cellular
suspension is allowed to percolate through. The enzyme which is specific for the
substrate
analogue binds to the gel becoming immobile while all other components move down
and out.
The technique has a very.high resolution power.
Figure 11.1 provides an overview of chromatographic procedures on the basis of t
he
stationary and mobile phases used.

-- LIQUID
BONDED LIQUID
SOLID
PLAIV
COLuMN
PLANAR
NATURE OF
NUMBER OF
MOBILE PHASE
STATIONARY PHASE
GAS
[-- LIQUID
CHROMATOGRAPHY --SOLID
SEPARATION
PHENOMENA
PARTITION -AI)SORFI'ION -PARTITION
TECHNIQUE
COLUMN -COLUMN -COLUMN -PI.ANAR
NAME OF CHROMATOGRAPHIC METHOD
GAS LIQUID CHROMATOGRAPHY (GLC)

GAS SOLID CHROMATOGRAPHY [GSC)

CLASSISCAL LIQUID LIQUID CHROMATOGRAPHY (LLC)
PAPER CHROMATOGRAPHY (PC)

CHROMATOGRAPHY
CHROMATOGRAPHY -COLUMN
MODIFIED
PARTITION -PLANAR -coLuMN --- ADSORPTION
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPTCL)
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPLC)
LIQUID SOLID CHROMATOGRAPHY (LSC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
THIN LAYER CHROMATOGRAPHY fI'LC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
PAPER CHROMATOGRAPHY (PC)
ION EXCHANGE CHROMATOGRAPHY (IEC)

"-- ION EXCHANGE

-THIN LAYER ION EXCHANGE CHROMATOGRAPHY (TLC)
PAPER IRON EXCHANGE CHROMATOGRAPHY
-- EXCLUSION
COLUMN
PLANAR

GEL PERMEATION CHROMATOGRAPHY (GPC)

THIN LAYER GEL PERMEATION CHROMATOGRAPHY (TLGPC)

-- AFFINITY
COLUMN
AFFINITY CHROMATOGRAPHY
Figure 11. I Class]Icatlon of chromatographic systems.

Chromatography
351
TECHNIQUES OF CHROMATOGRAPHY
There are two basic techniques of chromatography: plane chromatography and
column chromatography. In plane chromatography the stationary phase is coated on
to a plane
surface. There are two variations of plane chromatography: paper chromatography
and thin
layer chromatography. In paper chromatography the stationary phase is supported
by cellulose
fibres of the paper sheet. In thin layer chromatography the stationary phase is
coated onto a
glass or plastic surface. As opposed to plane chromatography, the stationary pha
se in column
chromatography is packed into a glass or plastic column. Each of these technique
s have their
specific advantages, applications, and modes of operation.
It is important to distinguish between types and techniques of chromatography.
Adsorption chromatography, partition chromatography, ion-exchange chromatography
etc., are
types of chromatography (based on different principles) which can be carried out
using any of
the techniques stated in the preceding paragraph. Thus, adsorption chromatograph
y can be
carried out either on a column or on a thin layer; the same is true for partitio
n and most other
types of chromatography. A discussion of the techniques, thus, will be fruitful
for
understanding various types of chromatography.
I. PLANE CHROMATOGRAPHY
A drop of liquid spotted on to a piece of paper or cloth will spread in a circul
ar pattern. If
the liquid possesses color, concentric rings of colors will be observed. Ancient
Romans used
this technique to analyse dyes and pigments. However, the modem day scientist re
alised the
value of paper chromatography only in the very recent past. A.J.P. Martin, the p
ioneer of liquid
partition and gas chromatography, described paper partition chromatography in wh
ich moisture
clustered around cellulose fibres served as the stationary phase. Within a few y
ears of Martin's
publication, this technique has helped to revolutionize biochemical research. In
more recent
times, Stahl popularised the concept of thin layer chromatography (TLC). A "thin
layer" of
adsorbent spread on a glass plate is used much in the same way as a piece of pap
er.
Apart from its obvious simplicity, plane chromatography techniques require only
minute
amounts of samples. Moreover, chromatography on plane surfaces, rather than with
a column,

offers the unique advantage of two-dimensional operation. This operation exploit

s selective
properties of two different solvents in developing a single chromatogram.
A. PAPER CHROMATOGRAPHY
Nature of the Paper
The paper commonly used consists of highly purified cellulose. Cellulose, a homo
polysaccharide of glucose, contains several thousand anhydro-glucose units linked th
rough oxygen
atoms. Many of the hydroxyl groups of glucose, however, become partially oxidize
d during
manufacture. The oxidation products usually are aldehyde, ketone or carboxyl fun
ctional
gr0ups.The paper also contains impurities through inorganic substances, adsorbed
salts and
mineral matter, which gets deposited on the paper while it is being processed. T
hese impurities
may be removed by washing the paper with 0.1 N HCI and drying it before chromato
graphy is
carried out. This gives better results. Cellulose fibres in the paper hold moist
ure tightly through
formation of hydrogen bonds. The cellulose itself takes a negative charge in com
pany of water.
The paper exhibits weak ion exchange and adsorptive properties. Modified forms o
f paper have
been produced in which the paper has been impregnated with alumina, silica gel,
ion exchange
resin, etc. While these modifications lead to different mechanisms of separation
, the technique
remains the same.

352
Biophysical Chemistry
Apparatus and Paper Development
The apparatus required for paper chromatography consists of a support for paper,
a
solvent trough, and an airtight chamber in which the chromatogram is developed.
The apparatus
illustrated in Figure 11.2 is good enough for simple chromatography.

C
"rrou wlO
developing solvent'
Cllp-per
-..
Equilibrating
solvent
M/grating
sample
Solvent

Figure 11.2 Methods of paper chromatography. (A) Descending. (B) Ascending

The sample is applied to the paper as a small spot. This is done before dipping
the paper
into the eluting solvent. Any device, which can transfer a small volume of sampl
e, can be used
for spotting. Generally used devices are platinum loop, capillary tube, or a mic
ropipette. Of
these, platinum wire is preferred because it can be reused with several substanc
es after heating
on a flame. A micropipette can also be reused after its tip has been disposed an
d a new tip
applied. For some methods the sample may be applied as a narrow streak at right
angles to the
flow of the solvent.
There are two main techniques, which may be employed for the development of pape
r
chromatograms - ascend/ng or descending techniques (Figure 11.2). In both cases
the solvent
is placed in the base of a sealed tank or glass Jar to allow the chamber to beco
me saturated with
the solvent vapour. After equilibration of the chamber is achieved, the developm
ent of the

chromatogram may be started. If the development is to be performed by the ascend

ing technique,
the paper is allowed to hang in'or is suspended in a manner that the base of the
paper is in
contact with the solvent at the base of the chamber: The sample spots should be
in a position
Just owe the surface of the solvent so that as th solvent moves vertically up th
e paper by
capillary action, separation of the sample is achieved. In the descending techni
que, the end of
the paper near which the samples are located is held in a trough at the top of t
he tank and the
rest of the paper allowed to hang vertically but not in contact with the solvent
in the base of the
tank. Development is started by adding the solvent to the trough. Separation of
the sample is
achieved as the solvent moves downward under gravity.
Ascending technique has two advantages. Firefly, the set up required for is very
simple. Secondly, the resolution of sample by ascending technique is somewhat b
etter as compared to
the descending technique. This is so because in ascending chromatography, two fo
rces are
acting on the solute: the capillary force, which makes it move up, and the gravi
tational force
which opposes this movement. Under the influence of these two forces, the sample
components
are resolved better than in the descending technique. The disadvantage of the as
cending
technique, however, is that it is very slow. The descending technique, on the ot
her hand is

much faster than the ascending technique. Based On the above advantages, one can
choose the
techlque which sts one's p'-pose most.
A thud, less used techrque is the .techrque of radkl development. In this method
the
sample is spotted at the center ofa crcularly cut disc of paper wlch is placed h
orzonty. The
center of the paper is connected with a wckto the solvent, wlch is placed at the
base of ajar.
The solvent rses up the wick and thence onto the paper through capillary action.
The smple
components now move outward radially formg concentric circles of increasing diam
eters. If
the components to be separated are coled, the chromatogram developed by t-s meth
od looks
pleasing to the eye. The resolution of components by tls techrque is sharper, Th
e apparatus
also is simple. One way is to press the pap6r between two glass plates with a ho
le in the center
through which the wick can be connected for solvent supply. Alternatively, a lar
ge circular
glass jar covered with a glass plate serves as a very good chamber.
A plane surface is amenable to sequential development in two directions using tw
o
differentsolvents. The paper, with the sample applied as a spot close to a corne
r, is developed
la the normal fashion by either ascending or descending procedure. The developme
nt is continued
until the faster moving component or solvent front approaches the. end of the pa
per. The paper
is then removed and the solvent is allowed to evaporate. This paper is then turn
ed 900 and
developed a econd time with another solvent having totally different eluting pro
pertiesl Since
the two solvents used have different eluting properties, the distribution coeffi
cients of individual
also differ. Thus, components which could not be separated using one
alone can be easily separated by this procedure as shown is Figure 11.3 (also se
e Box
This technique is known as two-dimenslonal chromatography.
Two-dimensional chromatography. (A} First development in the direction indicated
b the arrow does
not resolve B and C mpletely. (B) Second development in a direction at rtjht anc
jles to the.first using
a different solvent system resolves all components completely.
of Solvent System
Usually in paper chromatography the stationary phase is water since it is very w
ell adsorbed
cellulose. The mobile phase, which is less polar flows over the polar stationary
phase The
, not be necessarily immiscible with water if water is being used as the station
ary

is because the stationary phase water i very tightly held by cellulose and will
not
the mobile phase on this account. The mobile phase is usually a mixture of vario
us
such as alcohols, acids, esters, ketones, phenols, amines, and hydrocarbons etc.
The
are selected in such a way that the resolution of sample components is satisfact
ory.
There are other factors, apart from optimum separation, which must be considered
before
of a solvent system"is made One important consideration is to limit the number o
f
in the solvent system used to the barest minimum. This is so since the more

354
Biophysical Chemistry
components a solvent system contains, the more difficult it will be to maintain
a saturated
atmosphere in the chamber. The components of the solvent system should be so cho
sen that
the extent of evaporation of each individual component is more or less similar.
Differing extent
of evaporation of the solvent components can change the composition of the solve
nt and can
lead to serious anomalies of separation. The temperature chosen for development
should also
be decided after serious deliberation because individual components of the solve
nt will differ
in their extent of evaporation at different temperatures. The temperature, there
fore, nust be
maintained within strict limits during the entire experiment.
Ideally, the solvent system should be so chosen that the two phases are immiscib
le.
Moreover, the sample components should have differing solubilities in the two ph
ases. Such a'
choice would lead to maximum separation. The time required will also be short an
d the spreadlng,
of the separated zones will also be milxtmal.
Desirable characteristics that a solvent should possess are listed below.
(/) Partition coefficient of substances to be analyzed should range from 1-100 i
n favouro
the aqueous phase.
(//) The solvent should be removable from the paper and to this effect its boili
ng point
should ideally be less than 200C. This property becomes all the more essential ff
two-dimensional
chromatographyis to be performed. In this case it becomes necessary to remove al
l traces 0
the first solvent since even small amounts of the first solvent remaining on the
paper will
change the characteristics of the second solvent and will therefore interfere wi
th the separation
process.
('i) The solvent should be stable. It should not become oxidized when spread ove
r paper. Some solvents, for example phenolic solvents (particularly in ammoniaca
l environment),
react with small quantities of copper present in the paper and get oxidized to p
roduce brown of
black tarry material. This material then migrates with the solvent front and dis
torts the
chromatogram. It is necessary to add metal-chelating agents to these solvents be
fore they ar
used.
A few solvent systems used to separate diverse compounds through paper chromatog
raphy

are given in Table 11.1

Table 11.I Examples of Solvents System Used in Paper Chromatography
Solvent systems (/)
Ratio
Compounds

Butan- 1-ol/acetic acid/water

Butan- 1-ol/pyridine/water
Methanol/pyridine/water
Butan- 1-ol/pyridine/water
Propan- 1-ol/petroleum ether
Chloroform/petroleum ether
(40/10/5
(33/33/33)
(25/12/63)
(50/28/22)
(4/96)
(30/70)
Amino acids
Amino acids
Amino acids
Mono and disaccharides
Plant pigments
Plant pigments

Detection
There are various methods of detection available. If the sample components are c
olored,
the analysis becomes simple as the distinctive color itself identifies the compo
nent. When the
components are colorless (usually they are), they can be imparted color by spray
ing the paper

C\
3H:O
II
O
OPURPLE PIGMENT
Chromatography
355
with color producing reagents. A case In point is the detection of amino acids.
Ninhydrin reagent
spread on the paper reacts with amines and amino acids to form a blue or purple
color.
0
OH
OH
O
NINHYDRIN
H
l
"
.
OH
+ R-- C-- COOH
*"
-.
NH,
II
AMINO
0

, ACID
HYDRINDANTIN

O
O
II
II

The spots due to amino acids can now be detected on account of the blue color th
at they
develop. If not sprayed, the reagents may be applied to the paper by either imme
rsing the paper
in the reagent or by treating the paper to the reagent vapours. Other methods of
detection are
(0 ultraviolet and Infrared absorption, (//) fluorescence, and (//0 radioactivit
y. Otherwise, the
components may be extracted and chemical and physical tests be performed on the
extract.
The identification of a given compound may be made on the basis of the distance
traversed by the solute relative to the distance moved by the solvent front. Thi
s ratio, which
reflects the distribution coefficient of the given solute, is known as the retar
dation factor (also
known as re/at/veJ/ow),/, and is constant for a given compound under standard co
nditions
(see Box 11.2)
the distance moved by the solvent
= Rf
the distance moved by the solvent front
In case of carbohydrates, the term R is replaced, by R for convenience. G stands
for
glucose.
the distance moved by a carbohydrate
the distance moved by glucose = R
Since each compound has specific R (Ru) value, this value can be exploited to de
tect the
unknown compound by matching its retardation factor value to those of the known
compounds.

AppHeations
The technique of paper chromatography has revolutionized biochemistry where diff
icult
analyses with vanlshingly small sample volumes are legion. The control of purity
of
pharmaceuticals, the detection of adulterants and contaminants in foods and drin
ks, the study
of ripening and fermentation, the detection of drugs and dopes in animals and hu
mans, the
analyses of cosmetics, and to top it all, the analyses of the reaction mixtures
in biochemical
labs are all performed routinely with paper chromatography technique.
B. THIN LAYER CHROMATOGRAPHY
This technique is more or less similar to paper chromatography as far as most of
the
operations involved are concerned. Thus, most of the previous discussion applies
here.
To briefly describe the technique, a thin layer of a freely divided substance is
deposited on
to a flat glass plate. The sample to be separated is spotted at one end. The pla
te is dipped into
the solvent in a glass jar and the development carried out by the ascending tech
nique. After the
development, the layer can be dried and the components detected by various metho
ds available.
Thin layer chromatography may be either carried out by the adsorption principle
(if the
thin layer is prepared by an adsorbent such as Kieselguhr, or alumina) or by the
partition
principle (ff the layer is prepared by a substance such as silica gel which hold
s water like the
paper). More recently, thin layer gel filtration chromatography is also performe
d as we will see
later. In this case, the layer is made up of controlled pore beads of various ge
ls.
Preparation of the Layer
The glass plate on which the thin layer is prepared should be even and is thorou
ghly
-washed and dried before layer application. Modern thin layer chromatography kit
s provide
plastic or foil plates in lleu of a glass plate. The material of which the thin
layer is to be made
(silica gel, Kieselguhr, etc., for more details see Table ii.2) is usually mixed
with water in such
a proportion that a thick suspension, known as slurry results. This slurry is ap
plied to aplate
surface as a uniform thin layer by means of a plate "spreader" starting at one e
nd of the.plate
and moving to the other in an unbroken uniform motion. The nature of the desired

chromatographic separation dictates the thickness of the slurry layer used. Thus
, for
analytical separations the thickness of the layer is usually 0.25 mm, while for
preparative
separations the thickness of the layer might be about 5 mm. Although thn. layer
technique can
be used for many different types of chromatographic separations such as adsorpti
on, gel

Silica Gel G
Silica Gel G
Silica Gel G
Ethyl acetate/propan-1-oi (65/35)
(Butan- l-ol/acetone/
phosphate buffer pH 5 (40/50/I0)
Petroleum ether/diethyl
ether/acetone (90/10/I)
Chloroform/methanol/
Water (65/25/4)
Carbon tetrachloride/
Chloroform [95/5)
96% ethanol/water (70/30]
Butan- 1-ol/acet!cacid/
Water (80/20/20)
Petroleum ether/Prop-1-ol (99/1)
.Kieselguhr G (sodium acetate]
Kieselghur G (sodium
Phosphate. pH 5)
Silica Gel G
358
Biophysical Chemistry
filtration, ion exchange etc., it is usually adsorption type which is most used
with this
technique.

Tablell.2 Certain Solvent System and Adsorbents Used in Thim layer Chromatograph
y
Compounds
Adsorbents
Solvent system (/)
Mono and disaccharides
Triglycerides
Phospholipids
Cholesterol esters
Amino acids

Plant pigments
Kieselguhr G

While preparing stationary phase for adsorption chromatography a binder such as

calcium
sulphate is mixed with the slurry. The binder helps in better adhesion of the st
ationary phase
to the glass or foil plate. The plates are dried after application of the slurry
(thin layer for gel
fltration is, however, not allowed to dry)..If adsorption chromatography is to be
performed, the
thin layer is activated by heating at 110C for several hours.
Sample application
This is absolutely similar to that described for paper chromatography, except th
at care
should be taken not to scrape the thin layer while applying the sample.
Plate Development
The choice of solvents (Table 11.2} and the methods of elution are much the same
as for
paper chromatography. The procedure must of course be conducted in a closed cham
ber to
prevent evaporation of the solvent and the technique used is ascending out of ne
cessity. Two
dimensional chromatography may also be carried out much in the same way as descr
ibed for

paper chromatography. One of the greatest advantages of TLC is the speed at whic
h the
separation is achieved. Generally I0-30 minutes are sufficient.. However, with c
ertain
compounds about 90 minutes may be required.
Detection
Several detection methods are available. Many of these have already been named i
n the
section on paper chromatography (for example, ultraviolet absorption, fluorescen
ce,
autoradiography, if the components are radio-labeled, or production of colors by
chemical
treatment). Those specific for TLC are: (/) spraying the plate with 25-50% sulph
uric acid in
ethanol and heating. This results in charring of most of the compounds, which sh
ow up as
brown spots, and (//) iodine vapour is used extensively as a universal reagent f
or organic
compounds. The iodine spot disappears rapidly but can be made more permanent by
spraying
with 0.5% benzidine solution in absolute ethanol. Iodine vapour is seen concentr
ated in the
form of a cloud over the region where the components have separated. These spots
Can then be
scraped out, eluted and analysed quantitatively.

Chromatography
359
On plate quantification of the separated components might be achieved by employi
ng a
densitometer, which not only measures the ultraviolet or visible absorption of t
he separated
components but also gives a complete absorption spectrum of the compound for ide
ntification
purposes. Precision made densitometers are now commercially available. In case t
he substance
has been radi0-1abeled, radio-chromatogram scanning might be employed to quantit
ate the
separated components.
Advutages and Applications
Compared to paper chromatography, thin layer is more versatile, faster and more
reproducible. It is often used as pilot technique to quickly determine the compl
exity of a mixture.
It may otherwise be used as an aid in order to find out the best conditions for
large-scale
chromatography. Because of its speed and simplicity, it is often used to follow
the course of
reactions. Thin layer technique has often been used to identify drugs, contamina
nts and
adulterants. It has also been widely used to resolve plant extracts and many oth
er biochemical
preparations.
2. COLUMN CHROMATOGRAPHY
Column chromatography is an often used and routinely carried out technique which
is
adaptable to all the major types of chromatography. Although such diverse operat
ions as column
adsorption, partition, ion exchange, exclusion and affinity chromatography are c
arried out in a
column, the apparatus and general techniques used share a lot in common. The com
mon
features are discussed below.
The columns are usually made up of glass or polyacrylate plastic. Different colu
mns differ
in their dimensions. Laboratory columns usually have a diameter of 2-70 mm and a
length of
15-150 cm; lengths shorter than 15 cm are also available. Choice of a column of
a particular
dimension is dictated primarily by the amount of sample; larger the sample volum
e larger the
column chosen. The ratio of the column diameter to its length also varies. Most
workers choose
a ratio of diameter to length between 1:10 and I: I00.
The commonly used glass columns have a sintered glass disc at the bottom to supp
ort the

stationary phase. Certain columns are provided with a suitable support under a r
eplaceable
nylon mesh. A cheaper alternative is to use a plug of glass wool with a small am
ount of quartz
sand and glass beads.
Temperature fluctuations may be harmful for certain chromatographic separations.
For
such experiments, columns with a thermostat jacket are used. Liquid from a therm
ostatically
controlled bath set at the required working temperature is circulated through th
e jacket
continuously for the length of the experiment to maintain the column interior at
a constant
temperature.
The columns are provided with an inlet and an outlet. The inlet, which may be si
mple or
fitted with a ground glass adaptor provides for the eluting solvent to enter the
column. On the
other hand, the effluent from the column is led by a capillary tubing to a conti
nuous monitor
and thence to the collectin8 system (Figure 11.4).
Packing the Column
This constitutes a very critical factor in achieving satisfactory separations. T
he column is
ttted in the upright position and its bottom is sealed with glass wool or such o
ther supports.
The column is now filled to about one third its height with the mobile phase. A
thick suspension,
called slurry, of the degassed stationary phase (gel, adsorbent, or resin) is ge
ntly poured into the column with its outlet closed. The upper part of the column
is all the while stirred to ensure

360
Biophysical Chemistry
Marlotte flask
luent
Adjustable plunger
-Nylon disc
Column packing
or slntered disc
[ :
Turntable /- -"
Counter-balancing
weight
Siphon tube
(Collection of fixed volumes}
--Electrical cable
to turntable

Figure 11.4 Equipment for column chromatography along with a fractlon collector.
even packing and to avoid air bubbles. The slurry is usually added till 3/4 of t
he column is full.
The outlet is now opened and the column is stabilized by washing it with mobile
phase. A filter
paper disc or nylon gauze is then placed on the surface of the column to prevent
disturbance
during mobile phase/sample addition. Many commercial columns provide an opening
at the
top, which can be fitted with capillary tubing through which solvent drips into
the column. This
provision decreases the disturbance of the column surface considerably. To preve
nt the column
from drying, a layer of solvent is always maintained above the column surface.
Introduction of Sample
It is necessary that the sample to be applied reachs the surface o the column be
low the
top layer of the solvent. This can be achieved by sucking the .top layer of the
solvent out and
then carefully pipetting the sample on to the column surface. The sample is allo

wed to Just run

into the column. Solvent is then added to the column to a height of 5-10 cm. The
column is
then connected to a suitable reservoir which contains more solvent, so that the
height of the
solvent in the column can be maintained to a height of 5-I0 cm. An alternative a
nd better
method is to mix the sample with sucrose or flcoll to a concentration of about 1
% to increase
sample density. The sample now sinks below the top layer of the solvent to the s
urface of the

Chromatography
36 ]
column. The dye bromophenol blue can be used in place of sucrose. Alternatively
it is possible
to reach the column surface directly with the help of a syringe or capillary tub
ing.
It is necessary to apply the sample in as less a volume as possible. This gives
an initial
tight band of material when the separation begins and results in a sharper inal
separation. An
additional precaution is to desalt the sample to avoid anomalous adsorption effe
cts.
Techniques of Elution: Column Development
Continuous passage of a suitable eluant (mobile phase) through the packed column
separates the components of the sample applied to the column. This process is kn
own as
column development. There are two main techniques of elution: (/) isocratic elut
ion, and
(iO gradient elution.
When a single solvent is used as an eluant during development, the process is kn
own as
isocratic separation. However, elution with single solvent is many times not suf
ficient and a
satisfactory resolution may not be obtained. In such cases the pH, ionic strengt
h or polarity of
the eluant is changed with respect to time. This process where the composition o
f the mobile
phase is changed giving rise to a gradient is known as gradient elution, This te
chnique leads to
a better resolution of the sample components. This is so since with numerous cha
nges in the
composition of the mobile phase the partition behaviour of the solute molecules
is also
changing and the cumulative effect of these changes in the partition behaviour i
s reflected in
the sharper resolution. To produce a suitable gradient, two solvents of differin
g compositions
have to be mixed in correct proportions before entering the column. This may be
achieved by
using a commercially available gradient maker. The gradients used could be conti
nuous or
stepwise.
Flow-Rate
Flow rate, Fe is expressed as the volume of mobile phase per unit time. This is
a very
important criterion for suitable column development. For a satisfactory resoluti
on it is
absolutely necessary that the eluant flow should be maintained at a stable rate.
The easiest
way to maintain a stable flow rate is to use a peristaltic pump to force the elu

ant on to or out of
the column. An increase in the flow rate of the mobile phase, through the column
leads to
shortening of the time necessary for separation. It may therefore be preferable
to work with the
maximum possible flow rate (indeed this is one of the reasons for the quicker re
solution obtained
in high performance liquid chromatography as we will see later). However, an und
ue increase in
the flow rate decreases the efficiency of resolution and time economy. The flow
rates used differ
with respect to the nature of the sample and the nature of the stationary phase.
However,
commonly used flow rates fall between 30-120 ml/hour cm2.
Column packing also influences flow rate. Unevenly packed column leads to distor
tion of
the flow leading to unsatisfactory resolution. More densely packed columns retar
d the flow of
the mobile phase and decrease the flow rate. It is therefore very important to p
ack the column
optimally to obtain a good flow rate.
Analysis and Collecting of Effluent
The .effluent, as it emerges from the column outlet is analysed. The properties
of a
particular compound, vlz., ultraviolet absorption, color or fluorescence are exp
loited in its
analysis. Alternatively th compound may be labeled before application of the sam
ple to the
column and its radioactivity exploited for its analysis.
There are two approaches to analysis of solutes in the effluent: The classical a
pproach
which may be taken recourse to in the absence of a continuous monitoring equipme
nt is to
collect the effluent in equal fractions and to subsequently analyse each fractio
n for the presence
and content of the solute. Clearly, this approach becomes untenable if the numbe
r of fractions

362
Biophysical Chemistry
is too high (many times, the number of fractions may be in hundreds) or if more
than one
parameter of the solute is to be studied in each fraction. The modem approach, t
herefore, is to
continuously monitor the effluent coming out of the column. The monitoring equip
ment is
programmed to read the inherent property of the desired compound such as the ult
raviolet
absorption or radioactivity. For example, if. the desired sample component is a
protein, the
monitoring equipment may be a UV monitor and may be programmed to read absorptio
n at
280nm. A wide variety of monitoring equipments each exploiting a different param
eter are
available. Some of these which may be cited as examples are (/) fluorescence det
ectors,
polarimeters, (///) voltameters, (/v) refractometric detectors, and (v) conducti
vity detectors, etc.
The electrical signal generated by the detector due to the property of the compo
und which it is
reading is displayed on a chart recorder. Thus as the compound of interest emerg
es from the
column in the effluent and passes through the flow cell it is depicted on the ch
art paper. The
area under the peak is proportional to the amount of the compound present. This
area may be
determined by measuring the height of the peak and its width at half the height.
Figure 11.5
shows an example of the elution pattern that may be obtained and the way the are
a of a peak
may be calculated. It is common to split the outlet into two streams; one small
aliquot diverted
to the monitor for analysis while the other stream being collected into differen
t fractions for
further work.
Elution volume
Figure 11.5 Diogrammatic representation of a typical elution profile. The profde
is p by plotting a measurable
property of the sample components {absorbance in this diagtum)against the eluted
volume as it emerges
from the column. The peaks in such a Ixlttem represent individual zones of sampl
e components (proteins
in case since the absorbance is measured at 280 nm) separated from each other du
ring column developmenL It is possible to identify each component in the pattern
on the basis of its elutlon
volume, i.e., the volume of effluent collected which corresponds to the apex of
the peak. To quoittate
ech component its peak hekjht at the apex (a) is multiplied with peak width at h
alf height (b).

In the absence of a method for continuously monitoring the contents of the efflu
ent from
the column, it is necessary to analyse it after it is collected in small fractio
ns. Fractions in
small volumes may be collected in different tubes in order to keep the column re
solved
components separate from each other. If the separation has been satisfactory, a
particular
component will be distributed in relatively few number of tubes. The contents of
these tubes,
which contain the same component, can then be pooled together for further study.
Collecting effluent fractions manually can be both boring and time consuming. A
range of
automaticfract/on collectors are available commercially. They are designed to co
llect a definite

(B)
(c)
Chromatography
.
363
volume of the effluent in each tube before a new tube is placed in position auto
matically.
Different fraction collectors are programmed to operate in different ways. Some
fraction collectors
are fitted with electronic device to measure the number of drops falling in a tu
be. This number
can be predetermined so that after a set number of drops have fallen into a give
n tube, a new
tube comes into position. Other fraction collectors allow the effluent to enter
each tube for a
interval of time. A better method of fraction collection is by programming the f
raction
collector for collection of definite volume per tube. The fractions collected ar
e subsequently
analysed.
Recent innovations in fraction collecting systems have led to production of frac
tion
collectors which are connected to the monitor in a way that the signal for the f
raction collector
is derived from the monitor signal. Thus, if the desired component to be separat
ed is a protein,
and an ultraviolet absorption monitor is being used, the absorption at 280 nm by
the effluent,
which is produced by proteins, will signal the fraction collector. The fraction
collector will now
place an empty vessel below the column outlet. The whole effluent, till the abso
rption signal
persists, will be collected in a single vessei.
Concept of Plates: Column Eiciency
Let it be assumed that a given column can be divided into a number of identical
segments
such that one equilibrium distribution takes place on each of these segments. Ea
ch of these
segments is called a theoretical plate. Thus, the number of equflibratio.ns taki
ng place in a
column is equal to the number of plates that the column possesses. If two solute
components
have very small difference in their partition coefficients they will need a larg
e number of
equilibrations before they can be completely resolved from each other. Thus the
column which
has a larger number of plates will be better suited for resolving these componen
ts whereas a
column which has small number of plates will not be as efficient. Therefore colu
mn efficiency

is directly related to the number of plates it possesses (Figure 11.6).

I
Column lengtl = 15 cm
n = 80 Plates
Column length = 30 cm
n = 160 Plates

Column length 15 cm
160 Plates
Elution volume
Column length and number of plates as related to c)lumn e.jciency. (A) Column wi
th small number of
plates resolves two substances incompletely. (B) Increasing the number of plates
by ncreasing the
column length increases the resolution but the peaks are not sharp. (C} Increasi
ng the number of plates
without increasing the column length gives a good resolution with sharp peaks.

364
Biophysical Chemistry
It would seem that the number of plates would
uilt.
However, we have already seen that the nature
temperature and
many other factors influence equilibration of
and therefore
these factors will also have a bearing on the
.

depend only on how the column is b

of the solute, the flow rate, the
the solute between the two phases
number of plates that a column has

How can one increase the number of plates to increase the efficiency of a column
? One
way is to.lengthen the column. This is so since the number of plates is directly
proportional to
the column length. Although this way one might get a better resolution, there is
also considerable
band broadening (Figure 11.6 B). We can, however, increase the number of plates
without
increasing the length of the column. This may be done by making a better column
and optimizing
other factors which affect the solute interaction with the two phases, vtz., flo
w rate, temperature,
method of sample introduction etc. This optimization of column development condi
tions would
lead to increased number of equilibrations taking place thereby increasing the n
umber of
theoretical plates in the column. This approach of increasing the number of plat
es is therefore
obviously a better approach because the band broadening due to longer columns wi
ll be avoided
and one may be able to get resolution in narrow peaks (Figure 11.6 C).
TYPES OF CHROMATOGRAPHY
1. ADSORPTION CHROMATOGRAPHY
A solid, which has the property of holding molecules at its surface, can be desc
ribed as an
adsorbent. No electrostatic forces are used by the adsorbent to attract molecule
s to its surface.
Adsorption can be fairly specific so that one solute may be adsorbed selectively
from a mixture.
Two differing factors are exploited in separation of components by adsorption ch
romatography.
One is the different degree of adsorption of various components on the adsorbent
surface and
the other is the varying solubility of different components in the solvent used
(mobile phase}.
Adsorbents
A range of materials is available but a few common ones are sufficient for most
purposes.. A short list is provided in Table 11.3.

Table 11.3 Some Common Adsorbents in Order of Increasing Adsorbing Power

Powdered cellulose
Starch
Sucrose
Calcium Carbonate
Magnesia
Silica Gel
Alumina

Adsorbents can possess some qualities, which might be detrimental for separation
, and
as such care must be exercised while selecting one. Certain adsorbents might not
be inert but
react and degrade components to be separated during experiment. Certain other ad
sorbents
might imbibe water from the atmosphere and become useless. In such cases, the ad
sorbent
has to be activated by heating it for several hours at 110C. Sometimes, however,
presence of
water results in better separation since adsorption is coupled with partition du
e to the presence
of water. The adsorptive activity of an adsorbent can actually be controlled by
varying the
amount of water it contains. Thus we can prepare a tailored adsorbent by dehydra
ting it at high
temperature (e.g. 360CI for about 4-5 hours and then allowing the dehydrated mate
rial to pick
up a suitable amount of water.
Solvents
Virtually any organic solvent can be used as the mobile phase. As far as possibl
e, the
sample should be introduced in a solvent in which it is highly soluble; this hel
ps to keep the
sample volume at a minimum. In addition to relative solubilities of the solute i
n the eluting
solvent, it is necessary to consider the competition between the solutes and the
solvent for the

Chromatography
365
adsorption sites on the surface of the stationary phase. Thus, a solvent which e
lutes the solutes
too fast will give a poor separation, while the solvent eluting the solutes very
slowly will lead to
uncomfortably long retention times which will result into excessive band broaden
ing and sample
dilution. It is dear, then, that the choice of the solvent has to be a compromis
e between numerous
mutually opposing factors affecting chromatographic separation. It is. therefore
, fortunate that
a long list of solvents is available (Table 11.4).
Tble 11.4 Some Common Solvents in Order of Inreasing Eluting Power fom Alumina

Petroleum Ether
Carbon Tetrachloride
Tri-Chloro Ethylene
Toluene
Benzene
Chloroform
Ether
.Ethyl Acetate
Acetone
Ethanol
Methanol
Water
Organic acids & bases

Adsorption chromatography can. be carried out using column, TLC, or paper

techniques. Traditionally used adsorbents are composed of irregular shaped,
From a column chromatography point ofvlew, these particles allow adequate
flow. However they also decrease the resolving power of the colunm since they do
not
close together. A balance has to be reached between a cl#ser pacing and adequate
eluant
successful separation through adsorption chromatography. '
Adsorbents for thin layer chromatography are often impregnated with various long
during

)reparation. This enhances the separation achieved.

Papers coated with different adsorbents are commercially available and the techn
ique
has already been described previously.
Adsorption chromatography has been extensively used for biochemical separations.
The
list includes amino acids, mono-, and disaccharides, neutral lipids, cholesterol
easters,
phospholipids, etc. The disadvantage with adsorption chromatography, however,
the fact that it is highly empirical and non-reproducible because of variations
in the
of adsorbents. Thus, recently, techniques such as ion-exchange chromatography, a
nd
adsorption chromatography because of their greater
However, adsorption chromatography has re-emerged as a powerful separation
times in the form of high precision liquid chromatography (HPLC, see later}
sometimes applies adsorption principle for sample resolution. -J
2. PARTITION CHROMATOGRAPHY
Liquid-Liquid Chromatography
We have already covered one form of liquid-liquid chromatography in detail previ
ously,
?aper chromatography. The stationary phase there was a polar solvent, water, tig
htly held
on which migrated the mobile phase which was another solvent of lesser polarity.
, when thin layer chromatography is performed by silica gel layer, the stationar
y phase
particles. This too then is a form of liquid-liquid chromatography.
true for column chromatography when silica gel or powdered cellulose is the soli
d
separation achieved in liquid-liquid chromatography is therefore based upon the
the immiscible solvent systems. Further discussion
chromatography is avoided here for fear of repetition since most of the aspects
been covered under paper chromatography.

To detector
Gas
(C)
Column
Figure 11.7
Carrier
366
Biophysical ChemistryI
One point, however, may be noted. If during the separation of predominan
tly non-polar
solutes through partition chromatography, the stationary phase is polar, the sol
utes will not!
have much solubility in it and will therefore migrate fast with the mobile phase
in which they!
are more soluble (since it is relatively non-polar). In such cases separation wi
ll not be achieved.!
Ir such situations it is advantageous to use non-polar organic solvent as the st
ationary phase
and aqueous solvent as the mobile phase. In other words, the nature of the phase
s is reversed,i
The technique is aptly known as reverse-phase chromatography. The non-polar soli
d support
for such chromatography is usually powdered rubber coated with benzene. The mobi
le phase,
which is aqueous, flows over this stationary phase.
B. Gas-Liquid Chromatography (GLC)
The basis for the separation of the compounds in gas-liquid chromatography is th
e
difference in the partition coefficients of volatilized compounds in the liquid
stationary phase.i'
Gas-liquid chromatography is a form of column chromatography where the stationar
y.
phase is a non-volatile liquid. The stationary phase here is known as the liquid
phase. Thi
phase is dispersed over a surface of an inert so//d support. The solid support w
hich is coated on
to the inside surface of a long column is inert to the sample components and doe
s not react
with them in any way; it merely acts to hold the liquid phase in a stable disper
sed form so as to
present a large contact area. It is the liquid phase that interacts with sample
components. A

gas stream termed as the carrier gas flows continuously through this column at a
flow rate
which is controlled (Figure 11.7). When a small quantity of the volatile sample
is introduced int,
the gas, the gas promptly carries it on to the column (hence the name "carr/er g
as"}. In the
Column
Gas "'.':: 'v":: .:. :" '
::"- To detector
{A}
Column
(B) "
To detector
Solid
Liquid
support
phase
Diagrammatic representation of gas chromatographic separation of sample componen
ts on the basis of
their partltkm coefficients. (A) Sample consisting of three components is introd
uced in the column. Vertlcl
position of the arrows indicates the distribution (partition) coefficient of the
compnents; the component
by theflrst arrow interacts more with the liquid phase and less with the gas pha
se. The situation is ex'tly reverse for the components indicated by the third ar
row. The component indicated
by the second arrow interacts equally with both phases. (B) As the gas continues
to flow over the liquid phase, the three components migrate on the column. (C)
The components have become widely separated
from one another.

I 1.8
i Chromatography
367
icolumn, the sample components become distributed between the liquid and the gas
phase.
These components therefore travel more slowly than the carrier gas because they
are being
retarded by virtue of their interaction with the liquid phase. The retarding, ef
fect is different for
different components; the component distributing more in the liquid phase is ret
arded more
and the component prefering the gas phase is retarded less.
In the long column, which is present in the GLC apparatus, these sample componen
ts
ecome separated from each other on the basis of differences in the retarding eff
ect. These
separated components eventually elute out of the column and reach the detector,w
hich reads
the concentration of a given component present in the carrier gas and converts i
t to an quivalent
electrical signal. The magnitude of the detector signal as measured by a continu
ous recorder
when plotted against the time taken by .the particular component to elute out of
the column
produces a pattern which may be called a chromatogram (Figure 118). A chromatogr
am is
made up of a number of peaks, each of which is due to a component of the sample.
The position
each of the peak is characteristic of the component to which it is due. The char
acteristic sition of the peak is measured in terms of the volume of gas that has
traversed through the
column between the time that the sample was applied and the time at which the pa
rticular
!omponent emerged from the column. This gas volume has been given the term reten
tion volume,
!V,. This term is. fundamental to gas chromatography and may be used for the ana
lysis of a
ple (see later). As said for column chromatography previously, the area of the p
eak is
proportional to the concentration of the component causing it.
A typkxzl gas chromatogram. (A), (B), (C), (D), and (E) are detector skjnals due
to different sample
components which emerge from the column at different times depending on their pa
rtition coefficients.
There is a variation of gas liquid chromatography which is knon as gas-solid
, (GSC). Here, the liquid phase is absent. The solid phase, which is coated on t
o

is not inert in this technique; it interactswith the sample components

by the gas by exerting adsorption forces. The sample components therefore become
between the gas and the solid surface on the basis of differences in the
the solid surface. This eventually leads to their separation
other.

368
The essential components of a gas chromatograph are shown in Figure 11.9. The in
ert
carrier gas stored in a gas tank passes through the pressure regulator into the
sample injection
chamber from where it carries the sample onto the column. The column is usually
packed with
highly porous solid onto which is coated a thin film of liquid used as the stati
onary phase. The
sample components separate in the column as discussed above and pass through the
detector
which gives signal to the recorder. The individual solute components can be coll
ected in a
fraction collector for further study. The final passage of the gas is through th
e flow meter (not
shown in the Figure 11.9) into the atmosphere, Since the procedure is usually ca
rried out at
high temperatures, a thermostatted oven is provided for the column, injector, an
d the detector.
Figure 11.9 Schematic of a gas chromatograph. (A) Carrier gas tank. (B) Pressure
regulator, (C} Sample
injection chamber, (D) Column, (E) Detector, (F) FractWn collector, and (G) Reco
rder
Carrier Gas
In GLC, the carrier gas constitutes the mobile phase and provides transportation
for the
sample components through the apparatus. The gas must be chemically inert and pu
re. Gas
used at high density gives a better separation but takes a long time to achieve
it. A low
gas might give a faster separation which will not be as satisfactory. However, t
he choice
gas usually depends on the requirements of the detector and also on the availabi
lity of the gas.
Most commonly used gasses are nitrogen and argon, but, helium, hydrogen and carb
on dioxide
are also used. Even steam is used for special purposes.
Purity of the carrier gas is critical since even a small impurity can give rise
to noise in the
detector. The usual contaminant is water vapour. Passage through a cold molecula
r sieve trap
is very effective for removing last traces of water vapour.
A soap film flow meter is used almost exclusively to measure the flow rate. The
gas is
usually passed through the column at a rate of 40-80 cmS/mmute.

Columns
Two distinct types of columns are commonly used, packed and open tubu/ar. The op
en
tubular colunms are also known as cap///anj columns. Packed colunms are stainles
s steel,
copper or glass tubing, 1.6, 3.2, 6.4, or 9.5 mm bore, and anywhere between 1-15
meters in

OH
e
Me
---SI---O--Si-- + (MeSi)2NI-I ---> Me--SI--Me
Me--SI--Me
O
Chromatography
369
length. For preparative purposes packed cohmms of several inches diameter might
be used.
They are filled with narrowly sieved inert support coated with a liquid phase fo
r GLC.
Open tubular or capillary columns have open unrestricted path for the gas within
the
column. These columns are about 15-30 meters in length with an inside diameter o
f about
0.25 ram. The inner wall of these columns is coated with the liquid stationary p
hase to about
1 tm in thickness, The open tubular columns are of two kinds. One is known as th
e wal/coated
open tubu/ar co/umn (WCOT) in which the liquid phase is coated on the column wai
l. These
columns have limited sample capacity and are unsuitable for large-scale separati
ons.
The second type is known as support doated open tubular columns (SCOT). In these
columns a
porous layer is formed on the inside wall of the tubing. The porous layer can ei
ther be formed by
chemical treatment of the inner wall or is deposited on the inner wall. The supp
ort is coated in
such a way that the inherent property of the capillary columns, i.e., the unrest
ricted gas flow
is retained. The inert porous layer is then impregnated with the liquid stationa
ry phase. These
columns have a higher sample capacity.
The geometric configuration of the column is of little concern as long as the be
nds are not
sharp. Columns are bent in U or W shape or coted to fit in the oven.
bltd Support
An ideal support should be chemically inert although wettable by the liquid phas
e so that
it will spread in a thin layer of uniform thickness. It should ideally have a hi
gh specific surface
{I m2/g). In addition it must be thermally stable and mechanically strong.
The most commonly used supports are derived from diatomaceous earth and teflon.
They

are sold under many trade names depending on their pretreatment. The diatomaceou
s earth
supports may be either Firebrick derived materials, such as Chromosorb P (a form
of crushed
commercial firebrick) and Gas Chrom R, or materials derived from filter aids, wh
ich include
Chromosorb W (diatomaceous earth heated with an alkaline flux), Anakrom ABS, and
Gas Chrom Q. Some other solid supports are known by the trade names Celite 545,
Phasep P,
Embacel etc., (all diatomaceous earth derived). Table 11.5 lists properties of s
ome principal
types of solid supports.
Deactivation of all diatomaceous supports is necessary for most applications. Ac
id
washing is effective in removing mineral impurities which might otherwise serve
as adsorption
sites. This treatment is all that is needed if the sample to be separated is non
-polar. However, ff
the sample is polar further treatment of the support might be required. The sfla
nol (Si---OH)
groups that cover the surface of the diatomaceous earths are very polar and tend
to react with
polar solutes. Treatment with hexmethyldisflazane (HMDS) sialinizes the surface
and makes
the surface inert to polar solutes.

Silicon rubber gum (SE-30)

Aplezon L (High M.W. paraffin)
Steroid, alkaloids, pesticides
Esters, ethers, high boiling
hydrocarbons
Amines, ketones, alcohols
All types
All types
Hydrocarbons '
Hydrocarbons, fluorides
Oleflns, cyclic hydrocarbons
370
Biophysical Chemistry
TabIe 11.5 Some Properties of Principal Types of Solid Supports Used in GLC
Type of solid
Support
Surface area
Approximate maximum
liquid phase loading
Percent by weight

Diatomaceous earths
Crushed firebrick
Glass micro spheres
Porous silica beads (PORASIL]
Polytetrafluoro ethylene
1-3
4-6
0.01-0.05
1 - 500
2-11
3O
1
20

2O

Special supports are popular for particular applications. Very lightly loaded gl
ass beads
are preferred when very rapid separations are desired. Very often powdered teflo
n is used as a
support for very polar solutes. Graphitized carbon and carborundum are other sup
ports for
special applications.
Liqu/d Phase
A good rule to follow when selecting liquid phases is "like dissolves like". A g
ood
separation will occur only when the sample dissolves well in the liquid stationa
ry phase. This is
so since the gas phase is inert; the separation occurs only in the liquid phase.
Thus, to select
the liquid phase it is necessary to match the polarity of the stationary phase a
nd the sample
components of intercst.
The requirements for a good liquid phase are: (0 it must be non-volatile at the
temperature
it is to be used; (it) it should be thermally stable; (i//) it should provide ap
propriate partition
coefficient values for the components.0f interest, and (/v) it should be complet
ely inert towards
the solutes.
Obviously one single liquid cannot meet all the requirements stated above. Some
are
needed for low temperature, others for high temperatures. A few liquids commonly
used are
listed in Table 11.6.
Table 11.6 Common Stationary (Liquid) Phases for GLC
Stationary phase
Typical ssmples
Max. Temp. C
350
Carbowax 20 M (polyethylene glycol}
Silicone off (DC-550)
Dmonyl phthalate
Sqalane

Hexadecane
Silver nitrate in propylene glycol
300
250
200
150
140

Coating the Support

To prepare the column packing, correct amount of liquid,phase dissolved in a low
boiling
solvent (pentane, acetone) is added to the solid support. The mixture is then he
ated very slowly
with continuous stirring in order to evaporate the solvent; the last traces of s
olvent are removed

Chromatography
371
under vacuum. Columns are filled by pouring the packing into straightened column
.
The column is gently shaken and tapped all the while to ensure an even packing.
Sometimes
pressure up to 5 p.s.L may also be applied. Both the ends are plugged with glass
wool and the
,column is bent or coiled to an appropriate shape that will fit in the oven.
Open tubular columns contain no packing. To apply a thin coat. of liquid phase,
a dilute
solution is forced through the column at a very slow rate. Excess solution may t
hen be
evaporated by passing a hot carrier gas. This results in a thin layer of the liq
uid phase.
Sample Preparation and Introduction
If the sampIes are non-polar or have a very low polarity they may not need any
pretreatment. However, if the sample possesses such polar functional groups as -OH, COOH,
--NH2, etc., pretreatment is needed (in the absence of pretreatment these sample
components
would be retained on the column for excessive time which will result in poor sep
aration and
peak "ta///ng"). These fuIctional groups are subject to derivatization methods s
uch a metylation,
sflanization, trifluoromethylsflanization etc. The above named methods are very
useful for
carbohydrates, amino acids and fatty acids and increase the volatility and distr
ibution coefficients
of these compounds.
Sample should ideally be introduced in the vapour form. If the sample is not alr
eady a
vapour, enough heat must be supplied to establish vaporization immediately. Size
of the sample
varies depending upon the column and the detector used. An average packed column
of 9.4
mm bore coupled to a thermal conductivity detector optimally utilizesa sample of
1-50 ml or
less. The open tubular column has a much lesser capacity and utilizes very small
samples of
the order of 10-3 to 10-2 ml. Liquid samples dissolved in a suitable organic sol
vent such as
ether, heptane, and methanol are injected from a syringe through a rubber septum
into a small
heated chamber immediately preceding the column. A swift, continuous motion of t
he plunger
is the byword. Since this part o the apparatus is under pressure, due Care must
be exercised
while operating the plunger.
Solid samples are much more difficult to introduce. The best method is to seal t
hem into
a thin vial which can then be introduced into the injection port and then crushe
d from outside.

Detectors
Located at the exit of the separation column, the detector detects the presence
of the
individual components as they leave the column. The detector output is then suit
ably amplified
and is traced on a strip chart recorder. This results in chromatogram of concent
ration versus
time. The temperature of the thermostatted detector should be maintained at a le
vel high
enough to prevent condensation of high boiling vapours. At the same time it shou
ld not be so
high as to cause decomposition of eluent material. Three most commonly used dete
ctors are
described below.
(i) ionization detector. This is by far the most widely used detector. It measur
es all organic compounds and it can detect as low as one nanogram of any given c
ompound. Hydrogen,
either used as carrier or introduced into the detector through elsewhere, is bur
nt to give a
nearly colorless flame, the jet of which forms one electrode. The other electrod
e is mounted just
near the tiP of the flame and consists of a platinum wire. The flame changes col
or the moment
a separated component comes out of the column and into the flame. The sample com
ponents
becomes ionized in the flame and give rise to a cUrrent between the two electrod
es. As the
resistance of the flame is very high and the current generated is weak, the asso
ciated electronics
are complicated and moderately expensive. The principle of flame ionization dete
ctor is illustrated
in Figure 11.10.

372
Gas flow
Biophysical Chemistry
lgure 11.10 Flame onrtion detector
(li) Electron capture detector. This detector has radioactive source (3Ni} which
ionizes the
carrier gas coming out of the column. The electrons produced give rise to a curr
ent across the
electrodes to which a suitable voltage is applied. When a sample component, whic
h has the
ability to capture electrons, comes out of the column, it captures the ionized e
lectrons thereby
causing a drop in the current. This change in the current is measured and record
ed. Obviously
only those substances having an electrons capture capability can be measured by
this type of
detector, It is mostly used to measure polyhalogenated compounds, particularly p
esticides
such as DDT, dieldrin, and aldrine, It is very sensitive and can detect as littl
e as one picogram
of these compounds, The detector design is illustrated in Figure 1 I. 1 I.
Radioactive source
Fure 1 I. 11 Desk3n of electron capture detector
(///) Therm/on/C errgss/on detector. This detector employs fuel-poor hydrogen pl
asma. This
low temperature source suppresses the normal flame ionization response of the co
mpounds
not containing nltrogen or phosphorous. A non-volatile rubidium silicate bead is
centered about
1,25 cm above the plasma Jet, This bead is electrically heated to about 600-800 C
. This design
allows adjustment of the bead temperature independent of the plasma as a source
of thermal
energy. With vanishingly small hydrogen flow, the detector responds to both nitr
ogen and
phosphorous containing compounds by greatly enhancing the ionic dissociation of
rubidium

Chromatography
373
chloride. Consequently it is used to measure organophosphorous pesticides. Less
than one
picogram of compounds containing nitrogen or phosphorous can be detected.
In cases where the identity of the compounds being separated is not known, the d
etector
may be replaced by a mass spectrometer. Special separators remove the bulk of th
e carrier gas
from the sample emerging from the column prior to its entry into the mass spectr
ometer,
Retention Time .and Qualitative Analsis
Much in the same manner as the retardation factor, Rf (see section on paper
chromatography), retention time aids in qualitative analysis in gas chromatograp
hy. Under
standard conditions of temperature, gas flow, gas compressibility etc., the time
taken for a
compound to emerge from a column is constant and iS-known as the retention time.
Unlike
paper chromatography, it is difficult to reproduce conditions exactly from one r
un to another in
GLC. This difficulty is tackled by analysing a standardcompound with the sample.
The time of
emergence of the sample relative to that of the standard is determined to give t
he
relative retention time ( t.
The value of tis constant for a given column under different conditions. One can
therefore
identify unknown compounds by comparing their tR with that of the known compound
s.
It would be more useful, from the point of view of a positive identification, if
it were
possible to relate the retention characteristics to the structural properties of
the molecule. The
simplest and the most reliable relationship of this kind is the one which depend
s upon the
number of carbon atoms. However, this relationship is limited to members of a ho
mologous
series, for example the methyl esters of the saturated fatty acids. Each additio
nal CH2
increment is known to decrease the vapour pressure of the components by a consta
nt amount.
There is thus a linear relationship between the logarithm of the retention time
and the number
of carbon atoms which can be exploited to some extent to the identification of u
nknown
compounds.
lure 11.12 Dependence of resolutn f two peaks n sepamtn factr and number f plate
s in clumn (A Pr

separatl factor, small number of plates. (B) Good separation factor, small numbe
r of plates. (C) Poor
sepamtlonfactor, large number of p/ares. (D) Good separatWnfactor, large number
of plates.

374
Biophysical Chemistry
The separation of two components of a sample is a function of the ratio of their
retention
times. This ratio is known as separation factor. The separation factor is a func
tion of the
stationary phase and can be varied in order to improve resolution by varying the
stationary
phase. In addition, the resolution can be improved by increasing the number of t
heoretical
plates. Inpractice, both, variations in stationary phase and the number of plate
s is used for a
successful resolution (see Figure I 1.12).
Quantity of a sample component might be determined by analysis of its peak area.
Preparative GLC
GLC can be made preparative by usage of suitable columns and collection syste, m
s which
make it possible to collect appreciable amounts of individual components of samp
le mixtures.
Repeated chromatography of the sample mixture is carried out for this purpose. F
or preparative
GLC, a splitting system is established at the end of the column. This splitting
system splits the
gas stream emerging from the column in such a manner that a small part of the st
ream goes
into the detector (to be monitored) and the rest goes to the collector. The coll
ector possesses a
liquid nitrogen trap (to liquify the vaporized components by cooling) in which t
he individual
components are collected.
Gas-Solid Chromatography (GSC)
Gas-solid chromatography actually preceded gas-liquid chromatography by several
years.
The two techniques are more or less similar to each other, the primary differenc
e being that the
partition within the column is caused by adsorption on a solid surface rather th
an solubility in
a liquid phase. Since the techniques are similar in most aspects, the instrument
ation for GSC
is identical to GLC with very minor differences.
GSC Versus GLC
The problems and advantages of GSC visa vis GLC are outlined below.
(/)
Distribution coefficients of components in GSC are much larger than the
partition
coefficients of GLC. Consequently the retention times are inconveniently long in
GSC.
However, retention times are considerably smaller when working with such gases a

s
hydrogen, nitrogen, argon, and oxygen {these gases give too short a retention ti
me in
(i
The upper temperature limit in GLC is limited by the necessity of retain
ing the liquid
stationary phase. However, since there is no liquid phase in GSC, considerably h
igher
temperatures may be used. The upper limit in this case is limited only by therma
l
instability of the sample or materials constituting the apparatus itself.
('t)
GSC should better not be carried out with high concentrations of samples
as this gives
skewed peaks and the r, esolution is poor. Small concentrations of samples are,
however,
resolved well.
(iv)
Specific areas of solid adsorbents may be as much as 100 m2/g (about 100
times in
excess of solid supports used for GLC). Thls often leads to pyrolyzation of samp
les in
GSC. In other cases the sample is irreversibly adsorbed and no resolution can ta
ke
place.
(v)
GSC suffers from lack of reproducibility, as solid surfaces are very dif
ficult to
reproduce. The size and shape of the area of solid surface and also the composit
ion of
the surface layer varies greatly from one experiment to the other. The retention
behaviour
consequently differs greatly leading to approximate theoretical interpretation.
Application of Gas Chromatography
Apart from the separation of components of tobacco smoke, atmospheric pollutants
,
solvents, plant extracts, essential oils, volatile vegetable otis and organic ac
ids etc., for which it
is routinely used, gas chromatography is being increasingly used as an analytica
l tool to study

Chromatography
3 75
many parameters. A short list of such analytical applications is provided below.
It needs to be
pointed out that although most of such analytical, applications have been carrie
d out on
substances of non-biological interest, the ongoing developments in the field of
gas chromatography
will allow the same applications to be performed on areas of biological interest
also.
(/)
An ever expanding number of workers in the fields of solution chemistry
including
study of polymers, Lewis acid-base properties, liquid crystals, and gas-liquid i
nterracil
adsorption have found GLC to be a tool of importance, if not indispensable, util
ity.
Thus gas chromatography is serving as an excellent tool to study thermodynamic
properties of solutions.
(/0
Gas chromatography is one of the most widely used procedures to study re
action
rates, energies and mechanisms. There are instances in which a solute has been
made to react or interact on the GC column in a rate controlled manner. Kinetic
data
in these cases become available directly from elution behaviour.
('gO
GC columns can be made to behave as isotope exchange vessels. Isotope la
beled
organic and inorganic compounds are used in many biological fields. However, the
preparation of small quantities of several such species by conventional techniqu
es is
difficult. One can, however, use a GC column in which mixture of unlabeled compo
unds
is injected. The GC column contains the exchanging nucleus (e.g., aCl). The sepa
rated
and labeled compounds can then be collected from the column exit.
(/v)
Gas chromatography is today being widely used to analyse such molecular
properties as (a) vapour pressure, (b) heat of vaporization, (c) molecular weigh
t, (d)
molecular geometry, (e)bond angle deformation, and 0 ionization pdtential and el
ectron
Gas Chromatography - Spectrometry (GC-MS}
The use of retention data for identification is useful in those cases where the
chemical type of the
sample is known and when there are a number of pure compounds of this type avail
able for the

determination of reference date. However, more often than not, in complex mixtur
es there are several
possible interpretations for each peak and so the agreement between the retentio
n characteristics of a
known standard and any component can never be regarded as conclusive evidence of
identification. To
remove this ambiguity, use of an ancillary technique for identification of compo
nents is required. The
ancillary technique most uded in modern instruments is mass spectrometry. This t
ype has become so
popular that directly coupled gas chromatography - mass spectrometry (GC-MS) uni
ts have become the
norm of the day.
There are a number of possible arrangements, depending on the type of column use
d and the mass
spectrometer. In general, the gas stream exiting from the coiumn is passed to th
e spectrometer ion source
via an intermediate stage that reduces the carrier gas pressure to the high vacu
um conditions required by
. the mass spectrometer. The intermediate stage might involve the use of molecul
ar separators, which
remove a large fraction of carrier gas by diffusion. The separators achieve two
ends; (/) they remove the gas
from the sample components thereby enriching the component, which reaches the ma
ss spectrometer,
and (//) they remove the gas molecules, which might interfere in sample identifi
cation. The molecular
separators are usually of three kinds (0 effusion separator, (/0 Jet/orifice sep
arator, and (t/0 membrane
separator.
Two types of mass spectrometers are mainly used for GC/MS work; magnetic sector
mass
spectrometers, and quadrupole mass filters. There seems to be no clear preferenc
e for one or the other
type of instrument. For GC/MS usually low resolution spectra are sufficient. The
molecular peak and the
fragment peaks form a typical pattern that can either beinterpreted by experienc
e or by comparison of the
unknown spectrum in the library. However, ff the comparison method fails in iden
tification of the unknown
s.pectrum, a high resolution magnetic sector field mass spectrometer is needed.

Molecule too large to

enter the pore
Intermediate size molecules
i
are partially excluded
Path of a
Path of a
large molecule;
small molecule;
elutes fast
(B}
elutes very sloWly
376
Biophysical Chemistry
1. GEL PERbIFTION CHRObiTOGIPHY
Gel permeation chromatography is a separation method dependent upon molecular si
ze.
The method is also known as molecular sieve, gel filtration, or molecular exclus
ion
chromatography. The many advantages that this method has over other separation p
rocedures
have resulted in its widespread application. The advantages are: (/) gentleness
of the technique
permitting separation of a labile molecular species, (ii) almost 100% solute rec
overy
(iii) excellent reproducibility, and (iv) comparatively short time and relativel
y inexpensive
equipment needed for its opcration It is a powerful separation procedure so that
samples
differing by about 25% in molecular dimensions may be totally separated by this
technique on
a single gel bed.
Principle
Gel permeation chromatography is based on a very simple principle. A column of g
el
beads or porous glass granules is allowed to attain equilibrium with a solvent s
uitable' forthe
molecules to be separated. If the mixture of molecules of different size is plac
ed on the top of
such an equilibrated column, the larger molecules pass through the interstitial
spaces between
Figure I I. 13 Illustration of prlnclple of moleculox sieving. (A) Schematic rep
resentation of exc.lusion of large molecule.

(B) Effect of partlcle size on their elution rates.

Chromatography
377
the beads. This is because the pores of the gel have smaller diameter than what
is needed for
the large molecules to enter. Large molecules, therefore, move down the column w
ith little
resistance. The small molecules, however, can enter the pores and are thereby ef
fectively removed
from the stream of the elutlng solvent. (Figure 11.13, also see Box 11.3). These
molecules are
thus retarded. The degree of retardation of a molecule is proportional to the ti
me it spends
inside the gel pores, which is a function of the molecule's size and the pore di
ameter. The
molecules with Stokes' rad//equal to or exceeding pore diameter, do not enter th
e gel and are
said to be excluded. One can therefore describe the exclusion limit of a gel as
the molecular
weight of the smallest molecule incapable of entering the gel pores. It is usual
to judge the exclusion
limit by molecular weights (although Stokes' radii are more accurate) because su
ch data is
easily available. It, however, does not mean that molecular weights are a true r
eflection of
Stokes" radii. This point can be better understood when we consider the fact tha
t linear
polysaccharides and ibrous proteins have a lower exclusion limit in a given gel
as compared to
globular proteins of comparable molecular weight. The lower limit for effective
use of molecular
sieve gels is usually about 10% of their exclusion limits.
A few simple and useful mathematical relationships about solute behaviour on mol
ecular
sieve gels are described below. For a given type of gel, the distribution of a s
olute particle
between the inner and outer solvent (solvent within and outside the gel bead) is
defined by a
dstrlbution coefficient, Kd, which is a function of its molecular size. Kd = 0 w
hen the solute
molecule is large and completely excluded from the inner solvent. K = I if the s
olute molecule
Is small enough to penetrate the gel pores and diffuse into the inner solvent. D
ue to variations
in pore size for a given gel, there is some inner solvent that will be available
and some that will
not be available to solutes of intermediate sizes. The K value in such cases wil
l vary between
0 and 1. This molecular dimension dependent variation ofKbetween 0 and I makes i
t possible
to separate solutes within a narrow molecular size range on a given gel.
The volume of outer solvent, Le., the solvent surrounding the gel beads is indic
ated as Vo.
the technical term for this is vo/d volume (Figure I I. 14). The volume of solve
nt inside gel,/.e.,
the inner solvent, is known as V (Figure 11.14). The distribution coefficient, a
s said above, is

symbolized by Kd. The effluent volume, V, is dependent upon these three variable
s. Thus.
Fig. 11.14 Diagramatic. respres .nuion of solvent insle the gel bead (V), vokl v
olume (Vo} and total volume VJ.

therefore,
378.
Blophystco Chemistry
The volume of inner solvent, Vcan be calculated if the dry weight of gel employe
d and the
water rega/n va/ue of the particular gel are known. Thus
where, a = the dry weight of the gel, Wr = water regain value. The value ofKd is
characteristic
of a given molecule and does not vary with the geometry of the gel bed. However,
the numerical
value of V is dependent upon the size of the column.
For two substances possessing different molecular weights and, therefore, differ
ent
distribution coefficients (Kd and Kd2) the difference in their effluent volumes,
Vs, is given by
v, = (K,,, - KJv,
Thus, the sample volume applied for complete separation of two substances should
not
exceed Vs. The distribution coefficient and the effluent volume are both related
to molecular
weight of the molecules being separated. One can therefore calculate molecular w
eight of
molecules if one determines the effluent volume by gel filtration experiment (se
e applications of
gel permeation chromatography).
Types of Gels
A gel filtration medium should possess the following characteristics:
(/) The gel material should be chemically inert.
(/0 It should preferably contain vanishingly small number of ionic groups.
(///) Gel material should provide a wide choice of pore and particle sizes.
(/v) A given gel should have uniform particle and pore sizes.
(v) The gel matrix should have high mechanical rigidity.
No such material is available which will fulfill all the above criteria satisfac
torily. However,
there are five principal types of media that fulfill the criteria to quite some
extent. They include
(0 cross-linked dextrans (trade name Sephadex), (it) agarose (Sepharose, Bio-Gel
A, Sagavac),

(rio polyacrylamlde (Bio-Gel P), (iv). porous glass and silica granules Bio-Glas
Porasil), and
(v) polystyrene (Styragel, BioBeads S). Other gels, which have been used for gel
filtration,
include macroreticular polyvinyl acetate (Merck-O-Gel OR), mircroparticulate alu
minas and
silica's (Spherisorb), and eellullosepackings in bead .form (bead celluloses hav
e not been developed
-specially for gel filtration but are known to have molecular sieving properties
}.
Sephadx. For proteins and most of the bio-molecules, Sephadex is by far the most
popular
of all the gels. When Leuconostoc mesenteroids indulge in sucrose fermentation,
large polymers
of glucose are the result. These polymers, known as dextrans, are used to prepar
e Sephadex.
Each of the glucose residues in the polymer possesses three hydroxyl groups givi
ng the dextran

CHOH
OH
l
o r o
OH
OH
Fjure 11.15 Structure of cross-linked polydextran (sephadex)
a polar character (Figure 11.15). The agent used for cross-linking dextran polym
ers is
epichlorhydrin, CHCHCHCI.
%/
Pore size is controlled by the molecular weight of the dextran and the amount of
epichlorhydrinused in the preparation. By controlling the cross linking reaction
, various classes
of gel beads with exclusion limits between .1 and 200,000 daltons can be produce
d. These gels
are identified by a number such as G- 10, or G-200, which refersto the water reg
ain value (Le.,
the amount of water taken up in the completely swollen gel granules by one gram
of Sephadex}
of the gel multiplied by a factor of I0 (Table 11.7). Sephadex gels are insolubl
e in water, and are
stable in bases, weak acids and mild reducing and oxidizing agents. In addition
to water in
which they are normally used, Sephadex gels will swell in glycol, d/methylsulfox
ide, and
formamide.
Table 11.7 13/fferent Types of Sephadex Gels
Eclus/on llm/t
Mol. Wt.
*
G-25
G-50
G-75
G-100
G-200
2.5
5.0

7.5
I0.0
20.0
5,000
I0,000
50,000
I00,000
200,000
I00 5,000
500- I0,000
1.000- 50,000
5,000 - I00,000
5,000 -200,000

*Manufactured By Pharmacia, Inc.

Proteins
380
Biophysical Chemistry
Polyacrylamide. This very popular medium is produced by polymerizing acrylamide
into
bead form. A brief discussion of the control of the bead and pore size of these
gels is provided in
chapter 12. Polyacrylamide gels are usually identified by a number such as P-10
or P-100
which if multiplied by a factor of 1000 will indicate the exclusion limit of the
gel in thousands
of daltons. Polyacrylamide gels can be used to separate molecules of up to 300,0
00 daltons.
However, the large pored gels used for such separations lack in mechanical rigid
ity. These
beads also tend to become compressed in the column. This detrimental property ma
y cause
very low flow rates. Polyacrylamide is insoluble in water and common organic sol
vents and
may be used in the pH range of 2-1 I. These gels, however, are unstable to bases
due to hydrolysis
of amide groups. Table I 1.8 lists the commonly used polyacrylamide gels.
Table 11.8 Commonly Used Polyacrylamide Gels
Water regain
g/g dry gel}
Exclusion lim/t
Mol, Wt.
Fraction range
Mol. Wt. Limits

*Bio-Gel P- I0
Bio-Gel P-60
Bio-Gel P- 100
Bio-Gel P-200
Bio-Gel P-300
5.1

6.8
7.5
13.5
22.0
I0,000
60,000
100,000
200,O00
300,000
5.000 - 17,000
30,000 - 70,O00
40.000 - lo0,O00
80.0O0 - 30O,00O
IO0,000 - 400.000

*Manufactured by Bio-Rad Laboratories

Agarose. Agarose gels areproduced from agar. They are linear polysaccharides of
alternating residues of D-galactose and 3,6-anhydro-L-galactose units, and owe t
heir gelling
properties to hydrogen bonding of both inter-and intra-molecular type. These gel
s are
hydrophilic and are almost completely free of charged groups. They are therefore
almost
completely inert to the solutes being separated. In contrast to Sephadex, which
cannot be used
to separate biopolymers larger than 300,000 daltons, agarose gels, by virtue of
their greater
porosity, may be used to separate molecules and particles up to a molecular weig
ht of several
million daltons. Thus, wide use has been made of these gels in the study of viru
ses, nucleic
acids, and polysaccharides. The-agarose gels are compatible with all aqueous buf
fers and are
completely stable within the pH range of 4-10. In fact reports are available whe
re these gels
have been used even at pH 13. High salt concentrations do not usually affect the
se gels adversely,
Care, however, has to be taken about controlling temperature when using agarose.
Freezing
temperatures and temperatures higher than 30C cause alterations in the gel struct
ure, The
chromatography has therefore to be performed between 0 and 30C. Table 11.9 lists c
ommonly
used agarose gels.
Table 11.9 Commonlv Used Agarose Gels

Approximate agarose
Concentration |%)
Approximate exclusion limits (mol.wt,}
Polysaccharides

*Sepharose 2B
Sepharose 4B
Sepharose 6B
2
4
6
40x I0
20 x 10
4 x I0
20x I0
5 x l0s.
I xl0s

tromraphy
381
$eL For completely non-aqueous separations, a gel that will swell in an organic
Ivent is required. Styragel provides this option. It is a rigid cross linked pol
ystyrene gel which
be prepared in a range of different porosities. The gel structure is unaffected
by
mperatures as high as 150C, The gel can be used with such solvents as tetrahydrof
uran,
,ene, trichlorobenzene, perchloroethylene, cresol, dimethylsulphoxide chloroform
, carbon
trachloride and others. It is, however unusable with acetone, alcohols, or water
.
Conto|led pore gl.s lad. These fine glass spheres are manufactured from
osillcate glass to contain large number of pores within a very narrow size distr
ibution. High
rates are permitted by their total rigidity. These glass beads, however, suffer
from a
iortcoming of adsorbing a significant amount of protein on their surfaces. The g
lass beads are
ated with hexamethyldisilazane to circumvent this problem. These glass spheres h
ave a
lecular exclusion limit ranging from 3000 to 9 million daltons. A list of some r
epresentatives
ithis material are listed in Table 11.10.
e 11.10 Commonly Used Porous Glass Beads
xeluslon limit CMol. Wt.)

* Bio-Glas 200
i
Bio-Glas 500
Bio-Glas I000
Bio-Glas 2500
lxI0
4x 10
I x l0s
4x I@

*Manufactured by Bio-Rad Laboratories

Gel permeation chromatography can be performed either by column or thin layer
Column preparation. The general principles of column preparation are the same as
previously under the section on chromatographic techniques. Prior to use, the ge
l
to the swollen form. This may be done by allowing a known weight of the gel
either in water or in a weak salt solution. Although the initial swelling is fai
rly rapid, the
taken to attain equilibrium may be longer. The greater the porosity, the more wi
ll be the
required, to reach equilibrium. Thus, whereas overnight swelling is enough for
Sephadex G-200 should be allowed to swell for three days. The swelling time
be reduced drastically by heating the gel slurry on a boiling water bath for 1-5
hours,
on the gel type (the higher the gel porosity, the greater the time required). Po
rous
on the other hand, need not be hydrated (swollen) at all.
The gel bed is supported in the column on a glass wool plug or nylon net and the
previously
the form of a slurry and allowed to settle. Air bubbles must be removed
the column to a vacuum pump and the level of the liquid must never be allowed
lower than the top of the bed. Sample is applied in a manner indicated previousl
y (see
The volume of sample that should be applied varies as per
column size and the type of the gel used. The eluant is steadily added and the e
ffluent
in various fractions th be analysed. Blue dextran, a very high molecular weight
times to help in the calculation of the void volume. A knowledge
effluent volume of a particular compound is useful for the calculation of its di
stribution
which might be useful for molecular weight determination. Elution is usually car
ried
constant hydrostatic pressure head to achieve a constant flow rate.

382
Bphystca Chef, sir
Detection. The common detection methods include collecting and analysing
fr-actio
and continuous methods with flow cells in which ultraviolet absorption, refracti
ve index, radioactivity is measured.
Thin layer gel chromatography, Determann, and Johanson and Rymo (in 1962) showe
that gel permeation chromatography could be conducted using thin layers of gel.
For
use, thin layer gel illtration (TLG) is ideal since very small sample volume is
required for
technique. In this technique, a layer of hydrated gel is applied to the plate. T
here
a fixative to adhere the gel beads. The plate is not dried at all and is placed
in an
contai/ler at an angle of 20. The plate is connected to reservoirs at either end
by
paper bridges. Equilibrium must be carried out for at least 12 hours. Such equil
ibrium
to normalize the ratio between the stationary and mobile phase volumes. The
applied either as a spot or as a band. The plate may then be developed for a sui
table
the separated components detected by suitable means.
TLG is used mainly for the study of hydrophilic substances which require
conditions (proteins, peptides and nucleic acids). TLG has been used to study ce
rtain
such as adenosine deaminase (separation of high and low molecular weight forms),
from human granulocytes, -glucuronidase from human urine, glyoxylate reductase,
acid oxidase, lactate dehydrogenase, methyl esterase, and RNA synthetase (all of
these
molecular weight determination), and polyphenoloxidase for size heterogeneity st
udies.
other enzymes have also been studied using TLG.
TLG has found numerous applications in clinical immunology and immunochemistry.
many cases TLG has been combined advantageously with immunological
technique has bee used as screening procedure for several Immunopathological
involving a!tered immunoglobulin levels.
Advantages of Gel Permeation
Gel permeation is the gentlest of the chromatographic techniques. It has the
advantages.not paralleled by any.0ther chromatographic technique.
1.
Gel permeation depends only on the molecular sizes of the macromolecules
.
chromatography therefore can be conducted under virtually any condition of
temperature, pH, ionic strength, and buffer composition. This is a boon for
separation of those macromolecules which are labile under certain conditions of
temperature, ionic strength and other parameters. These macromolecules can

separated under the conditions where they are stable.

2.
Conversely, those macromolecules that can be renatured after they have
destabilized may be purified from other macromolecules of similar molecular size
s
deliberately destabilizing them. Such instability causes large scale changes
sizes making them migrate differently from other molecules which ma)
molecular weights. After purification, the macromolecule of interest may
by providing necessary conditions.
3.
Apart from the above named conditions, macromolecules can be damaged
adsorption too. Other chromatographic techniques do suffer from some
Such macromolecules can be separated by gel chromatography
is almost nil.
4.
For reasons which we know nothing about, there is less zone spreading in
chromatography as compared to the other techniques.

As is evident, gel permeation chromatography is chiefly used for the purpose of

separation of biological molecules leading to. their ultimate purification. Prot
eins,
enzymes, hormones, antibodies, nucleic acids, polysaccharides, and even viruses
have been separated in various experiments which have used different types of ge
ls or glass
granules. Lest it is thought that the method is only useful for large molecules,
it
should be.made clear that gel filtration has been used for separation of such lo
w
molecular weight compounds as amino acids, small peptides, and
oligonucleotides. The method is especially useful for the separation of 4S and 5
S
t-RNA. It is also the most satisfactory method for separating DNA (from bacteria
,
usually Gram positive) from the invariable contaminants, the teichoic acids.
(//)
One of the common separation problems in biochemistry is the removal of
salts and
small molecules from macromolecules, This can be easily performed using gel
filtration since the distribution coefficients of salt molecules will be largely
different
from those of macromolecules. For example, in a Sephadex G-25 column,
molecules above 5000 daltons will elute very quickly but the molecules less than
1000
daltons will be retarded. One of the advantages of this method of desalting is t
hat the
macromolecules are eluted with essentially no dilution.
('I)
Dilute solutions of macromolecules with molecular weights higher than th
e
exclusion limit may be readily concentrated by utilizing the hygroscopic nature
of the
dry gel. Sephadex G-200 absorbs 20 times its weight of water, although G-25 is '
preferred for its rapid action. This treatment leaves the macromolecular solutio
n
concentrated but at the same time unaltered in pH or ionic strength.
(/v)
Perhaps one of the most important applications of gel permeation chromat
ography is
in the determination of molecular weight of macromolecules. Due to the extreme
importance of this application, it is separately discussed below (also see Box 1
1.3).
Molecular Weight Determination by Gel Filtration
Gel chromatographic separations are achieved by means of differential distributi
on of sample

between the stationary solvent within the pores of a gel arid the mobile eluting
solvent
the pores. The distribution characteristics of a given macromolecule are a funct
ion of the size and
the hydrodynamic particle formed by the solute molecule, and the size range of t
he gel pores
It then logically follows that the distribution coefficient of a solute can be r
elated to its molecular
a fair degree of certainty. In a previous section relating to the principles of
this method we
already derived a mathematical relationship between the effluent volume of a par
ticular compound its distribution coefficient. Thus, in a gel filtration experim
ent if one determines the effluent volume
one can calculate its distribution coefficient. If distribution coefficients of
standard
of known molecular weight are plotted against the log of their molecular weights
, the position of
coefficient of the unknown protein on the plot will lead to the determination of
its molecular
of course, one presupposes here that all macromolecules, both standard and known
, have the same
such that their dimensions are a unique function of molecular weight. In most of
the cases, shapes
proteins vvaT widely and may lead to erroneous assignments of molecular weights.
This
can be obviated by choosing a solvent whose properties are such that it confers
identical shapes
protein molecules regardless of their original structure. Since guanldinium chlo
ride makes the
behave like randomly coiled linear homopolymers, it might be the ideal solvent f
or molecular

384
There is a classical relationship between the average dimensions of flexible lin
ear
and their molecular weights, M.
Where Re is the radius of a sphere of equivalent.hydrodynamic properties, is
of gyration of the polymer, a is the empirical factor related to a function
is related to effective moment of unit length, and Mo is the monomer unit molecu
lar
Viscosity measurements on proteins in 6M guanidinium chloride have related Rg wi
th Min
following manner
R = (constant} M
Effective hydrodynamic radius, Re, dictates solute partitioning in a gel. This c
an be
to the number of units in a flexible polymer. It is then a corollary that partit
ioning of a
polymer is a function of its molecular weight. This has been proved experimental
ly leading
popularization of gel chromatography in 6M guanidinium chloride as a useful meth
od of
molecular weight determination.
The gels usually employed for this purpose are 4% (limit of molecular
determination I0,000 to 300,000 daltons) and 6/6 (limit of molecular weight
1,000 to 80,000 daltons) agarose.
The solvent used is 6M guanldinium chloride in water, pH 6. The sample is prepar
ed
dissolving it at a concentration of about 1% in 6M-guanidinium chloride and 0.I
2-mercaptoethanol (mercaptoethanol is used to disrupt disulphide bondsbetween
The pH is adjusted to 8.6. The sample is then incubated for about 8-10 hours.
the sample is carboxymethylated by addition of iodoacetate and the pH readjusted
to
Chromatography is carried out and the effluent volume of the desired macromolecu
le
determined. This is used to calculate the distribution coefficient of that molec
ule.
5.2 " Globulin (Hi.nan). MW = 158,000
/
" Transferrln (Human). MW= 88,000
I 4"9J
"Serum albumin (Bovine). MW = 67.000

.= |
Ovalbumln. MW = 45,000
eC (H . = ,
4.0
60 70 80 90 100 I I0
Eluflon volume
Figure 11.16 The orrelation of molecular weight and elution volume for proteins
from a gel permeation column.

Cmatography ' 385

distribution coefficient of a number of polypeptide chains of known molecular we
ights have
been obtained, column calibration curves can be constructed. The most popular ty
pe of calibration
curve is a plot of log molecular weight vs. distribution coefficient. The positi
on of distribution
coefficient of unknown polypeptide on such a plot then gives its molecular weigh
t. Altematively
log molecular weight is plotted against elution volume. Such a,plot is shown in
Figure 1116.
It should, however, be pointed out that under the reducing, denaturing condition
s of this
method, only the molecular weights of the constituent polypeptide chains of a pr
otein can be
measured. To determine the molecular weight of the native protein, recourse to o
ther methods
is required.
Analytical Uses of Gel Permeation
Apart from determination of molecular weights of macromolecules, there are a few
other
analytical uses that gel permeation can be put to.
1.
Binding affinities of small molecules with macromolecules (e.g. substrat
e : enzyme)
can be studied easily with the help of gel permeation chromatography. Two differ
ent
techniques may be employed. Either one can use a gel permeation column equilibra
ted
with the small molecule and then passing the protein through it. Or one can try
separating a small molecule bound to protein by gel permeation. A simple calcula
tion
then is required to determine the binding constant. To be sure, there are other
methods
of determining such binding. However, none of the other methods has the advantag
e
of operating under any given condition of pH, temperature, or ionic strength.
2.
Less albumin in blood plasma may indicate liver disease. Likewise, measu
rement of
different plasma proteins is of great diagnostic value since alterations in thei
r
concentration may alert the doctor and help him reach a conclusion about the dis
ease
that the patient suffers from. Special dextran gels have been developed for this
purpose.
These give quick and reliable measures of plasma proteins.
3.

It is necessary during study of RNA metabolism to distinguish various fr

actions of
RNA. This can be easily done using gel permeation.

4. ION-EXCHANGE CHROMATOGRAPHY
Ion exchange rny be defined as the reversible exchange of Long in solution wtth
long
electrostaticalty bound to inert support medium. The governing factor in ion cxc
ha-ne reactions
is the electrostatic force of attraction, which in turn depends mainly on the re
lative charge, the
radius of the hydrated long, and the degree of non-bonding interactions. Ion exc
hange
separations are carried out usually in colums packed with an ion exchanger. Ion
exchangers
can be divided into two groups: anion exchangers and cation exchangers. The ion
exchanger is
an inert, insoluble support medium. This medium may be covalenfly bound to posit
ive (anion
exchanger) or negative (cation exchanger) .functional groups. Long bound electro
statically to
the exchanger are referred to as the counter/ons. This technique is extremely us
eful in the
separation of charged compounds (even uncharged molecules can be *charged" by va
riance of
pH as we will see later). The value of this technique lies in the fact that cond
itions can be so
manipulated that some compounds are electrostatically bound to the ion exchanger
whereas
the others are not. The basic process of ion exchange is illustrated in Figure 1
1,17. The exchanger
is prepared in a way that it is fully charged (Figure 11.17A). The sample contai
ning the ionic
species to be separated is allowed to percolate through the exchanger for such a
length of time
as will be sufficient for the following equilibrium to be achieved (Figure 11.17
B):

E-Y+X E-X+Y
where E- is the charged cation exchanger and Y is the counterion of the .opposite
charge
associated with the exchanger matrix. X is the charged molecule (bearing charge s
imilar to the
counterion) in the sample to be separated. This molecule can now exchange sites
with the
counterion as shown inthe above relationship. The neutral and anionic molecules
will not bind
at all. Once the exchange of counterlon with the sample ion has been achieved, t
he rest of the
uncharged and like charged species can be washed out of the column. Bound long,
X can now
be eluted either by percolating the medium with increasing concentrations ofY (th
is increases
the possibility that Y+ will replace X+ in the above stated equilibrium because
the former is more
in concentration|, or by increasing the pH of the solvent and hence converting X
to an uncharged
specie (Figure 1 I. 17C, D). Concentration of Y required to elute X will depend up
on
the quantity of charge possessed by X. The greater the charge possessed by X, the
higher the
concentration of Y required to elute it. In the second case, when the pH is being
changed, the
higher the pK of X, the higher will be the pH required to elute it. .

X X xxx
y Y y
+ +- .
X X X X
X

F/ure 11.17 Bascprocess oflon e.Jchange (exoxnple cation eJchange), (A) Exchange
r has been prepared with Y as
tlaz cotmtedorc The on to be separated from the samp!e IS X'. (B) Catlon X+ has
bound to column
dLsplacing Y'. (C) Elution by Increasing the pH of the solverrL X is converted to
an uncharged species,
X, and is dlsplcmed. (D) F-Jution by Increasbw the concentmn of Y in the.solvent
. Y displaces X,
The principles discussed above also apply to macromolecules such as proteins and
nucleic

acids, which are capable of possessing both positive and negative charges. These
large
n]olecules, then, can bind to both. anion and cation exchangers since they posse
ss both types
of charges. The macromolecules can, howeer, be made to bear more negative charge
s by
increasing the pH {resulting in a stronger binding to anion exchanger} or more p
ositive charges
by reduclng the pH {resulting in a stronger binding to cation exchanger}. The am
photerlc nature
of macromolecules, particularly proteins, can be exploited for purification purp
oses by using
ion exchange chromatography. For example, the desired protein may be made to beh
ave as a
ation by lowering the pH of the protein mixture {the pH is lowered to a limit whe
re most of the
other proteins in the mixture behave as anions}. This preparation, if chromatogr
aphed on a
cation exchanger will remove many of the anionic protein species. This process w
ill remove

@
and so einte out quickly. (C} (D*) (E} (F & (G) hind to the bead and are
retarded. These can be eluted later and collected. Two unwanted proteins
have been removed and we have a mixture of five proteins.
Changing the pH,
of the protein
solution to 8.0
F & G become
positively charged
while (C)
& (E} assume
negative charge Buffer pH 8,0
.@ @
Performing anlon-exchange chromatography. {F*) & (G*) are not
ex.snged, elute quickly and are discarded. {C-), (D-) & |E-) bind to beads
and csn be eluted later. The desired protein {D) is now in a mixture of
only three proteins. Thus the protein solution has been enriched for (D)
Ftgure 11.18 Sequent use of cation and anion exhange chromatoraphy results in a
large degree of purtfumflan
of the desired protein from a mixture of several

Chromatography
389
many of the unwanted proteins from the starting mixture and the resultant mixtur
e is rich for
the desired protein. If the pH of this resultant mixture is now increased the de
sired protein will
exist predominantly as an anion (the pH is increased to a limit where most other
proteins in the
mixture still behave as cations). If the preparation is now chromatographed on a
n anlori
exchanger, many cationic protein species will be lost. It should thus be clear t
hat anion and
cation exchange chromatography used sequentially can afford a large degree of pu
rification.
This is better explained by the example in Figure 1 I. 18.
Types of Ion Exchange Resins
Two main groups of materials are used to prepare ion exchange resins: polystyren
e, and
cellulose. Resins made from both of these materials differ in their flow propert
ies, ion
accessibility, and chemical and mechanical stability. Selection of one or the ot
her type of resin
is done on the basis of compounds being separated.
Polystyrene resins are prepared by polymerization reaction of styrene and diviny
l benzene-CH = CH2
CH2CHCH2---CH---CH2
+
CH2--CH--CH2-CH ffi CH2
CH
Diviny Ibenzene
Polystyrene
The above structure is repeated in three dimensions, with the number of cross li
nkages
determined by the ratio of divinylbenzene to styrene. A higher concentration of
divinylbenzene
produces higher cross linkages. 8% cross linkage finds wide'spread use. Increasi
ng the cross
linkage increases the rigidity, reduces swelling, reduces porosity, and reduces
the solubility of

the polymeric structure. Selection of high or low cross linkage resin depends up
on the
compound being separated.
Acidic functional groups are easily introduced, for example, by sulphonation in
which a
sulphonic acid group is attached to. nearly every aromatic nucleus. Sulphonic ac
ids are strong
acids with essentially completely dissociated protons. These protons, however, a
re not free to
leave the resin unless replaced by other positive long. The total number of equi
valents of
replaceable protons per unit volume of resin determines the exvhan capac of the
resin.
Resins substituted with sulphonic acid groups are strong cationic exchangers. To
prepare weakly
acidic (weak cationic exchangers) exchanger, carboxylate groups can be attached
to the aromatic
rings instead of sulphonic acid groups. Weak acid groups usually result in high
capacity. The
exchange distribution fhnction, however, depends upon the strength of the acid.
If basic functional groups are introduced, the resin can exchange anions rather
than
cations. Strong anion exchangers are prepared with a tertiary amine, yielding a
strongly basic

Dowex 50
Amberlite IR 120
SP-Sephadex
Amberlite IRC 50
CM-Sephadex
Dowex 1
Amberlite IRA 400
QAE-Sephadex
Dowex 3
Amberlite IR 45
DEAE-Sephadex
DEAE-Sepharose
390
Biophysical Chemistry
quarternary ammonium group. Weak anionic exchanger can be prepared with secondar
y amines,
yielding a weakly basic tertiary amine.
Polystyrene resins are very useful for separating small molecular weight compoun
ds. They
are, however, unsatisfactory when macromolecular separations are to be carried o
ut. A list of a
few common resins is given in Table 11:11.
The unsatisfactory separation of macromolecules on polystyrene type of resins pr
ompted
development of the cellulose-based exchangers. Cellulose resins have much greate
r permeability
to macromolecular polyelectrolytes and possess a much lower charge density as co
mpared to
polystyrene exchangers. Cellulose is a high molecular weight compound which can
be readily
obtained in a highly pure state from such common raw materials as cotton, softwo
od, and
hardwood. Carboxymethylcellulose' {CM-cellulose, w.ealdy acidic, cationic exchan
ger} where
the CHOH group is converted to CH2OCH2COOH, and DEAE cellulose CHOCH2CHN
{CH2CH3}, weakly basic, anionic exchanger} are examples of main derivatives of p
ractical value.
Strong cationic cellulose exchangers {sulphoethyl} and strong anionic exchangers

{guanidoethyl}
are also available. Each form is commercially available in gel and bead forms wh
ich possess
good flow and exchange properties. Typical examples of cellulose based ion excha
ngers are
given in Table 11.11.
Table 1 I. 11 Select lon Exchange Resins
Nature
Trade Name
Strong cation Sulphonated polystyrene

Weak cation
Strong anion
Weak anion
Sulphopropyl cellulose
Condensed acrylic acid
Carboxymethyl cellulose
Poly.styreri with -CH2NMe3CI
Diethyl {2 hydroxypropyl}
quarternary amino cellulose
Polystyrene with secondary amine

Diethylaminoethyl cellulose
Diethylaminoethyl agarose
Cellulose may be treated in such a way as to remove the amorphous portion. Thus,
rather
than being fibrous in nature, the modified cellulose is known as microgranular c
ellulose. This
modified cellulose has even better resolution than the natural fibrous cellulose
. It, however,
does not give high flow rate.
Other alternative resins in use are derivatives of cross-linked agarose. Both Se
phadex and
sepharose types are particularly valuable for the separation of high molecular w
eight
proteins and nucleic acids.
Chromatographic papers are based on cellulose. It is logical then, that if the c

ellulose of
the paper is modified to DEAE-cellulose, one can carry out ion exchange chromato
graphy on
paper also. These papers are commercially available. Other modified papers inclu
de cellulose
phosphate (strong cation exchanger), cell-ulose citrate (weak cation exchanger),
and
aminoethylcellulose (weak anion exchanger).

hromatography
'
391
Prepmtion of the Ezchene Medium
Conversion of the exchanger from the form in which it is supplied to the
form in which it
is to be used is known as the preparation of the exchange medium and is
essential for
satisfactory performance of ion exchange chromatography. Apart from remo
ving the impurities
there are three other.steps that are of absolute importance in exchanger
preparation. (i)Swelling
the medium.. This is also known as precycling. This is necessary since t
he dry exchanger
contains densely packed polymers, which results in the burial of many ch
arged functional
groups which are then unavailable for exchange. Swelling makes these fun
ctional charged
groups to become exposed. Cross-section of such a swollen cation exchang
e bead is shown in
Figure 11.19. Swelling of anion exchangers is usually carried out by tre
ating it first with an acid
Figure 1 I. 19 Cross-section of a suxlen cation exchange bead with the functiona
l charged groups exposed. H is the
(0.5N HCI) and then with a base (0.5N NaOH}. Exactly the reverse is the case wit
h cationic
exchangers. Finally, ff impurities like metal long are present, the matrix can b
e treated with a
chelator such as EDTA. (//) Removal of very small particles of the exchanger (fi
nes). It is
necessary to remove fines since the presence of a large number of such fines wil
l result in a
decreased flow rate and unsatisfactory resolution. Such fines are usually genera
ted during
swelling ff the exchanger is subjected to vigorous washing and stirring. To remo
ve fines, the
exchanger is repeatedly suspended in a large volume of water and after the large
r polymers
have settled down, the slow sedimenting material (fines) is decanted. (//0 Final
ly the exchanger

"has to be equilibrated with the suitable counteri0ns. This is accomplished by w

ashing the
exchanger with different reagents depending upon the desired counterlon to be in
troduced
(NaOH, in case the counterion to be introduced is Na; HCI ff H is the counterion,
NaNO3 ff
NO is the counterion; formic acid if formate is the counterion,etc.). Following
conversion of the exchanger to the desired form, excess counterions are removed
by washing the exchanger with
large volumes of water or dilute buffers.
Choice of Buffers
The choice of buffers, which maintain the pH of the column, is dictated by the c
ompounds
to be separated and the type of ion exchange being carried out {anionic or catio
nic}. Anion

392
Biophysical Chemistry
exchange chromatography should be carried out with cationic buffers. Reverse is
true for cation
exchange chromatography. If anion exchange or cation exchange is carried out wit
h anionic or
cationic buffers respectively, the buffer long will indulge in ion exchange and
hamper sample
component exchange. The pK of the buffer should be as near as possible to the pH
at which the
system is buffered. This results in high buffer capacity which can withstand the
local changes
of pH in the column easily. The pH of the buffer should impart the same charge t
o the sample
long as is present on the counterion. A llst of volatile buffers used in ion exc
hange chromatography
is given in Table 11.12.
Table 11.12 Some Volatile BuYers Used in lonExchange Chromatography
Buffer
pH range

Ammonium acetate
Ammonium formate
Pyridinium formate
Pyridinium acetate
Ammonium carbonate
4-6
3-5
3-6
4-6
8- I0

Practical Procedure
We have already stated that the choice of the ion exchanger depends upon the sta
bility of
the sample components, their molecular weight and the specific requirements of t
he
separation. Many biological components, especially proteins, are stable only wit
hin a fairly
narrow pH range. Therefore, the exchanger selected should operate within this ra
nge. General
rules could be formulated for the choice of exchanger depending upon the stabili
ty of the
components to be separated. Thus, ff the sample is more stable at a pH below its
isoionic point,
use of a cation exchanger is advocated. Conversely, for a sample exhibiting more
stability above
its isoionic point, an anion exchanger might be more useful. Samples exhibiting
stability over a
wide range ofpH may be separated using either type of the exchanger. The volume
of exchanger
used for separation is usually 2-5 folds greater than that needed to bind all of
the sample.
However, excesses greater than this are avoided. Since cross linked gel material
is used as an
exchanger, the diameter of the pores is usually controlled and some molecular si
eving effects
might also play a role in separation. Thus, the cross linkage, although it might
not affect the ion
exchange mechanism as such, might have an effect on the exchanger capacity. The
degree of
cross linkage should therefore be fixed taking into account the molecular weight
s of the sample
components.
The columns used also have an effect on the resolution obtained. Columns of a hi
gh
diameter to height ratio usually give a better resolution, whereas columns long
and narrow,
might impose a peak broadening effect. This isso since in a long column, during
elution, the
free long have to traverse a long way down before the fractions are collected. I
n the long column,
they will have enough opportunity to diffuse and therefore will be eluted in a l
arge volume
leading to peak broadening. However, if the elution is being done with a solutio
n of one
concentration, rather than a gradient elution, use of a long column might in fac
t be beneficial.
The pH of the buffer used is usually maintained at about one pH unit more or les
s than
the isoionic point of the sample components. Generally, cationic buffers such as
Tris, pyridine,
and alkylamines are used when anion exchange chromatography is being performed.

When

Chromatography 393
cation exchange chromatography is to be done, anionic buffers such as acetate, b
arbiturate,
and phosphate are used. Initial buffer pH and ionic strength should be adjusted
so as to just
allow the binding of sample components to the exchanger.
Size of the column and the capacity of the exchanger are the two factors which d
ictate the
amount of sample which can be applied. If isocratic separation is to be performe
d, the sample
volume is about 1-5% of the bed volume. Sample volume is not an important consid
eration
when gradient elution is to be performed.
i Gradient elution is more commonly performed than Isocratic elution. Gradient e
lution
|could be stepwise or continuous. The latter gives a better resolution. During a
nion exchange
|the pH gradient decreases and the ionic strength increases. On the other hand d
uring cation
exchange both pH and the ionic strength increase.
lleatlon
Ion exchange chromatography has several applications some of which are listed be
low.
(/)
Perhaps the most stimulating use of ion exchange chromatography is in am
ino acid
analysis. In fact the amino acid "autoanalyser" is based on ion exchange princip
le.
This significant advance in instrumentation did wonders for protein chemistry (s
ee
Box 11.4).

394
Biophysical Chemistry
(//)
Ion exchange has been extensively used to determine the base composition
acids. The mixture of nucleotides as a result of treatment with DNAses and
can be readily separated by ion exchange chromatography. Throughout
which established the equivalence of adenine and thymine, and
Chargaff used this technique for the purpose.
(///)
This is the most often quoted application of ion exchange, since it is u
sed
all laboratories as fast and effective method of water puriflcatlon. Complete
of water or non-electrolyte solution is performed by exchanging solute cations
hydrogen long and solute anions .for hydroxyl long. This is usually
water in a mixed bed of anion and cation exchanger. The same method is also used
f
softening of drinking water. Rather than complete deionisation, all multiple
cations like calcium, magnesium, iron, etc., are exchanged for sodium.
(iv)
For many biological applications, ultrapure, metal ion free reagents are
is commercially performed by ion exchange chromatography.
(v) There are many situations where
is below the limit detected by atomic emission or atomic absorption spectrometry
.
These samples are passed over an ion exchange resin that holds the metal
enough sample has been passed over the resin, enough trace metal long
concentrated over the exchanger. These long can now be eluted,
in the eluent is considerably higher than that in the sample.
be "read" by atomic emission or absorption spectrometry and the results
to the original concentration in the sample.
(v0
Apart from the above applications, ion exchange chromatography has been
used
the separations of many vitamins, other biological amines, and organic acids and
bases,

396
5. AFFINITY CHROMATOGRAPHY
As opposed to all the chromatographic procedures described thus far which exploi
ted
small physicochemical differences of molecules in a mixture for their eventual s
eparation, a./aity
chromatography exploits the capacity of biomolecules for specific, noncovalent b
inding of other
molecules called ligands.
Any student of biochemistry will be familiar with the concept that a given enzym
e will bind
and react'with only a group of substrates and will not react with others. This a
mazing biospecificity is not limited only to erymes; a given hormone will bind to only a s
pecific glycoprotein,
known as receptor, situaten the plasma membrane surface; a given antibody will s
pecifically
bind to only a given antigen and not to others. Let us suppose that an enzyme is
to be purified
from a mixture of thousands of proteins. Let the substrate analogue (molecule re
sembling the
substrate but not capable of reaction] specific for this enzyme is coupled to a
column matrix
which is immobile (e.g., agarose). When the mixture of proteins containing the e
nzyme to be
purified is allowed to percolate through such a column, only the desired enzyme,
which is
capable of binding specifically to the immobilized ligand (substrate analogue),
will be retarded
by the matrix. All other molecules, which have no specificity for the ligand wil
l pass down and
out of the column. After all the undesired components have been flushed out of t
he column, the
conditions of the wash solution are altered so that the enzyme dissociates from
the ligand and
elutes from the column, largely in the purified form (Figure I 1.20].
Buffer
Protein
mixture layered
at the top of
the column
obd
column
] I. LLLLL]
[LLLLLL
[LLLLLU
LI. LL

Proteins not
specific for
the ligand elute out
Buffer . ---.-.LLL.L
L I.L.L.L
C]anging the Composition
of eluttng buffer:
The desired protein elutes out

Ftgure 11.20 Principle of affinity chromatography

Thus, affinity chromatography exploits the biological affinity of the macromolec
ule for its
- characteristic ligand. As said earlier, macromolecules other than enzymes are
also separable
by affinity principle; thus, if the ligand immobilized is an antigen, one can is
olate the
corresponding antibody, or if the ligand immobilized is a hormone, one may be ab
le to purify its
corresponding receptor. Affinity chromatography is not limited to proteins only;
it is currently
used to purify nucleic acids, and even whole cells and cell fragments. Isolation
of messenger
RNA from a crude RNA preparation is one of its often used special applications.
In short, it may
be said that all such biomolecules involved in specific interaction with other m
olecules can be
purified with the help of affinity chromatography. A short list of separations c
arried out employing
affinity chromatography is provided in Table I I. 13.

Specific Ligands Commonly Used in Chromatography

Macromolecule/Cell
Llgaud

Avdm
a-Chymotrypsin
Thrombin
Coagulation factor
Interferon
Poly (A] messenger RNA
Ribosomal RNA
Glycoprotelns, Glyc01iplds
Fat Cells
mocyUn
Tryptophan
Benzaxnidine
Heparln
Antibody
Poly (U) or Poly dT ,
Lyslne
Concanavalin A
Insulin, Concanavalln A

The picture of affinity chromatography presented in the above discussion would m

ake one
that the desired macromolecule will be totally purified in one single step. Howe
ver, it is an
picture of affinity chromatography. In practice, a number of complications of th
is
picture are always present. Most such complications arise because of nonspecific
of sample components other than the desired one on to the matrix. Usually ionic
hydrophobic interactions are involved in such non-specific adsorption. This comp
lication
, be taken care of by judicious choice of operating conditions (e.g., pH, temper
ature, or ionic
,. Another type of complication arises when one uses ligands, which interact wit
h more
macromolecule present in a given mixture. For example, when a coenzyme like NAD
used as a ligand, many dehydrogenases rather than the specific one, which is to
be isolated,

to the ligand-matrix conjugate. In such cases it becomes necessary to use specif

ic
procedures to effect the desired separation. Inspite of all these complications,
affinity
remains probably the most powerful tool for purification of biomolecules and
an amazing degree of purity impossible to attain by other procedures.
Before
initiating specific laboratory procedures for affinity chromatography, s
ome
', is required. Major variables, which need careful consideration, are
(0 The type of matrix used,
(//)
Selection of the ligand, its nature, and the means ofcovalenfly binding
it to the matrix,
and
(///}
The conditions exploited to bind and dissociate (elute) the macromolecul
e from the
column.
A general discussion of these points is provided below.
matrix
Characteristics desired of an ideal insoluble matrix for affinity chromatography
have much
common with those desired for gel filtration. They are discussed below
(0 The matrix should be inert to other molecules to minimize non-specific adsorp
tion.
(/0 It should possess good flow properties.
(it0
It should be chemically and mechanically stable at varying pH, ionic str
ength, and
denaturatlng conditions employed for binding and elution. This allows flexibilit
y in
the design of experiments.
{iv) It should contain large numbers of suitable chemical groups for ligand atta
chment.

298
Boptu3scol
(v)
It should preferably be highly porous. High porosity provides a large su
rface area
attachment of the ligand and allows better interaction of the desired
with the immobilized ligand.
In practice, particles which are uniform, spherical, and rigid are used.
used ones are agarose, polyacrylamide, and controlled porosity glass beads. Of t
hese
agarose beads are by far the most often used because they possess most of the
cited above. Agarose beads, however, suffer from a major disadvantage; they
to contraction when denaturant solutions are
possess many of the desirable criteria outlined above, they suffer from a lack o
f porosity.
undesirable trait is heightened even further when they are substituted by ligand
s.
pore glass beads provide mechanical rigidity and chemical inertness in addition
to
very good flow rates. Their most serious drawback is a high degree of nonspecifi
c
adsorption. This problem can be circumvented to some extent by treatment
hexamethyldisilazane. Controlled pore glass beads might very well be the matrix
of future.
Llgand Selection
The ligand to be used in construction of an affinity column is selected after
consideration. Substrate analogues, effectors, enzyme cofactors, receptor antago
nists,
synthetic agonists, antigen epitopes, etc., are the usual candidates. It is not
usual to select a
substrate as a ligand. Under certain circumstances, however, it might be the onl
y choice.
these circumstances, the conditions are so arranged that the enzyme substrate re
action
not occur; these might be achieved by omission of required metal long, change in
pH, or
omission of the second substrate if the enzyme catalyzes a bi-molecular reaction
.
The selected ligand should meet two most important requirements. (t) The ligand
interact strongly with the desired macromolecule. The interaction, must however
not be
strong. A very strong affinity will require a drastic treatment to dissociate th
e complex
elution which might damage the desired macromolecule. (to The ligand to be bound
possess functional groups that can be modified to form covalent linkage with the
matrix. The linkage modification should, however, be done in such a way as to no
t to impair the
binding of the ligand with the desired macromolecule.

Ligand Attachment
Covalent coupling of the ligand to the supporting matrix involve the following s
teps:
(/} activation of the matrix functional groups, and (/0 covalent attachment of t
he ligand to these
activated groups. The chemical methods used should be mild so that the ligand or
the matrix Is
not damaged. After the reactions establishing the linkage are over, the support
matrix must be
washed repeatedly.
The most common method of activation of polysaccharide supports (agarose) involv
es
treatment with cyanogen bromide at alkaline pH (pH = 11.0). The reacUon conditio
ns and the
relative proportions of the reagents determine the extent of activation of the m
atrix particles.
Usually 300 mg of powdered cyanogen bromide is used per ml of packed gel gives t
he maximum
substitution. For lesser activation a lower amount of cyanogen bromide suffices.
The pH of the
gel suspension is raised to 11 and all the cyanogen bromide powder added at once
. The activation
reaction (Figure 11.21A] begins. The reaction is exothermic and therefore there
is a need to
maintain the temperature constant at 2O0 at all Umes. Moreover the reacUon gener
ates
protons decreasing the pH. To maintain the pH at 11, the mixture is continuously
stirred and
an electrode dipped into it at'all times. The pH is maintained by the addition o
f 2M NaOH.
Usually just 10-15 minutes are required for the reaction to be completed. Due to
the toxicity of
cyanogen bromide, the entire reaction should be carried out in a fume hood. The
activated
suspension is nowwashed with about 20 times the gel volume with a buffer [buffer
s such as

pH-.g11.0
Epoxides
Periodate
hro raphy
399
rls. ammonium acetate, glycine should be avoided because amino groups compete wi
th the
gand being coupled] at a pH of 9.5-10. Sodium bicarbonate and borate buffers are
the usual
liolce. It is known from chemical and spectroscopic evidences that activation of
polysaccharide
apports by CNBr gives rise to the reactive iminocarbonate structures(Figure 11.2
1A). Table
I, 14 summar/z methods of activation other than CNBr method. CNBr activated poly
saccharide
apports which are freeze dried are commercially available.
-CH
HO "0 + Br---C -= N
Cyanogen
OH '
bromide
Iminocarbonate
ljure 11.21 (A) Activation of polysaccharlde support with CNBn and (B) Coupling
of amlno llgands.
I I. 14 Alternative Methods of Activation of Polysaccharlde Supports
Active functionality for
Reac.tion with
Ligand coupling
type of Iigand
Reacts with amines
And other nucleophiles
Reacts with amino groups
and other nucleophiles

I
-- CHO
Reacts with amines
or hydrazines

The requisite ligand may be attached to the matrix immediately after the activat
ed suport
been washed. Coupling of amino-containing llgand to CNBr activated support is nr
mally
suspending the support and the ligand in a basic buffer solution at pH 9 (usuall
y is the same as that used for washing the activated support; e.g., 0.25M NaHCOa
, pH
suspension is gently stirred overnight in a cold room or for a couple of hours a
t room
During this time the ligand is covalenfly attached to the support medium as
in Figure 11.2 IB. It is likely that the ligand is attached through an isourea l
inkage. The
pH sensitive and therefore the pH must be maintained around 9.0 all the time.

400
Btophyscca
After the reaction is over. the matrix is washed with O.1M solution ofpH 9.0
This treatment assures destruction of any extra activated groups. Following this
the
extensively washed to remove any unboand llgand.
It Is useful to determine the number of ligand groups bound to the matrix as
us how useful will the eolurrm be in separation of the desired macromoleeule. Th
is
can be eas.fly performed if a radioactive ligand of a low but known specific act
ivity is used.
number of llgand groups bound is usually expressed in terms of capacity per ml o
f
matrix rather than in terms of its dry weight.
Figure I 1.22(A} shows that if a ligand is directly attached to activated groups
of the
the macromolecules might encounter steric restrictions due to which they might
adsorbed to the matrix. To obviate this difficulty, it is usual to introduce a s
pacer
activated groups of the support and the ligand. This spacer is known as the arm.
Due to
the ligand is projected at a distance from the matrix and the desired macromolec
ule will bind
it without facing any steric hindrance (Figure 11.22(B). A large variety of spac
er
investigated. Examples include hexamethylenediamine, 3,3' 1,6 - diaminohexane, 6 - aminohexanoic acid, and 1,4-bis - (2,3-epoxypropoxy)
spacer arms must possess two functional groups, one to react with the functional
groups
matrix and the other to which the ligand could be attached easily. The
procedures involved in attaching the ligand to the spacer arm frequently involve
the use
succinic anhydride and a water soluble carbodilmide. Gels attached with differen
t arms
commercially available.
Figure 11.22 Need of a spacer arm. (A) Ligand without a spacer arm is closely he
ld to the matr particle.
nmcronlecule cannot attach due to steric h6ndrance. (B) llgand wtth a spac arm.
The rrmcromoleade
binds without any sterlr hlndmnce.
Chromatography Procedure

The procedure for affinity chromatography has many similarities to other forms o
f liquid
chomatography. The gel beads are swollen much in the same way as described for g
el permation
before loading onto a column. The buffer which encourages adsorption of the desi
red molecule
on the gel surface is used. The buffer chosen must be supplemented with any cofa
ctors
(example, metal long) required for llgand-macromolecule interaction. The buffer
should also
possess a high ionic strength so as to minimize non-specific polyelectrolyte ads
orption onto
charged groups in the llgand. The sample is applied at the top of the column and
the buffer flow
started. Once the macromolecule is bound, the column is eluted with more buffer
to remove
non specifically bound unwanted molecules. The purified, bound component may now
be eluted
(diss, oeiated) by taking recourse to either specific or non-spectfic elution. N
on-speeific elution is

C ocjraphy
401
carried out by changing either the pH or the ionic strength of the buffer. Chang
e in any of these
two parameters causes destabilization of the ligand-macromolecule link and cause
s the
macromolecule to separate and elute out of the column. Specific or affinity elut
ion is carried
out by (/} addition of compounds for which the macromolecule has more affinity,
or (//) by
addition of compounds for which the ligand has more affinity than it has for the
desired
macromolecule, The purified material is eventually recovered in a buffer solutio
n which may be
contaminated with specific eluting agents or high salt concentrations. These mus
t be removed
(usually dialysis for a long time) to bring the macromolecule in its native conf
ormation.
*
Morphine. a psychotroplc drug binds to specific receptors on neurons. On
the basis of the foregoing,
design a procedure to purify these receptors.
Variations of Affinity Technique
With biotechnology coming centrestage, the use of alrmity chromatography is incr
easingly
required. This is so because most of the biotechnological applications involve e
ither use or
production of proteins/enzymes, Affinity separation still remains the most power
ful technique
for protein separations and hence its increasing use. However, the technique suf
fers from two
drawbacks. It is exceedingly costly. The high cost is mainly attributable to the
process of
conjugation of the ligand to the matrix, The ligands used here are quite costly
and the incubation
process also is fairly long leading to a further increase in costs. This problem
,cannot be solved
unless an economy of scale is achieved by more frequent use of these ligands.
The other problem isthat affinity chromatography cannot be used as a first proce
dure.
The given protein has to be purified to quite an extent before it can be applied
to affinity
columns. If affinity is used as a first step, other contaminants will soon clog
the column rendering
it useless. This problem has been addressed in several ways in recent years allo
wing affinity
principle to be utilized right in the initial stages of protein purification. In
a book of this size it
is impossible to discuss these variations in detail..A brief description of thes

e techniques is
being given with a good list of relevant references at the end so that the desir
ous student can
supplement his knowledge.
Affinity Cross Flow Ultraflltration " Membranes used here are mostly microporous and are usually made up of cellulose
acetate, polyvinylidene fluoride, or polytetrafluoroethylene. Functional groups
are made
.available on which the ligands may be bound. Preprepared activated membranes ha
ve also
become commercially available. The pore size is usually 3-5 ram. When the whole
mixture
containing the protein of interest is incubated with it the membrane retains the
protein-ligand
complex while the contaminants pass through the membrane pores. Later the protei
n can be
released from the ligand by a change of buffer. The flow rates can be very high
due to the
microporous membrane, The faster flow rate also enhances and greatly improves al
l the steps
of the protocol : washing, elution, and regeneration. The ligand used must be ve
ry specific and
th membrane should be of a material that shows very little non-specific adsorpti
on.
Affinity Precipitation
The technique has evolved mainly due to the development of what have come to be
called
.the 'smartpohmers.' What is smart about these polymers is that they can exist b
oth in solution
and suspension (precipitate] form depending on the environment, A simple change
in conditions
like the pH. temperature, ionic strength, or even the addition of a metal ion ca
n bring the
polymer down from a solution form to a fairly compact precipitate form.
Imagine the ease of purification by this method now, The ligand is bound to a ch
osen
smart polymer and this whole complexis allowed to exist in solution form. The mi
xture from
which the protein is to be separated is, now dumped into this solution. The prot
ein sticks by

402
Biophysical Chemistry
affinity to the ligand which is in solution form. Once this has happened, one of
the above
named condition is changed and the protein-ligand complex is made to precipitate
. It is easier
to separate this precipitate from the mixture now. Later, the target molecule is
recovered from
the polymer by changing the buffer.
The column is not present. In fact, no rigid matrix is present either, There is
no danger of
any clogging and the affinity principle can be used right as the first step of p
urification
shortening the time and cost of purifying the protein of interest.
Matrixless Affinity Separations
There is another variation of this technique which is quite interesting. As stat
ed above, the
cost of affinity chromatography is high mainly owing to the costly ligand as wel
l as the long
incubation period required to attach this ligand to the matrix. This is not all.
However well you
may attach the ligand, some of the ligand leaches off the matrix.
The variation that we are going to talk about does away with the above problems
by simply
doing away with the ligand itselfl Let us understand this variation with the hel
p of an example,
The wheat germ lectin has an affinity for N-acetylglucosamine. Conventionally, o
ne can
try separating wheat germ lectin on an affinity matrix to which N-acetylglucosam
ine has been
covalently linked as a ligand. Poly N-acetylglucosamine is a natural component o
f the
exoskeleton of arthropods. Chitin can be partially deacetylated to give rise to
what is called
chitosan. Chitosan can act as a smart polymer. What is more, it also has an affi
nity for wheat
germ lectin. No ligand therefore is required to be covalently linked to this pol
ymer for wheat
germ separation. The strategy is then to incubate wheat germ extract with chitos
an at, pH 5.5,
At this pH chitosan exists in the solution form, After sufficient time has been
given for incubation,
pH is changed to 8.5 at which the chitosan-wheat germ lectin complex precipitate
s. The precipitate
is separated easily and redissolved by lowering the pH to 5.5. After this a simp
le gel filtration
step can separate chitosan from lectin.
USE OF AFFINITY CHROMATOGRAPHY IN MOLECULAR BIOLOGY
Purification of mRNA

In order to study gene expression, molecular biologists frequently have to isola

te mRNA
from a total RNA preparation which contains other types of RNA, viz., rRNA. and
tRNA, Affinity
chromatography is routinely used for this purpose. Oligo dT (many units of deoxy
thymldylllc
acid) is immobilized on an agarose matrix. When total RNA preparation is allowed
to percolate
through this column, only mRNA is retarded while the other RNA molecules elute o
ut. This
becomes possible because most mRNA molecules have a poly A tail at their 3'-ends
. These tails,
through complementarity, recognize and bind to the oligo dT molecules immobilize
d on the
matrix. Other RNA molecules do not have such poly A tails. After other RNA molec
ules have
eluted out, mRNA molecules bound to the matrix are eluted by changing the wash c
onditions.
A few years back a company called Stratagene came up with what is known as a pus
h
column. The oligo dT attached matrix comes packed in a 1 ml syringe, This syring
e is the
column. The kit provides the eluting buffers. Total RNA preparation is layered a
t the top of the
gyringe column. After this one fixes the plunger at the top of the syringe colum
n and pushes.
The liquid coming out of the syringe contains the RNA molecules other than mRNA,
The column
is washed twice again by iilling the syringe with buffer and pushing the plunger
. After this the
eluting buffer is dispensed at the top of the syringe and the plunger is pushed
again. The
eluting liquid contains mRNA which can be collected. The entire procedure takes
barely 10
minutes.

Chromatography
403
Isolation of DNA-binding Proteins
A transcription factor is a protein that recognizes ttnd binds to a specific DNA
sequence lying in the regulatory region of a given gene. This binding may promot
e the transcription
of the gigen gene. To study the mechanism of action of transcription factors, it
is necessary that
they be purified. However, for quite a length of time, scientists could not succ
eed in purifying
these proteins owing to the fact that these proteins are present in extremely lo
w concentrations
in the ceil. To give you an idea how low the concentrations may be, most transcr
iption factors
constitute just about 0.001% of total ceil protein.Conventional biochemical tech
niques cannot
purify such proteins.
In the 1980s Tjian and Kadonaga were working with the monkey virus SV40. These
researchers found that one of the sequences occurring in the regulatory region (
promoter) of a
given gene bound to a protein specifically. The protein was called Sp I and the
sequence was
called GC box owing to the fact that it was formed entirely of the two nucleotid
es. They also
demonstrated that the binding of Sp I to the GC box stimulated the transcription
of the gene.
The problem was to purify this protein in order to study it. Kadonaga and Tjian
decided to
exploit the high affinity of this protein to the GC box sequence GGGCGG. They sy
nthesized
oligonucleotides containing multiple repeats of the above sequence and coupled t
hem to solid
beads. A crude nuclear extract was passed through a column consisting of these b
eads coupled
to the oligonucleotide sequence. The column was then washed to remove non-specif
ically binding
proteins. Finally the beads were washed with high salt buffer which disrupted th
e Sp I binding
to DNA. This resulted in the elution of the Sp 1. A further gel electrophoresis
experiment proved
that while the nuclear extract contained thousands of different proteins, the pr
otein recovered
after two cycles of DNA-affmity chromatography contained only Sp 1.
Since then several transcription factors have been purified using DNA-affinity
chromatography. The application of DNA-affinity chromatography is not limited to
purification
of transcription factors. All DNA binding proteins which show sequence specifici
ty cn be
purified by this technique.
ppllcations ..
(/)
Affinity chromatography has been used to purify a large variety of macro

molecules
such as enzymes, immunoglobulins, membrane receptors, nucleic acids, and even
polysaccharides.
(/0
Whole ceils have been purified using this technique. Affinity fractionat
ion has an
advantage over other conventional types of fractionation (centrifugation etc.);
the
fractionation is carried out under physiological conditions, without involvement
of
much trauma, and the cells remain viable. Cells separated by using alllnity frac
tionation
include fat ceils, T and B lymphocytes, spleen ceils, lymph node cells, oocytes,
and
chick embryo neural cells. To illustrate the technique in brief, let us consider
the case
of fat cells. Fat ceils have a large number of insulin receptors on their plasma
membrane
(about 10 receptors/sq, micrometer). An aft'unity matrix is designed which has i
nsulin
as the immobilized ligand, a mixture of ceils when passed through these columns
comes out of the column deficient in fat ceils which become bound to the matrix
due
to insulin. Many other types of ceils cited above are fractionated by immobilize
d
antibodies for which their membranes are antigenic.
('t//)
Metal chelate affmlty chromatography is a logical extension of the basic
technique.
Many proteins which have similar molecular weights and even isoelectric points c
annot
be separated even by such high resolution techniques as gel filtration chromatog
raphy,
or electrofocussing. These proteins might, however, differ in their metal bindin
g abilities.
This property may then be exploited if the particular metal ion is immobilized o
n a gel

BiophsicaZ Cheraistr
matrix by chelation. Binding of proteins to such metaJ long as Zn=, Cu+, Cd=, Hg=,
Co=*, d Ni=* is pH dependent. e sple is apped at neu pH d e compod
eluted by reducg e pH d ioc seng of e buffer. temaflvely eflon c be
done by cludg EA (which .as a good met chelator detely have better for e oh met
ion e macromolecule ) in e buffer.
()
Use of maetic gel beads is oer eension of chromatoaphy. ese gels beads,
usury polyaclde or aglow, have a core made up of FeO (maec]
d e checly coupled to a prote gd. e cells suspension e owed to interact e crosph
eres. e ure is en passed roll a malefic field. e des ces (which have become atta
ched to e malefic crospheres) move
towds e poles of e manet. e oer cells, however, e not ected by e
maec field as ey do not teract e mec crospheres. e desed cells e thus purified d
c be coect by simply remog e magenflc field.
Iunoobu negave oces d neoblastoma ces have en ped by
is me. e meod ves a roue puH of about 95%.
(v)
Iobed ees is oer ension of e i pciple. e ees may
be atmch to gel beads which e packed in a colu. e subsates a stable reaction ure
c now be conuy passed roll one end of e colu d
e product removed from e oer end. ese colus e extensively used duses because of
e obous advmges. Iobed ees (so o as
sod-ste ees) e o to be more ey sble, less prone to protea
digestion d easily stored as comp to e ee ees, ere is so dence
at some cases e obed ees e cannery more acve e ee cotes.
Sd -d Syste
Ce ma-gd systems have become stdd for sepag cen es of
macromolecules shang a coon stcturaHfuncon chactestic. l of these e
erciy avable too. Some of em e as foows.
1.
Cron-bl-ose. Cibacron blue is a dye at sctuy resembles nucleodes
spite of e fact at it is f more complex en s dye is used as a gd,
ees hang a gh for nucleofldes bd to e colu. Eples of such ees e e dehydrogenases
, ases, DNA polerases, d ate reducmses.
Blood coagaflon factors so bd to such colums.
.
He-se. Hep is a nat coult found e blood. It teracts a lge numr ofblo com
nents. However, when used as a gd, oy a few componen of e ce eact bd to it. me s
ubsces wch have been ped
usg s stem e as foows: DNA lerase, bosomes, hepafls B surface
gen, ne coagenaae, utee es&d receptor, etc.
3.
lyn-. scussed above, ogo-dT colums e used for pcaon of A. Poly-U cols ma
y so be used stead of oligo-. Sly, scffic
polucleotide sequences covalenfly nked to agose may be used to pur
scHpflon factors d oer DNA-bdg proteus.

4.
s-ose. Lyse-agoseds notbd to algenumberofsubsces.ong
e subsces at have been pued s system e m d e prote plasogen.
5.
te-A-. is prote, derived from ce w of Sphyloccus aureus bds
we to e F reDon of e oobu G. It has n ensively used fly pucaflon.

ChromatoraplJ
6.
405
Lectin-sepharose. Lectins are compounds that bind to a variety of polysaccharidc
s"or
glycoproteins. Normally they recognize a specific sequence of sugars or sugar de
rivatives
in the given polysaccharideYglycoprotein. Lectins are normally isolated from pla
nts
and a few other sources. There are several kinds of lectins and each lectin has
its own
specificity. The most used lectin is Concanavalin A. It is routinely used to iso
late
glycoproteins and polysaccharides. Wheat germ agglutlnin is used to separate dif
ferent
types of lymphocytes. Lentil lectin is used to separate histo-compatlbility anti
gens.
HIGH PERFORMANCE LII/ID CHROMOGRAPItN"
"- perlormance liquid chromatography is a product of the scientific effort towar
d optimization of the conventional column chromatography. Ordinarily, a conventi
onal
chromatographic experiment takes an inordinately long time. To reduce the time o
f experiment,
one can increase the flow rate thereby reducing the retention time of the solute
components.
Although this manoeuvre reduces the time of experiment, it also reduces the effi
ciency of the
column because the solute components undergo lesser number of equilibration, i.e
. the plate
number is reduced. To circumvent this, if one uses a longer column, the diffusio
n effects
increase and the peak is broadened. To increase the flow rate, the pressure has
to be Increased.
The conventional supports can tolerate pressure only up to a particular limit; a
t higher
pressures their structure is affected thereby causing flow rate anomalies. All t
hese factors were
efficiently resolved with the development of high performance liquid chromatogra
phy (HPLC).
This method uses an extremely high pressure (up to 8000 psi). The flow rate ther
efore is high
and the experimental time is shortened considerably. There is no loss in efficie
ncy also because
the supports that are used here are different inasmuch as the support particles
are very small
and more or less uniform in size. The lateral diffusion is less because of two f
actors, the smaller
particle size and the enormous pressure, which reduces the time that a solute sp
ends in the
column. Due to this, band broadening is minimized, HPLC is therefore highly effi
cient and has
a very fast speed of resolution. The technique may be used with vanishingly smal
l amounts of
sample (pico or even femto gram). The technique is primarily suitable for analyt

ical purposes
but can be used as a preparative technique also. It is particularly popular for
the separation of
polar compounds such as drug metabolites, which, in general, are poorly resolved
by other
techniques. Perhaps the greatest advantage of HPLC Is that tt may employ the pri
nciples of
adsorption, partition, ion-exchange, exclusion, and affinity chromatography. Thi
s makes it an
extremely versatile technique and explains its emergence as the most popular chr
omatographic
technique. None of the other techniques discussed in the previous pages are so v
ersatile. Table
11.15 summarizes the differences between the conventional column chromatography
and HPLC.
"tble 11.15 Experimental Differen BetwRn Coaventional Chromatzogzaphy end HPLC
Conventional*
Number of plates/second
Pressure
Flow rate (mm/min)
Time of experiment
Equipment
Purpose
Quantity chromatographed
0,02
Negllgble
5-50
Hours to days
Simple; column and accessories
Predomanfly preparative
mgto kg
5
Upto 8000 psi
6OO
Minutes to hours
Integrated chromatograph
Predomlnanfly analytical
ng to mg

*Values for Gel-F11teration have been used.

406
Column
Biophysical Chemistry
[ Solvent
ervolr
microfllter
High Pressure
pump
Sample
--- Injection port

Guard column
Detector
Recorder
Figure 11.23 Schematic diagram of an HPLC system
Figure 11.23 presents a schematic diagram of the instrumentation required for HP
LC. Six
major components needed to perform HPLC are
(/) A solvent reservoir to store the mobile phase.
(//) High pressure pump to push the mobile phase through the column.
(//0 A device to inject-the sample in.to the mobile phase.
(/v) A column in which the separation will take place.
(v)
A detector used in detecting the concentration of the sample components
as they
come out of the column.
(vi) A potentiometric recorder to produce a chkomatogram.
Solvent Resesoir and the Solvents
The solvent reservoir should meet the following criteria:
(0 it must contain volume enough for repetitive analysis;

(//) it must have a provision for degassing the solvents;

(///) it must be inert to the solvent.
Solvent degassing witPn the reservoir is performed usually by heating or by appl
ication
of vacuum or by treating it with ultra sonic sounds. Occasionally sparging with
helium might
be used for degassing. Generally, glass and steel containers of 0.5-2.0 liter ca
pacity are suitable
as solvent reservoirs. Glass bottles in which the HPLC solvents are sold also ma
ke for a very
good solvent reservoir. The solvent container should preferably be insulated aga
inst
contamination through laboratory atmosphere,
The type of separation desired dictates the choice oRhe mobile
separations
can be carried out with a single solvent or a fixed proportion
lvents. For
gradient elution, however, the development solvent composition
sly. This is
achieved by a gradient programmer usually attached to the HPLC

phase. Isocratic
mixture of two so
changes continuou
assembly.

Chromatraphy
407
All solvents to be used in HPLC must be extra pure since even the smallest impur
ity
interferes with the detection system. This is more so if the detection system is
measuring the
absorbance below 200nm. Thus, even the extra pure HPLC solvents are passed throu
gh a
I- 5 mm filter placed before the pump. Since air bubbles interfere with continuo
us monitoring
of the effluent and with resolution itself, all the solvents are degasscd by met
hods specified in
the preceding paragraph,
Pumping Systems
Pumping system can be said to be the heart of HPLC. By producing reproducible hi
gh
pressures, the pump is a major factor in obtaining high resolution, high speed a
nalyses, and
reproducible quantitative analyses, A .good pump should have the following quali
ties:
(/) A pulseless stable flow. Absence of pulsations minimizes detector noise.
(//)
A suitable pump should provide solvent flow-rates of 0.5-10 mYmin, which
is compatible
with most HPLC modes.
('/) A constant volume delivery.
(/v) Amenability to high pressures of up to 6000 psi.
(v) The pump should be adaptable to gradient operation,
Pumping systems available for HPLC are:
(/) Liquid displacement by compressed gases (holding coil).
(/0 Pneumatic amplifier
(///) Piston/diaphragm driven by a moving fluid.
(/v) Reciprocating piston.
(v) Syringe pumps.
(0 Holding coil. This unit is usually available with less expensive HPLC systems
, A large
' holding coil made up of stainless steel tubing is filled up with the solvent.
Compressed gas from
a cylinder forces the liquid at constant pressure from the holding coil into the
chromatographic
column. Flow rates are dependent upon column permeability and the gas pressure a
pplied.
These pumps can at best provide pressures up to 1500 psi., and cannot be used fo

r gradient
elution separations, Another disadvantage of these pumps is that many times the
driving gas
can inadvertently get dissolved in the mobile phase and cause problems in resolu
tions. These
pumps are consequently not very popular.
(i0 Paumakie ampler, This pump also uses compressed gas for pressure. The pump
has a piston driven by the compressed gas (Figure 11.24). The pump uses gas at c
omparatively
To column

Column
Mobile
phase
Piston
Reservoir.
Hydraulic
fluid
Flexible
diaphragm
To column
MoWr
408
Biophysical Chemistnj
low pressure of about 200 p.s.i. The gas is in contact with a large surface area
of the piston. A
smaller surface area of the piston is in contact with the solvent. The pressure
of the gas is thus
increased 10-20 fold when it is applied to the solvent. This pump gives a pulsel
ess flow and is
ideal for quantitative purposes.
I||/)/ov/ng fluhi type. These pumps use either a piston or a diaphragm driven by
moving liquid. These pumps give a pulseless flow and they are adaptable to gradi
ent elution.
Figure 11.25 is schematic for this type.
Check valves
Figure 11.25 Schematic of a moving fluid type pump
Iis Reciprocating piston. These pumps use a piston that is in direct contact wit
h the
solvent. The piston may be driven with motors and gears or by solid-state pulsin
g circuits. A
piston moves rapidly back and forth in a hydraulic chamber. On the backward move
the piston
sucks in solvent from the reservoir, which it pushes into the column on the forw
ard move. The

outlet to the columns close during the backward move to maintain the pressure in
the column.

Chromatography
409
The pump, however, fails to produce a pulseless flow. In order to suppress the p
ulses a pulse
dampening system has to be employed. The pump is not very popular.
(v$ Syringe pump. As shown in Figure 11.26, these pumps operate .by a screw gear
displacing a plunger through the solvent reservoir. These pumps provide a stable
flow rate high
pressure. The pump is well suited to gradient operations.
Sample Injection
Sample introduction on to the HPLC column is, as for all conventional types of
chromatography, an important factor in achieving a satisfactory resolution. Two
methods are
available to introduce the sample as a narrow band.
(/) The first method employs a micro syringe designed to withstand high pressure
s. With
help of this micro syl ge, the sample is introduced either directly onto the col
umn or onto
directly above the column. Preferably the sample is injected when the pressure
dropped to almost one atmosphere after switching the pump off. This technique is
known
0jectWn. Alternatively, the sample can be injected while the system is under hig
h
(/0
The second method employs a small volume metal loop which can .be filled
with the
An appropriate valve then channels the eluant fom the pump through the loop dire
ctly
the column. The sample is thus carried spontaneously with the eluant to the colu
mn.
The columns for HPLC are usually made up of stainless steel, glass, aluminium, c
opper,
Stainless steel columns are preferred since they can withstand pressures up to 8
000
Straight columns of between 20-50 cm in length are generally used. Short
uired for liquid adsorbent and liquid-liquid chromatography, whereas for other
longer colunms are necessary. The internal diameter of the columns is usually 14 ram.
columns usually possess an internal mirror finish, which allows efficient packin
g.
packing material is supported by a porous stainless steel or teflon plug/disc at
the end of
column.
Packings

Maximal separation without or with minimal band broadening is a function of the

and mobile phases chosen. Coarser particles induce increased band broadening. To
(s)
11,27 Schemat/c representation of the strcture of three types of supports common
ly used for HPLC.

410
Biophysical Chemistry
minimize this unwelcome phenomenon, stationary supports for HPLC have been so de
signed
that the individual particles are as small and as uniform as possible. However,
as the particle
size decreases the resistance to solvent flow through the column increases and a
higher pumping
pressure is required to maintain a satisfactory flow rate. Clearly then the smal
l particles should i
also be able to withstand the increased pressure that they will be subjected to.
The stationary
phase is a compromise between the above factors. Three forms of column packing m
aterial are
available'based on a rigid solid rather than a gel structure:.
(/) m/croporous supports where micropores ramify through the particles. These pa
rticle
are generally 5-10 mm in diameter (Figure 11.27(A)).
(//) pe///cu/ar supports consist of a solid inert core onto which are coated sev
eral
particles (e.g. a glass bead of about 40 mm diameter). These supports are
superficially porous (Figure 11.27(B)).
(i//) bonded phases where the stationary phase is chemically bonded to an inert
{Figure I 1.27(C)).
Individual sta.tionary supports for different modes of HPLC have been described
in
11.16.
Table II.16 A Lis-of Stationary Phases used in Various odes of HPLC
Mode
Adsorption
Partition
Exclusion
Ion exchange
Nature
Commercial name Type of support Applications
Silica
Alumina
Silica

Alumina
Octadecylsilane
Octadecylsilane
Alkylamine
Glass
Polystyrene
divinylbenzene
Agarose
Strong base
Weak base
Strong acid
Weak acid
Corasll
Pellumina
Partisll
MlcroPak A1
Bondapak Cs
Bondapack
. . Bondapack-NI-I
Blo-Glas
Styragel
Sephadex
Partisfl-SAX
Mlcropak-NH2
Partisfl-SCX
Zipak-WAX
Pellicular
Pellicular
Mlcroporous
Microporous
Pellicular
Porous
Porous
Rigid solid
Semi-rigid gel

Soft gel
Porous
Porous
Porous
Pellicular
Steroids, vitamins,
chlorinated pesticides,
polar herbicides,
triglycerides.
Dansylated amino acids,
drugs, pesticides.
aflatoxins, fatty acids.
Proteins, peptldes,
nucleic acids,
nucleotides,
polysacchar/des.
Amino acids,
peptides, proteins,
nucleot/des,
polysaccharides.
Column Packing Procedure. The packing has to be uniform without any crocks
channels for obtaining optimum separation. Rigid solids and hard gels should be
packed
densely as possible. Care should, however, be taken to not to fracture the parti
cles.
method known as high pressure slurry technique is used for packing the column. A
suspension of the packing material is prepared in a suitable solvent. The columr
a porous plug at the bottom. The slurry is now pumped into the column at high
column so packed is then equilibrated for a long time by passing the developing
solvent
it. The technique can be extended to hard gels. The hard gel, however, has to be
swollen
before it is pumped under pressure. For soft gel, however, this technique cannot
be
because pressure results in the fracture of gel particles. These gels, therefore
, have to be
into the column under gravity in a way similar to that of conventional chromatog
raphy.
Prepacked columns are also available and can be purchased from the market.
The Guard Column
The resolution power of HPLC is so high that an elaborate sample preparation

chromatography is not necessary. Thus, sera, or other biological materials can b

e applied

411
This, however, clogs the column after a few applications
during separation retains many undesirable components of the biological samples.
Crcumvent this problem, a short column (2-10 cm} precedes the main column. This
short
is known as guard cownn and its function is to retain those biological component
s
would otherwise clog the matn column. The guard column has they same diameter an
d
same packing as the main column. The packing of the guard colunm can be replaced
at
intervals.
Since the quantiW of the sample applied to the analytical column is small, it is
imperative
the sensitivity of the detector is sufficiently hlgh and stable.
UVNIS photometers can be used for HPLC. These detectors are inexpensive, sensiti
ve,
normal flow and temperature fluctuations, and well suited for gradient elution.
, are however, sample selective, as only those sample molecules which absorb at
254 nm or
nm can be detected. Thus, liplds, carbohydrates, fatty acids, hydrocarbons etc.,
are
by these detectors.
UVNIS spectrophotometers with wavelength selection range of 200-800 nm are very
popular
detectors. These can be either recording spectrophotometers or manual wavelength
spectrophotomcters. The advantage with these is that a range of biological subst
ances
detected by selection of appropriate wavelengths.
Apart from the above two, refractive index monitors and fluorescence detectors a
re also
with HPLC systems.
of Separation System
Since HPLC can be used in many modes (e.g., aflLnlty, ion-exchange etc.), it is
important
discuss how the choice of a mode is made for a given sample. Flgure 11.28 ttlust
rates the
Affinity]
F Non-ionic
Reverse

low polarity

phase

/ Large differences.
Biological
Aaueous
in molecular size I
Molecular
specificity
/- lvent
]
exclusion
Large derences.
Organic
in molecular size

Molecular

solvent
"[
exclusion
Different J Homologous
Normal
Adsorptlon ------. functlonal, series

phase

go ups
Flgure11.28 Gulde W HPLC, mode selectlon
It is usual to separate compoUtlds soluble in organic solvents by either partiti
on or
chromatography. Compounds possessing different functional groups are ideally

412
Biophysical Chemlstr
separated by adsoHon comatoaphy on sca a non-pol solvent as e
ioc or non-ioc substces which e water soluble eld to sepation
phase pHon mode. On e oer hd songly ioc solutes soluble water
by e ion-exchge mode. However, fever phase ption mode c so be
to these subsces. is techque is o as on-paa (see later).

mobile phase. Wey

by rerse
should be sepated
applied

epative
If the study involves chacteaon d fuher studies on e sepated component,
it is perave that a lger qu of the pure components is needed. For is prepative
HP may be peffoed.
epaflve HPLC c be ced out using these pacing mater,s d solvents. e
oy difference being at e colums is of a lger term dieter (10-20 ) so that a
1ger volume of e sple c be processed. Repeated appleton of e sple d coec0n
of effluent fractions contng e desired components c we a f eld ofe component d
interest.
Specialized Techniques: Reverse Phase Chromatography
As opposed to the usual polar stationary phase and a less polar or non-polar mob
ile
phase, the stationary phase in reverse phase chromatography is hydrophobic (hydr
ophobic
bonded phase usually possessing Cs or Cs functional groups) and the mobile phase
is pohar
(fully or partially aqueous). In this case, polar substances will interact more
with the polar
mobile phase and elute first. As the non-polarity of the solute components incre
ases, their
retention times will also increase since they will interact more with the non-po
lar stationary
phase.The reverse phase system is therefore very useful for separation of non-po
lar solutes,
Water, an extremely polar solvent becomes the weakest eluent here. Methanol and
acetonitrfle
are stronger eluents than water. Solvents of intermediate eluting strength can b
e obtained by
mixing one of these solvents with water.
We have already seen while discussing the nature of partition forces that non-po
lar
substances are squeezed out of the polar phase. Therefore the driving force for
retention of a
component is not its interaction with the stationary phase but the effect of the
mobile phase in
forcing the component onto the hydrocarbon bonded stationary phase. Moreover, th
i$ 'squeezing
out' is proportional to the non-polar surface area of the solute. Therefore homo

logs show a good

resolution by reverse phase chromatography. When the sample has many components
covering
a wide range of polarity, a gradient elution has to be carried out for optimum r
esolution. These
gradients are prepared by continuously decreasing the polarity of the eluting so
lvent. This can
be achieved by gradually increasing the content of organic solvents (methanol or
acetonitrile) in
water/organic solvent mixture.
Ion suppression. For separating weakly ionic substances also, reverse phase
chromatography Is preferable. In this mode the stationary phase is non-polar (hy
drocarbon)
and the mobile phase is a polar (water/acetonitrile, water/methanol) mixture. In
many
applications, especially when ionogenic substances (dissociable solutes such as
acids and bases)
are to be resolved, the eluent pH must be controlled by using a buffer. If the p
H is adjusted on
the acid side, ionization of acids will be suppressed. This will allow the acids
to be separated by
the reverse phase technique. Adjusting the pH on the basic side, bases will rema
in unionized
and therefore will yield themselves to reverse phase resolution. Thus by adjusti
ng the pH we
can alternately suppress the ionization of acids and bases and other dissociable
so!utes and
make them separable by reverse phase technique. This approach is termed ion supp
ression and
Is useful for resolving weak'acids and bases within the pH range 2-8. The buffer
concentration
in such experiments is usually maintained at a high level. This allows protonic
equilibria to be

413
rapidl"and therefore does not allow asymmetric peaks and band splitting due to t
he
of secondary equilibria involved in the chromatographic process. Phosphate buffe
r is
for moderate to high pHs. At low pH. perchloric acid is usually the choice. Sinc
e
phases are not stablein high alkaline pHs, use of alkaline solvents is precluded
.
lon.pa|ri. Strong acids and bases do not yield to resolution by ion suppression
sensitivity of siliceous bonded phases to extreme pH values. However,
can also be resolved by reverse phase technique ff they can be converted to
' neutral species. This can be achieved by fonnlng an ion pair (a coulombic asso
ciation
arising when two long bearing opposite charges associate) with a suitable counte
rion.
trick is to add a large organic counterion to the mobile phase. This counterion
forms a
ion pair complex with the sample long. The complex now behaves as a neutral,
The e.A-tent to which the ionized sample and counterion form an ion pair
the retention is increased. By adjusting the pH in a manner
sample components are present in their ionic forms and by choosing a strongly io
nic
group attached, the situation can be expressed by the following
Solute" + Counterion* = (Solute+, Counterion+ }o pair
Flgure 11.29 illustrates the pl'inciple ofion-palring. The commonly used counter
ions include
I chain aliphatic amines for negatively charged sample long and perchloric acid
for positively
sample long. The ion pair formation and consequently the resolution of sample
is affected by (/) the size of the counterion, (to its concentration, and (rio t
he pH of
solution.
{B}
POLAR PHASE (MOBILE)
{HA)
Weak
Weak
base
acid

(BH1 aq (F-) aq {HBF} {AF) (l) aq-,. (A) aq

Conjugate + Counter Ion pair .Ion pair . Counter. + Conjugate' acid ion .lipophi
lic lipophillc ion base
NON-POLAR PHASE {STATIONARY)
Figure 11.29 Diagmmmatlc representation of prlnciples of ion-pairlng

Separator
column
WoMe
ample Eiuent Injection
414
Biophysical Chemistry
Ion chromatogrcphg. Although strongly ionic substances can be separated by
ion- exchange mode, there are ]imitations to this process. These limitations are
usually due
the limitations of the detectors employed. Unless the ion coming out of the colu
mn has
directly measurable property such as radioactivtyr UV absorption, continuous mo
the effluent becomes difficult. Ion chromatography solves this problem of detect
ion by using an
additional suppressor column situated downstream of the separator column. This s
uppressor
column is so manipulated that it neutralizes the undesired long allowing
to pass through the detector; which is usually a conductance cell. Thus., for an
ion analysis,
anion-exchange column is followed by a cation-exchange suppressor column. The su
ppressox
column then removes the long of the eluting base:
CATION ANALYSIS
Y+-m No+, NH, tthylsmlne trlethylsmine
Resin- SetH ++ Y + Cl'q==Resin-SOY +. H + Cl"

Suppressor
oolumn
Resin-N+ O1'+ H +Cl'---'Resln-N + C" + H20
Resln'NOH-+ Y+CI'Resin-NCI" + Y+OH"

Conductivity
cell
Eluent : 0.01 N HCI

Flow:
0 5 10 15 20 25 :.oppmethylmlne
Min : rio ppm trlethylamine

415
Resin-N+ HCO + NaX'Resin-N+X" + NII+HCO
1
Reeln-80H + NHCC--Resln-SONa + FICCj
Resin-803"H + NaX'.-> Resin.SONa + HX"
1
-Waste
Fluent :0.003 M NaHCO3.
Flow : 138 ml/hr
DMector : 10p mholcm full scale
Peak Identity :
t
a. 1.4 ppm Fb. . ppm Cl,
. 2.9 ppm NO
d. 143 ppm P043"
Min
f. 8.6 ppm NOs.
g. 14.3 ppm SOt,
Fjure 11.30 Flow scheme of ion chromatography (reproduced from Dione. Corporatio
n).
resin - H + Na* + OH- resin - Na + H20
e anions to be separated get converted to their respective acid forms:
resin-H+Y+A- resin-Y+H+Athrough the monitor where they are detected by the conductance cell.For cation
situation will be reversed and the separator column used will have a cation exch
anger
whtle the suppressor column used will have an anion-exchange resin. The eluent u

sed
HCI.

416
Biophysical Cherntstr
The separator columns ususlly employ special low capacity resins while t
he
columns employ conventional resins with high degree of cross linking.
The choice of eluent is a compromise between eluting power and
The choice of the eluent is dependent on the relative affinities of the eluent i
on and the
long for the resin. Both the ion species should have roughly equal affinities fo
r
separation. AgNOz, HCI, NaHCO , and NaOH are most commonly used eluents.
Routinely separated long include F-, CI-, NO , SO -, CrO -, PO-, Ca=, Mg=, Na,
and NH etc.
Applications of HPLC
In recent times HPLC has emerged as a method of choice for analytical purposes.
biggest advantage that it. has over other techniques is the speed of analysis wh
ich, is
times more than other techniques except, perhaps, for GLC. The sample requiremen
t is
very low for this technique and as less as a few femtograms of the sample will b
e
satisfactorily. On top of all this the detectors that are employed in HPLC
nature and thus the separated components can be recovered for further study.
HPLC has been successfully applied to the separation of proteins, nucleic acids
polysaccharides, plant pigments, amino acids, pesticides, steroids, drugs and
animal and plant hormones and complex lipids.
SOME SPECIALIZED TECHNIQUES
Hydroxyapatite Chromatography
Hydroxyapatlte is a crystalline bone mineral. Its composition may he given
{Caz(PO4)213.Ca(OH)2]. Arttflclaliy it may be prepared from CaI-IPO4.2H20 crysta
ls. Columnsc
hydroxyapat/te interact and bind those substances which interact with calcium, f
or
phosphoproteins, DNA, RNA, and chromate. It is assumed that atleast for those
wh/ch contain phosphate, the binding is an attr/bute of these phosphate groups.
Thus,
compomds bind at a low phosphate concentration and c be eluted out by increas/r
concentration in the eluting solvent. There are several other theor/es about the
binding and we kow that electrostatic effect is not the only factor involved.
Hydroxyapatite chromatography is extremely usefulin nuclelc acid esearch where i
t

ch/ef use is in the separation of single-stranded DNA from double-stranded DNA.

If a mixture
containing both single- and double-stranded DNA is appl/ed to a hydroxyapat/te c
olumn, both
forms will in/tially be adsorbed. A sl/ght increase in phosphate concentration o
f the
result in selective desorption of the single stranded DNA. The double-stranded D
NA can now l
separately eluted with further increase in phosphate concentration of the eluant
.
Hydroxyapatite chromatography coupled with temperature var/at./ons can be used f
or
fracUonating DNA according to base composition. This procedure is known as therm
al
chromatography. DNA from different sources dLfers in the base composition, i,e,,
with respect
to its contents of A.T and G.C base pa/rs. Double stranded DNA rch in A,T base p
airs is known
to melt (conversion to single strands by denaturation) at a lower temperature as
compared to
the double stranded DNA r/ch in G.C base pairs. Im.e a system where DNA from var
/ous
sources d/ffering in their base compositions has been appl/ed to a hydroxyapatit
e colurnn: Al/
these double-stranded DNA will bind to the column. The concentration of phosphat
e in the
eluant is now ra/sed sl/ghtly only to a level wch is sufficient to desorb single
-stranded DNA,
Now the temperature of the column is increased steadily. Since melting temperatu
re for DNA
r/ch in A.T base pairs is low, these DNA molecules wfl/be denatured and converte
d to single-

Chromatography
417
molecules first and wi/l elute out at a low phosphate concentratlon Tile G.C rlc
h
DNA molecule still remains adsorbed to the column. Only when the temperature
olumn has risen sufficiently high to denature DNA molecules, will these molecule
s
elute out. Thus, thermal chromatography can achieve fractionati0n
to the base composition.
Hydroxyapatite columns are useful for separation of phosphoproteins also. Howeve
r, what
is that even those protein molecules which possess no phosphate groups can
on such columns. Needless to say that the.mechanism of such binding is not
Nevertheless, this method is very useful in protein separation and many times wh
en no
gives the desired result, hydroxyapatite chromatography,givs a good esolution.
use of hydroxyapatite chromatography in protein research is to Concentrate lrote
ins
a very dilute mixture.
An Affinity Bystem for Base Composition Dependent Fractionation DNA
A polysaccharide gel bound to the dye malachite green is used. Malachite green h
as a
of binding strongly to A.T pairs as compared to its binding to the G.C pairs. Th
e
l)olyacrylamide is made so that it has a large pore size to minimize molecular s
ieving. If DNA
fragments (most of the DNA fragments generated by restriction digestion) are loa
ded onto a
polyacrylamide immobilized malachite green, all DNA fragments bind tightly. Elut
ion
is now carried out by increasing concentration of sodium perchiorate (NaCIO4) in
the eluant.
DNA fragments which are poor in A.T content elute out at a lower perchiorate con
centration as
to those fragments which are rich in A.T pairs. This sequential elution effectiv
ely
DNA on the basis of base composition. The perchiorate can then be removed by
or by passage through a desalting gel.
Affinity System for Fractionating Supercoiled and Non-Supercoiled DNA
This affinity system consists of the dye phenyl neutral red immobilized onto
gel of large pore size. This dye binds more tightly to single-stranded regions o
f
is an attribute of supercoiled DNA. At low salt concentration both supercoiled a
nd
coiled DNA bind to the column. As the salt concentration is increased, the nb

auper coiled DNA elutes out first. A higher salt concentration is required to el
ute super coiled
DNA. This has made this affinity system very popular for fractionating these two
The phenyl neutral red column is also used to separate DNA on the basis of base
As opposed to the property of malachite green binding preferentiallyto A.T pair,
dye binds preferentially to G.C base pairs. Thus after binding DNA mixture to th
e column,
poor and G.C rich DNA fragments can be eluted out sequentially by increasing the
salt
the eluant.
Chromatography
DNA-cellulose columns are used to separate DNA-binding proteins from non-binding
Preparation of DNA-cellulose consists of mixing cellulose powder with a solution
of
(single- or double-stranded) and drying the mixture under vacuum. The dried powd
er is
to remove unboundDNA. To couple the DNA more tenaciously to cellulose, the dried
light. The procedure chemically
the two substances.
The procedure of DNA-ceIlulose chrornatography is as follows. A mixture of diffe
rent proteins
a low ionic strength buffer is layered on a DNA-cellulose column. As the mixture
passes
the column, the DNA-binding proteins adsorb to the immobilized DNA, The low ioni
c
of the buffer helps in such and adsorption. Washing of the column at this stage
elutes
The ionic strength of the eluant in now gradually raised; this results in

418
BWphys.al Chemtr
desorptioIi of DNA-binding proteins in a manner that the strongly binding protei
ns elute
last at higher ionic strengths. This method has become the byword for purificati
on
proteins as the nueleases, repressors, polymerases and many other enzymes and
which bind to DNA.
Chiral Chromatography
Many of the biological and pharmaceutical compounds are chiral in nature.
compounds contain at least one asymmetric carbon atom or are molecularly asymmet
ric. Thes
have identical physical and chemical properties and differ only in their
in their optical properties. It is usual that Just one isomeric/enantiomerlc
is biologically active. For example, it is the L-isomers of amino acids which on
e finds in proteins
and the D-isomer is biologically rejected. It is the D-glucose, not L-glucose th
at has been preferred
by nfe.
It is thus a challenge to the pharmacists as well as biologlsts to separate the
racemi
mixtures of the two isomers/enantiomers so that the biologically active amongst
them may be
obtained in pure form. Till sometime back this was impossible. But the advent of
chlral
chromatography has made it possible to a certain degree,
Chromatographic techniques, which allow mixtures of enantiomers to be resolved,
are
collectively called chiral chromatography.
Diastereoisomers are optical isomers without an object image relationship. These
isomers
differ in physical properties even though they have the same functional groups.
These
stereoisomers can be separated from one another using the conventional chromatog
raphic
techniques.
The diastereoisomer approach means that enantiomers contain a functional group t
hat
can be derivatised using a chemically and optically pure chlral derivatising age
nt (CDA) so that
they become d mixture of diastereoisomers. Thus
(D + L)
+
D
DD + LD

Mixture of
Chlral derivatising
Mixture of
enantiomers
agent
diastereorners
Note that the two diastereomers will have different physical properties and can
now be
separated by conventional chromatography.
In spite of the basic simplicity of the above approach it has suffered on accoun
t of its being
slow and not really quantitative.
A variant of the above technique is to use a mobile phase that itself is chiral.
This leads to
the formation of a transient diastereoisomeric complex between the enantiomers a
nd the chiral
mobile phase agent. Chiral mobile phase agents that have given good results are
10camphorsulphonic acid, albumin, and N-benzoxycarbonyglycyl-L-proline. It is usua
l to prefer
reversed phase chromatography for chiral chromatographic separations.
Perhaps the most successful method for chiral chromatography has been to use a c
hiral
stationary phase, This approach is based on the basic premise that there would b
e a threepoint interaction between the stationary phase and the stereoisomers. Due to the
different
spatial arrangement of the functional groups at the chiral center of the enantio
mers, different
transient complexes of stationary chiral phase with the enantlomers will form. T
hey will eventually
allow racemic mixture to be resolved. These transient complexes may usually be f
ormed by
hydrogen bonding and wander waals forces. One form of the stationary phases used
here is
proteins (bovine serum albumin) since proteins themselves are optically active.

Matrix
Matrix
Matrix
Matrix
Chromatography
419
Covalent Chromatography
This chromatography procedure was speclflcally designed to fractionate thiol-con
taining
proteins. The procedure which can be classified under affinity chromatographies
takes
advantage of the fact that a thlol (--SH),containing protein will interact with
an immobilized
ligand containing a disulphide group. The scheme given below illustrates the chr
omatographic
procedure.
The most used ligand in this chromatography is disulphide 2-pyridyl group attach
ed to
an agarose matrix. When a thiol containing protein interacts, pyridine-2-thione
is formed and
the protein becomes covalenfly attached through formation of a disulphide bridge
, Pyridine-2thione absorbs at 343 nm and therefore the binding of a protein to the matrix ca
n be
spectrometrically followed, The column is now washed to remove non-thiol protein
s, A weak
solution of dithiothreitol is used subsequently to remove unreacted thiopyridyl
groups.
Alternatively, one may also use mercaptoethanol for this step.
The covalenfly bound protein is now displaced by using a solution of a mucl high
er
concentration of dithiothreitol (50 nM). Alternatively, cysteine or glutathione
may also be used.
The last step is to regenerate the ligand for further use. This is achieved by t
reating the matrix
with 2,2'-- dipyridyldisulphide.
The ligand : 2'-pyrldine

poSH
thlol protein

--/ S--S-- P
Protein attached to the matrix

Add
dlthlthreltl
Puttied protein

2. 2'-Dlpyridyl disulph/de
The ligand
N SH H
+
2-Thiopyridine
Thlone

420
Craig, L. C. and Craig, D., in Techniques of Organic Chemistry {A. Weissberger,
ed.), Vol. Ill,
part I, pp, 52-392, Interscience. New York, 1956.
2, Lederer, L. and Lederer, M., Chromatography, 2"d ed., Elsevier, New York, 195
7.
3.
Martin, A.J,P. and Synge, R.L.M., Biocherru J., 35:91 {1941). The classi
c paper which described
partition chromatography.
Modern Reading
I,
Ettre, L. S., and Horvath, C., Foundations of Modem Liquid Chromatograph
y, Anal.Chert,
47:422 A, 1975.
2. Kirchner, J. G., Thin-layer Chromatography in Techniques of Chemistry, Vol. 1
4 (Perry, E.S.
"ed.}, Wiley Interscience, New York, 1978,
3. Grant, R. L., Gas-Liquid Chromatography, Van Nostrand, London, 1971.
4. Grob, R. L., Modem Practice of Gas Chromatography, Wiley, New York, 1977.
5.
Laub, R. J. and Pecsok, R. L., Physicochemical Applications of Gas Chrom
atography, Wiley
Interscience, New York, 1977.
6.
Snyder, L. R.. and Kirkland J., Introduction to Modern Liquid Chromatogr
aphy, 2"d ed,,
Wiley Interscience, New York, 1979.
7.
Journal of Chromatograph. Library, Vol. 3: Liquid Column Chromatography
(Z. Deyl, K. Macek
and J. Janak, eds.), Elsevier, Amsterdam, 1975.
8. Helfferich, F., lon Exchange, McGraw-Hill, New York, 1972.
9.
Khym. J. K., Analytical lon Exchange Procedures in Chemistry and Biology
--Theory, Equipment
Techniques, Prentice-Hail, Engelwood Cliffs, N. J., 1974.

10.
Brewer, J. M., Pece, A. J. and Ashworth, R. B., Experimental Techniques
in Biochemistry,
Prentice-Hail, Engelwood Cliffs, N. J., 1974.
11.
Freifelder, D., Physical Biochemistry: Applications to Biochemistry and
Molecular Biology,
W. H. Freeman, San Francisco, 1982:
12.
Cuatrecassas, P., Wilcheck, M. and Anfinsen, C. B., Selective Enzyme Pur
ification by Affinity
Chromatography, Proc. Natl. Acad. Sci., USA, 61:636-643,. 1968. The original pap
er on affinity
chromatography.
13.
May, S. W., Affinity Chromatography in Separation and Purification {Perr
y and Weissberger,
eds.) 3 edition, John Wiley and Sons, New York, 1978.
14.
Afftnlty Techniques, Methods in Enzymology (W. B. Jacoby, and M. Wflchec
k, eds.) Vol. 34,
Academic Press, New York, 1974.
15, Lowe, C, R. and Dean P. D, G,, Affinity Chromatgraphy Wiley, New York, 1974.
16.
Karger, B, L, and Giese, R. W,, Reversed Phase Liquid Chromatography and
its Applications to
Biochemistry, Anal, Chem,, 50:1048 A, 1978.
17,
Willare, H, H,, Merritt, L. L, Jr,, Dean, J. A, and Settle E. A. Jr, Ins
trumental Methods of
Analysis, 6 ed,, pp. 529-564 (a good text on HPLC) CBS, New Delhi, 1986.
xercise
I.
A solution containing glutamlc acid (pl = 3.22), alanine (pl = 6.02), le
ucine (pl = 5,98), vallne
{pl -- 5.97), and histidine [pl -- 7.58) in a pH 3.0 citrate buffer was applied
to a cation exchange
column that was equilibrated with the same buffer. Elution was started and the f
ractions
Biophysical Chemistry
Suggestions for Further Reading
Classics

1.

lotein
Mol. Wt.
421
collected. State the order in which you think the five amino acids will elute fr
om the column.
Give reasons.
2.
Three pure preparations of different proteins are mixed together and the
mixture is run on a
gel filtration column. Rather than getting three peaks as one would expect, only
two peaks
are seen during elution. What are your conclusions?
3.
You have a protein preparation which contains two enzymes of similar mol
ecular weights.
By activity studies you determine that while one enzyme retains its activity at
a temperature
of 41C, the other seems to have become denatured at the same temperature. Devise
a
chromatographic procedure for the complete separation of these two enzymes, Give
reasons
for your answer.
4.
A given solution contains three proteins A, B, and C. The pIof the three
proteins are 5.1, 6.2
and 7,6 respectively, Protein A is unstable above its isolonic point while prote
in C is unstable
below its isoionic point. Can you use ion exchange chromatography for their sepa
ration?
5.
Protein A has a molecular weight of 62,000 D, B has a molecular weight o
f 91,500 D and
protein C has weight of 90,000 D, Protein C has 11 cysteine residues while the t
wo proteins
lack cysteine. Devise a procedure for their complete solution.
6.
It is normal to select a competitive inhibitor as ligand for affinity ch
romatographic separation
of an enzyme. Suppose your objective is to separate an enzyme that carries out a
bisubstrate
reaction. Will it still be necessary for you to select an inhibitor as ligand?
7.
If the enzyme carries out a single substrate reaction requiring the pres
ence of a metal ion,
what ligand, other than a competitive inhibitor, can you choose for its alilnity
chromatographic
separation?

8.
Malate dehydrogenase carries out a reversible bisubstrate reaction using
NADH as one of the
. substrates. Can you use NADH as a ligand for the separation of the enzyme by a
ffinity
chromatography?
9.
A molecular exclusion column is designed with a protein fractionation ra
nge of 5,000 to
4,00,000 Daltons. In this column myglobin, serum albumin, catalase and an unknow
n protein
were migrated. Following is the elution volume data obtained from experiment
Elution volume (ml)
Myogiobin
Serum Albumin
Catalase
Unknown Protein
6900
68500
221600
?
118
58
24
37

What is the molecular weight of the unknown protein?

10.
I work with a particular enzyme during my research, Rather than buying t
his enzyme, I
have been purig/ing it from a given source by gel filtration. The results are al
ways good
and have been standardized. A few days back I thought that I can use my expertis
e in
preparing this enzyme commercially and selling it. I used a much larger amount o
f the
source than I usually do with a view to increase the yield of finally purified e
nzyme.
Rather than getting that, I found that the enzyme activity was eluting very quic
kly. What
may have gone wrong?
11,
A tetrapeptide and a heptapeptide ofglutamic acid have been bound to an
anion exchange
column by using a buffer of pH 6. Elution is now started by lowering the pH grad
ually.
Which of the tw6 peptides will elute first?

12
ELECTROPHORESIS
Electrophoresis is the migration of charged particles or molecules in a medium u
nder the
influence of an applied electric field. The usual purposes for carrying out elec
trophoretic
experiments are () to determine the number, amount, and mobility of components i
n a given
sample or to separate them, and (tO to obtain information about the electrical d
ouble layers
surrounding the particles. The modem day scientists, however, use it for purpose
s as diverse
as determination of molecular weight of proteins on one hand, and DNA sequencing
on the
other.
Although Reuss had observed the flow of water through clay (electro-osmosis) and
the
migration of clay in the opposite direction due to an electric current, the firs
t recorded
measurements of electrophoretic phenomenon were performed in 1861 by Quincke, Th
e most
important early theoretical studies in electro-kinetics were made by Helmholtz,
Smoluchowski,
Gouy, Debye, Huckel, Abramson, and Maclnnes. Tlselius, by describing his moving
boundary
apparatus in 1937, was instrumental in popularizing, the utility of electrophore
sis to the
biochemist. In modem days, methods employing stabilized media have become very u
seful to
the biochemist and will be discussed in the present chapter.
Upon suspension in an aqueous solvent, almost all particles (e.g,, red blood cel
ls, bacteria
etc.) and many important biomolecules (e.g., nucleotides, nucleic acids, amino a
cids, peptides,
proteins etc.) acquire either positive or negative charges. The acquisition of s
uch charges
depends upon the nature of the particle/molecule and the solvent. Example may be
cited of
protein molecules, which have a large number of ionizable amino and carboxyl gro
ups on their
surfaces. These groups determine the net charge density of the protein molecule
which makes
it move in an electric field in a direction and at a velocity dependent upon the
sign and quantity
of this net charge density (Figure 12.1). This net charge density, although depe
ndent uPon the
Figure 12. I A charged particle rngrates towards the oppositely charged electrod
e. Arrow 1 tnd:Icates move
ment of a particle bearing a net charged derts{tj of -1. Arrow 2 indUrates faste
r movement of a
particle bearing a net charge density of-2. Arrow 3 indicates the dtrectlon of f
rlctWnal force.

Electrophoresis
423
groups and their number present in the molecule, is modified by the nature of th
e
solvent. Thus, if the acidity of the solvent is increased (H long} the molecule w
ill tend to become
more positive and vice versa. Electrophoresis in not llmited to charged molecule
s only. Even
typically uncharged biomolecules such as carbohydrates can be made to wear weak
charges
through derivatization as. borates and sometimes as phosphates. Evenif two molec
ules have
the same charge, they might not migrate together because if there is difference
in their molecular
weights, they will have different charge]mass ratio (this differences is of more
use in
electrophoresis on gels). Taken together, these differences are sufficient to en
sure differential
migration when the long in solution are subjected to an .electric field. This is
the basis of
lectrophoresis.
MIGRATION OF AN ION IN AN ELECTRIC FIELD
Consider a situation where a spherical molecule of net charge q Is placed in an
electric
field. The force Fwhich will act upon this particle will depend upon (f) the net
charge density of
molecule, and (f0 the strength of the field in which it is placed. The above rel
ationship may
be mathematically described as
where A E/d is the field strength applied (AEis the potential difference between
the two electrodes,
the distance between them). Since the particle has been suspended in a solution,
we
friction occurring between the accelerating molecule and the solution, to
relationship of electrophoretic migration. The extent of the friction, as descri
bed
Stoke's equation,-will depend upon (0 the size and shape of the molecule, and (/
/) on the
viscosity of the medium through which the molecule will migrate. Thus
F=6rv
the friction exerted on the spherical molecule, r is the radius of this molecule
, is the of the solution, and v is the velocity at which the moleculeis migratin
g. This frictional
oppose the accelerating force generated by the electric field (Figure 12.1). Equ

ating
force of acceleration with Stoke's equation, we get
--q=6nrv
d
rearrange the. above relationship we get the expression
6rld
thus be seen that the velocity {v} of the molecule is proportional to (/} the fi
eld strength
.and (/0 charge (q) on the molecule but is inversely proportional to (/) the par
ticle size (r),
(/0 viscosity of the solution ().
FACTOR8 AFFECTING ELECTROPHORETIC MOBILITY
Saznple
Charge/mass ratio of the sample dictates its electrophoretic mobility. The mass
consists of
' the size (molecular weight} but also the shape of the molecule.

424
Biophysical Chemistry
(i)
Charge. The higher the charge, greater is the electrophoretic mobility.
The charge,
however, is dependent on pH of the medium.
(//)
S/ze. The bigger the molecule, greater are the frictional and electrosta
tic forces exerted
upon it by the medium of suspension. Consequently, larger particles have a small
er
electrophoretic mobility compared to the smaller particles.
(///)
Shape. Rounded contours elicit lesser frictional and electrostatic retar
dation compared
to sharp contours. As an example consider the case of globular and fibrous prote
ins.
Given the same size (molecularreight) the globular protein will migrate faster t
han
the fibrous protein.
The Electric Field
We have already seen that the force acting upon an ion of charge q is E q/d. The
rate of
migration under unit potential gradient is referred to as mob///ty of the/on. An
increase in
potential gradient increases the rate of migration.
The current (total charge carried per second to the electrode) in the solution p
laced between
two electrodes is carried mainly by the buffer long, only a small proportion bei
ng carried by the
sample long. An increase in the potential'difference therefore increases the cur
rent.
Resistance to current flow is an important point to bear in mind while performin
g
electrophoresis. Resistance shares an inversely proportional relationship with t
he rate of
migration and the current. The importance of resistance becomes clear if one con
siders the
following situation. During any electrophoretic run, some power dissipates with
consequent
generation of heat. The temperature within the medium rises. This results in a d
ecrease in the
viscosity of the solution (see Chapter 5). Consequently the long present can mov
e faster. More
charge is carried per second to the electrode, Le., the current increases while
the resistance
decreases. One might think that this rise in current would also make the separat
ion of sample

components faster. This is not so. Because, as the temperature rises, some of th
e solvent
evaporates increasing the buffer ion-concentration. This results in more charge
being carried
(A) Initial situation
l
Ewporon
i
/./.i/.
Temperature .-----#
, = solute long ; 0, = Buffer long ; = Evaporation
(All solute long are negatively charged and the movement is toward anode)
Figure 12.2 If current is not controlled, the temperature within the electrophor
etic cell rises leading to decrease in
viscosity of the solvent. Due to this the buffer ion mobility increases. Increas
e in temperature also leads
to a decrease in solvent volume and therefore increases buffer concentration. Th
is leads to more curren
being carried by the buffer long. Consequently the buffer long move faster. The
sample long move
slowly and their separation is slowed down.

, Electrophorests
425
by the buffer long rather than the sample long; consequently the buffer long mov
e faster, while
ithe sample long move slowly and the separation slows down (see Figure 12.2). Si
nce this should
not be allowed to happen, power packs have been made available which can control
either the
voltage or the current to a constant level despite unavoidable changes in resist
ance due to
temperature fluctuation.
Ideally, one should maintain a constant current, because, even with a constant v
oltage
heat will be generated and all the events described above will take place. Howev
er, it has been
seen that with low voltage paper electrophoresis, the heat generated is small an
d dissipates
easily. One may therefore use either a constant voltage or current in these case
s. However, with
cellulose acetate, and gel electrophoresis, heat dissipation is not easy and one
should therefore
maintain a constant current. If the voltage used is high, the whole apparatus mi
ght be shifted
to a cold room, or a cooling system installed (see section on high voltage elect
rophoresis). The
evaporation of the solvent due to heat generation might be minimized by enclosin
g the apparatus
in an airtight cover.
Fhe Medium
An inert supporting medium is chosen for electrophoresis. But even this inert me
dium
can exert adsorption and/or molecular sieving effects on the particle thereby in
fluencing its
of migration. The medium may also give rise to electro-osmosis, which may also i
nfluence
of sample migration.
(0 rpt/on. Adsorption, here, means retention of a component on the surface of
medium. Such a component has two forces acting upon it; the applied electric fie
ld
adsorption. The component is, thus, not resolved as a sharp band but as a band w
hich
a tail, rather like a comet. Thus the rate and resolution of the electrophoretic
separation
be effectively reduced by adsorption.
(iiJ Molecular sieving. Supporting media such as polyacrylamide, agar, starch an

d
have cross-linked structures giving rise to pores within the gel beads. In Sepha
dex,
than the pores are excluded from entering the gel beads and these molecules
faster. In case of polyacrylamide, starch, and agarose, the larger molecules als
o are
to squeeze through the pores. The smaller molecules here pass through the pores
easily,
larger molecules are retarded. The electrophoretic mobility is thus modified by
molecular
effects of the supporting medium (for a detailed discussion see molecular sievin
g under
Buffer
Apart from maintaining the pH of the supporting medium, the buffer can affect th
e
mobility of the sample in various other ways.
(i) Composition. Commonly used buffers are formate, citrate, phosphate, EDTA, ac
etate
"Iris, and barbitone etc. The choice of buffer depends upon the type of sample b
eing
buffer can affect electrophoretic mobility if it is able to bind to component(s)
sample being separated. [A case in point is the example cited below. An artifac
t due to
Cann (Cann. J.R,, B/ochem/stry, 5:1108- I 112, 1966) when
electrophoresced pure albumin in the presence of a borate containing buffer. Rat
her than
one band, he observed -two. He extracted the protein from one of the isolated ba
nds
Again two bands were observed. He averred that the multiple bands
the result of an interaction between borate and some of the albumin molecules].
Care
be taken to avoid such buffers. On the other hand, such buffers can be deliberat
ely
for certain separations. Examples may be given of carbohydrates, which are uncha
rged
therefore inseparable by electrophoresis. Borate buffer can, however, interact w
ith
to give charged complexes, which can then be separated by electrophoresis.

426
Biophysical Chemistry
(ii) Ionic strength. As we have already seen above, increased ionic strength of
the buffer
means a larges share of the current being carried by the buffer long and a meage
r proportion
carried by the sample long. This situation gets translated into a slower migrati
on of the sample
components. Since the overall current will also increase there will be heat prod
uction (we have
already seen that heat production is detrimental to proper electrophoresis).
A deci-ease in ionic strength, on the other hand, would mean a larger share of t
he current
being carried by the sample long leading to a faster separation. Since the overa
ll current will be
low. less heat will be produced. However, in low ionic strength buffers, diffusi
on (especially of
smaller molecules) tends to be high with concomitant loss of resolution.
The chosen ionic strength of the buffer is, therefore, a compromise. The ionic s
trength
used is usually between 0.05-0.1M.
(ll/) l/f, Since pH determines the degree of ionization of organic compounds, it
can also
affect the rate of migration of these compounds. Increase in pH
acids and a decrease in pH increases the ionization of organic bases. For an amp
holyte such as
an amino acid, which has both acidic and basic properties, both the above effect
s apply.

(lgure 12.3)
Z
H-----OOH
-- Migration NHs
R
Migration
Migration.
H,.-O0 Migrations,
Basic pH
Molecule is an anion

Acidic pH
Isolo.nic point
Molecule is a cation
Molecule is a zwitterion
ljure 12.3 Dfgram llvtratfng the effect of pH on the fonfzatn and migration of a
n amfno acfd.
The direction and also the extent of migration of ampholytes hre thus pH depende
nt and
buffers ranging from pH i to 11 can be used to produce the required separation (
also see
Chapter 1).
*
On the basis of the principles discussed so far can you answer the follo
wing qu.estions?
(a) Why should electrophoresls be done in solutions of low salt concentration?
(b) Two proteins have the same molecular weight and the same charge. Will they h
ave the same
mobility? Discuss.
TYPES OF ELE .CTROPHORESIS
Electrophoresis can be divided into two main techniques: free electrophoresis or
electrophoresls without stabilizing media and zone electrophoresls or electropho
resis in
stabilizing media.
1. FREE EEC'ROPHORF.IS
Free electrophoresis has two main techniques: m/croe/ectrophoresls and mov/ng bo
undary
electrophoress. Both the techniques have now become obsolete and are at best of
historical
significance. They are therefore discussed below very briefly.
Mlcroelectrophoresis
-This electrophoretic technique involves the observation of motion of small part
icles in an
electric field with a microscope (such as IB.Cs, neutrophils, bacteria etc.). Th
e suspension is

427
0ntained in a closed system composed of a thin-walled section for optical observ
ations and of
table electrode compartments. In principle any microscope with a graduated fine
focusing
tdJustment and an ocular micrometer may be used in conjunction with a flat elect
rophoretic
kind shown in Fi'ure 12.4. The ocular micrometer serves for the measurement of t
he
in conjunction with a stopwatch. In modern
this technique is applied only for measuring the zeta potentials of cells such a
s IB.Cs,
bacteria etc. -.
F@ure 12.4 Abramson'sJ/at micr0e/ec-rophoretlc ce//.
mndsry FAectrophoresis {m.b.e,)
This is the prototype of all modem methods of electrophoresis and was first deve
loped by
Tlselius of Sweden in the 1930s.
X
+Z
Buffer Prote/v.s x,y,z
x + y + z
char at the pH of th b. x has the hhest and z ha the lowet mob. N

428
Biophysical Chemistry
In moving boundary electrophoresis a buffered solution of macromolecules is plat
ed under
a layer of pure buffer solution in a U-shaped observation cell (Figure 12.5 A).
The whole cell
may then be immersed in a constant temperature bath insulated from vibrations. T
he power is
switched on generating an. electric field between the electrodes. Normally in a
complex sample
containing many macromolecules, there will be species, which will bear a net neg
ative charge
and therefore move towards the anode while at the same time the macromolecules b
earing a
net positive charge will move towards cathode. This situation of movements in mu
tually opposite
directions will not be good for a satisfactory resolution. The pH of the buffer
is, therefore,
chosen that all the macromolecules bear a net negative charge. The movement of
macromolecules consequent to generation of electric field between the electrodes
will then be
towards the anode. As they do so, they migrate from the macromolecule solution t
o the pure
buffer or into the zone of macromolecule free buffer and form a boundary or fron
t. As a result of
this there is sharp change in the refractive index of the solution at this bound
ary (the index of
refraction of macromolecules is different from that of pure buffer). The refract
ive index changes
along the electrophoretic ceil, when measured by appropriate optical devices (us
ually Schleiren
optics or Rayleigh interference optics), yield electrophoretic patterns that sho
w the direction
and relative rate of migration of the major molecules in the sample. Figure 12.5
B shows such
a pattern.
Albumin
-SpikeA
' II - GIobulkn
Globulin
"
I l I
glectrophoretic mobility

ljw'e 12.5 (B) Representation of an electrophoretlc pattern of serum proteins ob

tained by botJlanj elec-trophoresis.
For many years moving boundary method was very popular for quantitative analysis
of
complex mixtures of macromolecules, especially proteins,e.g., those in blood pla
sma. In recent
years, however, it has been superceded by techniques collectively known as zone
electrophoresis.
2. ZONE ELECTROPHORESI$
The same year (1937) that Tisclius described his moving boundary electrophorcsis
, Konig
published the first experiment on the use of idter paper as stabilizing medium i
n electrophoresis.
It, however, took ten more years for filter paper electrophoresis to become popu
lar as an efficient,
inexpensive, routine technique. This paved the way for several other porous stab
ilizing media,
most of which are gels such as agar, starch, and polyacrylamide. Zone electropho
resis is the
name given to the separation technique employing these stabilizing medi It is al
so known as

x
Y
429
tn stabItr.ed media. A great advantage of this analytical tool lies in the fact
that
are often quite sufficient for a complete electrophoretlc separation.
separation the molecules are Immobilized by fixation in different zones. The mol
ecules
detected by staining them on the supporting medium. Other methods to detect the
molecules are (/) visualization by ultraviolet light, (tO detection by virtue of
enzymic
or (t) detection by radioactivity, ff the molecules are radiolabeled. Alternativ
ely, the
components can be eluted from the medium and thus become available for further
Zone electrophoresis can also be utItlzed as large-scale or preparative method w
hereby
amounts of a component can be purified for further characterization.
i Comparison of Free and Zone Electrophoresis
Figure 12.6 depicts the difference between the separations that can be obtained
with free
zone electrophoresis. In boundary separation, .e,, in free electrophoresis, the
migration of
can be observed. The separation is carried out usually in a 'U' tube with the sa
mple
i layered under a buffer solution. The system does not use any stabilizing mediu
m but is
the density difference. As a result convection currents pose a big problem. As t
he
progresses, more heat isproduced and the convection currents increase.
mobility and lead to an inadequate separation. The only
can be isolated in substance are the slowest and the fastest migrating
other intermediate components overlap and can only be observed as separated but
be isolated.
x
'y
A comparskn ofseparatlon obtained with (A)free electrophoresla and (B) zone elec
trophoresis. X and
Y are two components resolved by the two technklues.

The zone separation uses stabilizing media (paper, gels such as starch, polyacry
lamide,
etc.). In zone electrophoresls, the sample components during their migration spl
it up
different zones as it contains differently grating components, each zone consist
ing princlple).which can be easily isolated. The zones have higher density
the medium, but the Use of stabfllzing system does not allow the zones to disper
se and
as is the case with free electrophoresis. The stabillzlng media might influence
and isoelectric points of sample long in a very unpredictable manner. However, i
f is separation and not a Study of electrical properties, zone electrophoresis i
s a much
usehfl tool than the free electrophoresis.

430
Biophysical
The apparatus for moving boundary electrophoresis itself and the associa
ted
optics are very costly. This is another restriction for the
the apparatus for zone electrophoresis is simple and cheaper.
As the zone separation resolves the sample components in a much better manner,
as a preparative technique is logical. The free electrophoresis on the other han
d cannot
modified for the purpose, as it is difficult to isolate individual components of
a sample by
method.
GENERAL TECHNIQUES OF ZONE ELECTROPHORESIS
1. PAPER ELECTROPHORESIS
Filter Paper
Filter paper as a stabilizing medium is very popular for the study of normal
abnormal plasma proteins. Paper of good quality should contain afleast 95% of ce
llulose
should have only a very slight adsorption capacity. Chromatography paper is suit
able
electrophoresis and needs no preparation other than to be cut to size.
Apparatus
The equipment required for electrophoresis consists basically of two items, a po
wer
and an electrophoretic cell. The power pack provides a stabilized
for both voltage and current output. Power packs, which have an output of 0-500
V and (
mA are available and can be programmed to give either constant voltage or curren
t.
The electrophoretic cell contains the electrodes, buffer reservoirs, a support
a transparent insulating cover. The electrodes are usually made of platinum.
The two buffer reservoirs are usually each partitioned into two interconnected
one cohtaining the electrodes and the other in contact with the supporting mediu
m.
compartments are necessary so that any change in pH occurring at the electrodes
does
affect the buffer in contact with the supporting medium. The supporting medium i
s
with the buffer prior to the start of electrophoretic process. Wicks are made up
of filter paper or gauze. Altematively, the paper can be made to dip
at both the ends thereby obviating the need for wicks. The paper is placed on an
material, usually a perspex sheet. The whole electrophoresis unit is then covere

d with
insulating material to minimize evaporation during the run and to
insulation.
Two arrangements of the filter strips are colnmonly used. The horizontal and
arrangements (Figure 12.7). Both the arrangements are equally viable and the cho
ice
depends upon personal preferences.
Sample Application
This is the single most critical procedure in the whole electrophoresis process.
The
may be applied as a spot (about 0.5 cm in diameter) or as a narrow uniform strea
k.
devices are available commercially for this purpose. There is no hard and fast
of application of the sample to the electrophoretogram. This can be applied befo
re the
has been equilibrated with the buffer or after it.
Eletrophoretic Run
The current is switched on after the sample has been applied to the paper and th
e
has been equilibrated with the buffer. Commonly used buffers are listed in Table
12.1. Although
deices providing stable voltage or current are available, the equipment has to b
e
intermittently throughout the run because overheating is a distinct posslbflity
(if not checked

431
EI trophoretic
"
filter paper.
I
perspex cover
/
"
I
.
-

I
-- -

.l,.Re,,,o-i

Bufe"
Insulattng plate
(A) Horizontal paper el'ophoresis
Buffer Electrodes
(B) Vertical paper electrophoresis
Figure 12. 7 Paper electroptwresis
heating may lead to charring of paper). Overheating can be avoided by placing th
e entire
in the cold room. The process .usually does not take longer than two hours. The
is switched off after the run and before the paper is removed.

7.7
1/15M
0.I
0.05
Barbital*
Barbital
Rheophor
Phosphate
Borate
Acetate
432
Table 12.1 Buffers for Electrophoresis of Different Substances
Biophysical Chemistry

Intended
Buffer
Separation
Proteins
Mucoproteins
Amino Acids
Phosphate
Barbiturate
Citrate
Phosphate
Phosphate
Michaelis

Nucleic acids
Borate
Tris acetate

Tris phosphate
pH
Ionic
Composition/Liter
Strength (}
8.6
8.6
8.6
7.4
9.0
4.5

4.4
8.6
4.5
4.6
7.2
8.6

9.2
7.8
0.05
0.075

0.05

7.63
0.I

0.2
0. I
0.13
0.15
10.3 g Na Barbiturate, 1.84 g Barbital
15.45 g Na barbiturate, 2.76 g Barbital
10.05 g Barbiturate, 90 ml 0.1N HCI
0.6 g NaH2PO4.H20, 2.2 g Na2HPO4
Na borate, 0.62g Boric acid.
3.51 g NaCl, 3.25 g Na acetate
pH adjusted with N HCI to 4.5
9.44 g NaHPO4, 10.3 g Citric acid
20.6 g Na Barbiturate, 3.68 g Barbital
28.46 g Na citrate, 20.6 g Citric acid
20.4 g KHPO
2.196 g NaH2PO. 8.622 g NaHPO4-2H20
9.8 g Na Barbiturate, 3.9 g Na acetate,
60 ml 0. IN HCI
3.3.g Na borate
24.1 g Tris base, 8.2 g Na acetate,
1.85 g Na2EDTA.2HO, adjust pH with
acetic acid (5x stock solution)
21.8 g Tris base, 23.4 g NaHaPO.2HO,

1.85 g NaEDTA-2H20 (5x stock solution)

Especially useful for hemoglobin.

Once removed, the paper is dried in vacuum oven at 110C (if the compounds are not
thermolabfle, in which case the paper is allowed to hang and air dried).
Detection and Quantitative Assay
To identify unknown components in the resolved mixture the electrophoretogram ma
y be
compared with another electrophoretogram on which standard components have been
electrophoresced under identical conditions. In this respect electrophoresis is
similar to
chromatography.
Individual compounds are usually detected and identified n sttu. Physical proper
ties like
fluorescence, ultraviolet absorption or radioactivity (if the sample has ben lab
eled) are exploited
for detection, Alternatively, the electrophoretogram can be stained to convert t
he resolved
components into colored derivatives.
(/) F/uorescence. Staining with ethidium bromide and subsequent visualization of
the
electrophoretogram under ultraviolet light makes DNA and RNA fluoresce and thus
facilitates
their detection. Similarly fluorescamine staining is utilized for detecting amin
o acids, amino
acid derivatives, peptides and proteins. Dansyl chloride may also be used in pla
ce of
fluorescamine.
(ii) Ultraviolet absorption. Proteins, peptides and nucleic acids absorb inthe r
an]ge of
260-280 nm, and this property can be exploited to detect these components on an
electrophoretogram.
(ill) Staining. Different dyes are used for detecting different components (Tabl
e 12.2).
Many times during staining, the zones of resolved components spread. As a safe m
easure, a

Acids
Proteins
Llpoprotelns
Comments
'trophoresis
433
xative may be applied before staining. Excess stain is eluted after st...aining
procedure is
mpleted.
lble 12.2 Visual and Fluorescent Dyes Used to Detect Components Separated by
Iietrophoresis
Polysaccharide
General
Bromophenol blue in acetic acid
Nigrosine in trichloro acetic acid/
acetic acid
Lissamine green in acetic acid
Coomassie Brilliant Blue R-250
Dansyl Chloride
Fluorescamlne
Aqueous anilinonapthalene
sulphonate (ANS)
Methyl green-pyronine
Lanthanum acetate + acridine orange
in acetic acid
Toluidine blue
Pyronine
Methylene blue
Ethidium bromide
Sudan Black in 60% ethanol

Periodate oxidation + treatment

with Schiffs reagent
/Iclan blue
Iodine
Stains-All
,
Visual, quantitative
Visual, very sensitive
Visual, quantitative
Visual, quantitative
Fluorescent, quantitative
Fluorescent, very sensitive
Fluorescent, very sensitive
DNA-blue, RNA-red sensitive
DNA. RNA-orange red
Visual, sensitive
RNA, sensitive
RNA, visual, sensitive
Fluorescent, very sensitive
Visual, senstive
Visual, quantitative
Vlsua,sensitlve
Visual. sensitive
DNA-bluish purple, RNA-blue,
Proteins-red, Phosphoproteins-blue,
Acid mucopolysaccharides -blue
to purple

(iv)Detection of enzymes in situ. If the component to be separated is an enzyme,

special
techniques may be used to detect it. A paper strip of the same size as the
is impregnated with the substrate for the enzyme desired to be separated.
substrate, however, is modified in such a way that it should become coloured upo
n reaction

the enzyme. The paper strip is now juxtaposed with the electrophoretogram and pl
aced in
suitable buffer. After a particular time the two sheets are separated. The posit
ion of the
)earance of Color on the paper strip gives the position of the enzyme on the
(Figure 12.8).
(v) ant/tat/ve estimation. A rough idea of the quantity of the components of a s
ample
had by visually comparing ("eyeballing") the color of different zones with their
standards
known quantity. But this is seldom accurate. To impart more accuracy, the c61or
density, of zone may be multiplied with the area.of the zone and the resulting v
alue would be a rough
component. However, for accurate estimation the compound
eluted from the electrophoretogram. For elutlon the zone is excised from the pap
er and
in a suitable solvent. After all the color has been extracted, the color density
of the

(C)
Figure 12.S DetocOnn of enzyme in situ.
(A)
A given sample containing the enzyme has separated into four bands as
tion. One if these bands is due to the desired enzyme.
(B)
A paper sheet of the same size as the electrophoretogram is imprnated wi
th a $bstrate
the enzyme to be separated. The substrate is so modified that it will develop co
lor
reaction.
(C) Substrateimpregnatedpaperjuxtaposadoverthelectrophoretogram.
(D) Upon removo at a certainposition colorwould be observed on Ore subetratepape
r.
ing band on the electrophoretogram (in this case) is due to enzyme.
solution is measured and compared with that of the standard of known quantity. T
his gives
fairly accurate idea of the quantity of the solute.
In some eases, the paper is made transparent by immersion in organic
refractive index (e.g., paraffin oil-bromonaphtha/ene, ardsol}. The transparent
paper is
placed between two glass plates and absorption of the colored region ie measured
by
densitometer, which gives an ideaabout the quantity of the resolved component.
If the sample was labeled before the run, the separated zone can be eluted and
a scintillation cocktail and counted. Alternatively the zone can be excsed and p
laced in
scintillation vial and solid scintii/ation counting can be carried out.
Example:
Separation of Protein by Paper Electrophoreeie
Sample preparation. Sample may range from crude tissue fluid and extracts to the
most highly
purified protein preparations. Most body and tissue fluids are applied directly
to the paper without diysis when
small samples are used, e.g. in most clinical work serum is directly placed on t
he paper.
//ter papere. Although a wide variety of papers can be used, so/t thick papers h
ave been found to give
better resolution.

/ffere. Best results, as a rule, are obtained at a pH on the alkal/ne side of th

e isoelectric points of the
proteins in the mixture. The commonly used buffers include (i) 0.05 M veronal bu
ffer at pH 8.6, and
(//) veronal/acetate buffer at pH 8.6 and ionic strength 0. I.
Teeha/qu. The origin is marked on the paper with the help of a pencil. Subsequen
tly, the paper is placed
on an insulting solid support with its ends hanging in the electrolyte (wicks ca
n be used to establish contact). It
takes about 30 minutes for the paper to equilibrate with the electrolyte..
The sample in small quantity (e.g., 0.1-0.4 ml of serum} is applied on the origi
n either as a spot
or a streak. The power is switched on. Usually 14-16 hours are required for sepa
rat/on at 100-200 volts.
If, however, the separation is being carried out in cold, a higher voltage of th
e order 400 volts may be used and
the time of separation gets reduced proportionally (the risk of overheating is p
ractically nil in a cold room).
Once the run is complete, the papers are dried m an air oven at 100C.
tInOW. One of the most commonly used dyes for staining proteins is bromophenol b
lue. The paper is
dipped in 0.1% bromophenol blue solution in ethanol saturated with mercuric chlo
ride. Excess dye cn be
washed offunder running water. Alternatively coomass/e blue can be used (Table 1
2.2).

Electrophoresis
435
Azocarmine B is also used. The paper is kept in the dye solution (100 ml dye sol
ution contains 50ml
methanol, 40ml aqueous solution of azocarmine B and 10ml of 5%acetic acid) for I
0 minutes and subsequently
washed for 5 minutes each in methanol and I0 % acetic acid. ,
Alternatively naphthalene black 1213200 is used. The paper is allowed to dip int
o a saturated dye
solution in methanol and 10% acetic acid. After 10 minutes the paper is washed s
everal times with methanol
containing 10% acetic acid.
t/ e/mtn. Eluting the colored zone and performing colorimetry of the extracted c
olor can
ive the most accurate idea of the quantity. Each colored zone is cut into 5mm wi
de strips at right angles to the
direction of separation and placed into a trough containing about 5ml NaCO3 solu
tion (1part ,methanol and 1
pert.10 % aqueous NaCO3 solution). About 30 minutes of extraction in.this solven
t is sufciet and the resultant
color is measured in colorimeter.
Serum analysis for disgnostic purposes is routinely carried out by paper electro
phoresis. Other proteins
that have been satisfactorily analysed using paper electrophoresis include muscl
e protein, egg white proteins,
milk proteins and snake and insect venoms etc.
2. CFLULOE ACETATE ELECTROPHORESIS
Cellulose acetate as a medium for electrophoresls was introduced by Kohn in 1958
.
It was developed from bacteriological cellulose acetate membrane filters and is
commercially
as high purity cellulose acetate strips, which are thin and have a uniform micro
pore '
- Although paper electrophoresis is still the choice for routine fast diagnostic
analyses, the
resolution of a given protein might suffer because of substantial adsorption on
paper. This
paper is completely taken care of if cellulose acetate strips are used instead o
f
paper. Additional advantages of cellulose acetate are (/)it is chemically pure;
it does not contain
hemicelluloses or nitrogen, (//} cellulose strips are translucent and this makes
them
for directhotoelectric scanning for separated bands of components, (iii) be.caus
e of
very low content of glucose these strips are suitable for Ciectrophoresis of pol
ysaccharides;

Schs reagent, background staining is negligible. Cellulose acetate is especiaJly

for clinical investigations such as separation of glycoproteins, lipoproteins an
d
from blood. The strips are suitable for immuno-electrophoresis (see later) and
Cellulose acetate is not very hydrophih'c end ..thus holds very littie buffer.
is conducive for a better resolution in a shorter time. Thi lower buffer capacit
y
however, is responsible for greater heat production and the electrophOresls has
to
preformed. Cellulose acetate is not suitable for preparative electrophoresls.
The buffers used for cellulose acetate electrophoresis are essentially the same
as used for
12. I). Solvents rendering the strips transparent are preferred rather
staining agents. Such solvents are glacial acetic acid, Whitmore oil 120, cotton
seed oil,
liquid paraffin or a special medium sold by cellulose strip manufacturers. The
of the strip helps in direct photoelectric quantitative determination of the
without elution being involved.

. 0 oa sxs
In all the types of electrophoreses we have discussed so far (free electrophores
ls, paper
cellulose acetate electrophoresis ) cmrge on the molecule was the major determin
ant for its
mobility and ultimate separation from the rest of the molecules. The gels,
are porous and the Size of the pores relative to that of the molecule determines
whether
t.he pore and be retarded or will bypass it. The separation thus not only
the chsrge on the molecule but also on its size.Needless to say that resolution
of a
sharper and better in a gel than in any other type of medium.

Acrylamide
436
Biophysical Chemistr
(i) Starch uel. Starch gel as a stabilizing medium for zone electrophoresis was
introduced by Smithies in 1955. Potato starch is hydrolyzed in acidified acetone
at 37C,
wherein temperature control and the timing of hydrolysis are extremely important
. The
suspension is then neutralized with sodium acetate and washed with large amount
of distilled
water and dried with acetone. This hydrolyzed starch when heated and cooled in a
n appropriate
buffer sets as a gel (the amylopectin chains intertwine). High porosity starch g
els are obtained
by using 2% (w/v) starch and low porosity gels are obtained by adding I0-15% sta
rch to the
buffer. The pore size in a starch gel cannot be controlled and this is the bigge
st drawback of
these gels. Starch gels are unsatisfactory for separation of basic proteins. Mor
eover, it is difficult
to prevent contamination of starch gels by microorganisms. Another disadvantage
of starch
gels is that upon staining to detect the separated components, the starch gel tu
rns opaque
making direct photoelectric determination impossible.-However, the resolving pow
er of starch
gels is very high and can be matched only by polyacaj1arnide gels. One of their
important
applications is the analysis of isocnzymc patterns (zymograms).
q/) Ar. Agar gel electrophoresis was first described by Gordon in 1949 and has b
een
since used increasingly for biochemical separations. Agar consists of two galact
ose-based
polymers, agarose and agaropectin. The latter is sulphated (and thus is charged)
and therefore
may give rise to severe electroosmosis which will be detrimental for electrophor
etic separation.
The sulphate content is however, removed by purification. A solution of agar at
80C is mixed
with an equal volume of 40% polyethylene glycol, which precipitates the agarose.
The latter is
collected, washed with distilled water and dried with acetone. The process is re
peated three
times and the agarose so obtained is virtually free of sulphate.
Agar solubilizcs in aqueous buffers above 40C and sets to form a gel at about 38C.
A low
quantity of agar in buffer gives a large pore size and almost no molecular sievi
ng action. Since
the molecular sieving action is negligible, these gels have low diffusion resist
ance and are

consequendy of much use in immunoelcctrophoresis for detection of antigenic prot

eins. Agar is
being used to separate high molecular weight macromolcculcs like proteins and nu
cleic acids.
In the latter case its use has become legion.
(/i) /rmd. The components used in the formation of this gel are known to be
neurotoxins and thus care has to be taken while preparing the gel. The most comm
only used
components to synthesize the matrix are acrylamide monomer, N, N'-methylencbisac
rylamide
(his), ammonium persulphate and tetramethylenediaminc (TEMED). Chemical structur
es of
these compounds are shown in Figure 12.9.

CH2 :ICH--C--NH2
HaC
. CHa
N--CH2---CH---N<
HsC
CH3
Tetramethylenedlamlne {TEMED}

O
O
II
II
CH = CH--C--NH--CH2--NH--C--CH = CH2
N, N'- Methylene-his (acrylamlde)
Figure 12.9 Components of polyacrylamide gel
Ammonium pcrsulphate when dissolved in water generates free radicals.

Electrophoress
43 7
These free radicals can activate acrylamide monomers inducing them to react with
other
acrlamide molecules forming long chains in the process, These chains become cros
s-linked if
the reaction is carried out in the presence of N, N'-methylenebisacrylamlde. The
whole reaction
is shown in Figure 12.10. TEMED acts as a catalyst of gel formation because of i
ts ability to
.xist in free radical .form. Ammonium persulphate and TEMED can be replaced by o
ther
compounds. For example riboflavin may be added to acrylamlde, bisacrylamide mixt
ure and
the whole mixture then irradiated with ultraviolet ]ight in presence of oxygen.
Riboflavin undergoes
photodecomposition and forms free radicals which act much in the same way descri
bed above
for ammonium persulphate.
H ONH2
SO " (persulphate)
CH2 -- + CH2(CH2 = CH---C--NH)2 -Acrylamide
N, N" - Methylene-his (acrylamide)
--CH,--HCH2--CH--CH2CH-'CH2
CONH]
c.O H
J
CH2.-H-.CI-.CH-CI.-.. H.-CH-.CH-.CH.-CH.
.
I
CON-H'
CONH
I
--CH2--CH--CI-I2Fgure 12. I0 ReactWns fnvolved fn polyacrylamfde get formatlon
The pore size of the gel is determined by the amount of acrylamide used per unit
volume of
the reaction medium and the degree of cross linkage, which is dependent upon the

amount of
bisacrylamide used (see Box 12. I). The pore size can be controlled by controlli
ng the amount of
these two compounds and a relationship between the concentrations of the two and
pore size
has been arrived at. This feature makes this gel particularly suitable for resol
ving mixtures of
proteins and nucleic acids in a very reproducible manner. Other desirable featur
es of these gels
are (f) a low adsorption capacity, (f0 lack of electroosmosis, and (f/0 suitabil
ity for in sltu
histochemical and qualitative analysis.

438
Biophysical Chemistry
(/v) A-Aerlam/de. The need to separate very high molecular weight nucleic
acids (200 Kd or more) prompted the development of this complex gel. Acrylamide
gels with
pores large enough for these large molecules remained liquid because the quantit
y of acrylamide
was 1-2.5% only. Agarose alone, on the other hand, has no molecular sieving acti
on necessary
for a sharper resolution. In a mixed gel, however, agarose provides physical sup
port to the low
percentage acrylamide gel which provides the molecular sieving action. The gels
were first used
by Peacock and Dingman in 1968. Agarose acrylamlde gels are prepared by dissolvi
ng 0,5%
agarose in bofllng water and allowing it to cool to 40C. At this temperature, co
mponents needed
for polyacrylamide gel formation are added and the mixture is transferred to eit
her a colunm or
a slab gel rig, which has been brought to the same temperature as .that of the s
olution. The gel
sets on cooling yielding a highly porous and rigid matrix. The acrylamide used m
ay be as low as
0.5%. These gels have been successfully tried for isolation of 3.5 x 10 daltons
DNA.
(v) Other gels, which have been used include pectin, gypsum, Sephadex, polyvinyl
chloride
and polyvinyl acetate. However, they are not used often and hence a discussion i
s avoided.
Electrophoretic Mobility in Gels
The discussion on electrophoretic mobility in previous pages did not take into a
ccount the
molecular sieving action of the gelsand its effect on the mobility of a macromol
ecule.
The pore size. thus the molecular sieving action and therefore the effect on ele
ctrophoretic
mobility of a molecule are functions of gel concentration. Rodbard and Chrambach
have developed
a set of mathematical relationships to describe the effects of gel concentration
upon a
macromolecule's mobility.
logE = log E'- K
where E is the electrophoretlc mobility, E' is the mobility in a sucrose solutio
n, Kr is the
retardation coefficient and G is the total gel concentration. The retardation co
efficient has been
described as follows
Kr=C(R+r)

where C is constant. R is the mean radius of the macromolecule and r is the radi
us of the gel
fibers.
Several proteins of biological importance contain more than one polypeptide chai
n. These
proteins are referred to as oligomeric proteins. The whole structure is stabfllz
ed by hydrogen
bending, disulphide bridges,-or by hydrophobic associations. These proteins migr
ate as a single
band during gel electrophoresis. If, however, the subunits of these proteins are
to be separated
from each other, a class of substances known to destabilize the quaternary struc
ture are
employed.. These substances are collectively known as solubil.ers. An example th
at can be
cited to illustrate the point is of core RNA polymerase. This enzyme has three n
onidentical
subunits, a, 6, and 6' but migrates as a single specie if subjected to zone elec
trophdresis.

Electrophoresis
439
However, if treated with SDS (see below), a solubfllzing agent, and electrophore
sced on a gel in
which SDS has been incorporated, the three polypeptide chains migrate as three d
ifferent
bands.
Use of gels containing substances capable of solubilizing certain macromolecules
, mainly
proteins, has thus.permitted a number of separations not achieved by any other m
ethod. Often
used solubfllzers are discussed below.
(i) Urea, Urea at concentration from 3-12 M is known to disrupt hydrogen bonds.
Thus
those macromolecular complexes or aggregates, the structure of which is solely m
aintained by
hydrogen bonding, disassociate readily in concentrated urea. In such systems, re
moval of urea
may be expected to lead to reaggregatioti. Thus the presence of urea in the gel
in which
electrophoresis is conducted is essential. Certain examples where use of urea ha
s provided
additional information about the macromolecular structure are myeloma y-globulin
, y-globulin
light and heavy chains, haptoglobins, a-and -caseins, E.coll ribosomal proteins
and a-crystallin.
In addition, double stranded DNA can be rendered single stranded by use of urea.
(il) Sodium dodecyl sulphate. (CH3(CH2)oCH2OSO Na). Sodium dodecyl sulphate (SDS)
is an anionic detergent and disrupts macromolecules whose structure has been st
abilized by
hydrophobic associations. SDS has been shown to bind to the hydrophobic regions
of proteins
and to separate most of them into their component subunits unless the subunits a
re covalenfly
bound.
SDS binding also imparts a large negative charge to the denatured polypeptides.
This
charge shadows any other charge previously present on the polypeptide. Since all
the
polypeptide chains now have an equal negative charge due to SDS,. they will migr
ate in gel
solely on the basis of their size. The differential migration of these macromole
cules is then a
index of their molecular weight and is empirically used for determination of rel
ative
molecular mass (see applications of gel electrophoresis).
Cetyltrimethylammonium bromide (CTAB) is used as a cationic detergent in place o
f SDS
ardonic. The principles remain parallel to those dexcribed for SDS.

(iii) -mercaptoethanol. Many proteins have their plural polypeptide chains linke
d
together by disulphide bridges. These bonds are broken by heating the protein so
lution in
of mercaptoethanol. A suitable alkylating agent like iodoacetate is also incorpo
rated
reformation of disulphide bond.
Note:
When protelns are insoluble in a concentrated aqueous solutlon of urea,
electrophoresis may be possible
in solvent systems of a different type. Phenol, acetic acid and water (2:1:1) ha
s been used. This solvent
. system has been used to separate proteins of ribosome sub-units.
Procedure
The equipment consists of two components (/) a buffer reservoir, and (ii) a D.C
power
A schematic diagram .of the equipment is shown in Figure 12.11. The buffer reser
voir
buffer reservoir connected by the gel. Save for the gel, there is
electrical connection between the two reservoirs. Platinum electrodes are positi
oned
reservoir and are connected to terminals extending from the top of the unit. The
whole
may be covered by a perspex shield.
The sample, prepared in a high density component such as glycerol or flcoll to p
revent its
with the upper reservoir buffer is loaded on top of the gel, A 'trac/ctng dye' (
usually
) is often mixed with the sample. The extent of migration of the dye gives an
of electrophoretic process. The dye migrates faster than all macromolecules. Thu
s if the

440
Biophysical Chemistry
electrophoresis is stopped before or just as the dye comes out of the bottom of
the gel, one can
be reasonably sure that all macromolecules are still within the gel.
The pH is usually fixed at 9, which gives a net negative charge to most
macromolecules. The anode, to which these negatively charged macromolecules woul
d
migrate under an electric field, is therefore placed in the lower buffer reservo
ir.
When fle power is switched on, a diligent observer will see that the amount of b
ubbles
generated in the reservoir containing the anode is much less than that in the re
servoir containing
the cathode. The reason for this lies in the reactions that permit the flow of c
urrent from
cathode to anode.. They are essentially the electrolysis of,water producing hydr
ogen at the
cathode and oxygen at fle anode (Figure 12,1 I). However, for each mole of hydro
gen produced
at the cathode only one-half mole of oxygen is produced at the anode leading to
less number of
bubbles being seen.
If the electrophoresis is going to take a long time it is necessary to replenish
the diminishing
level of buffer in the upper reservoir. This is done by recirculating the buffer
from the lower
reservoir to the upper reservoir with a peristaltic pump. The bufferis allowed t
o enter drop by
drop to avoid short circuit. An over flow tube is positioned in the upper reserv
oir which prevents
an undue increase in volume and the extra buffer flows back to the lower reservo
ir, again, drop
by drop (Figure 12.11).
CATHODE REACTIONS
2e- + 21-IO .. 2OH- + H
HA + OH- A- + I-IO
Upper
I i Psupply
0 2H*+O+2e-H
lure 12.11 Schematic dlagram of an electrophoretlc system with buffer recyding s
ystem. Also shown are the
Modes of Gel Electrophoresis

Gel electrophoresis is usually carried out in any one of the two modes (0 column
electrophoresis, or (//) s/ab gel electrophoress, Other modes are given in Box 1
2.2.

441
Electrophorests
Column electrophoresls, The gel is set or polymerized in a column. This column i
s then
between the upper and the lower buffer reservoirs. An apparatus is commercially
available
has between 8-12 columns fitted into buffer reservoirs (Figure 12.12(A).

442
Figure 12.12(A) Photograph of a Colurnn gel electrophoresis apparatus.A column i
s shown n the foreground.
Slab gel electrophoresis. The gel is set or polymerized into a thin slab between
two plates. The thickness of the slab of the gel can be adjusted by placing spa
cers of various
Upper Buffer
Reservoir
Gel between
Two Plates
Lower Buffer
Resewoir

.Electrophoresis
443
thickness between the two glass plates. Sample wells are made at one end of the
gel by placing
a comb-shaped jig into the gel before it sets or polymerizes. After the gel has
set, the comb is
removed leavingthe sample wells etched into the gel (Figure 12.12(B)). Sinee a n
umber of wells
ican be east side-by-side, number of samples can be loaded.simultaneously and co
mpared under
conditions which remain essentially identical. This is a great advantage of this
technique over
the column mode. The technique is becoming extremely popular, especially in the
field of molecular
Recovery and Estimation
The principles and major methods for detection and estimation of separated compo
nents
the same as described for paper electrophoresis previously. Only those points sa
lient to
below (also see Box 12.3)
Gels from a column are removed by forcing water from a hypodermic syringe around
the
of the column, allowlng the gels to be extracted under gentle pressure. Slab gel
s are
by introducing a thin metal plate between the two gel plates and coaxing the pla
tes
Before staining the gels may be immersed in a fixative (7% acetic acid) to guard
against
of separated components.
The same stains as described in Table 12.2 are used for staining components in g
els.
gel becomes opaque after staining. This makes it difficult for direct densitomet
ry to
estimate the quantity of a component. The problem may be obviated by treating
stained gel with glycerol and acetic acid which renders it transparent. Direct u
ltraviolet
may also be performed. However, polyacrylamide absorbs in the UV range, thus
some background absorbance.
If one desires accurate quantitative analysis or requires the separated componen
ts for
the compound has to be removed from the supporting matrix. Compound from
starch gel can be removed by slicing the appropriate portion of the gel, macerat
ing it and
it with amylase which solubfllzes the starch and leaves the compound in solution
.

acrylamide gel is frozen and then cut at the requisite size. The rubbery constit
ution of the
forbids any attempt to macerate it. The gel strip is put into a syringe and extr
uded
a thin gauge needle with force. Alternatively it can be put in a small Eppendorf
tube
small holes. The small tube is then put in a larger Eppendorf tube and microfuge
d for a
at high speed. The gel is extruded through the holes into the larger Eppendorf t
ube.
may then be solubillzed in appropriate solvents.
If the sample has been radioactively labeled prior to electrophoresis, autoradio
graphy may
performed to detect the position of the separated components in the gel. The sla
b or tubular
in a thin polythene sheet and is placed in a folder and exposed to a X-ray film.
Radioactive compounds can be eluted into vials containing a suitable scintillati
on cocktail
Ghapter 13) for counting. The gels are however, pretreated with H202 at 60C to el
iminate
quenching by gel polymers.

444
Biophysical Chemistry

Gel Electrophoresis of Nucleic Acids: FAeetrophoresis in Denaturing Media

Although. all the basic features described above for gel electrophoresis apply e
qually well for separation of
acids, problem arises when the a/m of electrophoresis is not separation, but mol
ecular weight
(see later). Although charge to mass ratio for a given RNA strand is always cons
tant, conformatlonal
' arise between RNAs of'similar molecular mass. These differences are mainly due
to the random
and base pairing of RNA molecule giving regions of double helix alternating with
the region of single
conformatlonal differences between similar molecular weight RNA strands give ris
e to frictional
in a gel and are reflected in electrophoretlc mobility anomalies. Thus all RNAs
do not adhere to the
e molecular weight calibration and gel dectrophoresis in conventional media migh
t provide erroneous data
of many RNA species. Therefore, to get a reliable data about the molecular weigh
t of
it is necessary to eliminate the conformatlonal differences by destruction of th
e base pairing. (It has to be
however, that even a chain devoid of base pairing will not be entirely structure
less, since it will
interactions between adjoining bases).
To eliminate conformational differences, electrophoresis is carried out in the s
o-called denaturing media.
first such experiment tried formaldehyde as the denaturing agent. Other reagents
, which have been used
: 8M urea and formamide. The latter is more frequently used since it has been fo
und to allow unequivocal
of molecular weights with considerable accuracy. In this technique formamide ser
ves as the
polymerization of the acrylamide. The supporting electrolyte may be sodium chlor
ide without any
or barbital may be used. The formamide method has been successfully used for mol
ecular weight
of many RNA species.
Another situation which calls for electrophorcsis in denaturing media is when th
e objective of the

of the DNA. Single stands are required for sequencing operations and this is ach
ieved
urea in the gel (SIN). Formamide is not popular for this application.
Of Gel llectrophoresis
Apart from separation and isolation of a large number of protein and other macro
molecules
this technique has been of immense use, and is still proving its indispensabilit
y, gel
is utilized for a larger number of analytical applications. The technique has
of such use in molecular biology that one can not think of any molecular biology
,.at some stage gel electrophoresis will not be carried out. Some of the analyti
cal
of this technique are discussed below.
in Molecular Biology
(O Determination of DNA sequences: The two methods of DNA sequencing (Maxam and
technique, and dideoxy nucleotide technique of Sanger} currently in use are both
high resolution polyacrylamide gel electrophoresis. Both these techniques have
basis. They both depend on generating specific sets of radiolabelIed fragments,
each
at a particular base. The use of high resolution polyacrylamide gels then allows
differing by only a single nucleotide to be resolved as a distinct band and the
sequence
Southern and Northern blotting'. The complementarity of the two strands of DNA
to find whether the DNA or the mutation in DNA in which we
is present in the sample or not. If the sequence of a portion of the desired DNA
is
one can synthetically prepare a complimentary oligonueleotide, radiolabel it, an
d make
react with the sample which has been separated by agarose gel electrophoresis an
d

446
Biophysical
transferred onto a nitrocellulose paper. The paper can then be autoradiographed.
Retention
radioactivity onto the paper shows us whether or not the sequence we are interes
ted in
present. As can be seen, gel eleCtrophoresis is central to this theme of hybridi
zation. It
central to northern blotting which is concerned with RNA rather than DNA.
(ilO ltrtetton mapping of DNA: During the study of genomic or cloned DNA it
essential to have some sort of map to differentiate one area from another. One
of mapping is known as restriction mapp/ng, and the use of electrophoresis is, a
gain,
it. In its simplest form, the technique consists of (A) digesting the DNA with d
ifferent
endonucleases. (B) separating the resulting nucleotide fragments by agarose or
polyacrytamide gel electrophoresis, (C) visualizing these fragments, while they
are in the gel,
ethidium bromide staining, and (D) estimating their size in relation to the stan
dards run on'
gel simultaneously.
(iv) Such important techniques as DNA footpr/n/ng (to find out the regions.of DN
A
interact with proteins) and restriction fragment length polymorphism (RFLP, used
to
mutations in various genes in carcinogenesis and other diseases) axe dependent o
n
electrophoresis in as much as that the final analysis is done on the gels after
has been carried out.
(v) Among the most-successful uses of analytical gels has been the detection of
molecules, which are processed (degraded) to give rise to the product mature spe
cies.
precursors differ very little in molecular weight from their products and
the gel is the decisive factor in the experiment. Precursors of t-RNA, r-RNA and
m-RNA have
been observed by their different mobil]ties on high-resolution gels.
(vi)The sensitivity of gel electrophoresls to variations in conformation of nucl
eic acids
also section on electrophoresis in denaturin4 media) has not only been applied t
o
nucleic acid molecules, but also been used to study the kinetics of interconvers
ions
conformation of many t-RNAs. .
Applieatimm in Protein Study
"k
5
glutamic dehydrogenase
, fumarase

x
4
[
"glyceraldehyde phosphate
.Mobility
Figure 12.13 Illustration of llneartty of the plot of electrophoretic
mobfly of vat.us SDproteba comp/es to the
of the moeu we@hts.
(t) Determination of
stoichiometry: The sub1
stoichiometry of an oli
protein might be determined by
electrophoresis of the SDScomplex after covalent
been introduced into the protein.
reagent dlmethyll suberlmidate NH =
(OCH3)--(CH2)e---(CH30)C = NH is
to produce cross ',
residues. Most of the cross-]
between lysyl residues will
intrachatn, but some can be
to be inter-chain. Ift
is now electrophoresced (not orfly
even cellulose acetate
has been tried with good
different electrophoretic bands will
observed depending on the
stoichiometry of the protein.
that the protein is
polypeptide chains. One would

447
corresponding to the monomer, dimer, trimer, tetramer, pentamer, and the hexamer
.
the absence of the cross-llnking agent one would observe only the monomer band u
pon
with SDS. This simple procedure is extensively used to determine the subunit
of oligomeric proteins.
i (li)Determination of molecular weight of proteins by gel electrophoresis:
geviously (see section on solubilizers] SDS can be used to dissociate oligomeric
proteins into
individual polypeptide chains. The degree of SDS-protein binding is very high (a
bout 1.4
i protein), and thus the charge on the .SDS-protein compl.ex is almost entirely
due to the
sulfate long. It can therefore be said that the surface charge of this complex p
er unit
regardless of the charge of the individual polypeptide chains. Moreover, it has
complexes tend to assume the shape of a long rod whose width is
the length of such complexes, therefore, becomes a function of the molecular wei
ght
polypeptide portion. These factors are at the base of.molecular weight determina
tion by
electrophoresis. It has been amply demonstrated (largely due to the initial work
by
and by Weber and Osborn) that in gels which exert satisfactory molecular sieving
,
exists between the electrophoretic mobility of an SDS-protein complex
the logarithm of the molecular weight of the protein. The method has gained extr
eme
' because of its simplicity, reproducible results, and easy interpretation; Figu
re 12.13
the linearlty of electrophoretic mobility of SDS-protein complexes to the log of
molecular weights. Molecular weight of quite a few proteins has been determined
by this
See Box 12.4 for some problems and answers regarding SDS-PAGE.

SPECIALIZED EROPHORETIC TECHNIQUF.

I. DISCONTINUOUS (DISC) GEL EOPHORESIS
In zone electrophoretlc techniques described so far, even ff all the
sample over the gel, the sample can never be loaded in a sharp band and
sample impedes in a sharp resolution of the components. Disc gel electrophoresis
because of the discontinuous buffer employed and discoid appearance of the
zones) is a modification of conventional zone electrophoresis, which allows the
sample to
the gel as a sharp band, thereby helping further resolution.
be analyzed is subjected to an electric field in a retarding gel support that is
separated
sections differing in porosity and buffered at different pHs. The macromolecular
m/xture
from the more porous into the less porous gel, a process accompanied by a change
in pH.
result, each macromolecular species becomes concentrated into a very thin, sharp
.
producing much higher resolution than can be achieved in a continuous buffer.
The gel that is preferred for disc gel electrophoresis is polyacrylamide. The tw
o
porosity gels used are known as the stacking gel (high porosity) and separating
(low porosity). The two gel system is illustrated in Flgure 12.14.

449

Chloride long
Stage
Current on
sample in
stacking gel
'Stage
Late stage of
electrophoresls;
protein separated
in many dlscold
bands. Trailing long
in running gel

Glycine
Sample

Figure 12.14 Schematic diagram of disc gel electrophorests showing migration of

lons at vaous stages.
Gel
The lower, separating or running gel is pre'ared using about 5-10% acrylamide wh
ich is
than that used in the stacking gel (the amount of acrylamide used depends upon
molecular weight of the macromoleculelbeing separated; Table 12.3 provides
about the differing compositions of the separating gel). Consequently the pores
are
and of a smaller diameter imparting molecular sieving property to this gel. It i
s in
gel that the macromolecules subsequently separate. The buffer used in this gel i
s usually
amine such as Tris, which is adjusted to the proper pH (Le., 8.3) using hydrochl
oric acid.
', gel constitutes about two-thirds of the length of the column or the gel plate
s.

Prote/ns
Table 12.3 Formulations fo Separating Depending Upon the Molecular Weights of th
e Macromolecules

Macromolecule
Molecular weight of the
Macromolecule to be separated
.-yhmdde in sepm'stin

I0,000
-- 40,000
40.000
n I00,000
I00,000
-- 300,000
300,000
n 500,000
> 500,000
15 20
I0 -15
5 -I0
5

2 m
4

Nucleic acids
Oligomer
I0,000
I0,000
-- 50,000

50,000
-- 200.000
200,000
2,000.000
15 20 I0
5
2.2 -- 3

Stcklg Gel
After the separating gel has polymerized, a second layer of gel is polymerized o
n top of it.
This gel, known as the upper or stacking gel is prepared using about 2.3% acryla
mide and is
consequently highly porous and devoid of any molecular sieving action. The buffe
r used here is
also an amine, mostly Tris. The pH is adjusted with hydrochloric acid and is abo
ut 2 pH units
lower than that of the running gel (e.g., 6.7).
The buffer used in the sample is identical to that used in the stacking gel. The
buffer used
in the lower reservoir is identical to that used in the lower or separating gel.
The buffer used in
the upper reservoir is also an amine. It however, differs significantly from the
rest of the buffers
in one important way. Its pH, which is kept slightly above that of the running g
el, is adjusted
not with hydrochloric acid, but with a weak acid whose pKa is at the desired pH.
Glycine is
commonly used for this adjustment.
Electrophoretic Process
Glycine in the upper buffer reservoir exists in two forms; as a zwitterion which
does not
have a net charge, and as a glycinate anion with a charge of minus one:
NHsCH2COO- NH2H2COO-I-I
Zwltter/on Negative charge
When the power is switched on, chloride, protein, and glycinate anions begin to
migrate
toward the anode. Upon entering the stacking gel, the glycinate long encounter a
condition of
low pH (pH of the stacking gel buffer is about 2 pH units lower than that of the
buffer in the
upper reservoir) which shifts the equilibrium towards formation of zwitterions.
As zwitterions
donot possess a net charge, they are immobile. This immobility of glycine zwitte
rions to migrate
into the stacking gel coupled with high mobility of the chloride long creates a
very high localized

voltage gradient between the /ead/ng chloride and the tra///ng glycinate long. S
ince proteins
have their mobility intermediate between the trailing and the leading long, they
carry the current
in this region and migrate rapidly in this strong local electric field. The prot
eins, however,
cannot overtake the chloride long as the strong local field exists only between
the chloride and
the glycinate long. As a result the proteins migrate quickly until they reach th
e region rich in
chloride long and then drastically slow down. This two speed movement of the pro
teins results

Electrophoresis
451
in piling up of the protein sample in a tight, sharp disc between the glycinate
and the chloride
long. It is in this sharp band form that the macromolecules enter the running ge
l. The smaller
pores of the running gel retard the movement of the sharp band of the macromolec
ules for a
time long enough for the glycinate anions to catch up. Since the running gel pH
is higher than
that of the stacking gel the glycine long become fully charged again and the loc
alized high
voltage gradient disappears. From this point on, the separation of proteins take
s place as in
zone electrophoresis. But since the macromolecules enter the running gel as a sh
arp band the
further resolution in disc electrophoresis is sharper than conventional zone ele
ctrophoresis.
Proteases are enzymes which break peptide bonds. They are also known as peptldas
es.
Ther are mainly two kinds of peptidases m the exopeptidases and the endopeptidas
es.
exopeptidases, as the name indicates, break peptide bonds that are at the periph
ery of the
polypeptide -- if they break it from the amino terminal end they are called amin
opeptidases,
and if they break it from the carboxyl terminal end they are called carboxypepti
dases.
The endopeptidases break peptide bonds that lie in the interior of the polypeptl
de.
Some endopeptidases are very specific in cleaving peptide bonds. For example, th
e
Staphylococcus aureus V8 endopeptidase will break only that peptide bond whose c
arbonyl
function has been contributed by glutamic acid ( in other words the peptide bond
must have a
glutamic acid residue lying towards the amino terminal end); bovine pancreas ela
stase will
break only those peptide bonds whose carbonyl function is contributed by alanine
, glycine,
serine, or valine.
Given the above, ttts conceivable that if one subjects different proteins to pro
tease
digestion, each of the proteins will be broken down into different number of fra
gments of different
sizes. If the protease digest is then made to migrate on polyacrylamide gel and
subsequently the
gel is stained for proteins, the pattern of bands will be different for differen
t proteins. This is the
prinle ofprotease mapp/ng. The banding pattern will be different not only for di

fferent proteins,
but also for the same protein with different proteases. Thus several different p
rotease maps can
be created for the same protein using proteases with different specificities. If
you have an
unknown protein on your hand, you can subject it to protease digestion of differ
ent kinds and
then compare the protease maps with protease maps of other known proteins. If th
ere is a
imatch, you have identified the unknown protein.
Protease mapping is a convenient method of identifying proteins and polypeptides
'
requiring no special equipment or technical facility beyond that necessary for S
DS-polyacrylamide
slab gel electrophoresis. The technique is performed mainly in two different way
s.
Suppose you have run a protein mixture on SDS-PAGE and have become interested in
one of the bands that shows up after staining. A slice of this gel containing th
e band is cut with
the help of a dean blade. It is then equilibrated with stacking gel buffer, and
set into a sample
well of a second SDS gel. The gel slice is overlayered with protease in sample b
uffer and
electrophoresis is performed in a slab gel apparatus in the discontinuous system
. Once the
protease and the polypeptide are compressed into a band in the stacking gel, the
power is
stopped for a time. This is to give time for protease action. The protease actio
n on the polypeptide
substrates produces fragments that resolve into a pattern of bands once the powe
r is switched
on again. This pattern can be compared with a similarly treated reference sample
in an adjacent
slot on the slab gel. As said earlier, the banding, pattern varies with site spe
cificity of the
protease.
The alternative method is to digest the polypeptide with protease in the sample
buffer
electrophoresis (not on the gel while doing electrophoresis as in the previous a
lternative).
is time consuming and burdensome. But this also gives rise to sharper bands and
allows

454
Cathode

(acidic)
ZwRterlons
Direction of
migration
of anions
(A) Sample
introduced at pH
above isoelectrlc
potnt
sample components |anions)
(C) Immobilized
sample components
at thetr
Isoelectrlc
0') Mlgarttng
sample components
(cations)

(A') Sample
Introduced at pH
below isoelectrlc
point
Direction of Increasing'
migration
pH
of cations

Flcjure 12.16 Schematic representation of the principle of Isoelectric focusslng

Protein molecules may have a net positive charge in an acid solution because mos
t amino
groups carry a positive net charge and most carboxylic groups are protonated and
electrically
uncharged. With a gradual increase in pH, the number of carboxyl groups carrying
a negative
charge increases, while the number of positively charged groups decreases. At a
certain pH
value, the isoionic point, the net charge of the protein molecule is zero. The i
soionic point of a
molecule is thus determined by the number and types of protolytic groups and the
ir dissociation
constants. Although there is considerable variation in the isoionic point of pro
teins
(see Box 12.5), they are generally in the pH range of 3-11. In conventional elec
trophoresis the
pH between anode and cathode is constant and the positively charged long migrate
towards the
cRthode and negative long migrate to the anode. In lsoelectric focussing (also k
nown as e/ectr
focussbng}, on the other hand, a stable pH gradient is arranged; the pH increase
s gradually
from anode to cathode. A protein introduced into this jstem at a point where the
pH is lower
than its isolordc point will possess a net positive charge and will migrate in t
he direction of
cathode. Due to the presence of the pH gradient," the protein will migrate to an
environment of
successively higher pH values, which, in turn. will influence the ionization and
net charge of
the molecule. Finally, the protein will encounter a pH where its net charge is z
ero and will stop
migrating. This is the isoelectric point of the protein. The consequence of this
is that every
protein will migrate to and focus at its respective isoelectric point in a stabl
e pH gradient,
irrespective of its origin in the apparatus at the time the current was applied.
Thus. the point of

Electrophoresis 455
application and the volume of the protein solution are not critical. Diffusion,
which is an obstacle
with every other method of electrophoresis, is not a problem with electrofocussi
ng, because
focussing effect works against diffusion. Thus, once a final, stable focussing i
s reached, the
resolution will be retained even if the experiment is continued for a long time.
The principle of
isoelectric focusing is illustrated in Figure 12.16.

R can be
Elecphoress
45
Establishing the pH Gradient: Carrier Ampholytes
The pH gradients may be obtained by electrofocusing special buffer substances kn
own as
carrr ampho/ytes. The carrier ampholytes must have the properties listed below.
(0
Since carrier ampholytes must dictate the pH course, they should have a
certain
buffering capacity at their isoelectric point.
(/ They should have a conductance at their isoelectric point.
(///)
They should have low molecular weight so that: macromolecules can be sep
arated
from them easily after electrofocusing.
(/v)
They should be soluble in water. This hydrophflic character will also pr
event their
binding to hydrophobic regions of proteins.
(v)
Ideally they should have a low light absorption at 280 nm. This would pe
rmit the
detection of proteins after electrofocusing by measurements at 280 nm.
Carrier ampholytes are isomers and homologs of aliphatic polyamino polycarboxyli
c acids.
general formula for a carrier ampholyte is:
R--N--(CH2).--N--(CH2)nOOH
(CH2),---COOH,H,
R
his usually less than 5. Carrier ampholytes are available commercially in mixtur
es covering a
pH band (e.g., pH 3- I0) or various narrow bands (e.g., pH 5-6). Cona-nercial am
pholytes
include Ampho//ne (LKB), Pharma/yte (Pharmacia), and Bio-lyte (Bio Rad). The pH
range of the
carrier ampholytes should be chosen such that pI (isoelectric point) values of t
he proteins
r lie well within the corresponding pH range. When making a first run with a pro
tein
it is often advisable to work with the pH range 3- I0. Generally an amount of ca

rrier
which gives a final average concentration of l%(weight/volume) in the columns is
For electrofocussing in gels, an average concentration of 2% (w/v) is reconmende
d since
electroendosmosis. When pH range is outside 6-8, ampholyte
of up to 10% have been used in order to obtain a more even distribution of
' between electrodes.
Convection
- As for all other electrophoretic techniques described thus far, electrofocussi
ng also needs
stabilization of separating protein zones against convective flow in the solutio
n.
ways are in use: (/) density gradient, (//) gel, and (///) zone convection elect
rofocussing. The
is not as popular as the other two, but is discussed as a special case at the en
d.
(0 Density rad/ents'.Density gradients suitable for electrofocussing can be made
with
uncharged solutes, which are dissolvable in water to a concentration that will i
ncrease
sufflcienfly. The compounds should not react with proteins and should have a low
metals. They should be of high purity. Sucrose is the most ideal compound for
of density gradients as it has a protective action on proteins. It has been used
with
(w/v) as the densest solution. Maximum solute concentration and thus the maximum
are placed at the bottom of the column. There is a linear decrease of the concen
tration
the solute.as a function of the column height giving rise to approximately linea
r density
However, nonlinear density gradients have also been used. Sucrose cannot, howeve
r,
at all pH ranges since it iS destabilized at pH range above pH I0. Glycerol is g
enerally

(c)
Bophvstcal ChenUstry
used at such pH rges. Oer compounds, which c be used foaflon ofdensl adlen, e mt
ol, sorbitol, eylene ycol, r, d coll.
(ti) , In electrofocussing, the gel sees only as an antlconvectant and not as
a molecul sieve. Obously the gel concentraon should be low (7.5% for polyaclde)
to
prode lger dleter pores. For lge proteus, exceedg 00 , lower concenaon of aclde
ght used in combaon th agose (0.5%). Aclde Is e prefeed gel for elecofocussg. os
e sch gels e not prefeed as these gels pH gradient considerably dng prolonged ee
ment.
(No n ef. e appatus, lusated gure 12.17, Is de upof o rectl boxes, e upr one bei
ng e cover. e upper surface of
lower x is co--gated fldges, a het of about 10 , sepated by depressions, d is fa
cing e upper box which has coesnd fldges. e fldges d depressions of e
o hves fit teer leag a space of a few eters beeen. us from one end to e oer ere
a now wave he chnel een the o ps wch c be descflbed as sees of tercoected broad
U-tubes. e cer mphole soluon is led in this space
odes situated at e two ends. Go e lid d the bottom p e hoow
chelst into em ou e coolt quid ses to m a const temperate.
en the cuent is on, a density adlent is foxed in each depression by e solute. en
prots come iobile at the islc pH, e densi increases Iocy d e proteus
settle do e depression of the bottom p. en e eeent is over, e cover is d e liqui
d coects in e depressions. Each depression cont a fraction sepat by a dge. e cfl
ons now cot out y ssib of conaflon by ne fracons.
Cer phoa
dg w cover
ling chela
Lid with .cpoling channels
Ridge
lalgure 12.17 Schematic dkgrarn of zone converctWn electrofocussu3 apparatus: (A
) Bottom partfllled with carrier
ampholytes; (B) The lld with correspondl ridges; (C) The apparatus assembled for
electrofocusstng.

P!
High pH
Separation can be carried out in a vertical coluinn (Figure 12.18) or on a horiz
ontal gel
but in both cases purpose-made equipment is required. The column system is in mo
dem
superceded by the plate system. For preparative purposes, the column mode is sti
ll
glass colunms are commercially available. These are filled with
mixture of carrier ampholytes suspended in density gradient solution (sucrose, g
lycol, etc.).
anode (upper) end of the colunm is connected to a reservoir containing an acidic
solution
phosphoric acid) and the cathode end is connected to a reservoir filled with an
alkaline
(e.g., sodium hydroxide). The valves of the reservoir are opened to allow the ac
idic and
alkaline solutions to diffuse through the column. This results in the formation
of a pH
the anode and the cathode. The valves are closed and the power is switched
L. The carrier ampholytes now migrate until they become immobile upon reaching t
he regions
their corresponding isoelectric pH. These compounds remain fixed in these region
s, and
buffering capacity the pH gradient is stabilized. Now the sample is applied at
end of the column. The charged components of the sample migrate in the electric
their net charge becomes zero, Le., till they reach their isoelectric pH. The co
mponent
of the sample remain focused at the regions of their isoelectric pH. The whole p
rocess
take 1-3 days. Once the experiment is over, the power is switched off and the sa
mple
are allowed to run off through a valve at the base of the column into a fraction
The fractions can be analysed further.
C
C
12.18
Schematic design of an isoelectrofocusing apparatus. (A) Anode platinum
rug, (B) Cathode platinum
ring, (C) Water jacket to maintain the temperature. (D) Power pack, and (E) Valv
e to empty the colum
The density gradient is shown by light and heavy shadowing. PI and P2 are two pr
otein bands
electrofocussed according to their isoelectrlc pH values.

460
Biophysical Chemistry
Instead of density gradient, polyacrylamtde gel impregnated with carrier
be used in the vertical columns. The time required for
less than that required in the density gradient, {2-3 hours). The carrier amphol
ytes are
with the unpolymerized gel solution and the mixture is allowed to polymerize. Th
e rest
process remains the same as for the density gradient. Gels are, however, used mo
re
plate mode rather" than the column mode. This is so since up to 24 samples can b
e
analysed using the plate system.
Separation of Protein" From Carrier Ampholytes
The average MW of ampholytes is about 800 whereas that for a protein would be
I0,000. This difference in the molecular weights immediately suggests two method
s
separating them from one another Dialysis against a buffer would effectively rem
ove
99% of the ampholytes; but it is a slow process. Gel filtration would give effec
tive se
a very short time. Sephadex G-50 is the general choice for the process. Other
methods for the purpose are ammonium sulphate precipitation of the proteins, ion
chromatography and partition chromatography by countercurrent.
Applications
Electrofocussing has been widely used for separation and identification of serum
proteins.
It is being used by the food and agricultural industries, forensic and human
laboratories, and for research in enzymology, immunology and membrane biochemist
ry etc.
5. TWO DIMENSIONAL GEL ELECTROPHORESIS
This powerful technique combines the resolving power of isoelectric focussing
gel electrophoresis. The resolving power of the technique is so acute that it ca
n resolw
containing 5000 proteins into individual species. The mixture is first subjected
to
focussing on a I mm diameter gel in a capillary tube. At the
the gel is extruded from the column and placed on top of a slab gel. The sample
is now subjected
to SOS acrylamide gel electrophoresis, which separates the proteins according to
their molecular
weights. Isoelectric point and molecular weight of a protein are in no way conne
cted to each
other and thus the technique exploits two properties of a protein for separation
and thus has a
great resolution power.

6. IMMUNOELECTROPHORESIS
The technique exploits the specificity of reaction between an antigen and antibo
dy and
molecular sieving of the gel in which this reaction is taking place for analysis
of components of
a given sample. The technique was developed by Grabar and Williams and is actual
ly a
modification of Ouchterlony double diffusion technique. To understand the techni
que better a
brief discussion of Ouchterlony double diffusion technique is given below.
The Ouchterlony method depends on the formation of a micellar structure during g
elation
of agar or sucrose. Molecular aggregates with molecular weights not exceeding 20
0,000 daltons
can diffuse freely through micellar channels. The aggregates with higher molecul
ar weights are,
however, retarded. Thus, the individual antigen and antibody molecules filled in
adjacent wells
cut into the gel can diffuse freely till they come into contact. On contact, /f
the ant/body/s
specific forthe antigen, they form complexes with larger molecular weight; these
complexes are
immobile. If the concentrations of correspoding antigen and antibody are suffici
ent to form a
visible aggregate, a precipitin band appears (Figure 12.19).

461
Well for
antiserum
Wells for
(c)

Preclpttn band
12.19
Schematic Diagram of a precipltin band formed due to double diffusion, A
n = Antigen,
Ab -- Anatx
Immunoelectrophoresis, a powerful modification of Ouchterlony method discussed a
bove,
i performed using a double diffusion chamber shown in Figure 12.20. The chamber
may either
purchased from the market or prepared readily by coating a glass microscope slid
e with
punching appropriate holes in the supporting medium. The antigen
in the small round well (1-100 mg). The current is switched on (8 mA, 4-8 volts/
cm] and
electxophoresis is allowed to continue for I-2 hours. Immediately after disconne
cting the
supply, the rectangular well is filled with appropriate antisera and the gel inc
ubated
temperature in a humid chamber to permit diffusion of antigen and antibody
each other. This leads to the formation of precipitin bands at the site lateral
to the
component has separated during electrophoresis from the rest of the
The major advantage of this method is its increased resolving ability due to the
of electrophoresis with immune specificity. The technique canbe made quantitativ
e
has been used to detect particular antigens in sera, tissue or cell extracts, an
d culture
It has also been used to derermine the purity of a given antigen.
Process of tmmunoelectropiwresis: (A] Chamber with pattern cut in agarose/agar.
[B} Round wells
.filled with antigens: Bovine serum albumin (BSA) and Bovine senan (BS). Eiectro
phoress separates the
sample nto different oampounds. (C) Addition of antisenun (cubit antt-BS and lea

ds to dtffu-sion of antlyens and antibody to Five preclpttln bands.

462
Biophysical Chemistry
7. PULSED-FIELD GEL ELECTROPHORESIS
Conventional agarose gel electrophoresis can not separate linear double
stranded DNA
molecules that have a radius of gyration which is larger than the pore size of t
he gel. These large
linear duplex DNA molecules migrate through agarose gels at the same rate irresp
ective of their
size. In other words agarose ceases to exercise any molecular sieving effect on
these large DNA
duplexes. This behaviour of large DNA molecules is because of a phenomenon known
as reptation
which means that the molecules migrate 'end-on' through the matrix as if they we
re migrating
through a sinuous tube.
The problem of separation of large size DNA molecules can be ameliorated to some
extent
by increasing the pore size of the gel. These large pore gels are capable of sie
ving larger size DNA
molecules, the molecular sieving being a function of the pore size. Thus, extrem
ely low'
concentration of agarose (0.1% to 0.2%) have been used to resolve extremely larg
e DNA
molecules. These low percentage gels, however, present problems of their own. Fi
rstly they are
very fragile and have to be handled extremely carefully. Secondly they have to b
e run very
slowly failing which the resolution might be poor. Moreover, they are absolutely
incapable in.
separating linear DNA molecules whose size is in excess of 750 kb.
Why do we need to separate such large DNA molecules? There is a straight forward
answer to this question. The importance of major histocompatibility locus of ma
mmals and therefore
the need to study it is well known. This locus occupies several thousand-kilobas
es of DNA
Naturally, in order to study this locus, it would be desirable if one can separa
te it from other
genetic material. This Is just one example. There are several molecules of DNA i
n the individual
chromosomes of lower eukaryotes which have a size in excess of 6000 kb. Thus, wh
enever one
has to study a single genetic locus spanning several thousand-kilobases of DNA,
one wants to
resolve these large molecules and it is at such a juncture that one painfully re
alizes the inadequacy
of convention.al ge! electrophoresis techniques.
A solution to this problem was evolved largely due to the efforts of Schwartz an
d Cantor in
1984 when they developed pulsed-field gel electrophoresis. As opposed to the con
tinuous
unidirectional electric fields applied in conventional gel electrophoresis, puls
ed-field gel

electrophoresis uses pulsed, alternating, orthogonal electric fields. When such

a field is applied
to a gel, large DNA molecules become trapped into their reptation tubes every ti
me the direction
of the electric field is changed. These molecules remain immobile till they reor
ient themselves
along the direction of the new electric field. It is here that different DNA mol
ecules adapt a
behaviour consonant with their respective sizes; large DNA molecules take a long
er time to reorient
themselves and are consequently retarded more in the new electric field as compa
red to the smaller,
DNA molecules. Thus, all those molecules of DNA whose reorientation times are le
ss than the
period of the electric pulse can be fractionated in a size dependent manner. Fac
tors, which are
of extreme importance for determining the limit of resolution of pulsed-field ge
l electrophoresla
are given below.
(/) The absolute periods of the electric pulses.
(tO The angles at which the two electric fields are applied to the gel.
(///)
The relative field strengths of the two electric fields and the degree o
f uniformity of
the two electric fields.
(iv)
The ratio of the periods of the electric pulses employed to generate the
two electric
fields.
The Schwartz and Cantor method was capable of separating DNA molecules of up to
2000
kb in size.
The original technique of Schwartz and Cantor has now been improved upon so that
DNI
molecules of size in excess of 5000 kb can be resolved with relative ease.

.tergents
mbedded.
mbedded
.s on the
ese DNA
horesis,
Electroplwresis
463
Instrumentation. The original apparatus used alternately pulsed electric fields
or
perpendicular orientations and linear electrodes. The apparatus of this type., h
owever, suffers
from certain drawbacks due to which the resolution is not so good. The electric
eld generated
in such apparatus is never uniform. This affects the speed and direction of the
DNA which
depend more and more on the position at which they are loaded into the gel. It i
s" typical of such
apparatus that towards the edge of the gel, the DNA path becomes skewed leading
to a
not-so-good resolution. Recent innovations have largely overcome these problems.
Two such
innovations of extreme importance are described below.
The first innovation is by Carle et. aL The apparatus designed by them does not
use
orthogonal or perpendicular arrangement of electric fields. Instead, the apparat
us utilizes
periodic inversion of a single electric field. For this reason it is also known
asfield-inversion gel
electrophoresis (FIGE). This arrangement produces an electric field which is uni
form in both the
directions (this is significant since it removes all the problems arising out of
nonuniform electric
fields in the original apparatus). The pulse in the forward direction is slightl
y longer than the
one in the reverse direction. This arrangement ensures the migration of the DNA
along an
absolutely straight track. While the ratio between the forward and reverse pulse
s is always
maintained as a constant, the absolute lengths of the individual pulses might be
varied to
improve the resolution. This innovation (FIGE) makes it relatively easy to resol
ve DNA fragments
of up to 2000 kb with fairly good resolution. The upper limit of separation migh
t be significantly
higher than 2000 kb.
Another innovation that needs to be discussed is that of Gradiner et. aL In the
apparatus
designed by them, a vertical gel apparatus with platinum wire electrode position
ed on opposite
sides of the gel are used. The electric fields are continuously switched between

the two electrodes

and the DNA moves alternately toward one and then toward another electrode in a
movement
which can at best be described as zigzag. However, the net result of such a zigz
ag movement s
a straight line and the DNA moves from the well in which it was loaded to the bo
ttom of the gel.
Significantly, all the lanes in the gel experience equivalent electric fields. T
his condition precludes
any horizontal distortion of the resolved DNA bands. The usual arrangement of th
e electrodes is
at an angle of 90. The length of the electric pulses varies depending upon the si
ze of the DNA
to be separated. Thus, while I0 second pulses give a good resolution when the ai
m is to separate
DNA molecules between 50-500 kb in length, 50-60 second pulses are required to a
chieve
a satisfactory resolution of DNA molecules larger than i000 kb. The system is ex
tremely useful and DNA molecules of up to 9000 kb length can be separated from e
ach other satisfactorily.
As pulsed-field gel electrophoresis is a relatively new technique, no single typ
e of apparatus
has gained broad popularity till date. Different laboratories use apparatus of d
ifferent designs
depending on their needs. There are several companies, which are marketing such
apparatus.
8. ELECTROPHORESIS ON CELLULAR GELS ]
" Long DNA molecules are fragile and molecules longer than I00,000 bp (0.1robp)
are
broken even by pipettlng. It is therefore quite difficult to isolate long DNA mo
lecules from cells.
method designed to circumvent this problem is to embed the cells in agarose bloc
ks or nicrobeads (diameter 50 mm) and then treating such embedded cells with ion
ic d,
such as SOS and proteolytic-enzymes. This leads to digestion of cells leaving in
tact DNA e
in agarose. The DNA can be manipulated and cut by restriction enzymes while it i
s still e
in the agarose. If one cuts this DNA with restriction enzymes, which have very f
ew sit
DNA (e.g., Not I),. large fragments to the tune of I mbp will be obtained. Howev
er, if tt
are extracted form the agarose to submit them to pulsed-field gel electro
' will be broken again. A new gel, known as the cellular gel, goes a long way in
sol ing this
The gel matrix, or 'cellular gel' is a composite containing agarose mirobeads (w
ith
i point) suspended in, and permeated by an interstitial, phase of easily melted'
agarose.

(B)
3' ... A. G. A. T. C.T... 5'
The process of electrophoresis is described in Figure 12.21. It is interesting t
o note
electrophoresis one handles DNA embedded in the mierobeads and not the naked DNA
further manipulation. It should also be noted that from the time of isolation of
DNA from
cell to its manipulation for further studies, never is the naked DNA handled as
such;
embedded form is used. For further electrophoresis the DNA, while it is still em
bedded in
beads can be used as the sample. When the current is switched on, the DNA
extricate themselves from the bead pores and move along on the gel. The trick is
to make
whole gel cellular. Thus, even after electrophoresis, one could just visuallz
zones under ultraviolet light and cut those zone off. This gel when heated will
result in
conventional agarose melting off quickly; the microbeads, however, will not melt
because
have a high melting temperature. The DNA has now been isolated in the
this form it is available for further study.
Beads Molten agarose
(c}
org
CelluFramL gel

(G}
Broken DNA
....:
hn molten agarose
f
1
.:.,,'.',
I I
I
(% } '
"'"
fragments
() IntactDNA

[ [

-embedded in
*
'*
unmelted beads
Figure 12.21 Ce ,.. gels
(A) Molten agarose of low melting temperature is with high melting temperatw (55
oc) mkTobea. The rnmre [B] Is then [C] poured tnto an agarose. The mlxture is no
w allowed|
set, (D) DNA fragments are now separated electrophoretlcally in th composlte gel
. (E) The regon j
which the desired DNA fragment has migrated is excised and heated. (F] MId heat
(below
releases agarose mlcrobeads containing embedded DNA. (G) Heating up to 55C releas
es DNA
ELECTROPHORESIS IN GENETIC ANALYSIS
Restriction Mapping
Restriction endonucleases are produced by many bacteria and hundreds of differen
t
of these enzymes have been purified from different organisms. These enzymes have
a
to cut double stranded DNA in a sequence specific manner, Le., any given restric
tion
will cut the DNA at one and only one base sequence. An example is given below.
5' ... T.C. T. A. G. A... 3'

465
X
5 15
35
(B)
A restriction enzyme known as XbaI makes a cut in the manner shown above. This e
nzyme
cut only at this sequence and will not cut at any other sequence at all. The seq
uence where
given restriction enzyme .can cut occurs very rarely along a DNA molecule so tha
t if a small
from a virus is subjected to the enzyme action we may expect it to be cut in not
five pieces. However, for a large DNA molecule such as that of a bacteria, hundr
eds
fpieces will be produced by the action of a given restriction enzyme. An interes
ting possibility
here. Different DNAmolecules differ from each other in their sequence, and since
restriction
sequence specific cuts we may expect that no two different DNA molecules
cut in the same fashion by a restriction enzyme. Thus, the number and size of DN
A
the action of any given enzyme will beflngerprint of the DNA molecule.
can therefore differentiate between two different DNA molecules by restriction a
nalysis.
will be made clear by the following example.
We are given two DNA molecules having the same molecular weight and want to know
they are from the same or different sources. We subject both these DNA molecules
to
digestion using the same restriction enzyme. Figure 12.22 (A) shows the position
s
both of these molecules are cut. If we take the molecular weight of both these D
NA
100 D, then the fragments produced from molecule X are 5D, 15D, 35D, 20D,
Likewise the fragments produced from molecule Y are 23D, 39D, 28D, and 10D. We
subject these restriction digests to electrophore$is. Since the molecular weight
s of the
produced from X and Y and even the number of fragments produced from each
differs, their electrophoretic patterns will also differ. From the electrophoret
ic pattern
Figure 12.22 (B) we can infer that the two DNA molecules were different.
20
25

23
39
28 10
I
Direction of
migration
X Y
(A) Two DNA molecules X and Y have the same molecular weight but have different
sites at which a
restriction endonuclease cuts. (B) Electrophoretlc pattern of X and Y digested w
ith the re-strlctlon enzyme.
Restriction analysis is used for many purposes including identification of delet
ion mutaions
point mutations in genes.
Transfer
Suppose that in the above example rather than using a small DNA molecule we woul
d
used a bacterial DNA and asked to identify a particular gene, it would be imposs
ible.
so because a large number of fragments would be produced and none will be resolv
ed
on the gel so that all portions of the gel would seem dotted with DNA. Edwin Sou
thern
a procedure, which enables us to identify the fragment containing a particular g
ene.
process is known as Southern transfer (also called Southern hybridization, South
ern
etc.) and has become one .of the most useful procedures in molecular biology.

466
The principle that is central to this procedure is that a given gene can be loca
lized
means of its hybridization to a radioactive DNA or RNA molecule ('probe') which
has
complementary sequence. However, it is not easy to hybridize DNA while it is in
the gel
previously this used to be done via the time consuming solution-hybridization
Secondly. DNA separated by electroph0resis is double stranded and again not amen
able
hybridization. Southern solved these complications by (/) denaturing the DNA wit
hin thl
gel by treatment with alkali, and (ii) transferring the denatured DNA from the g
el
nitrocellulose paper by means of capillary action. The whole procedure is descri
bed below,
Figure 12.23).
Agarose gel of DNA
Photographic plate
Neutralize
Nitrocellulose

Tissues
Filter paper

AutoradioRraphy / i i ii

Transfer assembly

Nitrocellulose

Figure 12.2,
Restriction digested DNA is loaded onto an agarose gel (mostly submarine
After the dye has run three fourths of the gel, the gel is removed and soaked in
a
solution which is usually NaOH. All DNA in the gel thus gets converted to single
-stranded I
This DNA is now transferred to the nitrocellulose paper. For
paper is placed in atray filled with a suitable buffer and allowed to imbibe the
liquid. The gel
carefully layered on top of this stack avoiding trapping of air-bubbles between
the
the gel. A nitrocellulose filter of the same size as the gel is now placed on to
p of the gel
air bubbles). On top of this is placed again a thick stack of filter papers and
these are
down by putting a weight on top. The liquid from the tray now rises through the
gel

it the DNA molecules (DNA is not bound in the gel as the gel is mostly liquid).
Once the
molecules reach nitrocellulose they become adsorbed tightly to the paper and are
remaining liquid passes through the paper and is absorbed by the filter papers p
laeed at
top. The positions of the DNA molecules adsorbed to the filter are more or less
identical to
positions they had attained in the gel upon electrophoresis. The paper is now
at 80C so as to fix DNA permanently onto the nitrocellulose. The dried paper is n
ow
inside a fight fitting plastic bag and hybridized with 32p labeled DNA or .RNA i
n a
solution volume at 68C. The solution has relatively higher ionic strength which
hybridization. The hybridization takes about 16-24 hours. The paper is then
plastic bag and washed in stringent conditions to remove unbound radioactive pro
be.
paper in then dried and subjected to autoradiography (see Chapter 13). The posit
ions
blackening on the film indicate the location of DNA fragments whose sequences
complementary to the sequences of the radioactive probes. Southern
purposes and one of the commonest use is in the screening of c-DNA libraries.,
The southern transfer procedure has also been extended to RNA now. The name
such transfer procedure is Northern blotting. There are some differences in the
between Southern and Northern procedures. The first major difference is that RNA
is
denatured by alkali because it becomes-hydrolysed with such treatment. Instead
is used for the purpose. Secondly, RNA does not bind to nitrocellulose
in Northern blotting diazobenzyloxymethyl (DBM) paper is used. This paper binds
both
and DNA.

468
Western llottln
It is by an analogy with Southern blotting that transfer of proteins from a gel
onto a
nitrocellulose paper has come to be called as Western blotting.
The first step in studying proteins fractionated by PAGE is to transfer them on
to a paper.
The paper .chosen is nitrocellulose, the same as in Southern blotting, There are
two methods
available to transfer proteins to this paper. The first method is absolutely sim
ilar to the one for
DNA and takes advantage of the capillarity. The gel on which protein separation
has been
achieved is placed on a wet block of buffe.r-soaked blotting papers. On top of t
he gel is placed
the nitrocellulose paper cut to the size of the gel. On top of this another wad
of dry blotting
papers is kept and the whole arrangement is weighed down by a weight placed on t
he top. This
whole arrangement is transferred into a trough of buffer to prevent drying and t
o assure a
continuous flow of buffer, The buffer flows due to capillarity from the trough,
through the pores
of the gel to the nitrocellulose and beyond into the wad of dry blotting papers.
The proteins
trapped in the gel also move along but are arrested by nitrocellulose to which t
hey bind irreversibly
by hydrophobic interaction. This process, like southern blotting, is carried ove
rnight. But this
method of transfer suffers from poor yield. Just about 20 per cent of the protei
n in the gel gets
transferred owing to the small pore size of the acrylamide gel
Another method, which is quicker and has a better yield of transfer is called el
ectroblotting,
Here a sandwich of gel and nitrocellulose is placed in a cassette and is immerse
d in buffer
between two parallel electrodes When current is switched on, the proteins extric
ate from the
pores of the acrylamide and get deposited on the nitrocellulose retaining the po
sition they had
achieved in the gel. This process takes just a few hours. The paper with protein
s transferred is
referred to as a blot.
The blot made by either of the methods named above can be used for further studi
es.
Suppose that there is a particular protein whose presence you were interested in
determining.
All you have to do now is to probe the blot with an antibody specific for this p
rotein. To do this
the first step is treat the entire paper with a 10 percent bovine serum albumin
preparation,

This is necessary because of the tendency of proteins to bind nitrocellulose thr

ough hydrophobic
interactions. So that the antibody, which too is a protein, does not bind throug
h non-specific
association, it is necessary that all open sites on nitrocellulose are blocked f
irst. Once this
incubation is over the specific antibody is incubated with the blot. The antibod
y will bind if the
corresponding antigen is present on the paper. Otherwise it will be washed off.
The next step is to use the anti-antibody. This is normally an anti-species anti
body.
Supposing the first antibody was raised in a horse, the second antibody is an an
ti-horse IgG.
The secondary antibody will bind provided the first one has bound the paper. Oth
erwise this
too will be washed off.
The second antibody is suitably labeled. Preferably it has been radiolabeled wit
h
radioactive iodine - Ix. If such is the case, the blot can be exposed to a photo
graphic film
overnight and the blackening due to radioactivity will be detectable upon develo
ping the film.
The blackening will prove that the antigen is present.
Altenatively, the antibody may be tagged with an enzyme. Normally the enzymes us
ed in
such cases are the horse-radish peroxidase or alkaline phosphatase. The paper is
then

Electrophorests
469
incubated
into an
insoluble
the paper
therefore

with a substrate solution and the tagged enzyme converts the substrate
colour product that is precipitated. The presence of a colour band on
is
indicative of the presence of the desired antigen.

Second antibodies may also be labeled with fluorescein isothiocyanate in which c

ase the
presence of an antigen is detectable by fluorescence upon irradiation of the blo
t with UV light,
Sometimes, gold labeled or even biotlnylated antibodies are used for the purpose
of probing.
Detecting Plasmids in Bacterial Cells
Antibiotic resistance is a worrying phenomenon. But the genes which confer antib
iotic
resistance to bacteria do not reside in its chromosome. These genes are present
on small,
circular, independently replicating DNA molecules, called the plasmids. Plasmids
possess a
replication origin and therefore replicate independently of the bacterial chromo
some. Often
they replicate much faster than the chromosome and therefore one may find bacter
ia containing
up to even 300 molecules of a given plasmid within a single cell.
There are two qualities in the plasmid that attract biotechnologists to these mo
lecules.
They are small and therefore one can easily manipulate them. Secondly, as stated
above, they
replicate faster and therefore a foreign gene incorporated into them will amplif
y much more
than it would if incorporated in the chromosome. Often the job. ofa biotechnolog
ist consists of
purifying a gene of interest and then incorporating it into a suitable plasmid..
Then this modified
plasmid is sent inside a bacterial ceil. The cells are made to grow onto an agar
medium. As the
cells grow, the plasmid replicates and the foreign gene incorporated in it also
amplifies.
Not all bacterial cells will pick up the plasmid. Some of them may pick up the o
riginal
plasmid--thaf is the plasmid which has not incorporated the foreign gene. How do
we find out
(I) the cells which have picked up a plasmid, and (2} the cells which have picke
d up the plasmid
containing the foreign gene?
The technique to do so is relatively simple. After your gene transfer experiment
, there may
be several colonies growing on the petri plate. You have to determine which of t
hese colonies
contains the bacteria carrying the modified/unmodified plasmid. An agarose gel i

s prepared
with about 16 wells. Fraction of each of the colonies growing on the petri plate
is picked up
using a tooth-pick or an inoculation needle and suspended in a drop of a solutio
n of sodium
dodecyl sulfate. This causes the cens to lyse. A fraction from each of such lyse
d preparations is
loaded onto a well in the agarose gel. The samples are then electrophoresced. Th
e chromosomal
DNA is very large and cannot penetrate the gel. It therefore does not move. The
plasmid, on the
other hand can move. At the end of electrophoresis, the gel is stained with ethi
dium bromide. In
thewells in which the cell-suspension does not contain a plasmid, a fl)Lcent ban
d will be
observed right at the origin and will be due to the chromosomal DNA/that hs not
rloved. In
some wells, however, one may observe another band far down in addition to the ba
nal at the
origin. This is the band due to the plasmld. In yet another well you may find a
band tat has
much less than the plasmid band. This may be the plasmid that has picked-up the
foreign gene. Because it has picked up the foreign gene, it is higher in molecul
ar weight and
therefore migrated slower than the original plasmid. This experiment tells us th
e colony of
has the modified plasmid. We can now go back to the petri plate, pick up this
r and grow it separately. This is our colony that has the foreign gene.
Sequencing
Just aRer 1975, several methods for DNA sequencing were developed Two of them, t
he
cleavage method of Allan Maxam and Walter Gilbert, and the chain terminator meth
od
, are today used. The chemical cleavage method is explained below.
The Chemical Cleavage Method : Rather than just discussing this method in a text
-book
let's try and actually visualize how we sequence a given piece of DNA. Let us ta
ke any

470
Biophysical Ctwmistry
arbitrary 10 nucleotide DNA sequence and see how iris manipulated and what the r
esullts are.
Following is the sequence that we randorrfly select.
5' P-'ITCAGCCGAT-OH 3'
The first step in the chemical cleavage method is to radioactively label one end
of the
single stranded DNA. In most cases the DNA fragment will have a phosphate at the
5'-end. To
remove this the DNA is treated with alkaline phosphatase which will carryout the
following
reaction
5' P-TI"CAGCCGAT-OH 3' + H20 - 5' OH-TI'CAGCCGAT-OH 3' + PI
Once this is done, the DNA is incubated with [-32p]ATP and the enzyme polynucleo
tlde
kinase from E. coli infected with bacteriophage T4. The following reaction Will
take place
5' OH-TI'CAGCCGAT-OH 3, +A-p-p-32p-------- 5' 32pTTCAGCCGATOH 3, +ADP
[7-32p]ATP
The DNA now carries a label at its 5'-end.
The basic strategy of the chemical cleavage method is to specifically cleave the
end-labeled
DNA at only one type of nucleotide. This is done in a manner that each DNA molec
ule is broken
at an average of one randomly located susceptible bond. This ensures production
of radioactive
fragments, all beginning at 5'-labeled nucleotide and extending up to any one po
sition in the
DNA strand. (If you do not understand how this is done or what exactly the above
means, don't
lose hope. Things will become clearer later in the discussion.) Let's see exactl
y how this is done.
The DNA solution is divided into four aliquots named "G only', 'G+A', 'C only',
and 'C+T'.
Let's see whathappens in each tube,
1. G only
In this tube the DNA is incubated with dimethyl sulfate (DMS). This compound has
a
property of methylating only G residues at position N7. Such methylation makes t
he glycosidic
bond of the methylated G residue susceptible to hydrolysis. Therefore when piper
idine is put
into this mixture, the DNA gets broken at those positions where the G residues h

ave become
methylated.
Now let's see the trick here. Very small concentration of DMS ts used. Concentra
tion so small
that it cannot methylate every G of evenj DNA strand. This ensures that in one D
NA strand a
particular G is methylated while all other G residues are spared. In another str
and some other
G will become methylated. The process is entirely random. Such a treatment will
result then in
DNA strands of varying length depending on which G has become methylated in a gi
ven strand.
To be sure let's see which fragments will result if we give the above treatment
to our
chosen DNA strand.
The original sequence of our strand is :
residues present here; one at position 5
om the labeled
end. In some of the strands the 5 G will
rands it will
be the 8. When piperidlne is mixed then,
.

5'P-TrCAGCCGAT-OH 3' . There are two G

and the other at position 8 counting fr
become methylated and in some of the st
the following fragments will be created

5' 32P-TrCA, and 5' 32P-TTCAGCC. One strand will be 4 nucleotides long and the o
ther
will be 7 nucleotides long.
Of course, in some strands both theG residues will be methylated. However, it is
easy to
see that in such strands the piperidine treatment will create a 4 nucleotide lon
g fragment.

Electrophorests
471
9.. A+G
Here the DNA is protonated rather than methylated. If DNA is treated
d, both A
and G residues are protonated at equal rates. Protonation also makes
dic bond
susceptible and therefore liable to break upon piperidine treatment:
trategy is the
same as in 'G only' : protonation of preferably only one of the A or
should take place
per strand. The following fragments will be created in this tube.

with an aci
the glycosi
Again the s
G residues

TIC, TrCA, TrCAGCC, TTCAGCCG. Four fragments, all labeled at the 5'-end, measuri
ng
3, 4, 7, and 8 nucleotides in length.
3. C+T
IfDNA is reacted with hydrazine (NH -- NH ) and this is followed with piperidine
treatment,
the glycosidic bonds before both C and T are destroyed. In this tube, therefore,
the following
fragments will be created.
T, Tr, TTCAG, TI'CAGC, TI'CAGCCGA. Five labeled fragments, measuring 1, 2, 5, 6,
and 9
nucleotides in length. Of course, in some strands no base will be destroyed and
therefore a
whole labeled piece will also be there. But these strands will be there in all t
ubes.
4. C only
If hydrazine treatment is carried out in presence of 1.5M NaCI, only C residues
react well.
The following piperidine treatment will therefore destroy glycosidic bonds befor
e only C. Following
fragments will be created..
TI', TFCAG, TTCAGC. Three fragments, 2, 5, and 6 nucleotides long.
The next step is probably the most important for sequencin4,. The four different
ly fragmented
samples of the DNA, A+G, G, C, and C+T, are simultaneously electrophoresed in pa
rallel lanes
(labeled G, A+G, C, C+T) on a sequencing gel. The sequencing gel is a long (up t
o 100 cm), thin
(just 0.1 mm) polyacrylamide slab. The polyacrylamide percentage is very high ma
king the
pore size extremely small. It also contains 8 M urea. Moreover, it is run at 70 C
. Together, these
three conditions ensure that (1) no hydrogen bonds can form between nucleotides,
(2) the DNA
fragments will migrate as straight rods, and (3) the DNA fragments will separate

only according
to their size. Even if two fragments differ in size by as littleas one nucleotid
e, the high
polyacrylamide percentage ensures that they migrate as separate bands.
Once electrophoresis is over, the sequencing gel is exposed to a photographic fi
lm. Since
the fragments are radioactive, the photographic film will be blackened at positi
ons where the
fragments have migrated to. The sequence of the DNA may be simply read off this
autorad/ograrn.
Let's try to picturize how this autoradiogram will look like for the fragments t
hat we have
created of our chosen DNA sequence (Figure 12.24).
Now just start reading the sequence from the smallest fragment onwards, that is,
from the
bottom. When a band appears only in the C+T lane and not in the C lane, it means
a T,
If it appears in both the lanes, it is read as a C. Likewise when a band appears
only in A+G and
not in G, it means an A. If the band appears in both the lanes, it means a G. No
w read.
The sequence that you can read is

472
5'-TCAGCCGAT-3'
Figure 12.24. Schematic autoradiogram of the sample DNA fragments as they migrat
ed on the sequencing gel on the
basis of their size. Sequence has to be read by the presence of bands in the two
lanes. A band present
in the A+G lane without a crresponding band in the G lane means an 'A': presence
in both lanes
means a 'G'. Likewise a band present in the C+T lane without a corresponding ban
d in the C lane
means a "F; presence in both lanes means a 'C'. Sequence has to be read bottom u
pwards,
If you compare this with the sample sequence you will find that the two are virt
ually the
same except that the first nucleotide (a T) has not been read. This is a norm. T
he 5'-nucleotide
cannot be read in the chemical cleavage technique.
For the sake of understanding we have taken a small, 10 nucleotide sequence. Nor
mally,
up to 300 bases can be read on a single gel.
What about the 5'-nucleotide? How do we read it? Remember that we took only one
strand
for fragmentation, DNA is double stranded and the two strands are anti-paralleL
Thus if you
take the other strand now, and develop it like above, you would be able to read
the 5'-nucleotide.
Of course, you have the option of ignoring to read that nucleotide, After all, w
hat is just one
nucleotide if you have been able to read 300.
DNA Footprlnting
There are proteins which bind to specific sequences within the DNA. By such bind
ing they
regulate the expression of genes - they can make the transcription of the concer
ned gene faster,
or even inhibit it. Transcription factors are such proteins. Moreover all enzyme
s concerned with
DNA synthesis or DNA directed transcription also bind DNA. It is very necessary
that we find
exactly what these sequences are if we have to understand the molecular processe
s underlying
gene expression. The technique that allows us to delineate these protein binding
sequences is
called 'DNA footprinting'. In fact this technique is just a variation of the seq
uencing technique
dscribed above.
Th basis of the technique is surprisingly simple. A piece of DNA is incubated wi

th a nonspecific DNase. The conditions are manipulated such that the DNase can make just
one random
cut per piece. This situation is reminiscent of the situation that was created i
n the sequencing
experiment, If the digested DNAis now electrophoresed, the fragments will migrat
e according to
their size if the gel has been prepared just as described for the sequencing exp
eriment.

473
DNA
5 10 15 20 25
Figure 12.25. DNA footpHntng. Nuclease cannot digest that portlon of DNA to whic
h a protetn is binding. Thus
when DNA Is fragmented and mjrated on electrophoretlc gel, there is a gap n the
ladder of segments
produced. The gap corresponds to that sequence where the protein was bound.
Consider that this piece has a protein binding sequence. If now in the solution
the
concerned protein is present then it would bind to that sequence. If now a DNase
digestion is
resorted to, then the protein will give protection to that sequence to which it
is binding. The rest
of the DNA will be cut just as before. Now if the resulting fragments are electr
ophoresed again,
there will be a gap in the ladder : the fragments for the protected region will
not be there (Figure
12.25). Comparing of :he previous gel where protein was not present and the gel
where the
protein is present tells us the region where the protein is binding. If we seque
nce the entire
piece with chemical cleavage technique, wewill be able to tell the exact sequenc
e of the protein
binding region.
Gel Retardation or Band-Shift Assay
Studying nucleic acids-protein interactions is of fundamental importance in unde
rstanding
the cell growth and behaviour. Such basic elementary processes as DNA replicatio
n, translation,
transcription, controls of gene expression etc. are due to specific interaction
between nucleic
acids and proteins. These interactions have been studied using X-ray crystallogr
aphy, circular
dichroism, and NMR. However, all these techniques are tedious, need expert analy
sis, and
require large amounts of material and quite expensive instrumentation. Gel retar
dation or
band shift assay on the other hand, is a technique which requires quite small am
ounts of
sample, no elaborate instrumentation and provides very good results about nuclei
c acld-protein
interaction which can be interpreted with comparative ease. Due to its simplicit
y, this technique
has become the most widely used for studying protein nucleic acid interactions.
Although this

technique has been used to study both DNA and RNA interactions with proteins, in
most of our
following discussion we would limit ourselves to DNA- protein interactions.
Linear DNA molecules migrate on an electrophoretic gel as a monotonic function o
f their
length, rather in a worm like fashion known as reptation. If before migrating on
the gel, DNA
fragments are incubated with a protein, which interacts with nucleic acids, a co
mplex might be

474
Biophysical Chemistry
formed. This complex would then migrate much more slowly than the DNA fragment w
ould
have migrated alone {naked). This is the principle of gel retardation assay. Thi
s retardation of
nucleic acid mobility might be because (i)the mass of the DNA-protein complex is
certainly
higher than that of the naked DNA leading to a lower mobility, e., retardation,
and/or (ii) the
charge of DNA .fragment has been modified due to ionic interaction between itsel
f and the
protein, and/or (iii) the interaction with protein has induced certain conformat
ional changes in
the DNA which impede its movement through the gel. Apart from these intrinsic fa
ctors, other
external factors such as the composition of the gel matrix, the composition of b
uffer, and the
temperature ofelectrophoresis can also affect the mobility of complex or naked D
IA. It, however,
seems that it is mostly the protein mass that influences the mobility of the com
plexed DNA (see
Figure 12.26)
Figure 12.26 Effect ofproteln mass on migration of DNAfragments. If proteln indu
ced changes tn DNA onfommtWn
oncl charge are ignored, the rrrigratlon of DNA ts is largely dependent upon the
mass of the
proteOl wffh which they complexed. S (A) and (B) correspond to bldng of multlple
copes of a
protein and thus the retardation is higher. Situans (C), (D) and (E) involve bin
ding of DNA to either a
- sgle copy or to truncated proten (truncated circles) and the retardan is corre
spondly lower.
The technique itself is .quite simple. The llrst step is to add the protein in w
hich one is
Interested to a solution of linear double stranded DNA fragments and to incubate
under
suitable conditions to promote complex formation. Since the complex formation is
mostly a
result of ionic interactions, it is important to use buffers which are of low io
nic strength and
thus do not interfere in c0mplexation. Many proteins need cofactors ( e.g., c-AM
P) must be
added to the incubation mixture. If the protein is a DNA binding protein, under
these
conditions it will complex with a given DNA sequence for which it is specific. H
owever,
non-speclfic binding can also take place. These non-specific interactions can be
avoided by
ad ding salt to the binding mixture so that non-specific ionic interactions will

be disturbed. Another way is to add excess of synthetic DNA copolymers such as

poly-dA-dT, or poly-dl-dC
etc. After the binding incubation is over the mixture is loaded onto the lanes i
n the gel. Normally
slab gel electrophoresis with polyacrylamide gels is routinely used. Alongside t
hese lanes, naked

Electrophoresis
475
DNA fragments are also loaded to run as standard. Electrophoresis is carried out
using low
ionic strength buffers. Standard electrophoresis buffers such as the THE (90 mM
Tris-borate, 2
mM EDT, pH 8.3) or the TASTE (40 mM Tris-acetate, I mM EDTA pH 7.9) are also use
d
routinely. In the lane where naked uncomplexed DNA is migrating, the migration o
f the individual
fragments will be strictly a function of their mass. In other lanes, however, .w
here complex
forming incubation was carried out with addition of a protein, those DNA fragmen
ts which
became bound to the protein will migrate at a reduced rate (retarded)while the u
ncomplexed
DNA fragments will migrate depending on their mass. After the electrophoresis is
over,
visualization is carried out. This visualization consists of autoradiography, if
labeled DNA was
used, or it involves ethidium bromide staining and visualization under UV light
if unlabeled
DNA was used. A comparison between the lanes in which the DNA-protein mixture wa
s loaded
and the lane in which the naked DNA was run aids in identification of the DNA fa
gment which
was retarded and therefore the fragment which interacted with the given protein.
The complex
can then be extracted from the gel and further studies can be performed.
Due to the simplicity of the technique and due to the quality of results obtaine
d using it
routinely, this technique has found many applications. The technique has been us
ed to
determine binding constants, co-operativity, and stoichiometry of DNA-protein in
teraction; it
2 nd DIMENSION
O
r
I
t
E
a
N
r

s
d
I
a
0
t
N
i
0
12.27
Two-dlmensl gel electrophoresls for genorne scanning. Gelretardan of res
trtlon dest of the
genome is performed in the jqlst dimension. The pmteln-DNA complex s denatured o
n the gel by
heating or by treating the gel with some denatwunts. Electrophoresls is now perf
ormed under denaturlng conditions in the second dimension 90o to the flrst. A retarded DNA fragm
ent (Heavy line), which
could not be detected tn the first dimension, runs ahead of th diagonal in the s
econd dimension
because it runs according to its octual sze. This leads to its identtJkUn. Arrow
ndUes the frag-

476 BiophysaI Chemistry

has been used to define binding sites, binding requirements, protein domains inv
olved in
interaction and in genome scanning.
The last named application, v/z., gen0me scanning is extremely important. Gel re
tardation
technique is quite sensitive so that it can select rare molecules from a large p
opulation.
This ability of the technique has been used to scan specific binding sites withi
n whole genomes.
The genome is fragmered and incubated with a protein(s). A problem, however, may
frequently
arise here. Due to the complexity and abundance of DNA fragments the retarded DN
A-protein
complex might co-migrate with larger uncomplexed DNA fragments. It would then be
very
difficult to resolve the DNA-protein complex from such gels. A two dimensional m
ethod has
been evolved for precisely such situations and this method allows one to resolve
the DNA fragment
involved in protein binding very easily. The DNA-protein mixture is rtln on the
gel in the same
manner as described above (Figure 12.27). The gel is now treated to denature the
DNA-protein
complexes either by heating or by incubating with denaturing agents. Electrophor
esis is
performed again in a second dimension, 900 to the first run. The protein'DNA bin
ding has been
disrupted and the DNA fragment involved in binding now migrates according to its
real size,
ahead of the diagonal (Figure 12.27). This technique has been used to localize p
rotein-blnding
sites in/ambda and Mu genomes and even E-colt chromosome.
Suggestions For Further Read/rig
Moving Boundary Electrophoresis
I.
Moore, D. H., Electrophoresls in Physical Techniques in Biological Resea
rch (D.H. Moore, ed.)
Academic Press, New York, 1968.
Paper Eleetrophorsis
1.
Peters, H., Paper Electrophoresis: Prlncples and Techniques in Advances
in Clinical Research,
Vol. 2, 1959.
Gel Electrophoresis and Other Techniques
I.
Charmbach, A., and Rodbard, D., Po/yacry GeIElectrophoress, Science, 172

:440-451,
(1971).
2.
Gordon, A. H., Electrophoresls of Proteins in Polyacrylamkie and Starch
Gels in Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 1, (T.S. Work and E. Work
, eds.)
North-Holland Publishing Company, 1974.
3.
Gould, H. and Matthews, H.R., Separation Methods for Nucleic Acids and O
llgonucleotides in
Laboratory Techniques in Biochemtry and Molecular Biology, Vol. 4 (T.S. Work and
E. Work,
eds.) North-Holland, 1976.
4.
Gabriel, 0., Analytkml Disc GeI Electrophoreses in Methods in Enzymology
, Vol.22, (W.B. Jakoby,
ed.) Academic Press, New York, 1971, pp. 565-577.
5.
Weber, K., Prlngle, J.R., and Osborn, M., Measurement of Molecular Weigh
ts of SDS-Acrylamlde
Gels in Methods in Enzymology, Vol. 26 C, (C.H.W. Hits and S.N. Tlmasheff, eds.)
Academic
Press, New York, 1972, pp. 3-27.
6.
Gaal, O., Medgyesi, G.A. and Vereczkey, Electrophoresls in the Separatio
n of Biological
Macromolecules. Wiley, Chichester, 1980.
7.
Catsimpoolas, N. and Drysdale, S, (eds.), Biological and Biomedh::d Appl
ications oflsoelectrlc
Focussing, Plenum, New York, 1977.
8.
Vesterberg, O., Isoelectr/c Focuss/ng of Prote/ns, in Methods in Enzymol
ogy, Vol. 22 (W.B. Jakoby,
ed.).Academic Press, New York, 1971, pp. 389-411.
9.
Sambrook, Fritsch, Maniatls, Molecular Cloning: A Laboratory Manual, Col
d Spring Harbour
Laboratory, 1988, pp. 650-652.

477
I0.
Dear, P.H. and Cook, P.R., Cellular gels: Purifying and Mapping Long DNA
Molecules,
Biochemical Journal 1991, 273: 695-699.
If.
Lane, D., et. aL, Use of Gel Retardation AnalySis, Microbiological Revie
ws, Vol. 56, 1992,
pp. 509-524. '
Exercise
I.
What will be the relative electrophoretic mobilities of alanine (pl = 6.
02), arginine
(pl = I0.76), glumatic acid (pl = 3.22), serine (pl = 5.68), and tryptophan (pI
= 5.88) at
pH 5.68 ?
2. Isoelectric pH of a few proteins are given below:
Protein
Isoelectric pH
Egg albumin
4.6
Serum Albumin
4.9
Urease
5.0
-lactoglobulin
5.2
Hemoglobin
6.8
Myoglobin
7.0
Chymotrypsinogen
9.5
Ribonulease
9.6
Cytochrome C
10.6
Lysozyme
11.0

At what pH would you carry out electrophoresis for the most effective separation
of the following
protein mixtures ?
(a]
Egg albumin and hemoglobin
(b]
Hemoglobin and ribonuclease
[c)
Egg albumin, serum albumin and urease.
Sickle cell hemoglobin migrates at a different rate than normal hemoglobin. You
are given
purified normal and sickle hemoglobin as standards. You have isolated hemoglobin
from 15
patients suffering from anemia. By comparing the electrophoretic mobllities you
want to find
out which of these are sickle hemoglobin. Which mode of gel electrophoresis will
you use?
E. Co//genome was isolated and a small DNA segment of it was arr/plifled a milli
on folds with
the help of suitable primers using PCR technique. When the whole mixture was
electrophoresced on a 2 per cent agarose gel and stained with ethidium bromide,
only one
band corresponding to the size of the amplified segment was visible under UV lig
ht. If the
same mixture was electrophoresced on a standard polyacrylamide gel, several band
s were
made visible by ethidium bromide. Give explanation for these observations.
A researcher is carrying out slab gel electrophoresis of 24 DNA samples. He has
completed
the assembly, poured the buffer which has a pH of 9.0 and has loaded the samples
. When he
switches on the current he observes more bubbles coming out from the lower buffe
r reservoir
as compared to the upper reservoir. Is he in trouble?
Aliquots from the same protein-containlng sample were subjected to SDS-PAGE and
cation
exchange chromatography. After staining SDS-PAGE reveals Just one sharp band whi
le one
obtains two distinct peaks at different elution volumes in cation exchange chrom
atography.
What are your onclusions about the composition of the protein solution?

478
7.
BWphys
X-ray crystallographic studies of a given protein have conclusively proved that
the
consists of four peptlde Chains in association with each other. Yet SDS-PAGE
of the protein gives just one band. How many explanations can you provide to jus
tify
above observation?
8.
You have a mixture containing two components. Given the choice of varyin
g either pH,
strength, or temperature of the medium, which condition will you choose to vary
separation? Why? Why will you not easily choose to vary the other two conditions
?
9.
Ybu are given a mixture containing two proteins having similar molecular
weights, and
Are you confident that they will migrate as a single band?
I0.
You have migrated a given mixture of proteins on two gels of same compos
ition using
of different pH. On each gel you get two bands. Wig you be sure that
proteins?
11. Why is electrophoresis done in solutions having low salt concentrations?
12.
You were given a piece of DNA. When you electrophoresed it on polyacryla
mide, it gave you
single band. You subjected this DNA to digestion with a restriction enzyme. Post
you still get a single band upon electrophoresis. In how many ways can you justi
fy
result?
13.
In the above experiment, if it was told to you that the given piece of D
NA contained a
site for the given restriction enzyme, which of the above conclusions would

13
.ISOTOPES IN BIOLOGY
The phenomenon of natural radioactivity was accidentally discovered by the Frenc
h
Henri Becquerel when he found that crystals of potassium uranylsulphate
emitted a radiation, which could blacken a photographic plate. Subsequently
r radioactive elements like radium, thorium, and radon were discovered. Of these
,
has become linked with the name of the legendary Madame Curie, whom we owe much
understanding of the phenomenon of radioactivity.
For obvious reasons, the aboe-described natural radioisotopes have a very limite
d use in
'. The biological applications of radioactivity had, therefore, to wait for the
development of
instruments as the cyclotron and the nuclear reactor that made possible producti
on of
radioactive materials. Using the nuclear reactor it became possible to produce
for those elements, which are commonplace in biology, for example, carbon,
sulphur, hydrogen etc. Since then the use of radioactive isotopes in biology has
legion, so much so that it is difficult today to imagine of an experimehtal setu
p without
However, before describing their use and the instrument involved, it would be pr
oper
consider the structure of the atom and ask why some atoms are radioactive.
An atom is composed of a positively charged nucleus around which the electrons s
ettle
, the charged clouds. The mass and the stability of the atom reside predominantl
y in the
Atomic nuclei are composed of two major components; protons which are positively
and neutrons which are neutral. The number of electrons (negatively charged) n a
n
is always equal to the number of protons in the nucleus thereby making the atom
neutral. This number is known as the atomic number (Z). Neutrons are uncharged
equal to that of a proton. The sum of protons and neutrons
given nucleus is known as the mass number (A). Thus, A = Z + N. Where N denotes
the
B an ItoI?
The word isotope is derived from the Greek /so (same) and topos (place) and thus
occupying the same position in the periodic table. It was the chemist Soddy in 1
913
first established the existence of atoms of the same element with different atom
ic weights
cdled them isotopes. Isotopes are atoms of a given element, with identical numbe
r of protons
nucleus, the same pattern of electrons in the clouds around the nucleus and ther
efore the

', characteristics, but with different number of neutrons and consequently dijer
ent
The number of stable isotopes that elements possess varies widely, for example,
possesses six, carbon has three, while sodium has only one.
There is a conventional form of describing isotopes The superscript to the left
of the
symbol indicates the mass number A, while the subscript to the left indicates th
e
number Z. For example, Na represents the sodium isotope, atomic number 11 and

lgure 13.1
480
Bioptujsical Chemis
mass number 23. Since the atomic number of any given isotope of an element is th
e same, th
subscript is often omitted. The sodium isotope may then be described merely as 2
3Na.
What is Radioactivity ?
In general the stability of an isotope of a given element is dictated by the rat
io of neutron
to protons in the nucleus. The relationship between the number of protons and ne
utrons in
nucleus can be seen by plotting the values of N against Z for known isotopes (Fi
gure 13. I). ]
will be evident from the graph that stable isotopes for elements with low atomic
numbers ten,
to have an equal number of neutrons and protons, whereas stability of elements w
ith high
atomic' numbers is usually associated with a neutron to proton ratio in excess o
f one. It thus be surmised that N : Z ratio of a stable isotope lies within narr
ow limits so that an isoto
outside these limits will be unstable. Such a nucleus will try to adjust its N :
Z ratio toward
stability, giving out radiation in the process. Thus the phenomenon of radioacti
vity can be see
as an attempt on the part of the isotope to achieve stability.
The first discovered natural radioactive elements were among the heaviest elemen
ts. Th
is logical, since only these elements could have existed for a length of time as
long as the age the earth. With the advent of the nuclear reactors, however, it
became possible for tl
scientists to bombard atoms with neutrons and alter their N : Z ratio, thereby p
roduci
artificial radioisotopes.
120
II0
I00
90
80
70:

O
50
10
.::.
. :-"
.#.
I0' 201 30 40 50 60 70
80. 90 100
Number of protons (Z)

Plot showing relationship between the atomic number (Z) and the number of neutro
ns (N) in
nucleus. The solid llne represents a neutron/proton ratio of unlty. Black dots r
epresent isotopes
are stable. Isotopes falllng away frorn the dotted zone would be unstable.
RADIOACTIVE DECAY
It is clear from the above text that the ratio of protons to neutrons is of impo
rtance
detenTdning whether or not an element is radioactive. An unstable radioactive nu
cleus

"
Isotopes in Biology
481,
reach or approach stability through emission of radiation' This emission of radi
ation is known
as rad/oact/ve decay. A radioactive compound may decay by any one or more of the
several ways
described below.
t Ngatron mulion. In this case a neutron is converted to a proton resulting in th
e
ejection of a negatively charged beta (() particle known as a negatron (-we).
NEUTRON PROTON + NEGATRON
A negatron is an electron, The term negatron is, however, preferred because it e
mphasizes
the nuclear origin of the particle. The negatron emission results in loss of a n
eutron and gain of
a proton. Consequently the N : Z ratio decreases while the atomic number (Z) inc
reases by one.
The mass number (A) remains unchanged. The isotope thus becomes a new element. 3
2p can be
cited as an isotope frequently used in biology which decays by negatron emission
.
32 P-"' 32 S we
15
16
(iO Positron emi,ion, The positron is understood to be a positively charged part
icle. Isotopes like 22Na and C decay by emitting positrons, During positron emis
sion a proton is
converted to a neutron.
PROTON NEUTRON + POSITRON
Positrons have a transient life and are annihilated on .coming in contact with e
lectrons.
The mass and energy of the two particles is then converted to two gamma rays emi
tted at 180o
Positron emission results in loss of a proton and gain of a neutron. Consequentl
y, the
: Z ratio increases, the atomic number decreases by one, while the mass number r
emains
example of positron emission is given below:

t||OAIpha emiion. The alpha particles are heavier than other emitted particles b
ecause
consist of two neutrons and two protons. Alpha emission results in a decrease in
atomic
number of two and a decrease in mass number of four. Decay of 2U is cited as an
example.
22U--- 234 Th + 4 He
90 2
(iv) Electron cttpture or EC-decay. This is similar to positron emission in that
a proton is
This, however, is accompanied when an orbital electron is captured the nucleus.
The capture changes the pattern of orbital electrons. The remaining electrons
themselves emitting gamma .ray or an X-ray in the process.
4.
4 + y/. a:'--ra-''-r^'251vlne ------> 24Jr
(v) EroSion of gamma ra. y-rays are usually emitted when the nucleus of an atom
is
This frequently accompanies alpha or beta particle emission. Gamma emission
not lead to a change, in atomic or mass numbers.
In addition to the patterns" of decay stated above, there are other patterns of
radioactive
which are of no practical interest to a biochemist. Some examples of such patter
ns are
transition and sontaneous fission.

482
Biophyslcol Chemistry
Radioactive Decay Energy
The principle unit of energy in nuclear physics is electron volt (eV): This is t
he energy
acquired by an electron when it is accelerated through a potential difference of
one volt and is
equivalent to 1.6 x 10-9 J. It is a very small amount of energy and usually KeV
(thousands of
electron volts) and MeV (millions of electron volts) are used. Isotopes emitting
a-partlcles are
most energetic, their energies falling in the range of 4.0-8.0 MeV, whilst - and
y-emitters
generally possess decay energies of less than 3.0 MeV.
Rate of Radioactive Decay
The disintegration of an unstable nucleus is an event so completely unpredictabl
e and
random that the only thing which can be said is that an unstable nucleus will de
cay within
a given period of time. For a particular isotope, the proportlon of nuclei that
decay in a given
time is a constant known as its disintegration constant. If the decline in activ
ity of a radioactive
isotope is plotted against time (Figure 13.2)'a tpical exponential form is obtai
ned.
II0- TOO .
701
2 3. 4 5
6
Time in half-I/fe
Figure 13.2 Radioactive decay. The units of tlme are in half-llves.
This may be mathematically be expressed as a simple first order process
-dN
- N

dt
-dN
where --di-- = the number of atoms decaying per small increment of time,
N =
= a
The
The

the total number of radioactive atoms present at any given time,

decay constant, characteristic for a given isotope, and t =time.
negative sign is essential because the activity is decreasing.
above equation may be rearranged to get a term for .
-dN
, N or k = -dN or = -dN/N
dt
N.dt dt

Isotopes in Biology
483
The above equation means that k is that fraction of radioactive atoms which deca
ys per
unit time. We may integrate the above differential equation
-dN
dN
-- .IN, o
dt
N
Carrying out integration between limits of NO (radioactive atoms existing origin
ally) and N
{radioactive atoms remaining after time t) and between the limits of zero time (
t = 0} and any
other time, t, we get
,!' d- -k S dtorln=
N
m = -kt
No

or InN = tt or 2.3031ogD NO = .t
N
N
or N =N e-U or IogN=- t+IogNO
0 2.3.3
Another constant for any given radioisotope is the time that is required
for the original
to fall by a half. This is referred to as the half-life and is used more
commonly than the
constant because it is of much more practical utility. The half-life, tI
/2, of a given
and its decay constant, , may be tied in a relationship as follows

or
or
2.3031ogm= N
Xt
or
2.3031og---1 = kt
N 0.5
2.303 log 2
= kt
or
2.303 (0.301} = t
0.693
kt
0.693 0.693
.'. t2 = orkffi
t2
The above relationships allow us to ca/culate half-life of a given isotope if it
s decay constant
krown and vice versa.
The curve in Figure 13.2 may be converted to a straight line by plotting time ag
ainst log N.
this curve touches the y-axis is,log NO and the slope is -/2.303.
Half-life is a widely varying property. For example, the half-life of 14C is 500
0 years, whereas
has a half-life of 15 hours only, When several different isotopes of the same el
ement are
for experimental use, the half-life is one of the most important factors in tell
ing us
to use.

484
Units of Radioactivity
The unit of radioactivit is the cur/e (Ci). It is defined as the quantity of rad
ioactive material
in which the number of nuclear disintegrations per second is the same as that in
I g of radium,
i.e, 3.7 x 10l (3.7 x 101s-). The curie is a large unit and only millicuries (mCi)
are needed for
tracer applications. The curie refers to the number of disintegrations actually
occurring in a
sample rather than the number of disintegrations counted in a radiation counter.
Normally in radiotracer experiments the radioactive isotope is added mixed up wi
th the
stable isotope. It therefore becomes necessary to express the quantity of radioi
sotope present
per unit mass. This is known as the specific acttv/ty and may be expressed as (0
disintegration
rate (ds- or d min-), ( count rate (ct s- or ct min-), or (/t0 curies (mCi or Ci
) per unit mass of the mixture (Ci or mCi/mole or gram).
The international system of units (SI) has specified the term bequerel (Bq) to r
eplace the
term curie, which is currently in use. The bequerel is defined as one disintegra
tion per second.
A curie is therefore equal to 3.7 10 Bq.
PRODUCTION OF ISOTOPES
Bombarding the target nuclei with alpha-particles from naturally radioactive sou
rces was
the way in which artificial isotopes were produced initially. An example is affo
rded by the work
of Cockroft, and Walton. In 1931, these workers bombarded lithium target nuclei
with hydrogen
nuclei and found that many such collisions, resulted in the emission of high ene
rgy alphaparticles:

Li+ H----- 2 [ He]

In the above example, the lithium nuclei and the proton have interacted to produ
ce a pair
of helium nuclei.
The cyclotron, an instrument that accelerates protons and other charged particle
s to very
high energies, was developed in the late 1930s. Although this development made a
large number
of nuclear transformations possible, perhaps the most important development was
the advent
of nuclear reactors. The significance of this development was that the scientist
s could now
bombard the target nuclei with uncharged particles like the neutron. Since it do
es not bear a
charge, it is easier for a neutron to interact with the target nuclei without ge
tting repelled.
After this development, the use of isotopes in all branches of industry and scie
nce became
legion. Most of the isotopes used in biology are today produced by neutron bomba
rdment. An
example of the kind of interaction that takes place in a nuclear reactor is give
n below:
:271 + on -- 2sI + Y
No chemical change in the target nucleus is involved in the above reaction. It i
s therefore
almost impossible to separate the radioactive isotope (lsI} from the stable isot
ope (127I). There
are, however, other reactions where the target is changed chemically. One such e
xample is
given below:
35
I
35
17CI +0n--- 16S p
The radioactive isotope here is chemically distinct from the target stable isoto
pe; the two
can be separated easily.
SYNTHESIS OF LABELED COMPOUNDS
A prerequisite for these isotopes to be used in biological studies is their inco
rporation in

the bio-organic compound which is to be studied. For example, 4C has to be incor

porated into
glucose if we want to study the fate of the glucose in the body..The glucose mol
ecule which has
one or more of its carbon atoms (2C) replaced by (4C) is known as a labeled mole
cule (glucose),
while 4C is the/abe/. It is also necessary that the label should be incorporated
in the position,
which is appropriate for the experiment being carried out. Thus, out of the six
carbon atoms
that glucose contains, the label should not get incorporated at any position fr0
.m 1-6 at random.
The labeling procedure should be such that the experimenter should be able to co
ntrol the
position being labeled. Two labeling procedures are widely used to prepare such
labeled
compounds. They are discussed below.
Organic Synthesis
The synthetic methods of organ/c chew/st are being used on an ever-increasing sc
ale to
prepare compounds with the isotopic label in defln/te positions in the molecule.
The synthetic
method used for labeling is usually an ordinary one. This method, however, has t
o be mod/fled
/n such a way that the y/eld of the labeled compound is the highest and the isot
ope, wh/ch is the
most costly mater/al in the reaction, is conserved. The preparation of acetic ac
id labeled with
'C in the carboxyl group is.cited as an example. The reaction consists of treatm
ent of Grignard
reagent from methyl bromide with 4CO2.
CH3MgBr + 4CO2 CH3,:'COOMgBr + H20 ----->CH.:'COOH + Mg(OH)Br
The reaction is actuaLhf earned out using Bat'COs which when subjected to the ex
perLmenta]
set up releases CO2. This gas is then del/vered to the container of Grignard rea
gent.

486
Biophystcal Chemistr9
The above example is very simple where the method of conventional organic synthe
sis did
not need to be modified much. There are, however, other labeled syntheses where
the organic
synthesis method has to be modified profoundly. One such example is the synthesi
s of DLlysine labeled in the z-carbon atom. One interesting problem arises here. Since
this synthesis is
a multistep process, the isotope can be introduced either at an early step in th
e experiment, or
in a later step. The latter option seems rosy from the point of view of conservi
ng the radioactive
isotope. However, it is experimentally found that introduction of isotope at a l
ater step in the
experiment does not yield the desired product. Therefore, even though the yield
would be less,
it is necessary that the isotope be introduced at an early step in the synthesis
. The reaction
steps are givenbelow.
(0 4CO2 + 4K + NH3
-----> KH + 2KOH + K4CN
(//) KCN + Br(CH2}3CI
CI(CH}4CN + KBr
/COOCH.
/'COOCH
(I) N'C'CH.CH'CH C] + NaCH
----> N'4C.CH2-CH'CH'C'H
+ NaCl
cooc,H cooc H
dlomalonie ester
/COOCH
N*C.CH.CH.CH.CH
.+ CH--O--NO N*C.CH.CH.CH..COOC2H
COOCH
Ethyl nitrite
NOH
Reduction
(v) NC.CHa.CH.CH..COOCH5 + 8H
-> HN-CH.CH.CH.CH.CH.COOCH + I-IO
Noa
Lysine ethyl ester
Lysine was finally isolated as lysine hydrochloride. The yield of this reaction

is fairly good.
Several labeled compounds are synthes/zed by way of organic syntheses.
Biosynthesis
Labeled compounds with increasing complexity, which cannot be synthesized by the
laboratory, are made through biosynthesis. Example can be cited of sugars, gluco
se, fructose,
sucrose and many others. These labeled sugars can be produced by photosynthesis
under
controlled conditions, with 4COin the gas phase. This biosynthetic method gives
high yields of
uniformly labeled sugar molecu!es. The sugars can then be extracted, separated b
y
chromatography and, crystallized by mixing with a carrier compound. Loss of the
isotope is
minimal in biosynthesis.
Biosynthesis has been employed to prepare such labeled pharmacodynamic agents as
digitoxin, morphine, and nicotine. Usually microorganisms are used in such label
ed
biosyntheses. However, ff synthesis of a metabolite peculiar to a particular spe
cie is required,
the specie concerned might be used for such labeled synthesis. Other examples of
biosynthesis
oflabeled compounds are (0 plant pigments such as chlorophyll, leaf carotenoids,
(/0 alkaloids,
(rio labeled cellulose and glycogen, (iv) haemoglobin with the help of radioacti
ve iron,
(v) thyroxine by tntroductlon of radioactive ''I iodine and (vi) cholesterol by
injecting .labeled
acetic acid,
The overriding concern during biosynthetic labeling is of assuring the purity of
the
desired compound, This is necessary so that erroneous results due to radiochemic
al
contamination can be avoided.

Isotopes in Bology
487
The nomenclature of the labeled compounds is based on the Geneva system. Thus, a
n
acetic acid molecule labeled by 4C in the carboxyl group (CH3.14COOH) is indicat
ed as
1-C-14 acetic acid. If the isotope used for labeling was 3C, the labeled acetic
acid would be
known as I-C-13 acetic acid. If the acetic acid is labeled tn the methyl group w
ith aC, the
nomenclature would be 2-C- 14 acetic acid. Similarly, ATP labeled in they-phosph
ate with 32p is
known as -P-32 ATP.
INTERACTION OF RADIOACTIVITY WITH MATTER
For the sake of simplicity, interaction of radioactivity with matter can be divi
ded into two
broad categories (0 excitation, and (//) ionization, depending upon the sfrength
of the interaction
as we will see below. Of the two, ionization constitutes by far the most importa
nt interaction.
Excitation. Radiation can interact with the orbital electrons of the intervening
matter that
it is p ..a sing through. This interaction may be weak, capable only of lifting
an electron to a higher
energy orbital from its ground state. This electron eventually descends to its g
round state and the
energy difference between the ground state and the higher energy orbital is emit
ted as
electromagnetic radiation. This type of interaction is known as excitation.
lon/zat/on. A closer interaction of radiation with matter can impart so much ene
rgy to the
orbital electron that it leaves the atom completely. This results in the formati
on of an ion pair (a
positive atom, i.e., ion and a negative electron). This process is termed/on/zat
/oru
We can now discuss the interaction of individual types of radiation with matter.
(/) Alpha-particles have a considerable energy (3-8 MeV). On account of their gr
eat mass,
such a kinetic energy means a relatively low velocity (Just 1% of the velocity o
f light). These
particles therefore spend a relatively long time in the vicinity of orbital elec
trons they approach.
These particles therefore have a high probabflity of interacting with the interv
ening electrons.
Moreover, they have a double positive charge and a great mass, All this ensures
that they will

frequently colllde with atoms in their path. Consequently, alpha particles cause
intense ionization/
excitation depending upon how strongly or weakly they interact with matter. Sinc
e they dissipate
their energy quickly they are not known to be very penetrating. Ordinarily, inte
raction of an alpha
particle with matter will result predominantly in inizatior
(//) Negatrons possess a single negative charge, have a small mass and high velo
cities. They
interact with matter to cause ionization and excitation in a manner exactly simi
lar to that of
particles. However, due to their extreme speed and vanishingly small size, their
probability of
interacting with matter is smaller as compared to a-particles. They are therefor
e/ess/onz/n9 but
more penetmteu3 tho the
Negatrons emitted from a given isotope may have different energies. The reason f
or
negatrons of a given isotope having different energies was explained by Pauli wh
en he stated that
the energy of a radioactive event is shared between a negatron and a neutrino. T
he proportion of
total energy taken by the negatron and neutrino can be different for each disint
egration,
Neutrinus are uncharged, have a negligible mass, and do not interact with matter
. Thus, two
types of negatrons, those having a relatively higher energy and those having low
er energy (soft
-particles) can interact with matter differently, Soj [-par/es may cause more n
than
(//0 Gamma rays are electromagnetic radiation
arge or
mass. Due to the above properties and because
ility of their
interaction with intervening .atoms is rather
great distances
before their energy is dissipated. This makes
with matter in
three important ways which lead to production
trons can in turn
cause ionuion and ex/tatn. (Figure 13.3).

and are therefore devoid of any ch

of their high velocity, the probab
less. These rays therefore travel
them highly penetrating. /nteract
of secondary electrons. These elec

488
Bophysica Chemistry
e- (Compton electron)
; ray (Reduced energy
longer wavelength)
Incident - ray (1.02 MeV) 7 e-(negatron}
7 - raY
180.
Y-raY
0.51 MeV
0.51MeV
Figure 13.3 Illustration of the ways Inwhlch -rois interact with matter
A PhotoelectrcabsOrpthmwheretheTraytmnsfersallltsenergytoanorbtd electron whlchm
oves
Photoe/ctr/c absorpt/on consists of the transfer of the ent/re energy of the rad
lat/on to a
s/ngle electron. The electron is then ejected as a photoelectron which behaves a
s a negatron.
lelat/vely low energy -rad/at/on/ndulges/n such/nteract/ons.
Compton scatter/n is the most common manner/n wh/ch -radlat/on loses its energy.
This tnteract/on results tn only a part of the energy be/rig transferred to the
interact/rig electron
caustng its eject/on. Thee-ray itself gets deflected and travels on with reduced
energy and a longe
wavelength. The ejected electron behaves as a negatron.
For pa/r production the 'rays must have energy greater than 1.02 MeV. The/nclden
t ray
reacts with electrons and is completely absorbed. Eject/on of a positron and an
electron (the
'parr') takes place. The energy of the ray, ff tn excess of 1.02 MeV, is transfe
rred to both the
members of the 'pa/r' as ktnet/c energy. The positron is not long-I/red. It comb
ines with a free
electron and is arm/hflated with -ray em/ss/on.

MEASUREMENT OF RADIOACTIVITY
It is because radiation reacts with matter that we have means to detect it. We h
ave seen how
all mdiatWns cause ionization .and excitation. Both these pes of interactions wi
th matter are

isotopes n Bogy
489
The simplest arrangement to measure radioactivity involves measurement of the
' radiation in agas filled chamber. The number of ionized particles generated
tn the gas by direct interaction (primary interaction) with radiation can be inc
reased several
by various methods discussed below. The photograplc methods of determation f
depend upon ionization action of the rys which makes the grains of sver
to the developer.
The scintillation counters, on the other hand, exploit the excitation caused by
interaction in order to measure radioactivity. Here, the electrons are not made
to
the atom completely, but are merely catapulted to lgher energy levels. When thes
e
return to their ground state, they emit electromagnetic radiation. This light ca
n be/
and the number of such flashes gives an index of the intensity of the radiation.
There can be various motives behind the measurement of radioactivity. In biochem
istry,
paramount reason for determining radioactivity is the wish to determine the acti
vity of a
Here it is important to distinguish between absolute act/v/ty and relative activ
ity.
activity refers to the number of disintegrations actually taking place in the sa
mple,
the relative activity refers to the number of disintegrations accounted for.
measurement of absolute activity is not necessary as the answers to bioclemical
problems
almost always be obtained by measuring the activity of the sample relative to th
at of a
which emits similar type of radiation(s). Obviously, relative measurement is eas
ier
the absolute ones. The standard usually is a known aliquot of the radioactive ma
terial
into the experimental system. For example, the amount of radio-element found in
a
formed by the body or otherwise present in an organ, is compared to the amount
r introduced in the body. Fhe nu. mber of 'counts per minute' (or counts per sec
ond),
with a certain experlmenal arrangement serves as a measure of the relative activ
ity.
:is, however, necessary to maintain the measuring arrangement constant during ea
ch series.
1. Method Baed Upon Ga Ionitlon

Alpha, beta, and gamma radiations have different capabilities of ionization. The
order of
r to induce ionization decreases in the order.
a > fl > (10,000 : I00 : 1)
From the above it becomes clear that although it is possible to detect a and par
ticles by ionization methods, these methods would prove incapable Of detecting-ra
diation and even
B-particles. .....
A. Ionization Chambers
-lonization chambers are not used in biochemistry on a larg scale (moreover, wit
h the
of seintllation counters these chambers have now become obsole). Yet a short
he/r mode of action is required for a good understanding of the-proportional and
counters.
Principle of the ion/zatiOn chamber I/es in the measurement of the number Of lon
g and
produced by the radiation in a as filled chamber. This is achieved by reating an
potential across the chamber by means of two electrodes. The electrons then rush
the anode, while the positive long travel tovrds the cathode. The migration of t
hese
particles gives rise to a current, which can be measured directly with the help
of
can be amplified electronically.
These
negative and positive long formed in the gas chamber attract one another
and Can
leading to formation of neutral atoms and molecules. To avoid this recombination
a

490
Biophysical Chemistry
strong electric field is created which accelerates the particles towards the res
pc., tive electrodes
giving them lesser opportunity to meet and recombine. Above a certain minimum va
lue of the
field strength essentially no recombination takes place, The current through the
chamber is.
then dictated solely by the number of long formed per unit time in the sensitive
volume of the
gas, This 'sammtWn current, therefore, is an unequivocal reflection of the inten
sity of radiation
and hence a measure of the activity, ff the nature of the radioisotope and the e
xperimental
conditions remain constant. When the current obtained is plotted against the app
lied voltage,
the region of saturation current shows up as a long plateau in the value of the
current (Figure
13.4), In practice, ionization chamber is always used in the region of this plat
eau.
Applied field
Figure 13,4 Relationship between the voltage applied and the number of long coll
ected (current], The regions at
whlch.d{ferent counters operate are indltxrted,
B. Proportional Counters
As obvious in Figure 13.4, the plateau discussed above ends ff the field applied
across the
chamber is further increased. Now, a rapid increase in the current is observed.
A new physical
effect, ionization by collision (or gas amplification) is responsible for this i
ncrease. This physical
effect is elaborated below.
The electrons rushing towards the electrodes are involved in several collisions
with
.intervening neutral atoms or molecules during their passage througl the gas. At
each collision,
they lose part or all of their kinetic energy. However, due to the applied field
, they regaln their
energy again, only to lose it again in a repeat collision. This process is repea
ted until the long
finally reach the electrode. At each collision, the primary ion pair produced du
e to radiation
ionizes more gas molecules to produce secondary long which are also accelerated
and which
also collide with other molecules to ionize them. Hence from the original evenJu

a whole torrent
of long reach the electrodes. This is the principle of gas amplification and is
known as the
Townsend avalanche effect after its discoverer. As a consequence of this gas amp
lification the
current flow increases. As is evident form Figure 13.4, in the proportional coun
ter region, the

sotopes n Biology 491

mmber of Ion pairs collected is.directly proportional to the number of primary e
lectrons produced
y the radiation until a certain voltage is reached where a plateau occurs.
Proportional counting has the advantage that particles of different energies can
be
listlnguished by pulse-helght analysis (see later)because the size of the curren
t pulse Is
roportional to the ener, of the original charged particle causing ionization. Th
us, if a sample
s labeled with two different Isotopes, both the Isotopes can be measured by thes
e counters,
,owever, counters operating in this region require a very stable voltage supply
since even small
anges in voltage result in large changes in amplification. Scintillation counter
s can also
ferentiate between different Isotopes and do not suffer, from the disadvantages
of the
proportional counters. It Is very logical then that the former have replaced the
latter.
C. Fundamentals of Geiger Counter
When the voltage supply Is still further increased, the proportional region Is l
eft behind
d the sizes of the pulses cease to be proportional to the number of primary elec
trons or the
01tage applled. The discharge produced by each ray does indeed increase as the v
oltage rises,
t the relative increase is the smaller, the greater the primary ionization. Fina
lly we reach a
where the size of pulse is totally independent of the amount of primary ionizati
on. All
produced by any type of radiation, are therefore nearly equal in magnitude. This
is
as the Geiger region (Figure 13.4) in which the Gelger counter operates.
The basic advantage of Geiger counter is derived from the amplification (each pr
imary ion
yields dose to 100 million secondary ion pairs). The amplification is so great t
hat an
simple external electronic amplifier is sufficient. This simplicity imparts stab
ility to
counters.
The disadvantages of Geiger counters are (/) the necessity of having high voltag
e, and
nability to distinguish pulses due to different physical processes. Sinc
e all pulses are alike,
or -particles give similar pulses, which cannot be distinguished from one anothe
r.

It takes some amount of time for the ion pairs to reach their respective electro
des. During
other ionizing particles entering the tube do not produceionlzation and thus esc
ape
Counter or
Anode
High voltage
Metaltlzed cathode
Window
Path of a
particle

492
Biophysical Chemistry
detection. Consequently, the time that an ion pair takes in reaching the electro
de is know as
the dead time of the Geiger tube. Usually this is about 100-200 s. Upon reaching
the electrodes, the long are neutralized. Some of these tons, however, might "e
scape" and produce their own
ionization avalanche. Thus, if care is not taken, a Geiger-Mfiller tube might gi
ve a
discharge. In order to overcome this the tube is quenched by the addition of sui
table
gas reduces the energy of the long. Some of the common quenching agents used are
ethyl formate and the halogens.
Let us try'and understand the importance of quenching. The tube is usually fille
d with
inert gas (helium, neon, argon) to which has been added a small amount of a
(butane, propane, ethanol, ethyl formate, chlorine, bromine). The quenching agen
t
continuous ionization. If continuous ionization is not prevented, the detector w
ill not
to any other particle except the very first which enters the tube. This is the s
ignificance
quenching.
How does quenching occur? Suppose that the gas filled in the tube is helium.
will give rise to a positive helium ion and an electron. While approaching the c
athode,
probable that the positive helium ion recombines with an electron and becomes ne
utral agair
In helium, such a recombination process is associated with the production of UV
and
X-radiations. Both these radiations are capable of causing ionization and produc
e
ion pairs. The process, occurring again and again, results in a continuous disch
arge. To
this process we need a quenching agent. Suppose butane is present as a quenching
agent.
Butane has a lower ionization potential compared to helium. Now the positive hel
ium
with a butane and physical and chemical properties of butane are such that exces
s
recombination is not thrown out in the form of UV or X-radiations. Instead it br
eaks the
bonds of butane resulting in the destruction of the molecule itself.
From the above it is clear that each quenching event results in the
quencher. The Geiger tube therefore should become useless after some time. This
is
Most commercial tubes become useless after 10s to 101 pulses.
The above limitation of conventional tubes can be addressed by what is called a

Such a tube has entrance and exit ports through which gas is madd to flow contin
uously.
the window here is eliminated for greater sensitivity. This is done to allow
those -particles which have very low energies and cannot enter even a thin windo
w.
other !-particles may also lose sufficient energy in penetrating the
occur if the window is removed and the measurement will have great
This modification is known as the windowlessJlow counter.
Although they are gradually being phased out by scintillation counters, Geiger
are still in use. A few more details about the counter tubes and counter charact
eristics
therefore provided below.
Counter tub. A few popular types of Geiger-Mfiller tubes are illustrated in Figu
re 13.5(B).
The choice of counter tubes to be used depends on the nature of the sample to be
(whether the sample is solid or liquid; whether it is soft or high energy -emitt
er etc).
(i) Solid mll, The end window tube is the most widely used type for solid sample
s,
The thickness of the window can vary according to the isotope being measured (th
in window
mica or mylar for soft [-emitters such as4C; thick window of glass for high ener
gy
such as P}.
(i/ L/quid tp/ Use is generally made of the Annular well, Thin wall dpplng.
L/qu/dflow type of tubes. Since these are all made of glass, they can be used to
measure high
energy -emitters only.

493
Insulated
base
Sample
bead
Insulated
internal .
metal cathode
Insulated
base
Internal.
metal cathode
or
metal window
for
liquid sample

End window
(Glass, Mica etc.)
(i)
(fl)
(fli)
base
cathode
I
Insulated
base

Long glass tube

Anode
'- cMaetolldie efu rface

(iv)
(v)
(vl)
13.5 (B) Different s types of Geger-Mfiller tribes: (0 End-window type; (ii) Thi
n-wall type; (iii) Annularwell type; (iv) Liquld-flow type; (v) Thin-tvall dipping type; (vO Needle probe
type.
(iii)
Spillage. Thin wall tubular type.is the most popular. The tube attached
to a suitable
can be moved along the laboratory table or other surfaces to locate the region o
f
(/v) D/agnost/c. The very thin, needle probe type is in current use to locate im
plants or
of radioisotopes in several types of diagnostic tests.
Although, gasJlow counters which are windowless, can be used to measure soft B-e
mitters,
counters are preferred for such isotopes.
Characteristics
13.6 illustrates a plot of count rate from a given radioactive source against
potential applied to a Geiger-Mtller tube. The characteristic curve at first sho
ws a
hike in counting rate, the starting potential. In this region only high energy B
-particles
tube discharge. This hike begins to taper and finally levels off into a plateau.
The precise
the plateau differs from tube to tube but is generally about 300 volts. If the p
otential
is further raised, another sharp rise in the count rate occurs.. Here the potent
ial
electrodes is so high that spontaneous discharges, not caused by radioactivity,
in the tube. The tube should never be allowed to operate in this continuous disc
harge
since it can lead to serious tube damage.

Region of
Threshold potenti continuous discharge
0
800
I000 1200 1400 1600 1800
Volts
lgure 13.6 The plateau of Gelger-Mller tube: the tube s operated in this regton.
Geiger Tubes: Certain Practic Aspects
Following points should be remembered while counting radioactivit
(0 Background count. A Geiger counter operated even in the absence of a
sample will register some counts. This is known as the background count. This co
uld be du
many factors such as natural radioactivity in the vicinity, nearby X-ray sources
, or
However, most commonly the source of this background count is the circuit noise.
background radiation might be reduced by shielding the instrument with lead. How
ever,
value of background count must always be subtracted from the actual count.
automatically subtract the background count from the experimental value.
(ii) Oomtrle q0"eCt. Steel planchets or ground glass discs are generally used fo
r
solid samples before they are counted with a thin end window tube. Such samples
always be placed in more or less the same position with respect to the tube, oth
erwise
fraction of the emitted radiation entering the tube may vary and may cause large
errors.
geometric effects are higher for soft [3-emitters. Thus, ff the position of soft
t-emitting
such as "C and varies by just one mm the count can vary by as much as 5%. For en
ergy emitters such as P, geometric effect is not significant. Secondly, care sho
uld
to ensure that all the samples have the same surface area.
(No j flmcwltom Consider a sample which is quite thick. The radioactivity from
surfaee will have to traverse the intervening air between the sample and the win
dow.
the radioactivity from its center will have to traverse the solid above it, then
the air and
window before it enters the tube. In such cases, particles emitted below a certa
in
distance are absorbed by the sample itself and never reach the counter. This is
so
soft -emitters like C. This is known as serf absorptlon and the
th/ck. This problem may be partially overcome by making samples as thin as
thin). However, it is best to measure soft 3-emitter by scintillation counting.

2. Photorphi Method
To photography we owe the discovery of radioactivity. The photographic emulsions
nowadays for detecting radioactivity contain 80% or more of silver bromide so th
at lltfle
radiation energy is lost by absorption in the gelatin and the grains of silver a
re in dose

in Biology
495
Ionizing radiations act upon a photographic emulsion to produce a latent image m
uch as
The radiation interacts with the emulsion to produce electrons which reduce
halides to metallic silver. This metallic silver during development shows up as
black
It is possible to measure the absorbed dose of radiation by densitometry of the
blackened
Although photographic method is no more used for counting radioactivity in routi
ne samples
task is increasingly being performed by scintillation counting), it still has a
speciallzed
known as autoradiography. Consider the following example. A radioactive isotope
of
activity has been administered to an organism.Our first objective is to ascertai
n
tissue 'traps' more of administered isotope. A photographic film is wrapped arou
nd the
After a suitable time the film is developed. The blackened area on tire film wou
ld
which has taken up most of the radioactivity (from our knowledge
!anatomy we can easily identify the organ). If the further objective is to deter
mine which
type of cell(s) within the organ is involved in accumulation of the isotope, the
organ
question is excised from the animal and wrapped in a photographic film. The cell
sin the
are not involved in the 'trapping' the isotope would show up as light spots, whe
reas
e cells which take up the isotope would show up as dark spots. If we further wan
t to determine
cell chiefly takes up the isotope, the cells are separated form the organ,
and after some development, chromatographed. The different components will
out on the chromatogram after a suitable time. If this chromatogram is now broug
ht
icontact with a photographic film, black spots will appear onthe film exactly at
the position of
component in which the isotopic label appears.
3. Methods Based Upon Excitation
We have already discussed excitation to be a process where a radiation, rather t
han
an electron, merely catapults it to a higher energy orbital of the atom concerne
d, from
when the electron descends to the ground state, it emits an electromagnetic radi
ation.
known as fluors, when excited, fluoresce. This phenomenon of fluorescence

by radioactivity is known as scintillation. The light emitted in scintillation c

an be 14) which converts the photon energy into
electrical pulse whose magnitude remains proportional to the energy of the origl
nal radioactive
fact that the pulse magnitude is proportional to the energy of the original radi
oactive
is a considerable advantage of scintillation counters over Geiger counters, enab
ling
counters to detect and measure two different isotopes in the same sample.
of Scintillation Counting
Scintillation can be counted by two different techniques (Figure 13.7). In solid
or externa/
counting,the sample is placed close to a fluor crystal (crystallized zinc sulphi
de for
sodium for y-emitters; anthracene for g-emitters), which in turn is placed adjac
ent
photomultiplier is connected to a high voltage supply and a scalar.
sc.intillation counting is particularly usef l for measurement of y-emitting iso
topes, This
since the y-rays are electromagnetic radiation and only rarely collide with neig
hbouring
or excitation. Obviously, the densely packed atoms in a crystal provide
of collision. Or/the other hand, solid scintillation counting is not so satisfac
tory
:weak g-.emitters (3H, C, S) since even the highest energy negatrons have a very
low
The soft gemitters will thus fail to cause sizeable ionization or excitation in
In liquid or internal scintillation counting, the radioactive sample is suspende
d in a
system composed of the solvent and an appropriate scintillator. Liquid
counting is extremely useful for quantitating soft g-emitters.

496
Figure 13.8
Light Ught
cover
Sample
Lead
shieldhng
Sample + fluor
Biophysical Chemistry
Light tight cover

Photomultiplier
High voltage Scalar High voltage Scalar
supply 'supply
Solid scintillation
Liquid scintillation
Figure 13.7 Solid and liquid scintillation counting, a schematic illustr
ation.
Since most of the radioisotopes used in-biology are soft [-emitters (3H, 4C, 35S
), we
discuss liquid scintillation counting in reasonable details. It may, however, be
pointed out
much of what transpires for liquid scintillation counting applies equally well t
o solid
counting.
A. Liquid Scintillation Counting
As said above, the radioactive sample is dissolved or suspended in a scintillati
on
composed of solvent and primary and secondary scintillators. The radiation from
sample molecules collides with a solvent molecule (Figure 13.8} imparting a disc
rete
its energy to the solvent molecule. This transfer of energy is most efficient if
an
is used. Different solvents with differing relative efficiencies of energy trans
fer are listed
Table 13.1. Collision with solvent molecule and concomitant loss of energy
which perpetuate until the radiation has lost most of its energy and is captured

. It
@ e-----O----- e------O-- e
200-aO0 nm
Primary fluor
Secondary fluor -d
330-400 nm
400-480 nm

Energy transfer in liquid scintillation counting and subsequent fluor excitation

, (R) represents radioactive
compound, e" represents emitted H-particle, @ indicates solvent molecule in grou
nd state, and
represents excited solvent molecule

Isotopes n liology 497

-noted that only about 5% of the total radiation energy.is finally observed as l
ight. The rest of the
energy is released as heat. The number of collisions with the solvent molecules
is a function of
the Initial radiation energy. Hence, the amount.of light emitted is directly pro
portional to the
radiation energy.
Table 13.1 Relative Counting Efficiency of Some Representative Solvents for Scin
tillation
Solvent
Relative counting efficiency%
Ethanol
0
Acetone
12
Ethyl glycol d/methyl ether
60
1,4 d/oxane
70
1,3 dlmethoxybenzene
80
Xylene
97
Methox-ybenzene (anisole)
I00
Toluene
100
Data adapted from J. D. Davldson and P. Felgelson. Int. J. Appl. Radial Isot., 2
:1 (1957).
The solvent molecule, which has become excited as a result of collision with the
radiation,
light as it comes back to ground state. This process is known as phosphorescence
. The
is due to the peculiar chemical nature of the solvent. The chemical configuratio
n
with aromatic ring structure is such that they have particular electrons, the which are relatively loosely bound to carbon atoms and can be raised easily to e
xcited
by radiation (also see Box 8.4). The solvent emits light of a very short wavelen
gth which

in the range of 260-340 nm. This range is too short to be detected by most exist
ing
To circumvent this problem, a second molecute is added to the system. This
known as a prtmaryfluor (also called sc/ntt//ators), absorbs light in the range
of 260nm
corresponds to the emission spectrum of the solvent} and emits light of
a longer
(340-430 nm). This longer wavelength light can be measured by most of th
e modem
However, older generation instruments are not sensitive to even this wav
elength
need an even longer wavelength. Such a shift to a longer wavelength can be easil
y obtained
what is calied a secondary fluor. The secondary fluor absorbs light emitted by t
he
fluor and emits light with a maximum in the visible region. Just like the solven
t, the
nature of the primary and the secondary fluors is dependent on their aromatic
and the availability of the -electrons. The structures of popularly used primary
fluor
) and secondary fluors such as bis-MSB and dimethyl-POPOP are given in Figure 13
.9.
CH:CH-H: CH
CH3 bls-MSB CH3
CH3
dlrnethyl-POPOP
CH3
Figure 13.9 Structures of popular prlmo and secondary flwars

498
Biophysical Chemistry
Very small amount of light is yielded by the fluors. Consequently, a highly sens
itive method
of detection is necessary to measure it. Most of the commercially available inst
ruments use
atleast two photomultpliers (for a detailed discussion of photo multipliers, see
Chapter 8). The
photomultiplier converts the light energy into an identical electrical signal th
at can be easily
manipulated and measured.
Advantages of Scintillation Counting
Advantages of scintillation counting over Geiger counting are discussed below.
(/)
It has already been discussed that soft B-emitters are not satisfactoril
y detected by
Geiger counters. Thus, while only 5% efficiency is possible in detecting 3H by G
eiger
counters, 50% efficiency is quite routine with scintillation counters.
(//)
It has been seen in the preceding sections that different types of Geige
r counters are
required for measuring different types of samples. Frequently different kinds of
accessories may also be required. On the other hand, virtually any kind of sampl
e
(liquids, solids, suspensions, emulsions, gels, chromatograms, etc.) can be
accommodated in a scintillation counter and its radioactivity can be determined
accurately.
(iii)
Geiger counters cannot differentiate between radiations of different iso
topes and so
cannot be used to measure dual or triple labeled samples. These kinds of samples
can
be measured easily in a scintillation counter.
(iv)
Since fluorescence decay is very rapid compared to the dead time in Geig
er-Muller
counters, much higher count rates are possible in scintillation counter.
(v)
Sample preparation for scintillation counting is generally devoid of com
plexities and
in any case easier than required for Geiger counters.
Disadvantages of Scintillation Counting
Most of the disadvantages in scintillation counting have been overcome by modifi
cations
in the instrument design. However, Some of the important disadvantages are liste

d below.
(/)
The greatest disadvantage of scintillation counting is quenching. This o
ccurs when the
energy transfer process, described previously, is interfered with at any point.
Because
of the importance of quenching, it is being separately discussed slightly later.
(ii) The cost per sample of scintillation counting is significantly higher than
Geiger counting.
(it0
The high voltage applied to the photomultiplier gives rise to electronic
events in the
system which are not dependent on radioactivity but which contribute to a high
background count. This effect is known as photomultiplier no/se and can be reduc
ed
by cooling the photmultiplier.
Quenching
As pointed out before, quenching means a reduction in the e/lclency of transferr
ing
energy from the -particles to the photomultiplier. In other words, the number of
photons
-reaching the photomultiplier per B-particle is reduced; this results in product
ion of a pulse of
reduced voltage. There are basically three types of quenehtng. They are (/) chem
ical, (ii) color,
and (///) dilution quenching. The three are discussed below briefly.
Chemical Quenching. A sample can at times contain substances which may absorb th
e
B-particles without emitting any photons. This means that these particles do not
excite the
fluor and get 'eaten' up even before they can reach the fluors. Alternatively, t
he sample may
contain substances that do not eat up the B-particles but absorb the photons emi
tted by the
fluors as a result of the latter's collision with the -particles. In either of t
he cases, sufficient

 I00
Isotopes in Biology
499
energy does not reach the detector and a weak pulse may be obtained which is not
a true
picture of tile energy of the radioactive particle. Such a process is called che
mical quenching.
Commonly encountered :chemical quenchers are water, dissolved oxygen, salts, aci
ds,
chlorinated hydrocarbons, and peroxides.
Chemical quenching can often be tackled by increasing the concentration of fluor
s in the
scintillation cocktail.
Co/or Quenching. This results when certain substances in the sample can absorb s
ome
of the photons emitted by the secondary fluor. It is called color quenching main
ly because
substances that are yellow, red, or brown normally cause severe quenching. This
should not be
taken to mean that if there is no color visible in the scintillation cocktail, i
t is free from the
menace of color quenching. The secondary fluor emits in the UV regions also and
color
quenching can result through absorption in that region also. Thus the enduring d
efinition of
color quenching remains absorption of photons from the secondary fluor. This que
nching can
also be reduced by increasing the fluor concentration.
D/lution Quenching. This is just what the title says. This is caused by the dflu
tlon of the
fluor by the sample. High dilution will reduce the probability of scintillation
event
Naturally, this type of quenching normally takes place when working with liquid
samples. It can be corrected by correcting the data obtained for the dilution fa
ctor.
In addition to the above there is another kind of quenching which has been calle
d opt/ca/
quench/ng. In this the sample components do not interfere. Dirty scintillation v
ials may interfere
with light emitted by the sample and prevent its passage to the detector.
Determination of Counting Efficiency
Counting efficiency may best be described by the mathematical relationship
Counts per minute of the radioactive standard

Disintegrations per minute of the radioactive standard

It will be a fallacy to believe that a given amount of radioactive sample will g
ive the same
count rate whatever be the conditions under which it is counted. Samples contain
ing same
concentration of radioactive samples might produce different number of counts an
d this
discrepancy is expressed in terms of counting ejiciency. Counting efficiency dep
ends upon
several factors, some of which are dependent upon the instrument sensitivity, st
ability,
temperature of the counting chamber and the volume of fluid in the counting vial
. All these
factors can be minimized with due care during the experimental set up. However,
the one major
which has to be taken care of, is the quenching (see above) which varies with di
fferent
Since, in practice, the efficiency frequently varies, it is necessary to determi
ne the counting
effieiency before one can compare different samples. A radioactive standard of a
ccurately known
activity is essential for the determination of the efficiency counting. The. use
of radioactive
standards is in principle similar to that of standards in colorimetric or spectr
ophotometric
assays. There are two types of standards (0 fntema/standard (this is usually a 1
3-emitter of
known activity which, when dissolved in unquenched scintillation mixture, provid
es
a reference standard), and (tl) external standard (a y-emitter incorporated in m
ost instruments).
(1) Internal standardization. In this method the first gtep is to count the samp
le. Suppose
that the sample gives X counts]minute. To .this sample a fixed amount of interna
l standard of
known disintegrations per minute is added. Suppose again that the disintegration
s of the internal
standard are Y/minute. The sample is now counted again. The counts that we get n
ow may be

500
Biophysical Chemistry
denoted as Z counts/minute. The counting efficiency of the sample can now be cal
culated by the
relationship
Counting efficiency = (Z - X).x 100
Y
The internal standard used should have the following desirable qualities.
(/) It should be readily soluble in the scintillation mixture.
(i/) The amount of standard used should not cause quenching.
(No It should be of a high specific activity.
(iv) It should be chemically stable.
(v) It should preferably be in liquid form so that small amounts can be dispense
d easily.
Internal standardization is an extremely simple and at the same time reliable me
thod of
determining counting efficiency and thus to correct for all types of quenching.
The drawback of
this method is that it is time consuming (one has to count each sample twice) an
d requires
accurate pipetting of the standard (failure in this might lead to a large error)
.
(ii) terna! standardizatin. The external standard is always a y-ray emitter built
into
the liquid scintillation counter. Its usual position in the instrument is far aw
ay from the
counting chamber. However, when required for efficiency determination, it is aut
omatically
brought adjacent to the counting vial. The y-ray from the external standard pene
trates the
counting vial and gives rise to scintillation.
The activity of the external standard is irrelevant to the preparation of a cali
bration curve
as it is used merely as a source of radioactivity. Its count rate is first deter
mined in each of a
series of quenched standards of known counting efficiency. The count rate of the
external
standard is then related to this efficiency. The external standard count rate ob
tained in a
sample of unknown activity can then be used to determine the efficiency of count
ing for the
particular sample.
With the advent of the scintillation counters with fully automated standard faci

lities, this
technique has become advantageous over the internal standard method in as much a
s that the
counting efficiency for each sample can be determined automatically. Moreover, t
he errors due
to pipetting are not there and the method does not consume tlme. It might, howev
er, be
pointed out that the internal standardization is a more accurate method.
(ii] Channels ratia. Quenching reduces the average energy level of the I-spectru
m.
This decrease in I-spectrum energy increases with increase in quenching. This re
lationship is
utilized in the channels ratio method of determining counting efficiency. Althou
gh a two channel
scintillation counter is preferable for this method, it is not mandatory since e
ach sample can be
counted twice at different channel settings in a single channel apparatus. One o
f these channels
is set to cover the entire unquenched I-energy spectrum, while the second channe
l coves only
half or a third of this spectrum, A sample containing a known amount of standard
(X dpm) is
prepared and counted in both the channels. Let us assume that channel A gives Y
counts per
minute and channel B gives Z counts per minute. The counting efficiency in chann
el A would
then be [{Y/X) x 100]. The ratio of the two channels will be Z/Y., Some known am
ount of quenching
agent is added to the sample and it is recounted again in both the channels. Aga
in the counting
efficiency and the channels ratio is determined. Further addition of quencher is
made and the
sample is recounted in both the channels. After many such additions of quenching
agent and
calculations of counting efficiencies and channels ratio at each stage, counting
efficiency values
are plotted against channels ratio values. This graph known as the channels rati
o quench
correction curve is utilized to determine the counting efflciencies of experimen
tal samples.

Isotopes in BIogy
501
Thus, channels ratio for each sample is determined and the efficiency can then b
e read off the
graph. The curve, however, applies only if the same isotope and scintillator is
used m the
experimental sample. For different isotopes and scintillators different calibrat
ion curves have
to be prepared.
The channels ratio method is satisfactory for all types of quenching. It is also
less time
consuming since only one count is required in two-channel scintillation counter.
Moreover, it is
the most accurate method for cotmting efficiency determination.

500
Biophysical Chemistry
denoted as Z counts/minute. The counting efficiency of the sample can now be cal
culated by the
relationship
Counting efficiency = x 100
Y
The internal standard used should have the following desirable qualities.
(0 It should be readily soluble inthe scintillation mixture.
(i0 The amoun of standard used should not cause quenching.
(i/0 It should be of a high specific activity.
(iv) It should be chemically stable.
(v) It should preferably be in liquid form so that small amounts can be dispense
d easily,
Internal standardization is an extremely simple and at the same time reliable me
thod of
determining counting efficiency and thus to correct for all types of quenching.
The drawback of
this method is that it is time consuming (one has to count each sample twice) an
d reires
accurate pipetting of the standard {failure in this might lead to a large error)
.
(iO xternal standardizagiom The external standard is always a y-ray emitter buil
t into"
the liquid scintillation counter: Its usual position in the instrument is far aw
ay from the
counting chamber. However, when required for efficiency determination, it is aut
omatically
brought adjacent to the counting vial. The y-ray from the external standard pene
trates the
counting vial and gives rise to scintillation.
The ctivity of the external standard is irrelevant to the preparation of a calib
ration curve
as it is used merely as a source of radioactivity. Its count rate is first deter
mined in each of a
series of quenched standards of known counting efficiency. The count rate of the
external
standard is then related to this efficiency The external standard count rate obt
ained in a
sample of unknown activity can then be used to determine the efficiency of count
ing for the
particular sample.
With the advent of the sclnt111ation counters with fully automated standard faci
lities, this
technique has become advantageous over the internal standard method in as much a
s that the
counting efficiency for each sample can be determined automatically. Moreover, t
he errors due

to pipetting are not there and the method does not consume time. It might, howev
er, be
pointed out that the internal standardization is a more accurate method.
(iii] Chtmael ratio. Quenching reduces the average energy level of the Is-spectr
tlm.
This decrease in Is-spectrum energy increases with increase in quenching. This r
elationship is
utilized in the channels ratio method ofdetermlning counting elciency. Although
a two channel
sctntt//ation counter is preferable for this method, it is not mandatory since e
ach sample can be
counted twice at different channel settings in a single channel apparatus. One o
f these channels
is set to cover the entire unquenched IS-energy spectrum, while the second chann
el coveks only
half or a third of this spectrum. A sample containing a known amount of standard
(X dpm) is
prepared and counted in both the channels. Let us assume that channel A gives Y
counts per
minute and c1anne] B glves Z counts per minute. The counting etclency ]n channel
A would
then be flY/X} x 1001. The ratio of the two channels will be 7-.. Some known amo
unt of quenclrg '
agent Is added to the sample and it is recounted again in both the channels. Aga
in the counting.
efficiency and the channels ratio Is determined. Further addition of quencher Is
made and the
sample Is recounted In both the channels. After many such additions of quenching
agent and.
calculations of counting efficiencies and channels ratio at each stage, counting
efficiency values,
are plotted against channels ratio values. This graph known as the channels rati
o quench
correction curve is utilized to determine the counting efficiencles of experimen
tal samples.

Isotopes in BWlogy
501
TIUs, charlllels ratio for each sample Is determlecl ad the ettclency" can then
be read olT the
graph. The curve, however, applles only Lf the same isotope and sctnt-fllator is
used In the
experimental sample. For different isotopes and scintillators different calibrat
ion curves have
to be prepared.
The channels ratio method is Satisfactory for all types of quenching. It is also
less time
consuming since only one count is required in two-channel scintillation counter.
Moreover, it is
the most accurate method for counting efficiency determination.

5O2
Biophysical Chernist3
Sample Iepaation for BeimtIIIatlon Comt
For solid scintillation counting the sample only need. be transferred to a plast
ic or glass
counting vial.
The nature of the sample to be counted dictates the mode of sample preparation f
or liquid
scintillation counting. There is a very large range of scintillation cocktails (
mixtures) available,
each useful for a particular type of sample. Generally, for non-aqueous samples,
toluene based
cocktails are preferred. For aqueous samples, cocktails based on 1,4-dioxane are
in use.
Alternatively, addition of a second solvent to toluene may make toluene-based co
cktails suitable
for aqueous samples. Compounds such as Triton X-100, when mixed with water form
gels
suitable for scintillation counting. Samples may be prepared in such gels also.
A good cocktail
should have the following desirable properties.
(/)
Even after addition of radioactive sample it should be clear, colourless
, and uniform.
However, in certain cases, suspensions or gels may be satisfactory.
(//)
It should have least possible quenching ability. This is even more neces
sary when one
is measuring soft -emitters.
('/)
It should be inexpensive.
(iv)
Its components should be reasonably stable. Its quality should not deter
iorate on
storage.
Contamination of solvents with chemiluminiscent substances might produce large e
rrors
in counting It is therefore necessary that the highest purity solvents and solut
es should be
used in all iquid scinti]lation counting. Keeping the samples in dark before cou
nting is
advisable in cases where chemiluminescence might pose a problem.
Some examples of toluene and 1,4,dioxane based cocktails are provided below.
Cocktail for toluene soluble materials:

503
Isotopes in Biology
If necessary, add 0. Ig POPOP/lltre toluene.
(//) Toluene based cocktail suitable for aqueous samples:
500 ml toluene + 500 ml Cellosolve (2-ethoxy-ethanol) + 5g PPO or 7g butyl-PBD.
If necessary, add 0. lg POPOP or dimethyl-POPOP/litre.
1 ml of water mixes quite homogeneously with I0 ml of cocktail.
(i//) Toluene based cocktail suitable for aqueous samples:
667mi toluene + 333 ml Triton K-100 + 5g PPO or 7g butyl-PBD.
If necessary, add 0. lg POPOP or dimethyl-POPOP/litre.
t ml of water mixes quite homogeneously with 10 rnl of cocktail.
(iv) Dioxane based cocktail for aqueous samples:
100 ml of absolute methanol + 20 ml ethylene glycol + 60g naphthalene + 4g PPO +
0.2g POPOP + 1,4-dioxane to make the volume 1 litre.
3 ml of water mixes quite homogeneously with 10 ml of cocktail.
If color quenching becomes a problem, the sample has to be bleached before count
ing.
Care has to be taken that the bleaching process does not give rise to chnflumine
scence.
A good alternative to bleaching is the combustion of sample. If thelsample is la
beled with
14C, the label can be collected in the form of 4CO2 which can be subsequently co
unted. If the
label is 3H, it can be converted to 3H20 which can be counted.
Alternatively, solid samples, like plant and animal tissues are frst solubilized
in strongly
basic solutions (Hyamine 10-X hydroxide, NCS solubilizer, Soluene, or Protosol).
Once the
solubilization is over, scintillation cocktail is added and the sample can be co
unted. Here also,
chemiluminescence may pose a problem.
If the compounds are separated by gel electrophoresis they may have to be eluted
or
extracted from the gel by slicing, grinding and centrifuging after addition of s
uitable buffer.
The supernatant can then be mixed with a scintillation cocktail.
Radioactive material adherent to solid supports such as glass fibres, discs,
chromatography paper or filter paper may be placed as a whole in a glass vial. A
simple cocktail
is then added to it and the mixture counted. This, however, may not give a good
reproducibility.

Double Isotope Analysis

As mentioned above, scintillation counters make it possible to measure two isoto
pes in a
single sample. It is, however, necessary that the energy spectra of the two isot
opes being measured
ar sizeably different from each other. There are many such pairs of isotopes who
se energy
spectra are very different. For example, 3P and 14C, aH and S, aH and asp, S and
asp, and
even two isotopes of the same element I and I. Figure 13.10 illustrates the prin
ciple of
double isotope analysis. It is evident from the figure that the energy spectra o
f two isotopes (A
and B) are almost non-overlapping. For dual isotope analysis, it is desirable to
have a two
channel apparatus. The trick is to set one pulse height analyzer to reject all p
ulses of energy
below W (threshold W) and above X (window X) while the second pulse analyzer is
set to reject
pulses of energy above and below Y and Z respectively. The two ch.annels (a puls
e height analyzer
consisting of a threshold and window is known as a channel) will now measure the
isotopes
A and B simultaneously. Alternatively one can make do with a single channel scin
tillation
counter, with the channel set differently each time, and measure one isotope at
a time.

Isotope A
'
I
Isotope B
I
Pulse energy
Figure 13.10 Diagrammatic ustmtlon of the p of double ,botope anolyss.
USE OF STABLE ISOTOPES IN BIOLOGY
Since radloactlvlty Is a magnltude so easlly measurable, use of radloactlve isot
opes has
galned an upper hand over the stable Isotopes tn blology. However, there exist n
o radloactlve
isotopes! with a long enough life tlme (half-llfe) of such Important elements as
nltrogen and
oxygen. Thus, studles Involvlng Oxygen and nltrogen have to use the stable isoto
pes of these
elements rather than the radloactlve ones.
Although measurement of stable isotopes is more cumbersome than that of the
radloactlve isotopes, It can nevertheless be performed with a very high efflclen
cy. The property
of Isotopes havlng different atomic masses by vlrtue of their havlng different n
umber of neutrons
Is ut111zed In the measurement of stable isotopes. The technlque of choice is ma
ss spectromet.
Sltrometay
Mass spectrometry involves Ionlzatlon of the parent molecule to glve poamt on an
d fragmentatlon of these Long to glve fragment ons. Thls Is achleved by bombardt
ng the
sample with electrons. All the long produced have a posltlve charge. These Long,
whlch possess
a certaln amount of klneUc energy, If subjected to a magnetlc field, wfllbe defl
ected to dlfferent
degrees depending upon their mass/charge (m/e) values. Thls Is the basls of sepa
raUon by mass

Ionization
chamber
Amplifier
reservoir
Recorder
fsotopes tn Bk>logy 505
spectrometry, lf the charge is kept constant, the long are deflected by agles wh
ich are inversely
proportional to the square roots of the masses of the long.
The trick in determining the relative abundance of an isotope label is to prepar
e the long
in such a manner that all the long have a singl positive charge, Le., only one e
lectron is
knocked out per atom (there are exception to this rule; many times an ion loses
another electron
spontaneously). The long produced then will differ only in
e being equal,
the mass]charge value, which is the basis of separation of
ometer, will
depend only on the mass of the ion which is the only basis
ng. The mass
spectrometer will then be forced to separate the different
e basis of their
masses.

their mass. The charg

long in a mass spectr
of difference remaini
isotopes solely-on th

The separation of long according to their rn/e values is as follows. They are ac
celerated to
a constant velocity in a vacuum by a series of negatively charged electrostatic
plates and are
then deflected from their original trajectory by a magnetic field (Figure 13.1 I
). For long carrying
the same charge the amount of deflection is dependent on the mass of the ion, so
that the long
of smallest mass undergo the greatest degree of deflection. By varying either th
e magnetic, field
or the accelerating voltage the ion trajectories may be varied so that fragment
long of a given rtY
e value impinge on the collector slit of the detector. The number of long of a g
iven mass impinging
on the detector is a measure of the abundance of that particular ion,

Electrostatic
field
trajectory

Light
long
: field
long

Towards
vacuum pump
Fure 13.11 Basic components of a mass spectrometer
Falling Drop Method for Deuterium Measurement
Deuterium is the stable heavier isotope of hydrogen and the density of "water"
deuterium, instead of hydrogen, is 1.10764 compared to water at 25C. Thus. the
of fall of a drop of water containing deuterium (heavy water) of standard volume
through
given height of a suitable immiscible liquid of slightly lower density would be
slightly more
that of normal water. This phenomenon is utfllzed to measure the deuterium conte
nt of a

506
Biophysical Chemistry
given biological sample. (Refer chapter on viscosity for mathematical treatment
of the
phenomenon and instrumentation).
There are very few liquids immiscible with water that have both the proper densi
ty and
viscosity to make them suitable for this purpose. A single pure compound is nece
ssary so that
there will be no change in the density due to differential evaporation. The liqu
ids that have been
satisfactorily used are ortho-fluorotoluene and meta-fluorotoluene; the former i
s used at 26.8C
and the latter at 19.8C. With both these liquids, the relatlon between the rate o
f fall and
density appears to be linear upto about 3% D20; above 7%, the departure from lin
earity becomes
significant and the method fails with these liquids. However, the water samples
from biological
systems usually have low deuteriarn contents; samples with higher deuterium cont
ents can be
suitably diluted.
Density Gradient Centrifugation
If biological samples containing the heavier stable isotope are.sedimented throu
gh a density
gradient (usually CsCI) they will sediment faster than the sample containing the
lighter isotope.
Thus solutions of CsCI have been used to distinguish between DNA with and withou
t 15N in its
molecular structure.
THE TRACER TECHNIQUE
What is tracer technique?
The reason behind biochemists' penchant for introducing radioisotopes in a syste
m is,
that while such labeled compounds remain chemically identical to slmilar compoun
ds within
the system with which they will mingle, they differ from unlabeled compounds in
as much as
that they emit easily measurable radiation or have slightly different masses. A
biochemist can
thus keep tabs on such labeled compounds and trace them in a system at a given t
ime. If such
compounds are metabolized, they will hand over the radioactive or stable isotopi
c label to the
next metabolite in the series, i.e, the pathway, and provide the biochemist with
a unique
possibility of tracing their history and thaLof their successors through a bioch
emical pathway.
This is the basis of tracer studies and/the experiments involving this principle

are known as
tracer experiments.

5O8
Biophysical Chemistry
General Tracer Requirements
The tracer studies in general consist of :
() Preparation of a labeled compound
(//) Introduction of the labeled compound into a biological system, and
(i//}
Separation and determination of labeled species in various biochemical f
ractions at a
later time.

in Biology
There are, however,
509
certain criteria which the label should satisfy before results
be correctly interpreted. Some of these criteria are listed

(i) The starting concentration of tracer must be sufficiert enough to withstand

dilution in the
of metabolism. Suppose that we are using a I+C labeled carbon dioxide to determi
ne the
of carbon dioxide during fermentation of glucose. The organism chosen is yeast.
Suppose
sample of labeled CO2which contains 5mg as Carbon, to an actively fermenting yea
st
During the course of experiment, through fermentation of glucose, 2000 mg of car
bon
the form of carbon dioxide evolves. The original sample of 14C02 (5 mg) has now
become
) times. This dilution poses two problems. Firstly, as the time elapses the radi
oactive
progressively dilute and if the initial concentration was not sufficient enough
stage might be reached where assay of radioactivity becomes a problem (radioacti
ve
are very sensitive and this problem is therefore not so important). The second
is of much importance. Let us review the above situation. The 4C label has becom
e
400 times. What thiSactually means is that for every 400 unlabeled molecules, on
e
molecule will take part in a metabolic reaction. If the level gets further dilut
ed, its
in the metabolic reaction would be so little as to be almost negligible. This is
since no information about the metabolic reaction will then be afforded by the
(ii) Throughout the metabolic pathway, the label must retain its ppsition in the
portion of the
in which it was originally incorporated. The 'active' hydrogen of alcohols, amin
es and
rapidly exchanges with hydrogen long in the surrounding aqueous media. Similarly
,
exchanges between two positions of the carboxyl group. Thus, if a compound is la
beled
such positions, it might lose its label before it undergoes a metabolic reaction
(Figure
The tracer will therefore yield no useful information about the metabolic pathwa
y. Of

if one wants to study exchange reactions, it is these positions one should isoto
pically
Figure 13.12A compound labeled in an atom which can be exchanged, loses its labe
l quickly.
liii) The half-life of the tracer isotope sho/hld be sufficiently long. Dependin
g on the length of
II
experiment, the tracer isotope of a givenlelement varies. Thus, while C is usefu
l for shortexperiments, it might not be very Useful for longterm experiments. "C loses its
by a factor of about I000 in as short a time as 3.5 hours. In long term experime
nts,
if one has introduced a compound labeled with "C, one might not get any useful
tracer has been removed through decay. On the contrary, ff one has used
label (half-life 5000 years), it will have suffered no diminution in its activit
y even after a long
Such labeled compound will then afford much information about the desired metabo
lic
The sensitivity of tracer methods is their most significant advantage over all o
ther chemical
physical methods. As an example let us consider the following. The specific acti
vity of

510
Biophysical Chemistry
carrier-free tritium is about 50 curies per millimole. This implies that a triti
um labeled compound
can be detected even ff it is diluted 1012 times. Many metabolic substances are
present in
tissues at such low concentrations that the most sensitive chemical method is un
able to detect
them. Radiotracer techniques can easily detect these substances. Whatever we
is true for stable isotopes also, since mass spectrometry is an extremely sensit
ive technique
and extremely small concentrations of stable isotopes can be assayed with relati
ve ease.
Another major advantage of using radiotracers (stable isotopic tracers are
that the studies can be carried out on a living organism.
Lim/tations of Tracer Experiments
There is no known technique without restrictions. Thus, certain restrictions abo
ut th,
of (radio) isotopes have also to be appreciated. Although, being chemically simi
lar the
are expected to undergo the same reactions, they might do so at different rates.
This effect is
termed as the ktnet/c/sotope effect. These different rates are more or less prop
ortional to the
differences in mass between these isotopes. One can, therefore, understand that
the isotope
effect would be considerably more pronounced in the case of isotopes of light at
oms, for example
hydrogen (all, as is obvious, is three times heavier than IH). As compared to th
is, the effect
would be smaller in the case of 2C and 14C, and almost non-exlstent for alp and
ap.
Although of lesser importance than the kinetic isotope effect, one should
that the equ///br/um/sotope effect also constitutes an important restriction on
For example, distribution coefficient of deuterium between water and gaseous hyd
rogen
25C has a large value of 3.7, whereas that for tritium is merely 6.3. The heavier
isotope
therefore more concentrated in water.
Another important restriction is with respect to the activity of the isotope use
d.
The activity should be so chosen that it is the minimum necessary to permit reas
onable
rates in the sample to be analysed. (one has to consider the dilution the activi
ty would
in the tissue). If an unnecessarily large activity is administered, the tracer m
ay
from the experimental organism thereby distorting the results.

When a tracer is added to a biological system, the normal chemical level of

compound in the system is automatically exceeded. We know that the metabolic
different concentrations of a given metabolite is different. Results obtained wi
th
experimental set up are, therefore, never unquestionable.
Box 13.5 shows howresults of radiotracer experiments can be misinterpreted.

512
LABELING PROCEDURES
Labeling procedures must be given careful consideration if full potential of the
tracer
technique is to be taken advantage of. The consideration to be given can be grou
ped under
three major categories : (I) the system giving ceils/cell free systems) being la
beled, (2) the chemical
nature of radioactive material, and (3) the labeling format.
System Being Labeled
Supposing you are using a bacterial culture to study replication or transcriptio
n. The
label you are using is 3H and incorporation of 3H-thymidine or 3H- uridine is yo
ur index of
replication or transcription respectively. You must see to it that the culture i
s In a state of
balanced growth throughout the course of your experiment. Since your aim is to s
tudy
replication, the culture must not be in a stationary or senescent phase. That wi
ll defeat the
whole objective as replication will either not be taking place at all or will be
taking place at a
rate that won't be much. It is better if the culture is in the log phase. Moreov
er, you must

Isotopes in Biology
513
ensure that the culture will continue to be in the log phase even beyond the tim
e-scale of your
experiment. This is to ensure that throughout your experimental course the growt
h situation
will remain the same. Moreover, you must ensure that the culture is not making a
transition
from lag to log phase during your experiment. For obvious reasons, this will ind
uce large scale
errors in your experimental observations.
Of course, if your objective is to study the effect of senescence on transcripti
on, or what
happens to transcription during the transitions, then you must choose these time
s deliberately.
In short, you must choose the system in a phase that fits your requirements and
that the
system continues to remain in that phase for at least the period of your experim
ent.
Chemical Nature of the Radioactive Material
Different cells have different capacities to take up the extracellularly provide
d label. For
example a given cell may be completely unable to take up tritium labeled uridine
, another cell
may take it up readily, and a third cell may convert it to uracil and then take
up uracil. In the
first case the experiment will fail as no incorporation of uridine in the RNA wi
ll take place. In
the second case the experiment may result in a linear incorporation of the label
and in the third
case a lag period may be there before incorporation becomes linear; the lag time
will correspond
to the time that has been spent by the cell to convert extracellular labeled com
pound into a
metabolite that it can take up. It may be a better idea to provide labeled uraci
l in the third case.
Labeling Formats
By labeling formats we mean the ways of labeling cellular constituents. There ar
e
basically three different labeling formats and each yields a qualitatively diffe
rent information.
It cannot therefore be overemphasized that depending on the kind of information
needed, one
should choose the format that is apt. These formats are known as pulse labeling,
equilibrium
labeling, and pulse-chase labeling.
Pulse labeling. Pulse labeling is done when the rate of synthesis of a particula
r
molecule is to be studied. Let us try and understand the method.

Let the objective of our study be transcription in a prokary0tic system. Let us

take the
system to be an operon. Let us assume that the operon is inducible, i.e., it is
normally not
transcribed, but transcription can begin if an inducer is added to the medium. L
et us assume
that the transcription begins immediately upon addition of the inducer. Let us a
lso assume
that initially only one RNA polymerase can sit on the operon. However, after 3 s
econds of
transcription by the first RNA polymerase, enough space is available for a secon
d molecule of
RNA polymerase to sit on the operon and begin transcription. Let us suppose that
a maximum
of 10 such RNA polymerases can sit on the operon. After a polymerase reaches the
end of the
operon, it falls offand releases free RNA into the cell. Lastly, let us presume
that the synthesized
RNA is not being degraded (this of course doesn't happen; but for the sake of un
derstanding
theae assumptions are necessary).
The experimental design is quite simple, The inducer is added to the culture. Th
e time of
.addition of inducer is taken to be 0 time. Subsequent to this, aliquots from th
e cnlture are
transferred to vessels containing small amounts of very high specific activity s
ubstrate and
allowed to incubate for a short time. The period of incubation is terminated by
killing the ceils.
The molecule being studied is then purified from the killed cells and the radioa
ctivity
in these molecules is measured (in our model the molecule is RNA and the label i
s
The radioactivity incorporated is then plotted against time. Here it must be
clearly that the transcription has' started after addition of inducer. However,
only
or part of RNA, will be radioactively labeled where the culture has spent time i
n the
containing the label. RNA molecules synthesized before the aliquots of culture w
ere
transferred to the vessel containing the labeled substrate, do not contain the l
abel.

30 sec
9
10
514
Biophysical Chemistry
Let's now see what happens during the experiment. Suppose that the first aliquot
was
transferred to a 3H-uridine vessel at 0 time and allowed to incubate for 3 secon
ds. At 0 time
only one polymerase can sit on the operon and therefore DNA is being transcribed
by just the
one enzyme at this stage.Suppose that 1 unit of RNA is made in 3 seconds and tha
t one unit of
RNA corresponds to 20 cpm. Therefore when the cells are killed, RNA isolated and
counted for
radioactivity' we would get 20 cpm. The second aliquot is transferred to the 3Huridine vessel
at 3 seconds and allowed to incubate for a further 3 seconds. Remember that when
the first
polymerase has spent 3 seconds on the operon, enough space has been created on t
he DNA
that a second polymerase can sit on it. Thus now, in this aliquot, two polymeras
e molecules are
synthesizing RNA. Since incubation here too is 3 seconds, 2 units of RNA are mad
e ( 1 unit each
by each. polymerase sitting on the operon) and when we kill the cells and count
radioactivity
incorporated in the RNA we should get a count of 40 cpm. Thus the rate of RNA sy
nthesis in
these 3 seconds is double that of the first 3 seconds. Remember that in this ali
quot the first
polymerase had already made one unit of RNA. But since the ceils were not transf
erred to the
radioactive vessel, this RNA is not radioactive (Figure 13.13 ). In the third al
iquot, which is
transferred to the H-uridine vessel at 6 seconds and allowed to incubate for 3 s
econds, 3
polymerases will be making RNA thus synthesizing 3 units of RNA incorporating 60
cpm. The
rate of RNA synthesis here is triple that of the first aliquot.
3 see
6 sec
Figure 13.13. Pulse labeling. Horizontal line represents the operon. Slanting li
nes are the mRNA. Solid slanting lines
represent radioactive portions of the mRNA while the dashed slanting lines refer
to the non-radiooctWe
portions. Between each slanting llne the time is constant ( in the case dlscsed
in the text, 3 seconds).

This logic Can be repeated 10 times because 10 polymerase molecules can sit on t
he
operon at a time. During the period between 27 and 30 seconds. 10 polymerase mol
ecules are
itting on the operon. 10 units of RNA will be made in the 3 seconds of incubatio
n and the
count be 200 cpm. The data obtained so far, if plotted Gill give rise to a linea
r relationship (Figure 13.14 ).
Let's now see what happens during the period between 30 and 33 seconds. The firs
t
polymerase that sat on the operon at 0 time will now fall off and a new one Gill
sit at the
beginning of the operon. Thus the totaJ number of polymerase molecules on the op
eron Gill
continue to be I 0 ( it will continue to be 10 from now on no matter at what tim
e we study the
system) and the count of RNA synthesized will be 200 cpm only. This means that w
eare going
to obtain a plateau now and that maximum rate of synthesis has been achieved.

515
24O
Seconds
2OO
120 I
6
12
18
24
30
36
42
lgure 13.14. Chart obtained with pulse labeling experiment as described in the t
ext.
It should be clear from the foregoing that pulse labeling monitors the rate of s
ynthesis of
a given molecule. Note that as the labeling period approaches zero, the observed
rate will approach
the true instantaneous rate of synthesis {ds/dt), This iS the reason why the lab
eling period is
kept as short as possible.
It must be noted that the period of labeling is decided by the half life of the
molecule being
labeled. Normally, it is no longer than I0 % of the half life; it may be less. T
hus ifa mRNA has
a half life of 5 minutes, the labeling period will be just 0.5 minutes or less.
F.uilibrium labeling. Equilibrium labeling measures th accumulation of a molecul
e
being synthesized. The experimental design is substantially different.
The system being studied is first incubated with large amount of low/moderate sp
ecific activity radioactive material (in this case 3H-uridine; we will continue
with our previous model
0fthe operon and synthesis of mRNA}. The incubation is continued till the precur
sor pool inside
the system has reached a constant specific activity. Once this has been achieved
, the inducer is
added to start the experiment. Thereafter, at specific intervals of time, alic!u
ots are removed
from the system, the ceils killed, the molecule purified, and the radioactivity
incorporated assayed.

Let us see what will be our results with this kind of labeling if we continue wi
th our
previous model. Supposing each aliquot here too is being taken after intervals o
f 3 seconds.
The first aliquot will give 20 cpm as only one unit of RNA is made by one polyme
rase. The
second aliquot, however, will not contain 40 cpm as in pulse experiment, but 60
cpm. This is so
because the first polymerase molecule has made 2 units of RNA in 6 seconds while
the second
polymerase molecule has made I. Total is 3 units and therefore 60 cpm. Please no
te that tn
equilibrium labeling, all the RNA made will be radioactive unlike the pulse expe
riment where
only that RNA was radioactive which was synthesized during the time the system w
as exposed
to short pulses of radioactivity (Figure 13.15 ). Thus, in the equilibrium exper
iment, the initial
increments in radioactivity counts at each time interval will be exponent/oL Thi
s situation will
till the end of 30 seconds. After that, the amount of RNA synthes.ized between 3
0 and
3 seconds will be the same as that synthesized between 27 and 30 seconds. From t
his point
on the increment in cpm will be linear (Pigure 13.16 ).

516
Seconds
Bphysa:al Chemistry
3 sec -6 sec 30 sec
ljure 13.15. Equilibrium labeling. Horizontal line represents the operon. Slanti
ng lines are the mRNA. All slanting
lines represent radioactive RNA. Between each slanting line the time is constant
( in the case discussed
In the text, 3 seconds).
Figure 13.16. Chart obtained with equilibrium labeling experiment as described i
n the text.
Two things must be noted here, It is absolutely necessary that the precursor poo
l of the
metabolite reaches constant specific activity. If this doesn't happen, large-sca
le errors will result.
Therefore, the preincubation with the label is a critical step in this experimen
t. Secondly, we
have assumed above that no degradation of the molecule being studied is taking p
lace. In
reality, this is never the case. The data obtained therefore, must be corrected
for the
degradation that is taking place. "ltLe-cha, t labeling. In reality this is just a variation of pulse labeling. Thi
s format
involves giving the cells a short pulse of high specific activity substrate and
then 'chasing' this
with non-radioactive substrate (or dflutlng the radioactivity -1000 fold). This
is followed by
sampling the labeled culture at various times after the .radioactive pulse was i
nitially given.

Isotopes in Biology
517
Although just a variation, this format gives a qualitatively different result :
it tells us about
precursor-product relationship.
Let us understand this with a famous experiment. Decades ago it had become known
that
the two anti-parallel strands of the DNA duplex were simultaneously replicate. H
owever, how
this could be achieved was not understood. All known DNA polymerases can only ex
tend the
DNA in 5' - 3' direction. This would mean that only one strand could be replicat
ed at onetime.
The evidence obtained with electron micrography and other techniques pointed to
the fact that
this was not the case. Both the strands were replicated together. This perplexin
g situation was
addressed by Reiji Okazaki in 1968.
In his initial experiments Okazaki pulse labeled a growing E. coli culture for 3
0 seconds
with !H-thymidine. Subsequent centrifugation of DNA under alkaline condition rev
ealed that
mucli of the radioactive DNA ( radioactive and therefore newly synthesized, as p
er the logic of
pulse experiment) had a sedimentation coefficient between 7S and 11S. This small
value can
only be given by very small DNA fragments. These small fragments subsequently ca
me to be
known as Okazaki fragments.
In another experiment, following the 30 second pulse, the E. coli were transferr
ed to an
unlabelled medium (chase). After sometime when the centrifugation was carried ou
t, the
sedimentation coefficient of the radioactive DNA was much larger. Also, the sedi
mentation
coefficient values increased with the time the cells were grown in the unlabeled
medium.
The inference was clear. The short DNA fragments observed earlier must have beco
me covalenfly
incorporated into larger DNA molecules.
Okazaki interpreted his experimental results in terms of the semidiscontinuous
replication model (Figure 13.17).
Motion of replication fork
5
,
I1 1 IV)kill IlY'a
5'
"
Parental strands

ljure13.17. In DNA replication, both daughter strands (dashed lines) are synthes
ized in their
5" -- 3' direction. The leading strand ts synthesized continuously, whereas the
lagging strand is
synthesized discontinuously.
The two parent strands are replicated in different ways. The newly synthesized D
NA strand
that extends 5' 3' in the direction of replication fork movement (leading strand
) is essentially continuously synthesized in its 5' - 3' direction as the fork a
dvances. The other newly synthesized
sfrand (lagging strand), is also synthesized in 5' - 3' direction, but discontin
uously as Okazaki
fragments. Sometime after, the Okazaki fragments are covalenfly joined together
by DNA ligase.
Synthesis and Secretion of Zymogens in Pancreas
In an experiment designed to follow the synthesis and secretion of zymogen, a
radioactive amino acid was injected into the pancreas of a guinea pig to label p
roteins
undergoing synthesis (pulse). Since most proteins in pancreas are synthesized as
zymogens,
the radioactive amino acid would be incorporated primarily in zymogens. Three mi
nutes after
the pulse, all incorporated radioactivity was found to be present in the rough e
ndoplasmic
reticulum (ER).

granule
complex
518
Bphysical Chemtry
After three minutes, large amount of non-radioactive amino acid was injected int
o the
pancreas (chase). This ensured that only the zymogen synthesized in the first th
ree minutes
would retain radioactivity. All other proteins, including zymogens, formed after
the first three
minutes would be almost non-radioactive. Thus one could follow the migration of
the earlier
synthesized radioactive zymogen.
Seventeen minutes after, most of the radioactivity was found in rough ER which w
as near
the Golgi complex. 117 minutes later, radioactivity was found neither in ER, nor
in Golgi bodies,
but in discreet vesicles near the plasma membrane called zymogen granules. That
these vesicles
contained zymogen was proved by isolating these radioactive vesicles and chemica
lly proving
the presence of zymogens within them. These zymogen granules later fuse with the
plasma
membrane surrounding a duct and release their contents into the duct.
Thus with the help of pulse-chase experiment the manner of synthesis and secreti
on of
zymogens can be ascertained. It appears that these zymogens are synthesized in t
he rough ER.
From there the proteins move into the Golgi bodies. Here they are concentrated a
nd packaged
into the zymogen granules. The granules then migrate toward the plasma membrane
and fuse
with it releasing their contents (Figure 13.18).
Lumen of a duct
Rough
retlculum
Figure 13.18. The synthesis and secretion of zymogens as determined by pulse-cha
se labeling.
USES OF ISOTOPES A TRACERS IN BIOLOGICAL SCIENCF.
Oufl/ned below are some applications of tracer studies in biological sciences. H

owever,
owing to the vast number of applications and complexlty of the exper/mental deta
ils, only a
br/ef treatment is possible.
Distribution Studies
The study prlmarfly consists of (0 admin/stering a radioisotope to a system or o
rgan/sm,
and (//) localization of the isotope by detection of radioactivity in dLfferent
parts of the system or

Illll
Isotopes in Biology
519
organism at a later time. Such studies can be made quantitative and the time-cou
rse of
distribution can be determined. The distribution may be detected at various leve
ls of
organization. The level of detection might be macroscopic (e.g., a whole organ),
microscopic
(e.g., sub cellular particles such as the mitochondria, ribosomes, or the nucleu
s), or molecular.
A wide array of applications of distribution studies can be cited in biological
and
medicine research: (0 Iodine is an important dement in thyroid physiology. 3I ad
ministered as
NaI has provided much information .about thyroid physiology. Thyroid functions h
ave been
measured by assaying the uptake of3q by the thyroid gland and the appearance of
the isotope
in saliva and the urine (Figure 13.19); (W Malignant tissues tend to have an acc
elerated
metabolism and hence take up more phosphate than the normal tissues. Consequentl
y ifP is
injected intravenously to a patient, after a particular time more .2p will be ac
cumulated in the
tumor, if there is one. This can be found by placing a photosensitive emulsion i
n contact with"
the tissue. The malignant tissues will show up as a spot blacker than the normal
tissues,
'"" " "
fill IIII
I
I
fill IIIIIIIIiiiii
ilil lllllill III IIIHIli]
tllll IIIIIlillllll
IIIII illltllH I'll
illii
Ifllillllllll
IIIII
IHIIHHIIII
IHI
IIIIIIIglill

ilfllHIll
IIIII
IIIIIIIHIIH
IIIIIIImi
illii
liiIIliliii
liIlilI/lliI
Sternum
Figure 13.19 Thyroid sctntkjram. Subject was given an oral dose of 75 mlcrocurs
of l 24 hours before. More than average distribution of I on the left b'Imtes a
normal left thyroid be. However, there s no on the r@ht side where the r@ht lobe
should have been. Thls shows that the rlght ttujrold
Isotope Dilution Studies
Radioactive isotopes are olten used to estimate the amount of a single substance
in a
mixture or even the volume of system (e.g., blood, lymph, cerebrospinal fluid et
c.). The technique
known as isotope dilution technique is especially useful when it is impossible t
o quantitatively
separate the given substance from a mixture or, in cases of fluids like blood or
lymph,
to completely drain the liquid out in order to measure its volume. The technique
involves
(a) introduction of a radioactive isotope of known specific activity into the sy
stem, (b) isolating
a small amount of this compound from the system after equilibration has been ach
ieved, and
(c) determining the specific activity of the isolated compound. From the specifi
c activity of the
compound isolated and the prior knowledge of the specific activity originally ad
ded, the quantity
of the non-radioactive substance in the given mixture may be calculated as shown
below.
Ao
= Counts per minute of the tracer added (care must be taken that the amo
unt of
radioactive cbmpound added (M is very small as compared to the a
mount of the
non-radioactive isotope in the system).
M u = Unknown amount of non-radioactive compound in the system.
A = S.A.u = Specific activity of the compound in the system after addition ofradi
oactlve
Mu isotope.

520
S.A.u =
S.A.r.
Bophgs.oX Chemtry
Mr -- Amount of pure compound reisolated from the system after equilibration wit
h the
radioactive isotope.
A r = Counts per minute of the re-isolated compound.
lr
M--- = S.A.r, = Specific activity of the reisolated compound.
However the specific activity of the compound in the mixture will be the same as
in the
reisolated compound. So,
S.A.r or A
=
Mu
Mr
.'. M = Mr orMu
=
..
" Ar S.A.r
It will be seen from the above that in all the mathematical relations, Mo has be
en totally
ignored. This is so since it has been assumed that the tracer added is of very h
igh specific
activity and consequently of an insignificant weight as compared to the non-radi
oactive
counterpart in the system. This is a valid enough assumption since most of the c
ompounds of
biological interest are avRilable in radioactive form with very high specific ac
tivities.
However, in certain cases where compounds of high specific activities are not ea
sily
obtainable, the amount of radioactive tracer added becomes significant and M has

to be taken
into consideration.
The equation (1) willthen take a slightly altered form.
Mu = Mr Ao Mo or IVL " Ao Mo

...{2}

Ar
S.A.r
As M0 becomes small, the equation (2} will reduce to equation (1}.
Let us now concern ourselves only with the specific activities. It is easy to se
e that the
specific activity of the re-isolated tracer is going to be less than the specifi
c activity of the tracer
added. This decrease will be higher if there is more amount of non-radioactive c
ompound present
in the system and lower if a small amount of non-radioactive compound is present
. In other
words, the degree of reduction in the specific activity is directly related to t
he amount of non
radioactive compound present in the system. Or, the dilution of specific activit
y is identical to
the dilution of the tracer added.
S.A.r
Mo
Dilution =
S.A.o
Mo +Mu
S.A.r.Mo + S.A.r.M = S.A.O.Mo or
S.A.r.Mu = S.A.o.Mo- S.A.r.Mo
S.A.o.-S.A.r.Mo
t, S.A.r ) --'t, - S.---'',r )

Isotopes in Biology
521
Mo
, S,A.r
There have been many applications of isotope dilution analysisin biochemistry an
d
medicine. Studies include measuremertt of erythrocytc volume with sCr labeled
erythrocytes; measurement of plasma volume with I labeled serum albumin; measure
ment
of body water with 3I labeled iodo-antipyrene or trltiated water; measurement of
extracellular
space with Br; measurement of total bbdy pools of cholesterol, urea and uric aci
d with
and sodium and potassium pool measurement with Na and 42K respectively.
Membrane Transport Stud/es
The classical method for the study of membrane permeability was limited in its a
pplication
with respect to both the cell types that could be studied and the materials whos
e diffusion
constants could be determined. Organic non-electrolytes which were not readily m
etabolized
were the only compounds whose permeability could be studied (such studies were n
ot of much
use anyway as these compounds were not biologically significant}. The use of ele
ctrolytes, on
the other hand, required such high concentrations in the medium that the possibi
lity of altering
the permeability of the plasmaimembrane was always present. Availability of radi
oactive isotopes
changed all this. Transport of electrolytes and of organic substances that are n
ormal cell
became subject to valid experimental approach because the quantity of tracer
substance necessary was so minute that the concentration in the medium was almos
t unaltered.
Transport of long into the tissues in the body has been extensively studied with
the help of
isotopes. Some examples are provided, (0 Studies with 32p have shown that in the
liver, kidney
and in the erythrocyte, the formation of ATP on the cell surface is the principa
l mechanism for
the entry of phosphate into the cell interior, (tO 45Ca, 24Na, and 4K have been
used to study the
of these elements through the plasma membrane of various cells. This is an impor
tant
since ionic fluxes are known to have a profound effect on cellular metabolism.
Thymidine permeability in bacteria was tested using radioactive isotopes 32p and
3H. The

is relatively simple. Incubate either a single or double labeled thymidine with

bacterial
the bacteria are harvested, washed and collected by filtration.
has entered the cell, the radioactivity will be retained on the filter since the
cells
been retained upon it. If thymicine has not entered, the radioactivity will be f
ound in the
In this experiment it was found that radioactivity was retained on the filter.
Should the above result be taken to mean that thymidine as a whole is permeable
to the
membrane? Not necessarily. It could also have been that the bacteria were breaki
ng
thymidine into thymine and phosphate and then taking up both the components, How
one test this possibility?
.Label thymldine with P and do the above experiment. After the cells have been
purify thymidine and see whether it is labeled. If so, thymidine itself is perme
able. if that is not so and the bacterial ceils show radioactivity due to P, one
can conclude
bacteria have broken thymidine to thymine and phosphate and have then taken thes
e
. Experiments like these confirm that the bacterial cells actually break the nuc
leotide and
the membrane.
However, similar results are not obtained with 2p or 3H labeled AMP. The radioac
tivity of
the double labeled AMP is not retained on the filter. It seems that the bacteria
cannot
AMP the way they cleave TMP.
of Cells and Sub-cellular Particles
Isotopes have provided much information about the behaviour, movement, activitie
s and
and other functioning particles in the body. For this kind of study it is necess
ary

522
Biophysical Chemistry
to incorporate a rad/oact/ve element or compound into the cell or particle in su
ch a way that it
will remain bound for the life or functioning period of the cell or particle. Me
asurement of the/n
vWo life span of labeled .erythrocytes can be cited as an example of this applic
ation. An aliquot
of whole blood drawn from the body is incubated with 5Cr in the chromate state.
The label
moves into the erythrocytes, where it is converted to chromic state and becomes
bound to the
haemoglobin. It remains bound till the death of the erythrocyte. After labeling
the ceils are
reintroduced into the blood stream. Sampling at the interval of one to three day
s permits the
determination of radioactive ceils in circulation. The half survival time for lo
ss of 50% of the
labeled cel/s from the c/reulat/on Is used as a measure of the/Ire span. One add
/t/oval Informat/on
that comes to light with this experiment is the fate of the erythrocytes. Since
5Cr can be
recovered from the spleen, it becomes clear that spleen must be directly involve
d in removing
ageing erythrocytes from circulation.
Isotope Incorporation and lsotope Exchange Studies
lotope incorporation studies yield irormation concerning the position of bond br
eakage
and formation during a reaction. A an exampJe consider the cleavage of glucosel-phosphate
as catmfzed by alkal/ne phosphatase. This react/on could conceivably occur with
cleavage of
either the CO bond or the PO bond. If this reaction is carried out in the presen
ce of H2sO
enriched water, the former reaction (CO cleavage) path yields glucose containing
one atom of
CH20H
0
The latter (P --O cleavage) is characterized by phosphate containing one atom of
;D
CH2OH .
OH +
HO

OH
During experimentation the label invariably appears in the inorganic phosphate i
dentifying
P--O bonds as the cleavage site.
Isotope exchange studies yield information about the existence of reactidn
intermediates. Sucrose phosphorylase provides an example of the isotope exchange
studies.
This enzyme catalyzes the following exchanges, each carried out in the absence o
f the second
substrate:
(I) Glucose-I-P + mPI Glucose-l-rap+ PI
(2) Glucose-Fructose + Fructose-4C Glucose-Fructose-C + Fructose
These findings are consistent with -the possibility of a glucosyl-enzyme interme
diate being
formed. Thus, the reaction seems to proceed via a two=step reaction path:

Isotopes n Btotogy
523
Glucose-1-P + enzyme Glucosyl-enzyme + P
Glucoseenzyme + Fructose Glucose-Fructose + enzyme
Both isotope Incorporation and exchange studies have yielded much information ab
out
the reactions of many metabolic pathways.
Biological structures are seldom stationary. The hydrogen bonds between the two
strands
of DNA are forming and breaking continuously. So are the hydrogen b6nds of prote
In. If one
incubates these two macromolecules In tritium labeled water, soon the tritium wi
ll come to be
located In these macromolecules. This is the proof that the hydrogen bonds are n
ever permanent
and the hydrogen Involved In these bonds is continually exchanged with the hydro
gen of the
water.
Metabolic Studies
It can be said without fear of ecaoogeration that the greatest area of applicati
on of radioactive
Isotopes In biochemlstry has been that of metabolism. Isotopes have been employe
d In the
study of almost every phase of metabolism; to name a few, amino acid metabolism,
proteIn
photosynthesis, carbohydrate degradation, the TCA cycle, nucleic acid synthesis,
heine biosynthesis, fatty acid degradation and biosynthesis, and cholesterol and
steroid
metabolism etc. Isolation and identification of metabolltes of major metabolic p
athways has
been made possible by use of isotopes. Moreover, minor metabolic pathways which
have a
potential to become major pathways In disease have been elucidated due mainly to
the use of
pathways exist, it has become possible to calculate the relative proportion
each route.
Consider this. A substance, A, containing 4C (C is chosen arbltrm-lly; it could
be any
other biological isotope) is administered to an anlmal. After a certain period o
f time a compound

524
Biophysical Chemistry
E is isolated and purified from the animal and is found to contain radioactivity
due to 14C. Since
14C does not naturally occur in an organism and since it was administered as par
t of a compound
A, it can be said that A is a precursor of E or that E is a product of A. Thus,
by means of
radioactivity a precursor product relationship has been established. Now, if in
the same set of
experiment after feeding IC labe/ed A, the process Is Interrupted at various tim
es, there is
every likelihood that we will be able to isolate intermediate compounds, B, C, a
nd D, all showing
radioactivity du.e to IC. It can then be surmised that A goes through the steps
B, C, and D
before it forms E. In this way a whole metabolic pathway can be established (the
actual
experimental set up has many complexities which have been ignored here for the s
ake of
understanding). A beautiful example of the above consideration is afforded by Bl
och's experiments
on cholesterol biosynthesis, where by using 3C, IC, and deuterium labeled acetat
e, he could
establish that all the carbon atoms of cholesterol are derived from acetate. He
along with his
colleagues went on to establish the pathway of cholesterol biosynthesis, which i
s a sort of
landmark discovery. The example is treated diagrammatically in Figure 13.20.
Mineral Metabolism
Isotopic tracer technique has yielded much information about the absorption,
distribution and excretion of the mineral constituents of the body. Extensive us
e has been
made of this technique to study the metabolism of calcium. The exchange reaction
s that take
place in the bone with respect to calcium and phosphorus have been gleaned from
the studies
made using gsC and a2p.
Studies on the metabolism of mineral elements present in large quantity in the b
ody have
been more successful in general than similar tracer studies of the metabolism of
the trace
elements. In many of the latter, not enough attention has been paid to the desir
ability of keeping
the total amount of material administered down to the level of the daily intake
of the element.
The results obtained with doses which are many times as high as the daffy intake
may not give
an accurate picture of the absorption, distribution, and excretion of the quanti
ties the body is
normally called upon to handle.
Metabolic Turnover Time Determination

For many years it was erroneously believed that cell proteins remained intact an
d stable
till such time as the cell lived. This concept could be challenged and disproved
solely by the use
of isotopes. When SN labeled amino acids were fed to animals, the label appeared
in the
polypeptide chains of liver proteins at a fairly high rate even when the total a
mount of protein
did not change. When the same animals were fed the normal or 4N amino acids, the
previously
labeled liver proteins rapidly lost their label, again with no change in the tot
al amount of liver
protein. The obvious conclusion of the experiment was that a relatively high rat
e of synthesis of
proteins in the liver was balanced by a relatively high rate of degradation. Thu
s, liver proteins
undergo metabolic turnover. On the basis of these experiments, it could be found
that the
proteins of the liver cell have a haft-life of about 5-6 days. On the other hand
proteins of muscle
tissue turn over quite sluggishly showing a half-life of about 30 days. The meth
od can equally
well be applied to carbohydrates, lipids, and other cellular constituents.
Mechanism of Enzyme Action
Enzyme action can also be studied using radioactive isotopes. In many reactions
there is
an ambiguity about which bond has actually broken or which has formed. A good ex
ample of
such an ambiguity is afforded by cleavage of glucose- 1-phosphate by alkaline ph
osphatase as
discussed above. Two different bonds in that reaction could be broken with the s
ame products
forming. In that case too isotopic studies gave the answer. Following is another
example where
mechanism of enzyme action is laid bare by isotopic studies.

Isotopes in Biology
525
Mechanism of DNA polymerization can be understood easily using radioactivity. If
DNA
polymerase I is incubated with DNA, Mg2+, a buffer, and all the four deoxyribonu
cleotide 5'
triphosphates labeled with 32p in the a- position, the 32P becomes insoluble in
10% trichloroacetlc
acid and can be collected on a membrane filter. Retention on the filter indicate
s that the label has
- become part of something that is big enough to not to pass through the pores o
f the filter; in this
case the growing chain of DNA. This means that the phosphodiester bond is formed
with the
a-phosphate of the nucleotides. This may be verified by other experiments. If th
e same incubation
as above is carried out with the four deoxyribonucleotide 5'-triphosphates label
ed in the -, or
y- phosphates, the 3p remains soluble in trichloroacetic acid and radioactivity
is not retained on
the filter. A sure indication that the P has not become incorporated into the DN
A. Thus, it is the
- phosphate which becomes a part of gr)wing DNA and the - and y-phosphates are e
xcluded.
..Additionally, if the reaction is carried out without Mg+ or with one deoxyribo
nucleotide 5'triphosphate less, radioactivity passes through the filter. This means that Mg+
is essential for
polymerization and that all the four triphosphate nucleotides are required to be
present in the
reaction mixture.
Clinical Applications
(0 i. Radioisotopes are very widely used for diagnostic tests. More than 100 dif
ferent
radioisotopes have been used for diagnosis. Among the most important ones are 13
I, 51Cr, SAu,
Fe, P, F(2, and 1Xe. II-iodohippuric acid is used for kidney function tests. Xe i
s used for
lung function tests. 5Cr is used for blood volume determination and investigatio
ns into certain
of anemia. A very recent use has been that of Thallium. This isotope mimics potas
sium
10n behaviour in its uptake from the blood stream into the normal cells of heart
. It however, does
enter the damaged heart cells. Its administration into the patient .and later ph
otographic
gives a clear idea of the area in which the heart muscle is damaged.
(iO Therapy. Radioisotbpes have been particularly useful in treating cancers. Co
radiations
given to tumors and many tumors have been known to regress to quite some extent
by this
In this therapy, known as the tele-therapy, a high intensity beam

(~ 1,000 curies) from a tiny Co source is collimated on to the tumor area.

Yittrium-90 has been used for cancer therapy. Tiny yittrium oxide ceramic beads
are
in a tumor. Since yittrium emits chiefly [-particles, it has effect only in the
vicinity. Thus, while not affecting the rest of the body, such implants result
tissue. Alternatively, yittrium oxide ceramic beads have also been implanted
the pituitary gland. The reasoning behind such implants is that since pituitary
stimulates
a destruction of this gland will slow down the tumor growth also. A great
ofyittrium implants is the short half-life if the isotope, just 64.2 hours. One
therefore
not have to surgically remove the implant at a later time; it loses its activity
by decay very
Modern science has reached a stage where it can utilize the energy involved in r
adioactive
' for therapeutic purposes. Thus, heat generated by Pu decay is converted to ele
ctricity
nuclear powered battery which is used to operate a heart pacemaker. This pacemak
er
be implanted in the body and needs replacement once in about ten years.
in Sterilization of Foods and Equipments
Strong y-emitters are ued to sterilize prepacked food (milk and meat) and surgic
al
such as syringes and needles. The technique obviates the need of maintaining an
environment. Drugs to be administered by injection and plastic disposables such
as
and syringes are also sterilized by such treatment.

1:2
(c]
2:1
Ratio
'
Free : Bound
Radioactivity
526
Biophysical Chemistry
Idiolmmanoy
Radioimmunoassay (RIA) is a highly sophisticated technique and can detect extrem
ely
small amounts of non-radioactive material. It can achieve this even if the mixtu
re contains j
huge amounts of other materials in which the investigator is not interested. Wit
h the development
of methods for labeling antigens to a high specific activity, very low concentra
tions (I0-12 g/ml}
can be detected easily.
The principle on which the technique is based is deceptively simple and is shown
in Figure
13.21 (A & B), The binding of radioactively labeled antigen (Ag*) to a fixed amo
unt of antibody
(Ab) Can be partially inhibited by addition of unlabeled antigen (Ag). The exten
t of this inhibition
Basline
Bound
antigen
Free antigen
*
3Ag" 2Ab
lag"
2 Ag- Ab
Concentration of test antigen funlabelled ]

Fure 13.21 (A & B)

Pritwiple of radloimmunoassay, lf 3 mol of radiolabeled Ag (.) are added to 2 mo
l of Ab, one
mol of Ag will remain free and two will bind to Ab. The ratio of free radloactWi
ty to that of
bound will be 1:2. This ts shown In the upper paneL The boffom panel shows what
happens
{ 6 moI Ag is added, 3 mol radlolabeled and 3 mol unlabeled (o) to 2 moI Ab. Aga
ln, only 2 mol
Ag will bind. However, because of the equal oncentration of the radlolabeled and
unlabeled
antjen, I mol of each will be bound to Ab. The ratlo of free to bound radloactW
in thls case
wfll now be 2: l. lt is easy to see that the b of radloactWlty to Ab la tnhlbed
by of unlabeled Ag. in other words, the ratio ts golng to vary deperultng upon t
he concentratn of
the unlabeled antigen added to the rrUxture. TtUs ratio can easily be plotted. (
C). A typloal
standard curve for radlomumoassay. If the x-axis were a logarithmic scale, a sIg
ht line
could be obtWne

Isotopes in Biology
527
is a measure of the unlabeled material added. Thus, if the medium consists of 10
0% antigen in
the radioactive form, all the antibodies will be found bound to the Ag*. If we n
ow make the
mixture 50% with respect to Ag* by adding Ag, only 50% of the antibody will be f
ound binding
to Ag*; the other 50% would have bound Ag. If the medium is made more poor with
respect to
Ag* - 25% Ag* and 75% Ag - only 25% of the antibody will associate with Ag*; the
other 75% will
bind Ag.
To measure the concentration of Ag in any given sample, a reference curve for Ag
must be
prepared. This is done by mixing a fixed amount of Ab and Ag* and placing the mi
xture in a set
of tubes, To these tubes a known amount of Ag is added; just the way we prepared
standards
for spectrophotometric assay, the amount of Ag in each successive tube is higher
. When the
reaction is complete, the Ab-Ag* complexis separated from Ag*. This done, the ra
dioactivity in
the collected Ab-Ag* is measured for each tube, The amount oi'radioactlvity so o
btained is then
plotted against the concentration of Ag (Figure 13.21C).
Once this standard curve has been obtained, it is easy to measure Ag in any unkn
own
sample. To do so, one adds an aliquot from theunknown sample to the same Ab-Ag*
mixture
used to obtain the reference curve. Ab-Ag* is collected after the required incub
ation period and
its radioactivity is measured. The value so obtained is fitted in the standard c
urve and the
amount of Ag is read from the reference curve.
How is Ab-Ag* complex separated from Ag*? Two methods are mainly followed. They
are
the dextran-coated act/vated charcoa/method, and the doub/e-anttbody method. The
first one is
given below.
Dextranoated activated charcoal method. You must have learnt from your
chemistry classes that activated charcoal can adsorb many small molecules. Even
large molecules
like large proteins and even larger complexes like the Ab-Ag* complex may bind.
However, the
binding for the larger molecules is considerably slower.
The method consists of covering the activated charcoal with cross-linked dextran
. This
material has molecular sieving properties (see chapters on chromatography and

CENTRIFUGE
=Ag* SUPERNATANT CONTAINS
].=Ag*-Ab ONLY
Flgw'e 13.22 The dextran coated charcoal method for separating bound radloactivi
ty from unbound radioactive

528
Bophysical Chemistry
electrophoresis). The pores here are sufficiently small to preclude entrance of
either Ab or AbAg* complex. These large molecules cannot get to the surface of actlvated charco
al and will not
adsorb. Free Ag*, however is sufficiently small to penetrate the pores and is ad
sorbed (Figure
13.22). Thus if dextran covered charcoal is added to a mixture of free Ag* and A
b-Ag* complex,
Ag* will bind and Ab-Ag* will not. If the mixture is then centrifuged, unbound r
adioactivity, i.e.
Ag* will pellet out while bound radioactivity, i.e: Ab-Ag* will remain in the su
pernatant. This is
the easiest and a fast way to separate Ag* from Ab-Ag* complex.
Labl|ng the antigen, Most of the time the antigen or the hapten used for radioimmunoassay is labeled with 12sI. There is a very good reason for this. 12sI is
a strong ?-emitter.
Other isotopes such as 4C or 3H are -emitters. -emitters have to be measured in
liquid
scintillation counters. Since the mixture containing the antigen contains many p
roteins,
quenching becomes a severe problem. There is no such problem with I since it is
a Y-emitter
and consequently radiates higher energy. Quenching, as such, does not remain a p
roblem with,
?-emitters.
Iodination of proteins is done on the tyrosine or the histidine residues. If the
protein does
not contain tyrosine or histidine, then it is conjugated with one of these resid
ues and iodination
of the conjugate is carried out. Of course, it has to be seen that iodination of
the antigen does
not cause it to lose its antigenicity toward the antibody.
Radioimmunoassay is a wonderful diagnostic technique and yet it suffers from one
shortcoming. This method cannot distinguish active protein molecules from biolog
ically inactive
fragments which may still be antigenic, The power of the technique has made it t
he one of
choice in determining the concentration of protein hormones, carcinoembryonic an
tigen, hepatitis
B antigen, and even such smaller molecules as steroids, prostaglandins, and morp
hine related
drugs.
Immunoradiometry
There are cases where a radioactive antigen is not available. A variant of radio
-immunoassay
technique, the immunoradiometric assay (IRA), can be adopted in such cases.

The logic here is as follows. The antigen cannot be labeled for a variety of rea
sons. So,
label the antibody. To label the antibody, the antigen specific for it is adsorb
ed or coupled to a
stable substance such as cellulose or sepharose. Anti-sera raised for the antige
n in a suitable
animal is obtained. Among other IgG, this also contains the antibody specific fo
r the antigen
concerned. This is added to the column of antigen adsorbed on cellulose or sepha
rose. The
antibody specific for the antigen will bind to the antigen and will become adsor
bed. Subsequent
washing of the column will remove the unadsorbed antibodies. The coupled antibod
y is now
iodinated with 51. Subsequently, it is eluted from the column by changing the co
nditions of the
wash medium sufficiently. This labeled antibody, Ab*, is now used for assay of t
he antigen.
For the assay, an excess of Ab* is mixed with the sample containing the antigen.
This
mixture is now added to the cellulose/sepharose to which Ag has been bound. The
Ab* which
has not reacted with Ag can only become bound to Ag in the cellulose column. The
Ab*, which
has already reacted is automatically eluted. Radioactivity in the eluent can be
counted and will
be a measure of the antigen in the given sample. The principle of immunoradiomet
ric assay is
described diagrammatically in Figure 13.23.
Other , Radioisotopes are used in many other areas such as pharmacological studi
es
involving development of new drugs, radio dating of rocks, fossils, and sediment
s, ecological
studie.s involving migratory and behaviour patterns of animals.

Iodir/ate
529
Isotopes in Biology
------- Antigen
Cellulose
IgG
Incubate to
adsorb
Specific
antibodies

Add
Add
Cellulose
'
antigen
antigen
Count
Radioactivity
Figure 13.23 Principle of immunoradiomery
Study of Protein $tractre
Most studies on protein structure using radioisotopes are based on iodination of
tyrosine
in the peptide chain. Students will recall from their studies on the thyroid pri
nciples, that
tyrosine is the only amino acid that can be iodinated readily. Just how this app
lication works is
being illustrated with the help of an example.
Suppose that sequence analysis of a protein has provided the data that the given
protein
residues of tyrosine. React the protein with radioactive iodine - 131 in a suita
ble
mixture so that tyrosine residues pick up the iodine. After this precipitate the
protein
the help of trichloroacetic acid. Count the precipitate. Suppose that the count
given is
cpm. Now denature another aliquot of the protein and subject this too to iodinat
ion
presence of radioactive iodine 131. After lodination is over, precipitate the pr
otein
count for radioactivity. Suppose now that the count is 9000 cpm.

The above data tells you that the native protein picked up less iodine than the
denatured
This goes to show that some tyrosines must have been buried in the three dimensi
onal
in a hydrophobic environment unavailable to the iodination. These same tyrosines
exposed upon denaturation and therefore the count given by the denatured protein
was

530
Ba>physa:al Chemtry
A small Calculation will also give you just how many tyrosines were buri
ed. [4000/9000]
7 = 3. Thus 3 tyrosines were available for iodination and out of total of seven,
other four were
buried in "the three dimensional conformation.
SOME INFORMATION ABOUT COMMONLY USED ISOTOPES
Deuterium. This is very cheap, readily available, and can be measured by the fal
ling drop
method very simply. It is possible to grow quite large organisms on heavy water
(D=0). Studies
of the results of such substitutions have been very informative about numerous m
olecular
processes.
Tritium. This isotope emits a very low energy -ray having a maximum energy of 18
kilovolts. It can be readily detected by the photographic emulsion, a Geiger cou
nter, or the
scintillation counter. It has been extensively used in the form of tritiated thy
midine as a means
of specifically labeling DNA and has provided much useful information in molecul
ar biology. It
has also been tried as a cancer cell killer (the cancer ceils replicate much fas
ter than the normal
cells and therefore take up more tritiated thymidine injected in the system as c
ompared to the
normal ceils leading to their death). It should, however, be understood that suc
h a compound
constitutes a very serious hazard because it is especially geared to enter and s
o affect the
genetic part of the cells.
Carbon-13, Nitrogen-15 and Oxygen-18. These stable isotopes, which are obviously
intimately related to biological compounds, are reasonably easily available. Car
bon, and nitrogen
are available up to 96% concentration, and so can be used in studies which need
very heavily
labeled material. The famous Messelson and Stahl experiment made use ofSN to pro
vide evidence
that the DNA replication might be semi-conservative. Bloch made use of3C to prov
ide evidence
that all carbon atoms of cholesterol are derived from acetyl CoA.
Carbon-14. This readily available isotope, with a long half-life of 5000 years,
and a single
beta-ray of maximum energy 130 kilovolts, is probably the most important and the
most
extensively used in biology. It has been incorporated into a number of important
biomolecules
and most of them are available on the market. It requires some care in counting
because the
beta rays travel just about 15 cm in air. Thus any thickness in the sample is de
trimental to
measurement. Although it has a fairly long half-life, it is relatively safe if t

aken internally.
About 250 microcuries are known to be tolerated well.
Sodium-24. This quite short-lived, extremely active element (half-life i5 haur,
1.4 MeV
beta-particle energy + two gamma rays of energy (1.4 and 2.8 MeV) is readily ava
ilable. It can be
used wherever a study of sodium is needed. Since sodium readily ionizes it excha
nges very
rapidly. Thus, studies of longer duration (such as a determination of pathway of
a particular
metabolite) are not normally possible with. this element. It has been used to st
udy sodium
transport across plasma membranes.
Phosphorus-32. More or less of equal importance with carbon- 14 is P (others hav
e called
it "almost the perfect /sotope to use"). Its maximum -particle energy of 1.6 MeV
makes it easy to
.detect. It has no gamma radiation and consequently can be handled in relatively
large quantities
without danger, The half-life of 15 days makes it possible to employ it in relat
ively long biochemical
processes (elucidation of metabolic pathways). This same half-life however, it t
oo short to store
it commercially; it is therefore necessary to use it immediately on acquisition.
It can be produced
by bombarding sulphur with neutrons of over 1MeV energy. The resulting P is tota
lly separable
from sulphur giving rise to very high specific activity 3=P.
Sulphur-35. With more or less similar characteristics as 14C (the half-life of o
nly 80 days
is an exception), 5S has been used as an important tracer in protein studies. Ne
arly every
protetn contains one or more of the sulphur containing amino acids {methlontne,
cystetne,

A
isotopes tn 531
cystlne) and thus can be labeled with S. S has been recently widely used in DNA
sequencing
studies.
Iron. Two forms are avzilable: Fe (half-life 2:9 years, decaying by electron cap
ture), and
bFe (half-llfe 45 days, two partlcle energies of 0.46 and 0.26 MeV + y-rays of e
nergy 1.1 and
1.3 MeV). Fe emitS mainly X-rays and is therefore hard to detect. Extensive use
has been
made of both the isotopes in many studies involving blood and its formation.
We have discussed that Iorflzation and excitation are the two events that take p
lace when
radiation interacts with matter. These events can also take place when radiation
interacts with
living tissues. This is precisely the mechanism by which radiation can have dele
terious effects
on life {see Box 13.7). Although every individual in the world Is exposed to a s
mall amount of
radiation arising from natural and man-made sources, exposure to radiation Is of
more than
mundane interest to a biologist who Is exposed to radiations from tracer experim
ents which
have become so common and useful. It Is therefore useful for a biologist to know
what are the
tolerable radiation doses and to limit his/her exposure to those levels or level
s even lower. .
of A is initially higher than the specific

532
Biophysical Chemistry
Let us define units by which we can quantitate exposure. One such unit in use is
the rad.
It is a measure of the energy liberated in a specific material by exposure to ra
diation. One rad
equals 100 ergs per gram of material. Although rad is quite useful, a unit of mu
ch better use for
biological exposure has been introduced and is known as the rein. The usefulness
of the rem
lles in the fact that it takes into account the varying effect of different radi
ations into account.
For example, alpha particles are about 10-20 times more damaging to the tissues
as compared
to the beta- and gamma-radiations.(alpha-particles have little penetrating abili
ty, and therefore
their effects become concentrated in the small area to which they are limited).
The different
radiations are assigned what is known as qualltyfactor (OF); the beta- and gamma
-radiations
have been assigned a OF of I while alpha,radiation has been assigned a OF of 10.
The rem is
defined as the dose in rads multiplied by the quality factor.
The whole.body tolerable dose of radiations has been set at a maximum of 5 rein
per year
(/.e., 0.1 rem per week). This whole body dose has been set up considering the m
ore sensitive
tissues such as the gonads and the bone marrow (as a general rule fast dividing
tissues are
more sensitive to radiation). There are, however, other tissues which are not so
sensitive. Thus,
hands and forearms, which are most involved in exposure while carrying out biolo
gical
experiments, can tolerate a higher exposure. The tolerable exposure limit for th
ese organs is set
up at 75 rem per year and up to 20 rem in any 13-week period.
Precautions
Certain precautions should be rellgiously followed when working with radioactive
isotopes.
One should always wear disposable gloves because radioactive compounds can be ab
sorbed
through the skin. The time .spent manipulating stock isotope solutions should be
minimized. If
the protocol is thought to be complicated, a dummy run before actually using iso
tope is advisable.
The experimental set up and the worker should be separated by a Perspex shield b
etween them,
allowing only the hands to be exposed. The stocks, partlcularly if they are gamm
a emitters,
should be shielded in lead shielding. If the experimental mix containing the rad

ioisotope is to be
transferred to other parts of the laboratory, it should be properly shielded.
The radiation workers should monitor their exposure radiation. They can wear af/
/m badge
on their lapel. The badge holds a photographic film which when developed at a re
gular interval
can provide an estimate of the exposure by the degree of blackening of the film,
One may also
carry apocket dos/meter. This instrument is a miniature electroscope: Its scale
is directly calibrated
in dose units and therefore one can determine the dose of exposure at any given
time. Its size
makes it easy to carry; it is roughly of the size of a fountain pen.

Isotopes in Biology
533
Accidental Ingestion of Radio-Isotopes
We have so far discussed the external exposure ordy. Radio-isotopes are much-mor
e daungerous
internally. Various factors have to be considered in assessing the danger from i
nternal radiation
from a given isotope. The space does not permit us to go into the details of the
se factors, however,
some basic aspects will be discussed.
The biological half-life (time taken for half of a given amount of radioisotope
to be excreted
from the body) of different radio-isotopes is one such factor. Different isotope
s can have widely
varying half-lives ranging from hours to several years. Obviously, the isotopes
having short biological
haif-llves will be less dangerous than the ones having a long biological half-li
fe. The values for
radioactive half-life and the biological half-life can be combined to arrive at
a term which is
known as the effective half-life. This term is much more useful and can provide
a correct index of
the danger of an isotope if ingested.
Different isotopes are distributed differently in the body. Thus, while ingested
radioactive
sodium will be distributed throughout the body more or less evenly, other isotop
es such as 3I,
43Ca, and 3p will accumulate primarily in the thyroid (3I) and the bones (4Ca an
dP), The
isotopes which are accumulated in a particular organ are much more dangerous tha
n those
which are distributed evenly.
The form in which the radio-isotope has been ingested also determines the extent
of danger.
Thus C ingested in the form of HCO- will not be so dangerous because it will be
rapidly excreted by the lungs as 4CO2. On the other hand, if 4C is ingested in t
he form of "C-thymidine,
it becomesvery dangerous because in this form it will be incorporated in the gen
ome and will
continue to send radiation in the tissue for a very long time.
The precautions that
disposable
gloves while working
ontaining radio
isotopes; always use
oke in a radioactive
laboratory, and (/v)
ly.

can be taken to avoid ingestion include (t) to always,wear

with radio-isotopes, (//) to never to mouth-pipette a mix c
the safety pipette. (iff) to not to eat, drink, and even sm
to carry out radioactive work in designated laboratories on

The disposal of radioactive material should also be done with due care. The soli
d radioactive
waste requires special storage before it is disposed off (incinerated); containe
rs to store solid
radioactive waste are provided in every designated laboratory. The liquid radioa

ctive waste can,

however, be suitably diluted and put down the sink.
DOSIMETRY
The techniques given below are used for the measurement of dose and dose rate to
which
biological systems are exposed.
(i) The ionization chamber. We have already discussed the principle of ionizatio
n
chamber. The same principle is used here. The only change is that the ionization
chamber is
calbrated so as to give the measure of radioactivity directly in dose units. ,
(ii) The Fricke dosimeter. This is a chemical dosimeter. It is based on the prin
ciple that
the energy of radiation (hv) can knock out an electron from Fe2 converting it to
Fe3+ in aqueous
solutions.
Fe2 - hv = Fe3 + eThe concentration of iron (Ill) so produced can now be determined in a spectro-p
hotometer
at 304 nm. This system has a good tissue equivalence and can be applied over a l
arge dose
range,
(iii) Film dosimeter. This is perhaps the most widely used type of dosimeter. It
uses the
of photographic film induced by ionizing radiations as its principle (see sectio
n on
methods). This dosimeter is mostly used for monitoring personal exposure by

0.693
534
Biophysicd Chemistry
workers in the lab; it is worn usually on the coat lapels and as a ring on the l
ingers wht/e
working with radioisotopes. At a later time when the film is developed, one can
measure the
exposure by the extent of darkening of the,film. This method has good sensitivit
y. But careful
standardization is essential, as the results depend strongly on the emulsion pro
perties and on
the procedure of development.
(iv} Thermluminescence doimetry. Certain crystals (e.g., CaF2) have the ability
to store
radiation-generated electrons In crystal defects over a very long period of time
, When such
crystals are heated, the trapped electrons return tO the ground state emittingll
ght in the process.
As the number of light flashes (excitons) is proportional to dose, the amount of
light emitted
gives a direct measure of the dose tO which one is exposed. These dosimeters hav
e excellent
tissue equiv,l'ence and can be made very small usually by mounting them on teflo
n. The system
is also usefhl over a very large dose range.
{v) Other t,hniques ofdoslmetry. Severa/other techniques of dosimetry are availa
ble.
They are based on colorimetrlc measurements, discoloration of crystals, turbidit
y formation on
glasses or organic polymers, mortality rate of bacteria on exposure to ionizing
radiation, and
conductivity alternation in semiconductors. These systems, however, are not used
frequently.
SOLVED PROBLEMS
1.
Mn4 has a half-llfe of 314 days. Calculate (a} the decay constant in ter
ms of days- and sec-]m and (b) percent of initial radioactivity remaining in a s
ample after 80 days.
0.693 0.693 6.93x I0
Arts. (a)) - tu2 - 314days 3.14xi02 days[) = 2.21x 10-days-]
314 days x 24 hr/dayx 60 min/hr x 60 sec/min

0.693
0.693
314daysx86400sec/day 3.14x101 (8.64x10')
69.3 x 10-2 sec
-

27.1xi0--'- or [ ) =2.6xI

No
2.3 log = -- = )t
N
let.N0 = 100%
2.3 = I0-012.21 x10-3)[80) = O. 1768
N
1o- 100- 0.1768 0 077
log 100 - log N = 0.077 or log N 2.000 - 0.077 = 1.923
Cesium - 137 has a half-life of 33 years. Calculate the fraction of ceslum - 137
that decays
(a) per year [b) per minute.

535
0,693
Isotopes in Biology
A. (a)
Calculating the decay constant
0.693 6.93x 10
yr-1 =2.110-2 yr33 yrs 3.3 x 10
This effectively means that 2,1 x 10-2 atoms per atom decays in a time period of
one year. Or, 1 atom out of 112.1 x 10-2 atoms decay per year.
1
= 0.4762x 102 = 47.62
2.1 x 10-2
Thus one out of every 47.62 radioactive atoms decay per year.
2.1 10-2 yr- 2.1xi0-2 yr-I
=
= 4.0x 10-a rain
(b) = (365) (24) (60)

0.5256 I0

1 0,25xi0s or 4.0 x 10-a

Thus one out of every 0.25 x 108 radioactive atoms decays per year,
K (ta = 1.3 x 109 yr) consUtutes 0.012% of the potassium in nature. The man body
contains about 0.35% potassium by weight. Calculate the total radioactivity from
K
decay in a 75 kg human,
To,al K = 0.012% x 0.35% x 75 x 10ag
= (1.2 x I0-) (3.5 x 10-3) (7.5 x 10) = 3.15 x 10-2 g
3.15 x 10-2 g x 6.023 x 1023.atms/g - atm
Number of K atoms =

40 g/g- atom
= 4.74x 102atoms
1.3x109 x365x24x60
=6.93" 10-min = 1,014x10-Is min-i
6.93 x I0-14

DPM = = kN = (1,014 x I0-) (4.74 x 1020) dt

= 4.81 x 108 DPM
Scientific data shows that the carbon compounds being synthesized currently by l
iving
systems contain enough C to give 13 DPM per gram carbon. Since C* has half-life
of
5700 years, it takes a long time to decay after the death of the animal. Given t
hat the
decay constant of C is 2.31 x 10-1 min-L can you calculate?
(a} the abundance of C of the carbon in living systems, and
(b) The age of a well-preserved animal which is emitting 1 dpnYg atom.
-dN
. (a) dpm/g = = N
dt

536
Bohsca Cheratstry
Where N = No. of C4 atoms per gram carbon and ,, as given is 2,31 x 10-min- 13 =
2.3i x 10-1 N
13
N =
= 5,63 x 10I atoms C141g carbon
2.3110-1
1 gram of carbon contains
1 g
x6.023 x 1023 atoms/g - at(m
12g/g - atom

= 5.02 x 1022 total atoms of Carbon.

5.63 x 10I atoms of C14
.-. abundance =
x 100/o = 1.12x 10-w%
5.02 x 1022 atoms Carbon
Decay constant is given in terms of min-I. Converting it to yr-I,
2.31 x 10-I min- x (365) x (24) x (60)
= 2.31 x 10-1 rain- x 525600 = 1.216 x 10- yrNow 2.31og13 =)t
1
or
2,3 log 13 = 1.216 x 10-t = (2.3)(1.1139}= 1.1216 x 10:t
{2.3)(1.1139)
2.56 ,
t =
-1.216x101.216x10--or t=2.1 x 104= 21000yr.
{a} Calculate the specific activity of pure C4 in terms of dpm/g.

(b)
What should be the maximum specific activity {ci/mole) at which uniforml
y
labeled L - leucine - CI may be prepared?
{c)
What proportion of molecule will actually be labeled in a preparation of
L - 0
leucine - C14 which has specific activity of 150 mCi/mmole? Given that the decay
constant ofC14 is 2.31 x 10- rain
Ig
I g CI" = 14g/g-atom = 0.0714 g-atom
N = 0.0714 g-atom x 6.023 x I02a atoms/g-atom
N = 4.3 x 1022 atoms
dpm -dN
.... AN
g dt
N means the number of atoms in Ig oft. dpm = 2.31 x I0- rain- x 4.3 x 1022 atoms.
Specific activity = 9.94 x 102 dprrdg.
(b)
Since the term of specific act/vity here is in Ci/mole, let us convert t
he dpm term of
the previous problem into Ci,
9.94x lO2dpm/g
= 4.48 C/g
2.2 x 1012dprn/Ci

Isotopes in Biology
4.48 Ci/g x 14 g/g-atom = 62.7 Ci/g-atom
L-leucine has 6 g-atoms of carbon per mole.
.-. maximum specific activity
= 6 g-atoms]mole x 62.7 Ci/g-atom = 376.2 Ci/mole.
150 Ci/mole
100 = 39.9
(c)
376.2Ci/Mole
.'. % of C14 - labeled molecules = 39.9%
Calculate {a) the number of radioactive atoms and (b) the weight in gms. of chro
mium in
1 Ci of pure chromium-51. (c) Also determine the specific activity of pure chrom
ium - 51.
Given that the half-life of chromium- 51 is 27.8 days and the decay constant per
minute
is 1.73 10-5 rain-I.
(a) 1 Ci z 2,22 10I dpm
-dN
dpm = = XN
dt
.'.
2.22 10I = 1.73 10-5 N
2.22 1012
N =
- 1.28 x 10v
1.7310-5
.'. Chromium - 51 contains 1.28 x 1017 radioactive atoms/Ci.
(b) 1 g-atom of chromium-51 (51 grams) contains 6.023 x 103 atoms.
.'. the weight of 1.28
atoms should be

1.28 1017
6.023 1023 X 51g = 10.9410-6g
Weight in gm. of chromium in 1 Ci pure Chromium-51 is 10.84 10-6m.
(c) Pure chromium-51 contains 1.28 1017 atoms/Ci.
1Ci
= 0,781 10-]7 Ci/atom
or 1.28 10v atoms
0.781 x 10-Iv Ci/atom 6.023 10 atoms/g-atom
= 4.703 x 106 CiJg-atom.
4,703 x 106 ',CiJg - atom
or,
= 92.2 Ci/mg
5 I000 mg/g - atom
Thus the specific activity of pure chromium-51 is 92,2 Ci/mg
A small tube given to you contains I m Ci of L-histidine-C14 in 3 ml aqueous sol
ution. The
amino acid is uniformly labeled. The specific activity of the amino acid is 200
mCi/m
mole. Find out -- (a) the concentration of histidine in the solution, and (b) th
e activity of
the solution in terms of CPM/ml if the counting efficiency is 65%.

(b)
Biophysical Chemistry
The problem states that 200 mCi is equivalent to 1 inmole.
the number of mmoles that correspond to
lmmole
lmCi -

= 0.005 mmolelmCi
200 mCi

The tube contains 1. mCi in 3 ml.

0.005 mmole
".
= 0.001667mmole/ml
3.0ml
= 1.667 x 10-." mmole/ml.
Thus the concentration of.L-histidine in the tube is 1.667 x 10-" mmole/ml.
1 Ci ---- 2.22 x 1012 dpm
.'. 1 mCi = 2.22 x 10 dpm
.'. total activity at 65% counting efficiency should be
(2.22 x 109) (0.65) CPM = 1.443 x 109 CPM
But then this is the activity in 3 ml. Activity per ml should be 1.443 109 CPM
= 0"481xl09CPM/mlor 4.81 x 109 CPM/ml
3.0mi
.'. Activity per ml = 4.81 x I0 CPM
8,
How will you prepare 100 ml solution of a 10-2 M solution of glucose (1C14) so that the
monosaccharlde has a specific activity of 2.0 x 10s dpm/] moJe. You have been pr
ovided

O. 1 M solution of unlabeled glucose along with a stock solution of ucose (1- C1

4]
(40 mCYmmole and 1 mCi/ml.)
The amount of radioactivity needed will be
10-2 M = 10 I moles/ml.
10 I moles/ml x 100 ml = 1000 l moles.
1000 moles x 2.0 x 106 dpnY/ mole = 2.0 x 109 dpm
2.0 x I0 dpm WIll be required to prepare I00 ml of the required solution with th
e
required specific activity.
Now, let us find out how much radioactive stock solution wig be needed to provid
e
2.0 x 109 dpm.
1 mCYml x 2.22 x lO dpm/mCi = 2.22 x ]09 dpm/ml
2"0xI0dpm = 0.91ral
2.22x 109 dpm/ml
.-. If 0.91 ml of radioactive glucose solution is added to 1000 I moles of unlab
eled
glucose and the solution diluted to 100 ml, the required solution wig be obtaine
d.
E. Colicells were grown on a defined medium which contained S3 sodium sulphite a
s the
sole source of sulphur. The specific activity of radioactive sulphur was 4.39 x
104
CPM/ mole. The ceils were allowed to grow for several generations at the end of
which
theywere harvested, lysed, and ractionated for purification of L-cysteine. Purifi
ed
L-cysteine gave a count of 160000 CPM. I0 gm. wet weight of ceils was used for

I0.
A cell free extract capable ofsyntheslzlng amylose was incubated with gl
ycogen primer
and radioactive Glucose- I- phosphate - C4. The radioactive isotope was uniforml
y labeled
and gave a specific activity of 23000 CPM] mole. Ir.terrupting the experiment at
various
tlme intezvals and assaying the radioactivity incorporated, it could be determin
ed that
initial rate of radioactivity incorporated into glycogen primer was 3600 CPM/min
. On the
basis of the above, can you calculate
(a) The rate of enzyme reaction in terms of I moles glucose incorporated per min
ute?
(b)
If it is given that the reaction mLx-ture contains 0.5 mg protein,, calc
ulate the rate of
enzyme reaction in terms of l moles per mg protein per minute. What is the speci
fic
activity of the enzyme?
Am. (a) The rate of enzyme reaction can be calculated as follows:
3600 CPMimin
=

= 1.57x10-] t mole/min
23,000 CPIV mole

.'. Rate of enzyme reaction is 0.157 I mole/mln

0.157 I mole/min
(b) u =
= 0.321 mole/rag proteinJmin
0.5rag protein
.'. The specific activity of the enzyme is 0.32 units/mg protein.
11.
B. subtl/is culture was grown in a defined medium with P as the only sou
rce of
phosphorus. After several generations, the cells were harvested and resuspended
in a
fresh medium containing unlabeled inorganic phosphate as the source of phosphoru
s.
The medium was so nazxipulated that further growth was improbable, l,om this cul
ture
aliquots were drawn at regular time intervals and their lipids extracted. Fracti
onation
procedures were performed, for phosphatldyllnositol which was assayed for quanti
ty as
well as radioactivity. The following table depicts the specific activity of phos
phatidylinositol
at dllferent tlme periods. From the data presented, calculate the rate of turnov

er of
phosphatidy.linositol (half-llfe} in B. subtf/fa.

540

Time elapsed after

resuspension
(mu}
Specific activity of
phosphatidylinositol
(CPM/, mole P)

0
30
60
120
150
180
300
60,000
45,456
34,442
19,771
14,977
11,347
3,744

From the data itself a rough idea of half-life of phosphatidylinositol can be ha

d. In
the first hour less than half the label has been removed as evident from the cou
nt.
However between 30 min and 120 min, more than half of the pa2 at 30 min has
disappeared, t/2 should lle somewhere between 60 and 120 rain. First calculating
the first-order rate constant, K:
34, 442
2.3 log ffi K (90rain)
14,977

(The numerator and denominator are counts removed by 90 rain)

K = 2"3 Iog2.299 = {0.0256)or K = 9.24 x 10-3/min
0.693
0.893
= 0.075x 103 5 min
t12 '
=
K
9.24xI0-3
.'. Half-life of phosphatidylinositol in B. subt///s is 75 min.
12.
To calculate the amount of NaSO in a given system, the researcher adds 3
x 10 CPM of
carrier-free SO - to the system. A small aliquot is drawn from the sample post e
quillbration. The label is found in Has. It is found that the aliquot gives a count o
f 1.7 x
10 CPM per mg of BaS3rD. What is the amount of non-radioactive SO42- as NaSO in
the system?
A. S.A,r, the specific activity of reisolated BaSO is 1.7 x 10 CPM/mg or 1.7 x 1
0'
CPM/mg x 233.5 mg mmole = 3.97x 106 CPM/m mole.
Now,
Mu = Ao
S..r
3 107 CPM
.'. M =
= 7.6 mmoles
u 3.97 106 CPM/mmole
But we have to express the amount of SO - in the system as NaSO.
.'. M. = 7.6 m moles x 142 mg/m moles = 1079 mg or 1.08 gm
Thus the amount of NaSO4in the system is 1.08 gm.

Isotopes in Biology
541
13.
A commercial manufacturer of heparin solutions is claiming that he is se
lling 0.1 grn
% heparln solution at half the price of other suppliers. To check his claim, 10
mgs, of C'4
labeled heparin sulfate (7.2 104 CPMImg) were added to 50 mls of heparin solutio
n
supplied by the manufacturer. A small amount of heparin was reisolated from the
solution
and it was found to have a specific activity of 1.8 x 104 CPM/mg, .Find out whet
her the
manufacturer's claim is true.
(S.Ao lMo (7"2x104 ) (4.0 1)10mg
. M, :S--r- )
=l-xl .,1 10mg = M = 30.Omg. "
Thus 50 ml of the solution provided contains 30 rag. heparin. This means that th
e
manufacturer is supplying 0.06 gm % solution of heparin while claiming
0.1 gm %. No wonder he is selling at such a low price.
14.
A substance containing C'4- and Ha - labels has been chromatographically
isolated from
the cytosol of E.coli. The compound gives 50, 000 CPM in channel 1 and 90,000 CP
M in
channel 2 of the scintillation counter. A C'4 standard known to have 24,000 dpm
gives
6000 C in channel 1 and 12,000 CPM in channel 2. An Ha standard having 40,000 dp
m
gives 8000 CPM in channel 1 and 20 CPM in channel 2. Background counts have alre
ady
been subtracted from the data. Determine the dpm of C and Ha. in the isolated
compound.
,
From the measurement of standards, it is clear that Ha contributes littl
e to the counts
in channel 2. Therefore it may be safely assumed that all 90,000 CPM in channel
2 is
due to C' alone.
Again from the standard measurements it is clear that the efficiency of channel
2 for
counting C4 islust 50% [ -- 0.5
90000

.'. The dpm of C4 in the given compound is 0.5 - 180000

The 50000 CPM of channel I are due to contribution from both - C' and H3.
Again, counting of standards in channel I tells us that the 50,000 CPM of channe
l
1 represents 25% of the C'" dpm and 20% of Ha dpm.
.'.
50000 = (0.25) (180000) + (0.20) (Ha dpm)
... (5oooo)-(o.25)(18oooo)
5OOO
0.2
0.2
.'. dpm due to Ha = 25000 dpm.
Compound A labeled with C4 was introduced in a perfused liver and the appearance
of
label in other compounds was followed.
Two compounds, B & C, were found to pick up the label. Given below are the speci
fic
activity charts of A, B & C as a function of time. Taking assistance of the rule
s given in
Box 13.5 can you determine from the charts whether B or C is the immediate produ
ct of
A?

542
ure
Biophysical Chemistry
Time -- Time
(a) (b)
Fig. Specific activity of A and two potential products (a) B & (b) C
Ans. C is the product of A. (Reason it out.)
16.
On the basis of elementary studies a group of scientists has proposed th
at a path way
A - B -- C -- D operates in the cytosol of most cells. On the basis of the fo-ll
owing chart
of specific activ/ties of A. B, C & D as a function of time, can you det-ermine
whether the
surmise of the scientists is true? If you have reasons to believe that the pathw
ay should
be different from the one suggested, please state those reasons along with the a
ltered
pathway that you believe is more apt to occur.
Arts. The pathway should be A -- D --> C --> B. (Reason it out)
Suggest/ons For Further Read/rig
1. Dyer. A., ed., 1972. Liquid Scintillation Counting. Heyden.
2. Horrocks. D, L.. ed., 1974. Application oflAquid Scintillation Counting. Acad
em/c Press,
3, Sacks. J., 1953. Isotopic Tracers in Bbchemistry and Physiology. McGraw-HI11.
4. Thornburn, C. C.. 1972. Isotopes and Radiation in Biology. Butterworths.

Isotopes in Bbology
543
1.
Half-life of Ca4 is 163 days. Can you calculate (a) the decay constant [
,) per day & (b) the
percent of original radioactivity remaining in a given sample after 90 days.
An. (a) 4.25 x 10S/day
{b} 63.1%
2.
C4 has a half-life of 5700 years. Can you calculate the fraction of C4 a
toms that decays (a}
per year (b) per minute.
An. (a) One atom per 8225 radioactive atoms decays/year.
(b} One atom per 4.32 x 10g radioactive atoms decays/rain.
3. The half-life of P is 14.3 days. Can you calculate
(a} the number of radioactive atoms in 1 Ci of pure P,
(b) the weight in gms. of phosphorus in I Ci pure pa2 &
(c) the specific activity of pure pa2?
An.
(a) 6.6 x 10Is atoms/Ci.
(b} 3.51 x I0-s gm,
(c) 9.125 x 10s Cl/g-atom.
4.
You are given a vlal containing 1 mCi of L-Serine -- C4 (uniformly label
ed) in 4.0 ml solution.
You are also told that the specific activity of the labeled compound is 250 mCi/
m mole.
Calculat.e |a) the concentration of L-serlne & (b) the activity of the solution
in terms of
CPM/ml. at a counting efficiency of 50%.
Arts. (a) I x 107M
(b) 2.78 x 10s CPM/ml
5.
How will you prepare a 50 ml 10-3 M solution of L-cysteine -S3s in which
the amino acid has
a specific activity of 2.0 x I0s DPM/m mole? Yot are provided a 0.1 M solution o
f unlabeled

L-cysteine & a stock solution of L-cysteine - Ss

An. (45 m Ci/mmole & I mCi/ml}.
6.
B. megather/um was cultured in a defined medium containing Ss - sodium s
ulphite as the
only source of sulphur, The initial concentration of. Ss, sodium sulphite in the
medium was
0.007 M, Every ml of the medium contained 2 x I0s CPM of radioactivity. After a
few days, the
cells were harvested, extracted & fractionated for L-methionine. The fraction co
ntained 53000
CPM of S35 per gm wet weight of the cells. Find out the intracellular concentrat
ion of

L-methionine in the organism, given that 1 gm wet weight corresponds to

0.2 gm dry weight
& 0.8 ml intracellular water.
.An. 2,31 x loS M,
A cell free extract which contained 720 mg protein was mixed with C4 labeled met
hylmercaptan of specific activity 2.4 x 10s CPM/m mole. O-acetyl homoserine was als
o added.
The total volume of the system did not exceed 1.5 ml. Since the cell-free extrac
t was from a
microorganism, it contained enzymes required for methionine biosynthesis. Aliquo
ts were
drawn and fractionated for L-methionine at different intervals. It could be dete
rmined that
the rate of incorporation of radioactivity in methlonine was 2240 CPM/min. Calcu
late the rate
of enzymatic reaction.
{a) in term of m moles/rain; &

544
xs. (a} 9.33 x lOm moles/min,
Biophysical Chemistry

(b) 1.3 x 104 p, moles/mg proteirYmin.

E. coli cells were grown on a defined medium with only Pia2 as the sole source o
f phosphorus.
After a few generations the cells were harvested and resuspended in a fresh medi
um containing
unlabeled inorganic phosphate as the source of phosphorus. Further growth of cel
ls was
disallowed. From this culture aliquots were drawn at regular time intervals and
their lipids
exacted. Fractionation' procedures were performed for phosphatidylglycerol which
was
assayed for quantity as well as radioactivity. The table below gives the specifi
c activities of
phosphatidylglycerol at different time periods. From the data presented, calcula
te the rate of
turnover of phosphatidyl glycerol (half-life) in E. coll.
Time after resuspension (min)
Specific activity of phosphatidyl
glycerol {CPM/mmole P)

0
30
6O
90
120
150
63261
42113
27996
18763
12502
8302

Ans. t% = 51.3 min.

9.
Milligrams of C14 -- labeled insulin (2.4 x 104 CPM/mg) were dded to a c
ommercial solution
of insulin containing an unknown quantity of the hormone. A small amount of insu
lin was
then isolated from the solutionand it gave a specific activity of 0.7 x 104CPM/m
g. Calculate
the amount of unlabeled insulin in the solution.
An$. 2119 mg.
10.
A sample of labeled RNA containing C14 -- adenine. & Ha -- Uracil yielde
d 25000 CPM in
channel 1 & 45000 CPM in channel 2 of a dual-channel scintillation counter. A C1
4 standard
containing 40000 dpm yielded 10000 CPM in channel 1 & 20000 CPM in channel 2. An
Ha
standard containing 200000 dpm yielded 40000 CPM in channel 1 and 100 CPM in cha
nnel
2. Background correction has already been taken into account. Calculate (a) Ha d
pm & (b)
C4 dpm in the sample.
Ans. (a) 12500 dpm (b) 90000 dpm.
11. Why do you use ass and not a2p to label nucleic acids?
12. Sulfur is not found in nucleic acids. So how do you label nucleic acids with
asS?
13.
If a particular isotope preparation is said to be 2 Ci, how many Becquer
els do you'think it
has?
14. Which of the following according to you will disintegrate faster - 'P or deu
terium?
15.
You are to study kinetic parameters of nucleotide incorporation in a gro
wing nucleic acid
chain. Which of the hydrogen isotopes - deuterium or tritium - will you use and
why?.
16.
Historical specimen can be dated with the help of radioactivity. Why do
historians prefer
14C dating?. Why don't they use ap or asS or aH dating? "

545
17.
A researcher is interested in studying glucose metabolism. He labeled gl
ucose with tritium
homogeneously. After suitable time he tried to detect the presence of tritium in
various
chemical compounds in the tissue. However, he found that every single compound t
hat he
picked up has homogeneous distribution of the label. He is enough of a researche
r to know
that every compound is not a glucose metabolite. Can you help him by pointing ou
t his
mistake?
18.
You have a 32p labeled compound and you have to measure it. Unfortunatel
y, you hage run
out of some scintillation cocktail components. Do you have a way out?
19.
Both phosphorus and hydrogen ,are integral parts of nucleotides, Why do
then researchers
prefer using [3HI thymidine to a aP labeled nucleotide while assaying DNA synthe
sis?
20.
Scintillation counting is based upon a phenomenon called phosphorescence
. How is this
different from fluorescence?
21.
A researcher tells you that he has
u take the substance
from him and electrophoresce it. You put a
oretic
apparatus to bar radioactivity from coming
utside the
shield gives out noise. Was the researcher

labeled a given compound with all. Yo

perspex shield covering the electroph
out. Still, the Geiger counter kept o
telling you the truth?

14
CERTAIN PHYSICOCHEMICAL TECH
NIQUES USEFUL IN
BIOCHEMISTRY
POLYMERASE CHAIN REACTION
History of science is replete with examples of obvious and simple discoveries be
ing missed.
When these obvious discoveries have been made at a later date many of them have
turned out
to be major advances. One such example is that of polymerase chain reaction (PCR
).
Inside the cell, within the nucleus, DNA polymerase carries out the following re
action
(dNMP)n + dNTP (dNMP)n +I + PPi
Thus, starting with a double stranded DNA molecule, the replication proceeds wit
h
(0 unwinding of the two strands, (//) provision of primer, and (fl0 addition of
nucleotides on
the primer in a sequence complementary to the template, the reaction being carri
ed out
by DNA polymerase. Obviously, the result of this cycle of reactions will be two
double
stranded molecules of DNA (see Figure 14.1). Can this reaction not be carried ou
t in vftro
if all the requirements, i.e., template, primer, dNTPs and purified DNA polymera
se are provided?
That it can was demonstrated by Arthur Kornberg in the sixties. If the reaction
can take place
tn vitro, can one not select the region of the DNA to be replicated by synthesiz
ing primer of a
particular sequence? It is a pity that this question was not asked until 1985 wh
en Kary Mullls
of Cetus Corporation, USA, asked it. The result was the development of PCR.
Polymerase chain reaction can be defined as recurring replication (amplification
) of
a particular region of the genome preselected with the help of primers flanking
the region
of the genome to be amplified (Figure 14.2). Within the test tube isolated doubl
e stranded
DNA is denatured by heating at 90-98C to separate the two strands. Two primers, ea
ch
recognizing one of the strands at sites bordering the sequence to be amplified a
re mixed
and the tube is cooled to 40-60C. The primers anneal to complementary sequences
(Figure 14.2). The tube also contains all four nucleoside triphosphates and a su
itable

buffer. If now DNA polymerase is added, synthesis Of the complementary strand wi

ll take
place starting from each primer. This process, or rather cycle, of denaturation,
annealing,
and synthesis of DNA, ff repeated again and again leads to amplification of sdec
ted regions
of DNA. Each cycle of synthesis generates DNA products, which are copies of a."t
arget"
DNA sequence, and these products become substrates of subsequent cycles of synth
esis;

547
Parent double
stranded DNA
A
G
G
TO
A
A
T.
A
G
A
AC
C
5'
"
3'
T
C
C
A G
T
T
A
T
C
T
T G
G
Prt.mer attachment
5A,
G
G
T C
A
A'T A G A A C
C3,
Septed
3' G

G 5'
DNA sds

A G G T pmem
5' atched
plee
A G G T C A AT A G A A C C
5' 3' o double
3'. 5' sded
T C C A G T T A T C T T G G
dauter
A G G T C A A T A G A A C C
DNA
3'
molecules
Fig. 14. I. Simplified diagrammatic representation of DNA replication.
this is why the method is known as a chain reaction. As should be obvious, the
concentration of the "target" DNA doubles every cycle (Figure 14.2) leading to e
xponential
accumulation of the reaction products. What this means is that after about 20 cy
cles, the
concentration of the target DNA will be about a million times more (22) than the
concentration of nonamplified DNA. Due to this considerable amplification, it be
comes
possible to detect and to analyse small quantities of nucleic acids even when th
e starting
material consists of a large excess of sequences irrelevant to the investigator.
Thus, to
be analysed easily, the starting material can be as little as a few nanograms.
So far we have discussed all procedures with respect to DNA polymerase. However,
at-each denaturation step, when the mixture is heated to about 94C, the enzyme is
destroyedthrough denaturation thus necessitating addition of a fresh aliquot at
each
synthesis step. This problem was solved with the discovery of Taq polymerase, th
e DNA
polymerase of the bacterium Thermus aquaticus, which thrives in hot springs. Nat
urally
taq polymerase is resistant to high temperatures. In PCR, therefore, taq poiymer
ase is
used instead of DNA polymerase, and Just one time addition of the enzyme is enou
gh for
any number of cycles.

{+)
1 copy
Separated
strands
2 copies
Separated
strands
4 copies
Biophysical Chemistry
(.--)
I
Heat denaturation
,
l
Renaturation
(Annealing of primers)
Primers (A & B}
annealed
Extend primers
with Taq polymerase

Completion.
of
first cycle
{-)

.Heat
denaturation

I
Renaturation
with primers
I
Extend primers
with Taq polymerase
Completion
(+)
of
(-):
second
,(+)
cycle
{-)
Figure 14.2 Basic PCR reaction which amplifies a target DNA sequence, Essential
components of polymerase
chain reaction are (i) template DNA, (ii} oligo-nucleotide primers, {iii) Taq DN
A polymerase, and
(Iv) dNTPs. DNA synthesis is initiated from two slngle stranded primers (A & B i
n the flgure} flanking
either side of target DNA and oriented in opposite directions. After the pr&ners
have been extended by
Taq polymerase, the DNA strands are heat denatured. Temperature is lowered to al
low the primers to
anneal to their complementary sequences. Extension can now again be carried out.
Note that the number
of copies increases exponentially every cycle.

Lid
Certain Physicochernical Techniques Useful in Biochemistry
549
Practical Procedure, the Instrument and Precautions
In a small eppendorf tube the following mixture (total volume mostly 50 gl or 10
0 gl) is
prepared.
10 x Taq buffer
excess dNTPS
excess 5' and 3' primers
water to dilute the Taq buffer to Ix
1 gg DNA containing the region to be. amplified.
About 4-5 units ofTaq polymerase.
After this mixture is prepared, a small aliquot (50-60 ml, this volume is not cr
itical) of
mineral oil is layered on top to avoid splashing of the mixture at high temperat
ures.
Many investigators prepare a mixture of everything except the DNA and layer the
oil. The
DNA is added later by allowing the tip of the micropipette to go below the coil.
This is
supposed to be good for avoiding contamination.
The tubes are now placed in the instrument, which is known as the DNA Thermal
Cycler (Figure 14.3). The instrument has several wells (the number varies with
instruments; the original instrument marketed by Cetus Corporation has 48 wells)
, which
accommodate the small eppendorf tubes snugly. Before putting the tubes in these
wells, a
few drops of mineral oil are put in these wells so that the tubes are placed vir
tually in an
oil bath, which maintains a uniform temperature throughout the tube. After placi
ng the
tubes in the wells, the lid is closed and the instrument is programmed. Programm
ing of
each step in the cycle, fie., denaturation, reannealing, and synthesis is done u
nder two
heads-the temperature at which each step is to be carried out, and the time that
each
step is to be allotted. While denaturation is usually carried out at 94C for 1-2
minutes,
extension or synthesis is always carried out at 72C for about 2 minutes. It is th
e
reannealing step where the temperature usually varies. The temperatt!re at this

step
varies depending upon the A-T and G-C content of the primer; a temperature, whic
h is
IC below the melting temperature of the primer, is selected for reannealing. The
time
allotted to this step is usually 2 minutes. One cycle consisting of all the abov
e steps can
be completed in about 4-5 minutes. Once programmed, the instrument will carry ou
t the
individual cycles automatically without any further act on the part of the worke
r. Usually
30-45 cycles are enough to achieve a satisfactory amplification. The instrument
also has
the ability to store the tubes indefinitely at 0-4C after the programmed number of
cycles
are over and till the investigator is ready to take the tubes out.
(Sample holder)
Key board

550,
The precautions to be taken revolve around one central aspect: to avoid contamin
ation.
The chances of contamination emerge out of the ability of the PCR to tremendousl
y amplify the
target DNA. It can be easily understood that the contamination of the reaction m
ixture with the
smallest possible quantity of unwanted DNA can give an absolutely erroneous ampl
ification.
On the other hand, after amplification, if appropriate precautions are not taken
, there isa
danger of contaminating other samples in the laboratory with the PCR amplified s
equence. It is
good to confine the DNA thermal cycler into a secluded space and It is better If
it is kept in a
chamber having the facility of UV radiation so as to destroy DNA that may accJde
ntally fall out.
The preparation or" reaction mixture for PeR is best done behind a Perspex shiel
d. It goes
without saying that the micropipettes used should be cleaned beforehand and afte
r the
preparation of the reaction mixture.
Compr/son of PCR With Gene Cloning
In many respects PCR has scored over gene cloning and it might be that PCR will
ulUmately replace gene cloning to a large extent (such applications of .gene clo
ning as
production of a protein in large quantities for human use of course will not be
replaced by
PCR), The final result of both PCR and gene cloning can be said to be a selectiv
e amplification
of specific sequences. To achieve this objective via gene cloning, the investiga
tor Joins a
fragment of DNA to a vector DNA which replicates and produces multiple copies (s
ee Figure
14.4), Joining of the fragment of DNA to the vector DNA is necessitated by the f
act that
the desired fragment cannot replicate itself while the vector can. The hybrid DN
A then
needs to be introduced into the host organism where it will replicate. Figure 14
.4 makes
it clear that there are many steps, both /n v/tro and /n v/vo in a gene cloning
experiment.
The starting material that is required for gene cloning is afleast a thousand fo
ld higher
than required for a PCR experiment. Moreover, the possibility of error in PCR is
much
less than in conventional gene cloning experiment. Some major points of comparis
on
between PCR and gene cloning are listed in Table 14,1.
Table 14. I PCR and Gene cloning: A Comparison

PCR
Clonin

Ultimate result
Selecting the target sequence
to be amplified
Concentration of starting
material
Requirement of biological
reagents
Manipulation
Automation
User
bb/,,. ,/'-,f
Labour intensive
Cost
Time required for a
typical experiment
Selective amplification of target sequences
First step Last step
Nanogram
Microgram
DNA Polymerase
Restriction enzymes,
ligase, vector bacteria
In vitro
"
In vitro and in vivo
Total
None
No
Yes
Less
More

3-5 Hours
3-5 days

551
Transform
Certain Physicochemical Techniques Useful in Biochemistry

Foreign DNA
{Restriction site
Digest with
restriction enzyme shown by the bar)

O O Vectorandforeign
DNA anneal at the
sticky ends
Recombinant vectors
carrying foreign DNA
{Foreign DNA
Indicated by wavy llnes)
Figure 14.4 Basic cloning procedure, Foreign DNA and vector DNA are cut with the
same restriction enzyme (let
us take Eco RI as representative example of restriction enzymes). Note that the
vector (plasmld) has
only one restrcthm site while the foreign DNA has several Restriction digestion
is sequence specJlc
and the cut ends are complementary (Eco RI cleavage site is given below) and upo
n restriction
ends of foreign and vector DNA can anneal through hydrogen bonding. The entire s
et of recombinant
plasmlds carrying different segments of foreign DNA are now introduced into the
bacterial cells
through transformation. Bacteria carrying the desired recombinant plasmld are no
w selected ago:inst
5'G A A T T C3'
3C T T AA GS'

Variation of the Basic PCR Technique

In the above pages we have Only outlined the basic PCR process. There can be var
ious
variations to this technique all serving different purposes. It may be said with
out the fear of
exoneration that PCR has many utilities which are limited only by the investigat
or's imagination.
A few of the variations of the basic PCR technique are described below.

I
Restrlct and
llgate to
circularize
0
552
Biophysical Chemistry
(0 The inverse PCR technhlue. In the basic technique described above, a knowledg
e of
the sequence of the gene to be amplified was essential in order to amplify it. H
owever, there are
times when the investigator has little idea about the sequence of the gene that
he is interested
in. The inverse PCR technique is useful in such cases and will allow amplificati
on of such a
DNA fragment without using primers specific for it. The In-st step in inverse PC
R is to clone the
DNA fragment in a circular vector DNA. The sequence of.the vector DNA is known a
nd hence
one can synthesise primers specified for it. If PCR is now carried out with thes
e primers, the DNA fragment of interest will be .amplified and it will have sequ
ence of the vectors at it ends
(Figure 14.5A). Inverse PCR can also be carried out on linear DNA as shown in Fi
gure 14.5 B.

Region to be amplified
cut with restriction
enzym
I
Clorflng
procedure
Oved Insert
e
Region of unknown
sequence to be
amplified
Region of known
sequence

Restriction
site

) Primers
annealed
Extension
Amplified (A} :product
Primers annealed
i, Extension
Amplified
(B)
product

Figure 14.5 Diagrammatic representation of inverse PCR

(A)
Foreign DNA of unknown sequence is cloned in a circular vector DNA. Sequ
ence of vector DNA is
known and hence it is possible to synthesize primers complementary to those vect
or regions which
flank the cloned foreign DNA. Amplification product will possess vector sequence
at both ends.
(B)
In a linear DNA molecule, if the sequence of the gene adjacent to the ge
ne to be amplified is known
(either partially or wholly}, it is possible to amplify the sequences flanking t
he known gene. The DNA
molecule is restriction digested and circularized by ligation. The primers which
are complementanj to
the ends of known sequence are annealed. The DNA sequence flanking the known seq
uence can
now be amplified. The amplt.fication product will possess known sequence at both
ends.

553
1st cycle
2nd cycle
(I)
[1+2)
Certain Physicochemical Techniques Useful in Biochemistry

Denature
aReanneal
nd extend
Figure 14.6
Site directed point mutation by PCR. Basic PCR reaction
is similar to that'shown in,figure 14.2. Here, the primers
contain the required base change ( indicates primer
with one base change at the position of the cross bar).
The base change is minor and does not alter the annealing
of the primer with the template. The base change is then
faithfully copied into the new DNA strands in the
subsequent, cycles.

B
D
Figure 14.7
Site directed mutagenesis by overlap
extension PCR. A single stretch of DNA
is amplified in two different segments
by using primer pairs A, B and C.D.
Internal ends of these two segments
i overlap because the internal primers
B and C are comple-mentary. When
denatured and reannealed, the
amplified segments of the two PCR
reactions form base pairing at the
overlap region and can be extended
in the next cycle to produce the whole
original sequence. The primer at the
overlap region (B and C} carry

alteration (s) is the base (s). Mutations

can be induced at any region of the
, altered internal
primers for that region.
(2)
I
Denature and
reanneal
I
Extend

Next cycle
(Mutated product
acts as the template)

554
Biophysical Chemistry
|0 SIt directed m,tcggesi b//. PCR c be used to reduce mutations at a
preselted site y ent of DNA d to p at mutated DNA. eension d s approach ows aesl
s of hybd genes d mosc proteins. gure 14.6 shows
flcy e prede for pg a sequence mumons oduced dung R itse. e ck is to use a per w
ch is not peecfly complement; e per us
cons a be wch ds not base p e template. Uge of s per is responsible for due t mu
tation at a site cmted e posion ofe non-complemen base e per. D e lese ch rcflon
s mutation is copied into e dauter
sd d er cles of R p e gene wch has e mumon. ers c be
es such a way at stead of a pot mutation. seons or deleons be oduc to e gene. s
meod, howler, is ted by e fact at see e pe
used e te. e mutations duced e gene so be at e ends of e gene.
A meod, howler, has been recently desired to overcome is on d is o
as e o en . Using s meod It is possible to oduce mutton at at y site e gene of t
erest (gure 14.7]. e meod consists of pg e pc gene as o sepate seents o sepate P
CR reactions. is is aceved by usg go e PCR reactions one per at e end of e gene
d oer per at a suence mtem to e gene (re 14.7). e term pers used e o reacons e c
omplen to ea oer. e o R products e su at overlap e re,on whe e mte p w sion d t
erpp o memen to each oer. It is ese t pers at a sequence vaflon(s) is Woduc depe
n un e of mutation desed. e o PCR products e now ed d detured. en re-eg is owed
to e place, e o overlappg sWds of e PCR pruc ne
d eff overlappg ends now act as pers. In e PCR at now be peHoed, e complete gene
be sesd berg e mumon (e 14.7). e site of e mtem pers be ved e gene depeng upon
e site where e mutton is to be uc. e me is ed to we 1 effi mu site see mumons.
(it0 $plg .e overlap enston me discussed m e prous pph ud to consct cec genes w
1 produce ceHc proteus. e meod Is tHusated gure 14.8. ere e ser uses of chec gen
es d e cec proteus. For ple, nscflon of cec r proteus have prodded
pot oaon about e reons of genes pot for eir oncogec role. oer eple is at of coco
n of a ceHc pepde hoone..produ..-spR
eff respecve gent. Such chec hoones eclt e acW of go e hoones
whose genes were spced.
() n Conveno R is not eately appRble to quen or footpg because e process reqes o
deed ends. Usury a sequence or footpt
ladder Is made up of related nuclelc acid frents. Each of ese ffen has one of e
ends made on by usg eler a per or a rescon cut. e oer ends e deteed
by vous chec cle mes or ch teaon (e.g., dldeo teaflon); ese
ends e efore que to each ffent. A sple Hgaon step c be Woduced e blc R.reacflon
wch ows suence deteaon of R products ecfly. s Hgaon
step attaches a co--on nucleoflde sequence to e get sequence. Usg a per to s
coon suence, nucleodes c be added upon d uque ends sesised just e conveno sequen
cg or foot-p tecqCes. e process Is sple d ows f
deteaflon of e sequence.

555
Certain Physlcochemlcal Techniques Useful in Biochemistry
A
Gene X
C
Gene Y

Denaturatlon
ednaturatio.n
J
Extension
G no x
Figure 14.8 Gene splicing using overlap extension PCR. X and Y are two d'erent g
enes and are to be spliced
together for constructfon of a chimeric gene. These genes are ampljcl usfng prlm
er parrs A, B and
C D. Primers A and D are dstnct but a part f prlmer C and B fs cmplementary t ea
ch ther (heavy
lfftes) The ampled products therefore overlap n thls regfon. Upon denatumn and r
enatumtWn
mes X and Y form base pairs in this region. These overlap re, forts act as prime
rs in the next
extension cycle and a chimeric gene formed. Further ampl{flcation fs possible us
ing primers A and D.
Applications There are numerous applications of PCR and since the technique is comparatively
recent,
new applications are being added with an amazing speed. We will not discuss the
applications
which are common and which can easily be imagined by the reader. Instead some of
the novel
applications of PCR are discussed in this section.
(1) Genetic diagnosis by PCR. In 1987 Embury and associates reported that rapid,
specific,
and highly sensitive diagnosis of sickle cell anaemia mutation can be achieved w
ith fetal DNA
samples using polymerase chain reaction. In that very year, Kogan et.al, describ
ed a method for
prenatal diagnosis of haemophilia A using PCR. Genetic diagnosis by PCR will soo

n become a
byivord in clinical studies because of (A) the easy nature of the experiment (PC
R is totally
automated), (B) lesser time requirement (see comparison of gene cloning with PCR
discussed
previously), (C) less sample requirement, and (D) little or no loss of cellular
DNA through prior
purification, since purification is simply not needed.
Although, techrticaliyspeaking, most of what can be achieved by. PCR was achieve
d by
conventional gene cloning; the worker expertise and sophisticated instrumentatio
n required
were not possible for small clinical labs. These conditions were not required fo
r PCR and this is
where PCR scores over cloning and will allow genetic diagnosis to become commonl
y available.
(i@ Study of/Ifetlous.dfsm uslrtg/W,,R. PCR allows direct amplification of trace
amounts of genetic material of infectious agents in blood, cells, water, food, a
nd other clinical
and environmental samples. Due to this abtlity PCR becomes especially valuable f
or detecting

556
Biophysical Chemistry
pathogens that are difficult or impossible to culture {examples - the HIV, causa
tive of AIDS; the
agent for lyme disease). Moreover, in the case of AIDS, it is known that many in
fected persons
do not test positive for AIDS antibodies. Nevertheless these same persons may te
st positive
later on. However, PCR can detect the presence of even a small number of HIV and
its genome
even though no immune response is detectable. PCR can thus be of much use in pro
viding a
very early and almost foolproof diagnosis of the deadly disease that AIDS is.
[iiO in m" research. Since clinical markers of predictive value are not available
for cancers, PCR is not yet of much diagnostic value for this human malady. Howe
ver, PCR is
being tremendously used to gain Lnsights into the molecular basis of cancer. Som
e examples of
this use are cited bekw.
The observation that activated (through single point mutation) ras genes are pre
sent in a
variety of human tumours has aroused tremendous scientific investigation of the
role of these
genes in tumorigenesis. In order that-ras genes be demonstrated as causative for
tumorigenesis
and not merely of secondary involvement it is necessary to prove that (A) mutate
d forms of
these genes are consistently associated with a particular tumour type, and (B) m
utation in ras
genes occur at very early stages of tumorigenesis. Since hotspots of ras mutatio
ns are known
(12t, 13t and 61at codons), PCR can be used to selectively amplify ras gene regi
ons around
these codons and the subsequent RFLP (restriction fragment length polymorphlsm)
or sequencing
allows demonstration of mutations fairly quickly. Using PCR it has therefore bee
n possible to
demonstrate that mutated ras genes can be consistently associated with pancreati
c carcinoma
(90% pancreatic tumours contain activated ras), colon adenoma or adenocarcinoma
(50%),
thyroid follicular carcinoma (50%}, and seminoma (40%). PCR has also been succes
sful in
demonstrating that mutated ras genes are present in benign adenomas and malignan
t
carcinomas in the same frequency potentiating the belief that ras genes might be
causative
rather than secondary genes for carcinogenesis. More importantly, chemical carci
nogenesis
studies on animals using PCR have been much more useful in demonstrating that ra
s genes

are activated early in carcinogenesis. When 2-day old rats are injected with nlt
rosomethyl urea,
ms mutations in the mammary tissue can be demonstrated as early as at the stage
of 15-days.
Earliest turnouts, however, develop only after onset of puberty.
Presence of viruses that are implicated as causative agents for human cancers ca
n be
demonstrated by the use of PCR. Samples contalnlngas less as I00 DNA molecules c
an be
screened for the presence of such viruses.
It was thought that in pituitary tumours the elevated levels of cAMP cause uncon
trolled
proliferation thereby contributing to malignant transformation. Using, PCR it wa
s recently
demonstrated that pituitary tumours contain mutated versions of the a-subu-nit o
f Ga (mutations
in the 201st and 227th codons), the protein that transduces signals from
[-adrenergic receptors to adenylate cyclase. Gs can therefore be viewed as a pot
ertial oncogene.
PCR is also being used to screen mutations in tumour suppressor genes such as th
e
retinoblastoma and thep53 genes.
Lastly, it may be said that once clinical markers of predictive value become kno
wn, PCR
will provide an excellent early diagnostic warning so that the patient can be tr
eated at a very
early stage.
(iv) l'io u ofFeR, DNA of trace materials such a semen, blood, ha/r found at the
icene of or/me can be analyled by PCR amplttlcation and can provide a clue to th
e ident/ty of
the crlmmal when matched with the DNA of the suspects, PCR has been used to iden
t rrstng

Physlcochemical Techniques Useful in Biochemistry

557

;rsons and to settle paternity cases through DNA analysis. Another area in which
the utility of
R is increasingly being realised is the matching of transplant donors and recipi
ents.
(v) Study of evolution using PCR. The mammoth, which is now extinct, is widely b
elieved
be ancestor of the modem day elephant. PCR amplification allowed DNA from Siberi
an
mnmoth (these mammoths have been well preserved since they have been buried in t
he ice
ra long time) to be compared with the DNA of modem day elephants. The mammoth se
quences
roved to be closely related to the modem elephants thereby proving the link betw
een these
o. The ability of the PCR to amplify ancient DNAs open up the possibility of stu
dying molecular
o|utlon by actually going back in time and dlrecfly approaching DNA sequences th
at are
cestral to their present day counterparts. This makes possible for us to relate
many extinct
les to other extinct or extant species. Evolution of population size, migration,
selection and
metic drift can be studied with PCR even if small number of preserved samples ar
e available.
(vO Archueology and anthropology using IR. Recently, using PCR and direct
uencing, mitichondrial sequences from 7000 year old brain excavated in Florida w
ere
mpared to mitochondrial sequences of more than 100 present day native Americans.
It
found that this ancient DNA sequence does not match any of the three mitochondri
al '
found to date in America. Further studies are on to find out the population
and geneological history of Amerindians. Similar studies involving amplification
direct sequencing studies are in progress with Egyptian mummies. When they arc
with the sequences of modem day Egyptians, the study will yield important
about the population structure and geneological history of the Egyptians.
A rough dating of the ancient sample m..ay be possible using PCR and direct
It is known that sequence changes in the exons of same genes of the same
are much rarer over a period of time as compared to the introns. Determination
the extent of change in the sequences of the introns of the ancient DNA as compa
red to
modem DNA of that specie might give a rough idea about the age of the sample.
ivlfJ Other applfatlana. PCR enables the researchers to determine the sex of an
tn
fertilized embryo at a very early stage of,Just 6-10 cell cleavage. What is more
. the

does not interfere with the life of the embryo at all, This approach will be ver
y
for genetic counselling and avoiding birth of a girl in those cases where the pa
rents
at a risk of X-linked disorders in their offsprings.
The sensitivity of PCR allows one to amplify a gene from a single isolated cell.
This
can be well employed to pick out a single antibody-producing cell and to
amplify the genes encoding the antibody. These genes can then be cloned
bacteria that will then mass-produce the particular antibody. The PCR technology
thus
hybridoma technique redundant for the production of monoclonal antibodies.
The many advantages of PCR technique are summarized below.
(A) .Ability of selectively amplifying a small segment of DqA from a sample cont
aining
numerous unrelated sequences.
(B)
The ability to amplify this DNA seglnent from an extremely small quantit
y of sample;
even a single cell may be sufficient sometimes.
(C) No skill required on the part of the worker.
(D) Easy and extremely quick operation.
These advantages of PCR have made it one o.f the most prime developments of our
The appl/eations of PCR are numerous even at this nascent ste oi the technology,
can only/mane the range of appl/caUons that would be ava/lable a decade later,

558
Biophysical Chern/stry,
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
ELISA is today playing an increasingly important role in diagnostic and research
laboratories
the world over. The principle of ELISA is as follows: one of the immunoreagents
is immobilized
through adsorption On the solid phase support (usually polyvinyl chloride or pol
ystyrene) n
such a way that there is no loss to its activity. The second immunoreagent is li
nked to an
enzyme in a way that there is no loss either to immunoreactivity or to the enzym
e activity. After
incubation and subsequent washing, a chromogenic substrate of the enzyme is supp
lied. If the
two immunoreagents have bound to each other, colour will develop because of the
presence d
the linked enzyme; ff not there will not be any colour. The basic principle of E
LISA is shown
diagrammatically in Figure 14,9.
.. 0 Subtrate
(color less)

"

Ev.zyme
A Product (colored)
Conjugate
Adsorbed

Reacting

"
antigen
antibody
Solid
phase
Fjure 14.9 Prlnctple of Enzyme Linked Immunosorbent Assay.
In Chapter 13 we have discussed radioimmunoassay, a technique used to detect
bioactive antigens and even antibodies. ELISA is fast replacing radioimmunoassay
s because
certain inherent advantages. Fir@t of all, ELISA does away with the need of
which are difficult to store because of their short shelf life. The reagents for
ELISA on the
hand are long lived. Dueto the problems the radiochemicals might pose to the hea
lth,

is limited by legislation. There is no such health risk with ELISA reagents and.
as
administrative inconveniences are circumvented. Moreover, ELISA lends itself
sophisticated automation and is quite adaptable to simple tests. Lastly, ELI.SA
can be,
with a variety of detection systems such as visual comparison, photometry, fluor
ometr, and
luminometry.
Discussed below are a few assays which are routinely used.
Assays for Antibody: Indirect Method

This method is largely used to measure antibodies in almost all human infections
. The
method has become popular because it requires only a single conjugate, for examp
le enzymelabeled antihuman globulin. Different immunoglobulin classes can be detected dif
ferentially by
using class specific conjugates, for example enzyme-linked antihuman IE.
Salient points of the procedure for indirect method are briefed below.
(i)
A suitable antigen is allowed to adsorb passively onto the wells of plas
tic
microplates. The plates are subsequently washed to remove unadsorbed antigen
and then dried, These plates can be stored,
(ll)
Diluted serum (or any other test sample) is then filled in these wells a
nd allow
to incubate. If the antibody specific to the adsorbed antigen is present it reac
ts

Techniques Useful in Biochemistry

559
and becomes immobile. Washing is now carried out to remove all unreacted serum
components.
(///}
Enzyme-llnked anti-human immunoglobulin conjugate (or any other class sp
ecific
conjugate) is now added to the wells and allowed to incubate. This conjugate now
binds to the antibody captured on the solid phase in step (ii). The unreacted
conjugate is washed away.
A chromogenic enzyme substrate is now added to the wells in a solution. The rate
of
degradation of the substrate (increase in colour intensity) is proportional to t
he
concentration the antibody in the serum. The reaction is stopped after a particu
lar
time and the colour intensity is measured photometrically.
A summary of the above steps is given below:
Solid phase-antigen test sample antibody anti-immunoglobulin enzyme conjugate +
substrate colour
An alternative to the indirect method is the so-called Sandwich methocL In this
method the
antigen is adsorbed to the solid phase. The test sample is then incubated with t
his
phase and subsequently the enzyme linked antigen conjugate is reacted. Finally,
the
is added to develop colour.
for Antigen: Sandwich Method
The sandwich method is the most largely used method for this purpose. It is summ
arized
(i) A specific antibody is immobilized on the solid phase by adsorption.
(ii)
The test sample containing the antigen is now incubated with the antibod
y
immobilized on the solid phase. Unreacted antigen is washed away subsequently.
(iii)
Enzyme-linked antibody specific for the antigen is now incubated. The un
reacted
antibody conjugate is then washed away.
(iv)
A chromogenic substrate is now added and the colour rnasured by photomet
ry.

The colour intensity is proportional to the concentration of the antigen.

Solid phase-antibody test sample antigen antibody-enzyme
conjugate + substrate , colour
A variation to this method to increase its sensitivity has been made through the
use
,biotin-avidin system. The above sandwich method is followed as it is up to the
incubation
'test sample reagent with the solid phase [step (ii)]. Subsequently a biotin-lab
eled antibody
,reacted. Finally an avidin-linked enzyme solution is incubated.
So, lid phase-antibody test sample antigen
conjugate
avidin-enzyme conjugate +
substrate colour

antibody-biotin

Practiczl Notes
Coating of the solid phase, is influenced by the material used for the solid pha
se, the
and the concentration of the immunoreagent, by the nature of the diluent, by the
and finally by the time of incubation. Two types of solid phases are in general
polystyrene and polyvlnyl chloride. Of these polystyrene binds much less protein
as
to the latter. Polystyrene is therefore the material of choice when non-specific
with serum components is to be minimized. On the other hand when a high

560
Biophysical Chemistry
coating level is required, polyvinyl chloride is the best solid phase. The purit
y of the
immunoreagent is a must since impurities, if present in substantial amounts, wil
l compete
for available space in the solid phase surface. The diluent used for proteins is
usually
carbonate or phosphate buffer used either at a neutral or alkaline pH. The conce
ntration
used normally for proteins varies between 1-10tg/ml. Usually overnight incubatio
n between 40
to 8C is sufficient to coat the solid phase. However, with polyvinyl chloride eve
n one hour
incubation at room temperature is enough. The coated phase can be stored in a de
ssicator in a
refrigerator for periods exceeding five years with little loss to the reactivity
.
The test sample concentration should be adjusted so that the measurement is opti
mum.
Usually the test samples have to be diluted to reach the optimum concentration.
Another problem
which should be avoided arises due to non-specific reactivity of the sample comp
onents which
get adsorbed directly to the plastic surface. To reduce this problem a wetting a
gent such as
Tween 20 is usually included in the incubation medium. Along with the wetting
a blocking agent has to be used to further minimize non-specific reactivity. Mos
t often BSA
used as the blocking agent.
The most often used enzyme to be conjugated with the immunoreagent is
peroxidase. This enzyme is cheap and can be attached to the immunoreagent by a v
ariety
of methods. Moreover many chromagenic substrates for it are also available.
enzymes are alkaline phosphatase, glucose oxidase, penicilinase, urease etc. 8"
galactosidase has become very popular now since it can be used with fluoro
substrates.
Conjugates are usually prepared with glutaraldehyde as the linking agent.
periodate, maleimide, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) have
also
used as linkers. If biotin avidin system is followed, biotin can be linked to th
e antibody
incubation with dimethyl formamide, and avidin can be linked to the enzyme throu
gh
glutaraldehyde.
The substrate is such that it is colourless before reaction and after degradatio
n
produces colour. The substrate used most often with horse radish peroxidase is o
rtho.
phenylenediamine (OPD) which produces a strong yellow product. When the
reaction is stopped by addition of acid, the colour changes to orange-brown. How
ever, this

substrate is light sensitive and there are doubts about its safeness. Alternativ
e substrates
for peroxidase are 5-aminosalicylic acid, 3, 3, 5, 5 -tetra methylbenzidine (TMB
) and 2,2azinodl (3-ethyl-benzithiazoline sulfonic acid-6) diammonium salt (ABTS). Normal
ly the
incubation time is about 30 minutes following which the reaction is terminated b
y
either alkali or acid. The absorbance of the solution can then be easily measure
d using an
ELISA reader.
Some fluorogenic substrates have become popular of late. These are methyl
galactoside for the enzyme -galactosidase and methyl umbilliferyl phosphate for
the enzyme
alkaline phosphatase.
FLOW CYTOMETRY What is Flow Cytometry?
Measurement of physical and/or chemical characteristics of cells or, in general,
particles is known as cytometnj. When such measurements are performed while the
cells
other biological material pass in a single file through the measuring apparatus
the process is known as flow cytometry.
The intense interest centred around flow cytometry is mainly due to the speed
which analyses are performed (even up to 3 x 105 cells per minute) and due to th
e

561
of simultaneous analysis of multiple parameters (see Table (14.2). It is very im
portant to note
that all the measurements are performed on a cell by cell basis. An additional f
unction that flow
cytometers can perform apart from cellular analysis is their physical sorting. T
his sorting takes
Just few minutes and gives purity of any cellular subtype in excess of 95%.
Pylieal Paxamete
Chemical Paxamete

Cell size
Cell shape
Cytoplasmic granularity
Cytoskeletal organization
Redox state
Membrane integrity
Endocytosis
Surface charge
Membrane fluidity
Structuredness of cytoplasmic matrix
Membrane potential of biological membranes
Animal and plant
pigment content
Total protein
Basic protein
Sulphydryl groups
DNA content
DNA base ratio
DNA synthesis
RNA content
Antigens
surface sugars

Enzyme activity
Membrane permeability
Intracellular receptors
Surface receptors
Membrande bound [Ca
Cytosolic [Ca2+l
Intracellular pH

The flow cytometer analysis and sorting instruments combine electrical and optic
al
sensing techniques thereby permitting several measurements to be performed
simultaneously on the same cell. Typical measurements inclOde electronic cell vo
lume,
fluorescence of endogenots cell constituents or from stains bound to cell consti
tuents,
light scatter due to extrinsic or intrinsic cellular features, absorption or los
s of extinction
of light due to cellular components,, and fluorescence polarization. If a hetero
geneous cell
population is being assayed, the simulteaneous measurements allow a biochemical,
functional, cytological relationship to be established between different cell ty
pes.
The basic process of flow cytometry is illustrated in Figure 14.10. Cells incuba
ted
with fluorescent or absorption dyes are suspended in. physiological, saline. Thi
s suspension
is allowed to pass through the flow chamber at a rate of about 1000 cells/see. A
s the cells pass,
they either scatter, absorb, or fluoresce the light.impinging upon them. This sc
attered,
transmitted or fluoresced light is then measured by appropriate optical sensors,
Additionally
electronic sensors detect the particle volume and other related parameters. Sign
als from
each of the sensors are processed on a ceil by cell basis and the resulting data
are displayed
as frequency distribution histograms.

562
Figure 14.10 A highly schematic representation of.flow cytometer
After measurement the stream which exits from the flow chamber is disturbed by a
peizoelectric transducer to give uniform droplets at the rate of 40,000/sec. Abo
ut 2% of such
droplets contain one cell each. Processed signals from cell sensors now activate
the cell-sorting
device. If the amplitude of the signals fall within a preset range a droplet cha
rging device is
activated. The droplets, which are so charged, are deflected by a static electri
c field and are
collected in a container. Other droplets not containing the desired cells are no
t charged and
these are therefore not deflected. One can achieve a cell separation rate of 100
0 cells/sec and
purity of such cell preparations is routinely about 95%.
Instrumentation
Although a variety of commercial flow cytometers are available, all of them are
composed
of the same basic units, v/z. fluid transport system, flow chamber, measurement
region, excitation
source and optics, optical and electronic sensors, collection optics, signals pr
ocessing electronics,
and data display, storage and analysis system.
The samples are placed in a sample chamber which is fitted with a Continuous sti
rring
device to mix the heterogeneous cell population and with a filter to remove cell
clumps
from flowing into the fluid transport system. The cells from here are pressurise
d for delivery
by tubing to the flow chamber. In the flow chamber the sample stream is injected
into the
centre of a ceil-free stream of sheath fluid (Figure 14.10}. The sample along wi
th the
sheath fluid now enters a constriction region, which increases the flow velocity
. In order
that the cells pass in a single file, it is necessary to adjust the sample conce
ntration and the
sample stream diameter. For optimum measurements it is necessary that a laminar
flow be
maintained.
The measurement region means the site where the cell stream intersects an excita
tion
beam of light (Figure 14.10}. Measurements on cells are made here. This region i
s usually
located inside the flow chamber but in certain instruments the measurement regio
n is placed
outside.
The source of light in most modern instrument is the argon-ion laser. Lasers hav
e the
advantage of extremely higll radiance, stability and spectral purity. In additio
n it is easy to

focus them to areas equal to the cell dimension A beam stop is placed to stop th
e laser after it
has irradiated the cells. Appropriately positioned detectors measure the fluores
cence and light
scatter due to the cells.

Mithramycin, chromomycln.
olivomycin, acridine orange, eth/dlum,
propldlum
Thlofalvln T, pyronine Y, oxazlne I.
Fluorescein isothiocyanate [FITC]
Certain Phtjsicochemical Techniques Useful in Biochemistry
563
Most of the modem flow instruments are capable of multiparameter detection. A se
ries of
detectors and filters are positioned appropriately to measure fluorescence at a
number of
wavelengths. Usual placement for fluorescence detectors is at 900 to the inciden
t laser beam.
The presence of multiple detectors allows use of several fluorochromes to detect
multiple cellular
parameters. Many instruments also use two or more sequential lasers to excite fl
uorescence in
cells at multiple wavelengths.
The cells passing through the flow chamber also scatter the incident light. Ligh
t scattered
at different angles reveal different information about various cellular characte
ristics. The higher
angle light scattering assists in the identification of specific cell sub-popula
tions. Low angle
forward light scatter is dependent upon (t) the cell size and shape, (tO the ori
entation of the dell
in flow, (/to the refractive indices of the cytoplasm and the nucleus, and (iv)
the internal structure
of the cell. Analysis of low angle forward scatter therefore reveals much inform
ation about the
above cellular parameters. In addition, orthogonal (90) light scatter gives impor
tant information
about the granularity and fine structure of the cell. Thus a flow instrument con
tains a series of
detectors to measure the light scattered at different angles.
Each detectr in the flow cytometer gives a signal in the form of an electrical p
ulse that is
proportional to either the concentration of a cellular substance or to a cellula
r feature. This
electrical pulse is amplified and converted to a digital value before being sent
to a digital computer
which analyses, stores, or displays the signal. For every pulse, its peak amplit
ude, width, and
area are recorded. If fluorescence measurements are being performed, the peak gi
ves us the
about the maximum fluorescent intensity of the cell; the width tells us about th
e width of
the fluorescing part of the cell and the area of the pulse informs us about the
total fluorescence
of the ell.
It might be stated here that fluorescent measurements are the most often used

measurements in flow cytometry. A list of fluorochromes used to assay DNA, RNA,

proteins,
or various cellular features is provided in Table 14.3.
Table 14.3 Some Fluorescent Probes Used in Flow Cytometry of Important Cellular
Parameters
Parameter
Fluorescent probes used
IJV excitation
Visible excitation
DNA content
4',6-dlamino-2-phenylindole
RNA content
Total Protein
Cytoplasmic [Caz']
Intracellular pH
Enzyme activity
Surface structures
Membrane potential
Membrane integrity
4-acetamldo-4'-isothiocyanatostilbene 2,2'-dlsulfonlc acid
(SITS) dansyl chloride
indo- 1
1,4-dlacetoxy-2-3-dicyanobenzene, 4-methylumbelliferone
Coumarin and naphthol
based substrate.
SITS, dansyl chloride
Oxycyanines
Prlmulln. fluoresceln d/acetate
Carboxyfluorescein dlacetate.
Fluorescein based substrates

FITC, phycoerythrin, ailophycocyanlne, Phycocyanine,texas red.

Oxacarbocyanines, indocarbocyanines. Rhodamlne 123
Propldium, fluorescein diacetate

564
Bphysal ChemLstry
APllieltions of Flow ome
Flow ome h numerous appcaons and as case of PeR it mit be sd at e appcations of
flow ome e ited oy by our o imaaflon. A deled discussion of
ese appcaflona is imp0ible here. However, major well documented appcaflons have
been
cury det .
[}
. sus measmen ofDN A, ce s d prote, it comes possible to dee ces' posion
e cell cle. It so becomes possible to so ce popaflons erent ce de phases d to s
ubject each popaflon to
bioche ysis.
(J
$ on hmom. It is possible to detee kaote of a cell usg
flow cytomet. For example Chinese hamster embo celia were treat th
the fluorochrome propidium iodide. e chromosomes were then alysed by
cig at 4 nm d studg e fluorescence at wavelena above 560 .
11 chromosomes gave a derent pe. By eension it c be sd at tered kaotype would gi
ve an altered fluorescent pattern. Such studies therefore
achieve great siificance in studying malignant celia that often demonstrate
altered kote. Moreover. genetic counselling can also benefit from flow ome as th
e altered kope c be studied we easily and quicy. Hi
speed comosome soers are now use to facilitate e task of mapping e hum genome.
(3)
uocte characterization. o colour fluorescence characterization of
acridine orange treated leukocytes makes it possible to dlstinghlsh between
lphoes, monoes, d granuloces and to so them out. Acdine orge Is
so ued to chactee leukoes from ents vaous nds ofneoplasma such as leukemlas, Iph
omas etc. In addlon flow come Is so utilized for
derenfl leoe countg where it ves we precise vues.
(4)
T of anul d plalt n. Tests for ulote function such
as NBT reduction, dichlorofluorescein oxidation, membrane potential,
measurement of deulation, d phagoic assays are I possible usg flow
ome. Using flow ome it has been demonsated at oplasc Ca*
concentration increases, membrane potentlal becomes more positive, and
superode is generated duflng phagoosis by neutrophils.
Platelet abfli c be assayed easily by eler a dye exclusion test or by measug
e membre poten usg a cboe dye. Platelet activation c also be
measured easily by measug crse oplasc Caa+ concentraon.
(5)
an of ell-tell ommunivan. Flow omet mes it easy to
quanfl cell-to-cell communication. As an example work of va may be
cited. Cells are scrape-loaded th Lucifer yellow th or thout rhodamine
labelled d. e sfer of e o dyes beeen donor d recipient cells
c be studied easily using two colour fluorescence flow omet.
Ofily a few representaUve appcations have been cited above. In addlUon It may be

sd that flow comet finds numerous applications in e fields of ce differentiation

,
immunolo, parasitolo, sperm analysis, food science, phaacolo and tocolo.
cancer blolo d carcinogenesis, bone mow analysis, tissue ting d lphoe
applications, d T-ce subset anysis.

Certain Physlcochemlcal Techniques Useful in Biochemistry

565
Flow cytometry is increasingly being used for sorting different cellular populat
ions. Thus,
different leukocytes, macrophages, and many other types of cells are routinely s
orted using
flow cytometry.
Suggestions For Further Reading
I.
Polymerase Chain Reaction, Current Communications in Molecular Biology,
(Erlich. H., Gibbs,
R. and Kazazlan, H. Jr. eds.), Cold Spring Harbor Laboratory Press, 1989.
2.
PCR Technology, Principles and Applications for DNA Application. (Erlich
, H., ed.) Stockton
Press, New York, 1989.
3.
PCR Protocols. A Guide to Methods and Appllcatlons (Innis, M.A., Gelfand
. D.H., Sninsky, J.J.
and White, T.J., eds.}, Academic Press, San Diego, 1989,
4. Guyer, R.L, and Koshland, D.E. Jr. (1989}: The Mo/ecu/e of The Year. Science,
246:1543-1546.
5.
Ho, S.N. and Hunt, H.D. et aL, (1989): Site Directed Mutgenesls by Overl
ap Extension Using
the Po/ymerase Chain Reaction, Gene, 77:51-59.
6.
Horton, R.M. and Hunt H.D., et al., (1989), Eng/neer/ng Hybrid Genes Wit
hout the Use of
RestrlctWn Enzymes: Gene Splicing by Over/ap Extension. Gene, 77:61-68.
7.
Muller. P.R. and Wood. B. (1989}, In vlvo Footprlntlng of Muscle Specifi
c Enhancer by Ligation
Med/ated PCR. Science, 246:780-786.
8.
Li. H., Gyllensten, U.B. et al., (1988), Amplification and Analysis of D
NA Sequences in Single
Sperm and D/p/o/d Ce//s. Nature, 335:414-417.
9.
Mullis, K.B. (I 990}: The Unusa/Or/gin of Polymerase Chain Reaction. Sci
entific American, Aprll,
42-49.

10.
Talwar, G:P., Non-isotopic Immunoassays and Their Applications, Vikas Pu
blishing House,
New Delhi, 1983.
I I. Engvall. B. and Pulmann, P., EIISA III QuantltalWn of Specific Antibodies b
y Enzyme Linked
Irnmunoglobulin in Antigen Coated Tubes, J. ImmunoL, 1972, 109:129-135.
12.
Manual of ClinicaILaboratory Immunology, 3" Ed., (Noel R.- Rose, H. Frei
dman, and J.L. Fahey,
eds.) American Society for Microbiology, 1986. pp.99-109.
13. SteinKamp, J. A. (1984): Flow Cytometry. Rev. Sci. Instrum., 55:1375-1399.
14. Shapiro, H.M. (1988): Practical leww Cytometry, 2"d Ed., Alan R., Liss Inc..
New York.
15.
Diagnostic Flow Cytometry (John S. Coon and Ronald S. Weinstein eds.) Wi
lliams and Wflkins.
Baltimore. 1991.

APPENDICES

Ammonium hydroxide
(stronger)
Sodium hydroxide
Isaturated]
Sulphuric acid
(concentrated)
Nitric acid
(concentrated)
Hydrochloric acid
(concentrated)
Phosphoric acid
(syrup)
Acetic acid
{glacial)
APPENDIX A
Specific Gravity, Normality and Per Cent Concentration of Common Acids
and Alkalies as Purchased
AcidAlkali
Specific Gravity
Normality
Per Cent
(room temperature)
by Weight
0.9
15.1
28 (as NH3)
1.5
19
50
1.84
35.9
96
1.42

15.6
69.5
1.19
11.7
36
1.71
45
85
1.06
17.6
99.8

570
APPENDIX B
Composition of Some Routinely Used Buffers
Table 1
Hydrochloric acid-potassium chloride
X ml 0.2 M HCI + 250 ml 0.2 M KCI, diluted to 1000 ml., Temperature 25C.
1,00
670
1.70
130
I, I0
528
1.80
102
1.20
'425
1.90
81
1.30
336
2,00
65
1.40
266
2.10
51
1.50
207
2.20
39
1.60
162
Table 2
Aconitic acid-sodium hydroxide
100 ml 0.5 M aconitic acid + X ml 0.2 M NaOH, diluted to 1000 ml, 23oC.

2.5
75
4.1
380
2.7
105
4.3
415
2.9
140
4.5
450
3,1
180
4.7
485
3.3
220
4.9
515
3.5
26O
5.1
54O
3.7
300
5.3
565
3.9
340
5.5
595
" Table 3
Citric acid-sodium citrate
X ml 0.1 M citric acid + Y ml 0.1 M Na citrate, diluted to 1000 ml, 23C.
pH
X
Y
pH
X
Y
3.0
465
35
4.6

255
245
3,2
437
63
4.8
230
270
3.4
400
100
5.0
205
295
3.6
370
130
5.2
180
320
3.8
350
150
5.4
160
340
4.0
330
170
5.6
137
363
4.2
315
185
5.8
118
282
4.4
280
220
6.0
95
405

Table 4
571
Table 5
8uccinic acid-sodium hydroxide
0.2 M succinic acid X nd 0.2 M NaOH, diluted to 1000 ml, 23oc.
pH
X
p/-/
X
3.8
75
5.0
267
4.0
I00
5.2
303
4.2
133
5.4
342
4.4
167
5.6
375
4.6
200
5.8
407
4.8
235
6.0
435
Acetic acld-sodium acetate
X ml I M actlc acid + Y ml I M NaOH, diluted to 1000 ml, 25oC. (I = ionic streng
th)
=0.05
1=0.1
=0.2

pH
Y= 50 ml
Y= I00 ml
Y= 200 ml
1 M NaOH
I M NaOH
1 M NaOH
X
X
X
3.6
650.0
--3.8
428.0
828.O
-4.0
288.0
559.0
-4.2
200.0
389.0
760.0
4.4
145.0
283.0
553.0
4.6
I I 0.0
215.0
423.0
4.8
87.7
173.0
.
341.0
5.0
73.8
146.0
289.0
5.2
65.0

129.0
256.0
5.4
59.5
I 18.0
235.0
5.6
56.0
112.0
222.0
5.8
53.8
107.3
-Table 6
Trls acid maleate-sodlum hydroxide
M tris acid maleate + X ml 0.2 M NaOH diluted to 1000 ml, 23C.
5.2
35
7.0
240
5.4
54
7.2
255
5.6
77.5
7.4
270
5.8
102.5
7.6
290
6.0
130
7.8
317.5
6.2
157.5
8.0
345
6.4
185
8.2

375
6.8
212.5
8.4
405
6.8
225
8.6
432.5

Table 7
Table 8
Biophysical Chemistry
Potassium d/hydrogen phosphate-d/sod/urn hydrogen phosphate
Xni; 0.5 M K/PO4 + Y ml 05 M NaHPO4, d/lUted to 1000 ml, 25C.
{I = ionic strength)
=0.05
I=0. I
I= 0,2
pH X
Y
X
Y
X
Y
5,8
--6.0
74,2
8,58
6,2
64,6
11.8
6,4
53,4
15,5
6,6
42,0
19,3
6,8
31,4
22.8
7,0
22,4
25.8
7.2
15,4
28,2

7,4
10,3
30.0
7,6
6,74
31.0
7,8
4,36
31,8
8.0
2,80
32,4
159
13,8
142
19,5
121
26,4
98,2
34.0
75,6
41,4
55,4
48,2
39,0
53,6
26,4
57,8
17,6
60,8
l 1,5
62,8
7,38
64,2
333
22,4
303
324
265
44,8
222
59,4

176
74.6
133
.
89,2
95,2
i02
65,8
111
.44,2
119
29,2
124
18,9
127
12,1
129

Potassium dihydrogen phosphate:sodlum hydroxide

500 ml 0.1 M KHPO4 + X ml 0,1 M NaOH, diluted to 1000 ml, 25C.
pH
X
pH
X
5,8
36
7,0
5.9
46
7.1
6.0
56
7.2
6,1
68
7.3
6.2
81
7.4

6,3
97
7.5
6.4
116
7.6
6.5
139
7.7
6.6
164
7.8
6.7
193
7.9
6.8
224
8.0
6.9
259
-291
321
347
370
391
309
424
435
445
453
461

Table 9
'lble 10
I= 0.05
I=0.I
I=0.2
X = 5O rnl I M HCl
X = I OO ml I M HC[
X = 2OO mI I M HCI
Y
Y
Y
7.0
53.6
117.2
214.0
7.2
55.7
111.3
222.0
7.4
59.1
117.8
235.3
7.6
64.4
127.5
257.0
7.8
72.9
144.0
290.0
8.0
86.1
169.0
342,0
8.2
107.0
208.0
421.0
8.4
141.0

270.0
550.0
8,6
194.0
367.0
738.0
8.8
279.0
524.0
-9.0
414,0
761.0
-9.2
627.0
--5:5' - Diethylbarbitur/c acid-sodlum 5:5 - diethyroarblturate
(Barbitone-barbltone sodium}
X ml 0.025 M barbitone Y nd 0.5 M barbltone sodium + Z ml 0.5 M NaCI, diluted to
1000 ml, 25C.
(I = ionic strength)
/= 0.05
I=0. I
/=0.2
X
Y
Z
X
Y
Z
X
Y
Z

648
I0
90
409
10
90
645
25
75

814
50
50
.514
50
50
648
I00
409
I00
-258
I00
-163
100
-639
I0
190
403
10
190
636
25
175
401
25
175
506
50
150
639
I00
I00
403
100
100
509
200
-321
200
--

--

275
5
395
348
I0
390
219
10
390
346
25
375
218
25
375
275
50
350
348
I00
300
438
200
200
277
200
200

--O'g19
I'Ol
-O'gL
O'OI
g'Ol
-O'gl
O'ILg
0"01
O'IL9
O'09g
O" 161
9"6
0"661
O'I9g
0"6gI
9"6
0"69g
O'IOg
0"90I
I'6
0"6I
0"9I
'9
'6
O'gLE
O" IF
I,'L
0"6
O'LIt;
0"9I
[ "I9
9"9
0"0
I,'9I I
6"9g
9"9
6"9I
'OI I
9"gg
6"II
--

-'9
I'6
0"6
6"8
9"9
'9
'9
;'8
0"9
X
Hd
x
Hd

575
Appendices
Table 13
Glycine-sodium glycinate
X ml 2 M glycine + Y ml 1 M NaOH, diluted to 1000 ml, 25oc.
(I = ionic strength)
I = 0.05
I = 0.1
I = 0.2
Y= 50 ml
Y= I00 ml
Y= 200 ml
1 M NaOH
1 M NaOH
1 M NaOH
pH
X
X
X
8.8
234.0
467.0
9.0
157.0
313.0
637.0
9.2
108.1
216.0
437.0
9.4
77.4
155.0
312.0
9.6
58. I
116.1
234.0

9.8
45.8
91.7
185.0
I0.0
38.1
76.3
153.0
10.2
33.2
66.5
134.0
10.4
30.1
60.3
121.0
10.6
28.0
56.4
113. I
10.8
26.7
53.8
108.0
Table 14
Sodium bicarbonate-sodium carbonate
X ml 1 M NaHCOs + Y ml 1 M NaCO8, diluted to 1000 ml, 25oc.
(I = ionic strength)
I= 0.05
I= 0.I
I= 0.2
X
Y
X
Y
X
y
9.0
39.8
3.41
9.2
35.5

4.83
9.4"
30.3
6.55
9.6
24.6
8.44
9.8
18.9
10.3
3.0
13.
12.0
10.2
9.58
13.4
10.4
6.29
14.5
10.6
3.86
15.2
10.8
2.01
15.7
II.0 ....
76.8
7.74
67.6
10.8
56.8
14.4
45.3
18.2
34.3
21.9
24.6
25. I
17.0
"
27.6
11.2
29.5

7.06
30.8
407
31.7
1.80
32.3
147.0
17.8
127.0
24.4
104.4
31.9
81.5
39.5
60.5
46.5
42,9
52.3
29.2
56.9
19.3
60.1
12.3
62.4
7,41
63.9
3.92
64.9

576
Bloptu3siccd Chemlstr
Table
Un/versa/buffer m/xture
1000 ml of a mixture of diethylbarbituric acid, citric acid, KIIPO,, and boric a
cid,
0.02857 I/with respect to each of the above compound + X ml 0.2 M NaOli, 25C.
pH
X
pH
X
2.42
0.0
6.90
50.0
2.90
5.0
7.30
55.0
3.36
I0.0
7.71
60.0
3.92
15.0
8.14
65.0
4.40
20.0
8.63
70.0
4.82
25.0
9.15
75.0
5.27
30.0
9.71
80.0
5.68
35.0
10.50
85.0

8.10
40.0
11.25
90.0
6.51
45.0
11.58
95.0
11.79
I00.0

"Img
1 g
Ing
Ipg
APPENDIX C
I Unit

Name
metre
second
kelvin
mole
ampere
Name of u.t
Joule
newton
pascal
watt
volt
coulomb
ohm
hertz
tesla
Symb
M
k
da
d
c
m
P
f
= 1000 grammes = 10s grammes
= O.O000000000000l grammes =
Symbol

J
N
Pa
W
V
C
Hz
T
Mu/tp
10e
10
10
10-1
10-2
10101010-2
10 grammes

=
I000 grammes,
=
I000 mg
=
I000 lg
=
I000 ng
=
I000 pg
=
1000 fg

578
1 nm = lO-Sm = 10mm = 10-Tcm = 10-m
Additionally
$I Units for Volume
The SI unit of volume is the cubic metr, ms. One lltre therefore means one cubic
decometre.
dm. Thus. the terms litre and millilltre should be replaced by the terms of Sl u
nits given below.
llltre(1) = Idms = I0-ams
Imfllflltre(ml) = Icms = I0ms
I microlitre (tl) = I mms = I0 ms
Conversion of Common Units into SI Unlt
angstrom (A
I00 pm = I0" m = I0" em
centigrade ('C)
|C + 273 } K
calorie
4.186 J
cycles/sec
1 Hz
curie. Ci
3.7 x 10Oserg
10-J
micron ()
I
molar, M ( I tool I)
I tool dm
gauss [G)
I0 T
pound-force/sq in ( Ib f In- ) 6894.76 Pa
[p.s.i.)

579
Y
alpl
beta
gamma
delta
epsflon
eta
theta
kappa
lambda
mu
nu
p!
rho
sgma
tau
phl
ch!
s!
omega
APPENDIX D
Greek Symbols and Their Pronunciations

313 351
251 288
189 225
158 193
127 162
12
43 74[ 107 142
31 63 94 129
31, 63 97
32 65
580
BophystcoJ Chentrg
Table Showing the unt of Ammonium Sulphate to be Added to 1L Solution to
.... Arrive at a Particulax % Saturation
Final concentrat/on of ammordum sulphate, % saturat/on
Grams solid ammonium sulphate to be added to 1L of solution
209 243 277

150 183 216

29 91 123 155
301
49i 61
93 125
19' 30
62 94
390 !430 472 1516 561 [562
326]365 4061449 494i592
'300 340 1382 ,520
230 267
307
348

390i485
198
235
273
314
356
449
177
214
252
292
333
426
164
200
238
278
319
411
132
168
205
245
285
3?5
99
134
171
210
250
339
66
101
137
176
214
302
33
67
103
141
179
264
34
69
105
143
227
34
70

107
190
35
72
153
36;115
77
100
767
694
619
583
546
522
506
469
431
392
353
314
275
237
198
157
79

N--H
o O-H
(b)
Inter molecularly hydrogen
bonded OH (changes on d/lut/on)
(I) s/ngle bridge compounds
{o} olymer assoe/aUon
()
a moary hydrogen
bonded OH-s/ngle br/dee
compounds
Amn
(o ry, free {2 bands}
(b) Secondary, free | I band}
and
Imbues |=N - H) { I band}
Amides
(o) Primary, free {2 bands}
2.74 - 2.79
(3650 - 3590}
Variable, Shsrp
2.82 -2.90 (3550 - 3450} Variable, Sharp
2.94 - 3,13 (3400. 3200} Strong, Broad
2.80 - 2;90 {3570 - 340} Var/able, Sharp
- 2,86
(~ 3500}
Medium
2.86 - 3.02
(3500 - 3310)
Med/um
2.94
(~ 3400}
Medium
2.94 - 3.03
(3400 - 3300}
Medium

(b) Secondary, free (I band}

(c) Pr/msry, bonded {2 bonds)
and
Atones
(a) Secondary, bonded (I band}
A/kene
(a Monosubst/tuted
} {2 bands}
(b) Disubst/tuted, germ
~ 2.86
(~ 3500}
Medium
and 2.94 (~ 3400}
~ 2.92
(~ 3430}
Medium
- 2.99
(- 3350}
Med/um
3.15
{~ 3180}
Med/um
3.0 - 3.2
(3320 - 3140}
Medium
~ 3.03
(~ 3300}
Strong
3.23 - 3.25
{3095 - 3075)
Med/um
and 3.29 - 3.32
(3040 - 3010}
Medium
3.23 - 3.25

(3095 - 3075}
Medium

R
CH = R
3.29 - 3.32
(3040 - 3010}
Medium
- 3.30
(- 3030}
Var/able
3.38 - 3.51
{2962 - 2853)
Medium
3.2 - 3,3
(3130 - 3030}
Medium

C=O
stretching
C=O
stretching
582
Biophysical Chemistry
Vbratons Absorbing Group
Absorption Band Range
Absorption
and its Envonment
(])
(cm)
Intensity
O--H
stretclu
A/coho/s and Pheno/s
(a) Intra n/ecular hydrogen
bonded chelate compounds
3.1 - 4.0
(3200 - 2500)
Weak
C--H
Aldehydes (characteristic)
3.45 - 3.55
(2900 - 2820)
Weak
stretching
(2 bands)
and 3.60 - 3.70
(2775 - 2700)
Weak
stretching
bonded, multiple bonds
3.70 - 4.00
(2700 - 2500)
Weak
S--H
Sulphur Compounds
3.85 - 3.92
(2600 - 2500)

Weak
stretch/rig
C = C
A/kyne-dlsubstituted
4.42 - 4.57
(2260 - 2190)
Weak
stretching
Variable
C = N
Isocyanates
4.40 - 4.46
(2275 - 2240)
Medium
stretching
Alkyl Nitrites
4.42 - 4.46
(2260 - 2240)
Medium
Aryl Nitrites
4.46 - 4.50
(2240 - 2220)
Medium
a, -Unsaturated
Alkyl Nitrites
4.47 - 4.51
(2235 - 2215)
Medium
C = N
Isocyankles
4.50 - 4.83
(2220 - 2070)
Medium
stretching
--N = C = N
Dflm/des
4.64 - 4.70
(2155 - 2130)
Medium
Azdes
4.63 - 4.72 (2160 - 2120)
Strong

and 7.46 - 8.48 (1340 - 1180)

Weak
Anhydrales
(a) Saturated 5-membered
5.35 - 5.49
(1870 - 1820)
Strong
ring
and
5.56 - 5.71
(1800 - 1750)
Strong
(b) a, -Unsaturated 55.41 - 5.56
(1850 - 1800)
Strong
membered ring
and
5.47 - 5.62
(1830- 1780)
Strong
(c) Saturated. acyclic
5.41 - 5.56
(1850 - 1800)
Strong
and
5.59 - 5.75
(1790 - 1740)
Strong
(d) a, Unsaturated and
5.47 - 5.62
(1830 - 1780)
Strong
aryl, acycllc
and
5.65 - 5.81
(1770 - 1720)
Strong
Acyl Halldes
(a) Acyl bromides
~ 5.53
(~ 1810)
Strong
(b) Acyl chlorides
~ 5.57
(~ 1795)
Strong

(c) Acyl fluorides

~ 5.41
(~ 1850)
Strong
(d) a, -Unsaturated
5.61 - 5.72
(1780 - !750)
Strong
and aryl
and
5.72 - 5.82
(1750 - 1720)
Medium
Esters
(a) Saturated, cyclic
(I) [3-]actones
~ 5.5
(~ 1820)
Strong
5.62 - 5.68
(1780 - 1760)
Strong

C=O
stretching
C=O
stretching
C=O
stretching
= 0
C=N
stretdUn
C=O
stretching
C--O
stretchg
Vibrations Absorbing Group
Absorption Band Range
Absorption
and its Environment
, ()
(cm)
Intensity
(3J 6-1actoncs
(and larger rings)
5.71 - 5.76
(1750 - 1735)
Strong
(b) Saturated, acyclic
5.71 - 5.76
(1750 - 1735)
Strong
(c) Unsaturated
(I) Vinyl ester type
5.56 - 5.65
(1800 - 1770)
Strong
(2) a, B-unsaturated and aryl
5.78 - 5.82
(1730 - 1717)

Strong
(d) Carbonates
5.62 - 5.75
(1780 - 1740)
Strong
(a) Saturated. allphatic
5.75 - 5.81
(1740 - 1720)
Strong
(b) Aryl
5.83 - 5.90
(1715 - 1695)
Strong
(c) a, -Unsaturated, aliphatlc
5.87 - 5.95
(1705 - 1680)
Strong
Note : .every aldehyde has
characteristic C m H
stretching vibrations
(2 bands} at
3.45 - 3.55
(2900 - 2820)
Weak
and
3.60 - 3.70
(2775 - 2700)
Weak
Ketones
(a) Saturated. acycllc
5.80 - 5.87
(1725 - 1705}
Strong
(b) Saturated, cyclic
(I) 6-membered ring
(or higher)
5.80 - 5.87
(1725 - 1"705)
Strong
(2) 5-membered ring
5.71 - 5.75

(1750 - 1740)
Strong
(c) a, B-Unsaturated, acyclic
5.94 - 6.01
(1685 - 1685)
Strong
(d) a, B-Unsaturated, cyclic
(1) 6-membered ring
5.94 - 6.01
[1685 - 1665)
Strong
(2) 5-membered ring
5.80 - 5.85
(1724 - 1708)
Strong
(e) Aryl
5.88 - 5.95
(1700 - 1680)
Strong
Diaryl 5.99 - 6.02
(1670 - 1660)
Strong
CarboxI/c Adds
(a) Saturated, aliphatic
5.80 - 5.88
(1725 - 1700)
Strong
(b) a, B-Unsaturated, aliphatic
5.83 - 5.92
(1715 - 1690)
Strong
(c) Aryl "
5.68 - 5.95
(1700 - 1680)
Strong
Amides
(a) Primary. dilute solution
~ 5.92
(~ 1690)
Strong
(b) Secondary. dilute solution
5.88 - 5.99
(1700 - 1670)
Strong

Imlnes, Oximes
(a) Alkyl compounds
5.92 - 6.10
(1690 - 1640)
Strong
Ketones
-Diketone
6.10 - 6.50
(1640 - 1540)
Strong
Esters
(a) -ketoesters (enolic)
~ 6.06
(- 1650)
Strong

C=O
C=C
stretch/ng
NmH
C = NO2
stretching
(b) Aromatic
Nitrates
Noso Co
584
Biophysical Chemistry
Vibratfons Absorbing Group
Absorption Band Range
Absorption
and fts Environment
. ()
(cm)
Intensity
0- NO2
stretchln
CmNO
setch/ng
(b) Carboxylate anion
6.21 - 6.45
(1610 - 1550)
Strong
stretchin
and
7.15- 7.69
(1400- 1300)
Strong
Amides
(a) Primary, solid and
concentrated solution

~ 6.06
(~ 1650)
Strong .
(b) Secondary, solid and
concentrated solution
5,95 - 6,14
(1680 - 1630)
Strong
(c) Tertiary, solid and all
solution
5.99 - 6,17
(1680 - 1620)
Strong
Alknes
(oJ NonconJugated
5.95 - 6.17
(1680 - 1620)
Variable
(b) Monosubstituted (Vinyl)
- 6.08
(- 1645)
Medium
(c) Disubstltuted, cfs
- 6.03
(~ 1658)
Medium
(d) Disubstituted, trams
~ 5.97
(~ 1675]
Medium
(e) Disubstituted, gem
~ 6.05
(~ 1653)
Medium
(]) Trisubstituted
~ 5.99
(~ 1669)
Medium
(9) Tetrasubstituted
~ 5,99
(- 1669)
Weak
Amfnes
(a) Primary
6.06 - 6.29
(1650 - 1590)

Strong
(b) Secondary
6.06 - 6.45
(1650 - 1550)
Weak
(c) Amine salts
6.25 - 6.35
(1600 - 1575)
Strong
and
~ 6.67
(~ 1500)
Strong
Amides
(a) Primary, dilute solutions
6.17 - 6.29
(1620 - 1590)
Strong
Aromatfcs
(4 characteristic bands due to
~ 6.25
(~ 1600)
Variable
skeletal carbon stretching
~ 6.63
(~ 1580)
Variable
vibrations)
~ 6.90
(~ 1500)
Medium
~ 6.90
(~ 1450)
Medium
Azo Compounds
6.14 - 6.35
(1630 1575)

Variable

N/tro Compounds
(a) Aliphatic
6.37 - 6.45
(1570 - 1550}
Strong
and
7.25 - 7.30
(1380 - 1370}

Strong
6.37 - 6.67
(1570 - 1500}
Strong
and
7.30 - 7.70
(1370 - 1300}
Strong
6.06 - 6.25
(1650 - 1600}
Strong
and
7.70 o 8.00
(1300 - 1250)
Strong
6.25 - 6.67
(1600 - 1500)
Strong

C---H
O--H
(d} Phenols
O--NO
sretch/ng
N--H
Appendices
585
Vrtons
Absorbing Group
Absorption Band Range
AbsorptWn
and its Environment
. ()
(cr)
Intensity
Nitrites
5.95 - 6.06 [1680 -i650)
Strong
Amkles
(a) Secondary amides
(dilute solution)
Alkanes
(oJ --CH=-- (scissoring]
(b) C--CH
(c) Gem dimethyl
(isopropyl)
(d Teary-butyl
.(e) C--H
A/coho[s and Phenols
Two bands - shorter
wavelength due to

O--H bending and longer

wavelength band caused
by characteristic C--OH
stretching vibration
(a) Primary alcohols
(b) Secondary alcohols
(c) Tertiary alcohols
and
and
and
and
and
and
6.45 - 6.62 (1550 - 1510) Strong
6.74 - 6.92
6.80 - 7.00
7.25 - 7.30
7.22 - 7.25
7.30 - 7.33
7.17 - 7.22
- 7,33 7.46
(1485- 1445)
(1470- 1430)
{1380- 1510}
(t385- 1380}
(]370 1365)
(1395" 1385)
(~ 1365)
(- 1340)
Medium

Medium
Strong
Strong
Strong
Medium
Strong
Weak

7.4 - 7.9
9.30- 9.90
7.4 - 7.9
8.93 - 9.05
7.1 - 7.6
8.55 - 9.09
7.1 - 7.6
8.13 - 8.77
(1350- 1260}
{1075- I010)
(1350- 1260]
(I 120 - I I05]
(1410- 1310)
(I 170 - I I00)
(1410- 1310)
{1230 - 1140)
Strong
Strong
Strong
Strong
Strong
Strong
Strong
Strong

C--O--H
stretching
C--O
and
OH
Aromatic
out of plane

{a) Unsaturated, Aryl

(b) Aliphatic
Carboxyl/c Adds
(a) C--OH stretching
(usually characteristic
doublet near 8.00 ,
1250 cm-j
(b) OH bending Aomatics
Substitution Type
(a) 5 adjacent
hydrogens
7.87 - 8.13
{1270 1230)
8.70 - 9.43
{I150 I060}
7,58 - 8,28
(1320 1210}
6.95 - 7.17
{14401395)
~ 13.3 (~ 750}
{not always present}
Strong
Strong
Strong
Weak
Variable, Strong

- 14.3
{~ 700}
(always present}
(b) 4 adjacent hydrogens
,- (dlsubstituted
~ 13.3
(~ 750)
(c) 3 adjacent hydrogens
(trisubstituted}
~ 12.8
(~ 780}
k: DisubUtuted, cis
~ 14.5
(- 690}
Ch-o

12.5 - 16.6 (800 - 600)

Variable, Strong
Var/able, Medium
Strong
Strong

Any compound where

n>4
~ 13.8
(- 722}
Strong

587
Appendices
APPENDIX G
Commonly Ued Molecular Weight Markers for Protein
1. Gel Filtration Chromtography
Protein s
Moleculor weight
Aprotlnin from bovine lung
Cytochrome C from horse heart
Carbonic anhydrase from bovine erth ocytes
Albumin from bovine serum
Alcohol dehydrogenase from yeast
-amylase from sweet potato
Apoferritln from horse spleen
Bovine thyroglobulln
Blue dextran
6,500
12,400
29,000
66,000
150,000
200,000
443,000
669,000
2000,000

2. SDS Gel Eletrophoresis

Mo/ecu/ar we@hi
a-Lactalbumin
-Lactoglobulin
Trypsin inhibitor from soyabean

Trypsinogen, PMSF treated

Carbonic anhydrase from bovine erythrocytes
Pepsin
Egg albumin
Pyruvate kinase from chicken muscle
Bovine albumin
Fructose-6-phosphate kinase from rabbit muscle
Phosphorylase b from rabbit muscle
-g.alactosidase from E.co/t
a2-macroglobulin from human plasma
Urease from Jack bean
14,200
18,400
20 I00
24,000
29 000
34 000
45 000
58 000
66
97 ,,400
116 O00
180,000
272,000

Several other proteins, ot.cited here, have also been used as molecular we/ght m
arkers.

588
1.4, 2.1
0.85
APPENDIX H
Radioisotopes Frequently Used as Tracers and Their Properties
Radioisotope
Type of
Half-life
Energy of radltion, MeV
Radiation
a - and particles

6Zn
82Br
mSr
OSr
1311
8121.1 yr
857oo yr
8%
2.6yr
8-,
14.8 hr
814.3 days
887.1 days
8-,

prays

12.4 hr
8180 days

310 days
x- y
8-, Y
47 days
8-,
5.3yr
X-ray, 8-, 8
12.8 hr

8, y
250 days
8-,
34 hr
855 days
825yr
8-,
8 days
0.017
O. 154
0.58
1.4
1.7
0.17
2.0 (25/)
0.26
0.26, 0.46
0.31
0.57 (8-)
0.66 (8+}
0.32

0.465
1.5
0.6
0.6
1.3
1.38, 2.7
I.I, 1.3
1.16, 1.32
1.35
I.I
0.55, 0.79, 1.35
0.37, 0.08, 0.65

Actinium
Alumlnlum
Antimony
Argon
Arsenic
Barium
Beryllium
Bismuth
Boron
Bromine
Cadmium
Calcium
Carbon
Ceslum Chlorine
Chromium
Cobalt
Copper
Europium
Fluorine
Gallium
Gold
Helium
Hydrogen
Indium
Iridium
Iron
Krypton
Lanthanum
Lead
Lithium
Magnesium
Ac
Al
Sb
Ar
As
B!
B
Br

Cd
Ca
C
Cs
Cl
Cr
Co
Cu
Eu
F
Ga
Au
He
H
In
Ir
I
Fe
L!
Mg
89
13
51

18
33
56
4
83
5
35
48
20
6
55
17
24
27
29
63
9
31
79
2
1
49
77
53

26
36
57
82
3
12
Atonrlc Weight
227
26.98
121.75
39.95
74.92
137.34
9.01
208.98
10.81
79.9
112.4
40.08
12.01
132.91
35.35
54.99
58.93
151.96
18.99
69.72
196.96

4
1
118.82
192.22
126.9
55.85
83.8
138.9
207.2
6.94
24.3
589
Table of Atomic Weights and Atomic Numbers
Symbol
Atomic Number

59O
Element
Symbol
Manganese
Mercury
Molybdenum
Neon
Nickel
Nitrogen
Osmium
Oxygen
Palladium
Platinum
Plutonium
Polonium
Potassium
Radium
Radon
Rubidium
Ruthenium
Selenium
Silicon
Silver
Sodium
Strontium
Sulfur
Tellurium
Thallium
Thorium
Tungsten
Uranium
Vanadium
Xenon
Yttrium
Zinc
Zirconium
Mo
Ne
Ni
N
Os
O
Pd
Pt
Pu
Po
K

Ra
Rn
Rb
Ru
Se
Si
Na
Sr
S
Te
Sn
U
V
Xe
Y
Zn
Zr
25
8O
42
10
28
7
76
8
46
78
94
84
19
88

86
37
44
34
14
47
11
38
16
52
81
90
5O
74
92
23
54
39
3O
4O
54.93
200.59
95.94
20.17
58.71

14.0
190.2
16.0
106.4
195.09
242
210
39.1
226.02
222
85.46
101.07
78.96
28.O8
107.86
22.98
87.62
32.06
127.6
204.37
232.03
118.69
183.85
238.02
50.94
131.3
88.9
65.37
91.22
Biophysical Chemistry
Atomic Number
Atomic Wejht

591
Appendices
APPENDIX J
Trmsmission/Extinction
Itelatlonship between the transmission in % ( %T ) and the extinction ( E )
( absorbance; optical density ).
%T
E
%T
E
%T
E
%T
E
lO0
0.000
75
0.125
50
0.30l
25
0.602
99
0.004
.74
0.131
49
0.310
24
0.620
98
0.009
73
0,137
48
0.319
23
0.638
97
0.013
72
0.143
47
0.328
22

0.658
96
0.018
71
0.149
46
0.337
21
0.678
95
0.022
70
0.155
45
0.347
20
0.699
94
0.027
69
0.161
44
0.357
19
0.721
93
0.032
68
0.168
43
0.367
18
0.745
92
0.036
67
0174
42
0.377
17
0.770
91
0.041
66
0.181
41
0.387
16
0.796
90
0.046
65
0.187

40
0.398
15
0.824
89
0.051
64
0.194
39
0.409
14
0.854
88
0.056
63
0.201
38
0.420
13
0.886
87
0.061
62
0.208
37
0.432
12
0.921
86
0.066
61
0.215
36
0.444
11
0.959
85
0.071
60
0.222
35
0.456
10
1.000
84
0.076
59
0.229
34
0.469
9
1.046
83

0.081
58
0.237
33
0.482
8
1.097
82
0.086
57
0.244
32
0.495
7
1.155
81
0.092
56
0.252
31
0.509
6
1.222
80
0.097
55
0.260
30
0.523
5
1.301
79
0.102
54
0.268
29
0.538
4
1.398
78
0.108
53
0.276
28
0.552
3
1.523
7
0.114
52
0.284
27
0.569
2

1.699
76
0.119
51
0.292
26
0.585
I
2.000

&
Absorbance, 178
Absorption coefficient, (see extinctioncoefficient)
Absorption maxima, 186, 187
factors affecting, 190-192
effect ofpH, 190(I)
effect of polarity, 191
orientation effect, 191
Absorption spectra, 201-205
interpretation, 203-205
structural analysis based on, 201-205
Absorption spectrum, 186-191
electronic transition and, 187-190
Absorptivity, 179, 206
Acid-base catalysis, 47
Acid-base properties of hemoglobin, 48, 49
Acrylamide, (see polyacrylamide)
Activity coefficient, 9
Adsorbate, 157(I)
Adsorbent, 157(i
Adsorption, 157-174
and dispersion forces, 158
and hydrogen bonds, 159
and induction forces, 159
and molecular orientation, 160, 161
and Van der Waals forces, 158
characteristics of, 160
from solutions, 171-172
importance of, 172-173
Adsorption chromatography, 349, 364-365
Affinity chromatography, 396-404
applications, 403-404
isolation of DNA-binding proteins, 403
lignd selection, 398
ligad etachment, 398-400
.purification of mRNA, 402
standard matrix-ligand systems, 404-405
supporting matrix, 397-398
variations of, 401-402
Affinity cross-flow ultraffltrtion, #01
Affinity precipitation, 401-402
Ag/AgCI reference electrode, 57-58
Aarose, 380 ( also see gels)
Agarose-acrylamide, 438
Alpha emission, 481

Alpha particles, 487

Amicrons, 76
Amino acid analyser, 393-394
Ammonium ion electrode, 71 (also see ion
selective electrodes)
Ammonium persulphate, 436
Amphiprotic compomds, 36
Ampholine, 457 (also see isoelectric focussing)
Ampholytes, 426, 457 (also see isoelectric
focussing)
Analytical centrifugation, 321-342
Analytical ultracentrifuge, 306-308, 307(i)
Anion exchanger, 389, 390, 391,392
Archibald method, 342
Arrhenius theory, 2-3"
Artificial isotopes, 480, 484
production of, #84-485
Ascending chromatography, 352
Association colioids, 83, 84
Atomic absorption spectrophotometry, 245
Atomic fluorescence, 247
Atomizers, (see nebulizers)
Autoradiography, 495
Auxochrome, 192
B
b-energy spectrum, 500
b-mercaptoethanol, 439
Band sedimentation, 334-339
Bend-shift assay, (see gel retardation)
Bathochromic shill, 192(i
Beer's law, 177-186
deviations from, 183-186
fluorescence, 186
spectral shift, 184
imperfect monochromacy, 185
instrumentation limitations, 184
isobestic point, 184
sample instability, 186
temperature effects, 185
turbidity, 186
Beer-Lambert law, 178
Bending vibrations, 210, 220
Bequerel, 484

594
Biological half-life, 533
Bioluminescence, 240, (also see luminometry)
Biosensors, 241
Birefringence (see flow birefringence)
Blank, 182
Blue-shift, 195
Bohr effect, 48
Bonding/antibonding orbitals, 188-190
Bouger-Beer law, 178
Bouger-Lambert law, 177, 178
Boundary sedimentation, applications of,
330-334
Bragg equation, 298
Bronsted-Lowry theory, 3-6
Buffer capacity, 28-29
Buffering of blood, 48-50
Buffers, 23-31
bicarbonate buffers, 48
carbnonate buffers, 29
EDTA buffers, 30
glycine buffers, 30
glycylglycine buffer, 30
Good buffers, 30-31 phosphate buffers, 29
triethanolamine buffer, 30
tris buffers, 29-30
Calomel reference electrode, 56-57
Capillary flow, measurement of viscosity by,
131-132
Carbon dioxide electrode, 73-74, 73(f)
Carboxymethylcellulose, 390
Carrier ampholytes, 457, 459 (also see isoelectric focussing)
.Carrier gas, 366, 367, 368
Cation exchanger, 398, 390, 391,392
CD/ORD spectra, 274-276
of carbohydrates, 279-280,. 27910

of nucleic acids, 280-282, 280(0, 281 If)

or'proteins, 275-278, 277(0,278(0
Cellular gel, 463-464
Cellulose acetate elcctrophoresis, 435
Centrifugation, 301-343
instrumentation, 305-311
in zonal rotors, 319-321
Cerenkov counting, 504
Channels ratio, 500-501 (also see scintillation
counting)
Biophysical Chemistry
Chemical quenching, 498-499
Chemical shifts, 251-253
Chemiluminescence, 240
Chemiluminescent assays, 241
Chemiluminescent substances, 502
Chiral chromatography, 418
Chloride shift, 49
Chromatogram, 352,367
Chromatography, 344-421
procedures, survey of, 346-350
Chromophore concept, 19 I- 192
Cibacron-blue-agarose, 404
Circular dichroism, 271-283
instrumentation for, 283
CM-cellulose, (see, carboxymethylcellulose)
Coefficient of viscosity, 122
Colloidal osmotic, pressure, 120.
Colloids, 75
classification of, 78-80
definition of, 76
properties of, 87-92
Color quenching, 499
Column chromatography, 359-364
columns, 359
elution, 361
effluent collection, 361-363
flow rate, 361
sample introduction, 360-361
Column electrophoresis, 440-442,442(f}
Compton scattering, 488
Conjugate acid/base, 5
Contact angle, 147-149, 148(i

of bacteria with neutrophils, 147-149

and phagocytosis, 147, 148
Continuous density gradient, 317
Continuous discharge, 490
Controlled pore glass beads, 381
Couette rotating-cylinder viscometer, 133 - 134,
133(
Countercurrent distribution, 347-348
Counting efficiency, (see under scintillation
counting)
Covalent chromatography, 419
Craig apparatus, 348
Cross of isocline, 286-287
CTAB, 439

Index
Curie, 484
Cyclotron, 485
D
Danieli and Davson model, 153-154
Dansyl chloride, 432
Dead time, 492
DEAE-cellulose, 390
Decay constant, 482, 483, 484
Densitometry, 444
Density gradient centrifugation, 313-317, 318,
319,320
density gradients, preparation of, 317 318
gradient materials, 316-317
rotors, choice of, 319
sample, application of, 318
sample, recovery of, 318-319
Desk top centrifuges, 305
Dextran gels, 378-379
Diamagnetic shielding, 251,252, 253
Dideoxynucleotide sequencing technique, 445
Difference spectroscopy, 208
Differential centrifugation, 31i-313
Diffraction grating, 195(1)
Diffusion, 100-109
Diffusion coefficient, 101
significance of, 103-106
Diffusion of electrolytes, 106-109
Diffusion potentials, 108
Dilution quenching, 499
Dimethyl POPOP, 497
Disc gel electrophoresis, (see discontinuous gel
electrophoresis)
Discontinuous density gradient, 317
Discontinuous gel electrophoresis, 448-451,
449{0
separating gel, 449
stacking gel, 450
Disintegration constant, 482
Dispersion forces, 158
Dissymetry constant, 106
Distribution coefficient, 377
DNA-affinity chromatography, 403
DNA-cellulose chromatography, 417-418
DNA footprinting, 472-473

DNA sequencing, 469-472

DNA thermal cycler, 549-550
Donnan effect, 79
Donnan equilibrium, 95-99
biological significance of, 98-99
Dosimetry, 533-534
film dosimeter, 533-534
Fricke dosimeter, 533
thermoluminescence dosimetry, 534
Double-beam spectrophotometer, 200(I)
Dual isotope analysis, 503-504
Dual wavelength spectrophotometer, 200-201,
201(1)
EDTA buffers, 30
Effective half-life, 533
Electric birefringence, 289
Electrical double layer, 87
E1ectroblotting, 468
Electrode contamination, 63
Electrode interference, 70
Electrode potential, 52-54
Electroendosmosis, 90
Electrofocussing, (see isoelectric focussing)
Electrolytes, i-2
Electrolytic dissociation, 1-2
Electromagnetic spectrum, 176-177, 177(1)
Electron capture, (also, EC decay), 481
Electron spin resonance spectrometry, 258-261
applications of, 259-261
instrumentation for, 258(1)
Electrosmosis, 90(0
Electrophoresis, 422-476
types of, 426-430
on stabilizing media, (see Zone electro
phoresis}
Electrophoretic mobility, 423-426
factors affecting, 423-426
buffer, 425-426
electric field, 424-425
medium, 425
sample, 423-424
in gels, 438
Stoke's equation, 423

Electroviscous effect, 130

ELISA, 558-560
Elliptical polarization, 272
Emission of gamma rays, 481

596
Emulsions, 162-165
HLB number and, 163
inversion of, 165
ion antagonism in, 165
oil in water, 164
water in oil, 164
Emulsoids, 79
Enzyme assay, 207
Enzyme electrode, 71(f}
ammonium electrode, 71
Enzyme-linked immunosorbent assay, (see
ELISA)
Equilibrium constant, 11, 12-14
Equilibrium isotope effect, 510
Equilibrium labeling, 515, 516 (also see
labeling formats)
Ethidium bromide staining, 432, 441
Exchanger capacity, 392
Exclusion chromatography (see gel permeation)
Exclusion limit, of gels, 377, 381
External standardization, 500
Extinction coefficient, 179-182
molar extinction coefficient, 179
Extrinsic fluorescence, 228
F
Facilitated diffusion, 100
Falling ball viscometer, 134-136, 134()
Falling drop method, 505-506
Fick's laws of diffusion, 101-102
Field inversion gel electrophoresis, 463
Flame ionization detector, 371-372
Flame sPectrophotometry, 242-247
nebulizers, 243-244, 244(1}
Flocculation value, 92
Flow birefringence, 284-289

apparatus for, 285-286

Flow cytometry, 560-565
applications, 564-565
instrumentation, 562-563
Fluidity, 123
Fluorescence, 221-223
Stolce shift,, 221-222, 222(]
structural factors, effect of, 222
Fluorescence, 3S8
Fluorescence ene ta'nsfe, 228-235
applications of, 232, 235
enz3nne conformation, changes in, 235
Biophysical Chemistry
pyruvate dehydrogenase complex,
studies on, 233-234, 234(I}
tRNA structure, 232-233, 223(1}
Fluorescence polarization, 235-239
Fluorescent dyes, 433
Fluorescent probes, 226
Fluorescent staining, 444
Fluorometry (see spectrofluorimetry]
Fluors, 495
Fraction collectors, 362-363
Fragment long, 505
Free electrophoresis, 426-428
Frequency, 175,,176
Frictional ratio, 106
Gamma rays, 487
Gas cells, 215
Gas flow counters, 493
Gas-liquid chromatography, 366-375
applications, 3?4-375
carrier gas, 368
detectors, 371-373
liquid phase, 370
retention volume, 367

solid support, 368-370

Gas-sensing electrodes, 71-73
Gas solid chromatography, 374-375
Gegenions, 87
Geiger counters, 491-494
background count, 494
continuous discharge, 492
counter tubes, 492-493, 493(1}
dead time, 492
Geiger region, 512(I)
geometric effect, 494
quenching, 492
self absorption, 494
tube characteristics, 493
Gel electrophoresis, 435-448
applications of, 445-447
detection, recovery and estimation, 443-445
modes of, 440-443
molecular weight, determination by, 447
of nucleic acids, 445
procedv-re oxr, 439-440
soIub'Iizers, 438-439
Gel Itrar'on claromatoaphy, (see, el ze
at/on .chromatography}

Index
Gel permeation chromatography, 376-386
advantages, 382
analytical uses of, 385
applications, 383-385
molecular weight determination by. 383-385
types of gels, 378-381
Gel retardation electrophoresis. 473-476
Gelatin filters, 194
Gels. 82-83, 436-438
agar. 436
agarose-acrylamlde, 438
polyacrylamide 436-437, 437(t}
starch gel, 436
Gel-sol transformations, 143
Gene cloning, 550-551
Genetic engineering, 241
Geometric effect, 494
Gibbs' adsorption equation, 168-171
Glass electrode. 58-62
Glass membrane electrodes, 67-68
Globar, 193
Gold sol curves. 76{i
Good buffers, 30-31
Gradient gel electrophoresis. 452
Gradient maker, 31
Graphite rod atomizer, 244-245
Gyromagnetic ratio, 249
H
Half-life, 483
Helmholtz double layer model, 87(fl
Henderson-Hasselbalch equation, 24-27
Heparin-agarose, 404
Hess viscometer, 132-133
High performance liquid chromatography, 405416
High speed centrifuges, 306
High voltage electrophoresis, 452-453, 453(i
HLB number. 163
Hofmeister series, 94
Hooke's law, 2 I0

HPLC, (see high performance liquid chromato

graphy}
column packings, 409-410, 409(i
guard column, 410-411
pumping systems, 407-409
sample injection, 409
597
separation system, selection of, 411-412.
411(fl
reverse phase chromatography, 412-416
ion chromatography, 414-416
ion suppression. 412-413,
ion pairing, 413
Huggin's plot, 123(t). 135
Hydrogen electrode, 54-56
Hydrogen ion activity, 58
Hydroxyapatite chromatography, 416-417
Hyperchromic effect. 192(i
Hyperfine splitting, 254-256
Hypochromlc effect, 192(f)
Hypsochromic effect, 192(i
I
Immunoelectrophoresis, 460-461
Immunoradiometry (IRA), 528-529
Imperfect monochromacy, 185
Infrared gas cells, 196
Infrared spectra, 212-215
of carbon skeleton, 213-214
of carbonyl grOuP, 214
of hydroxy compounds, 214
of nitrogen compounds, 214
Infrared spectrophotometer, 215
Infrared spectroscopy. 209-220
applications of, 216-220
bending vibration, 210
fingerprint region, 212
infrared dichroism, 219
modes of vibration, 211-212
overtone bands, 211
sampling techniques, 215-216
stretching vibration, 210
vibrational frequencies, calcttlation of, 210211
Interfaclal tension, 147. 153-154
Internal standardization, 499-500
Intrinsic viscosity, 123
Inverse PCR, 552
Ion antagonism, 165
Ion chromatography. 414-416
Ion-exchange chromatography. 386-395
applications, 393-394

counterlons, 387
exchange medium, preparation of. 391
ion-exchange resins, types of, 389-390
procedure, 392-393

598
Ion-pairing, 413
Ion product of water, 13
Ion-specific electrodes, 66-74
basic principles, 6667
electrode interference, 70
method interference, 70
applications, 70-71
Ion suppression, 412-413
Ionization,
of amino acids, 36-41
of proteins, 42-43
of strong acids, 20
of water, 11-12
of weak acids, 17-19
dissociation constant, 18
Ionization chambers, 489, 490
Ionization constant of water, 12-14
Irreversible colloids, 93
Isobestic point, 184
Isoelectric focussing, 453-460, 454(f)
carrier ampholytes, 457
Isoelectric pH, 44
Isoelectric point, 44
of protein, determination of, 455
Isoionic point, 38
Isopycnic centrifugation) 314, 315, 335, 336
nucleic acids, separation of, 335
plasmid, purification of, 336
Isotope, 479
definition of, 479
Isotope dilution, 519-521
Isotope effect, 47
Isotope exchange studies, 522-523
Isotope incorporation, 522
Isotopes, uses of, 518-530
clinical applications, 525
distribution studies, 518-519, 519(i9
fate of cells, 521-522
isotope incorporation/exchange studies,
524-525
mechanism of enzyme action, 524-525

membrane transport studies, 521

metabolic studies, 523-524
metabolic turnover time determination, 524
mineral metabolism, 524
sterilization of foods, 525
study of protein structure, 529-530
Jhonston'Ogston effect, 322-323
Biophysical Chemistry
K
Kelvin equation, 155
Kinetic isotope effect, 510
Knoop's experiment, 506-508
Krasny-Ergen equation, 130
Kronig-Kramer transform, 275
L
Labeled compounds, synthesis of, 485-487
Labeling formats, 513-517
equilibrium labeling, 515-516
pulse labeling, 513-515
pulse-chase labeling, 516- 517
LanEmuir trough, 165-166, 166(f)
Lanthanum fluoride electrode, 68
Lattice theory, 103
Laue pattern, 293
Law of mass action, 10-11
Laws of absorption, 177-186
Lectin-sepharose, 405
Lewis acids and bases, 4
Light scattering, 290-292
Limiting viscosity number, 123
Linear dichroism, 219
Liquid-liquid chromatography, 365-366
Liquid-membrane electrodes, 69-70, 69(t]
Luminometry, 240-242
Lux genes, 241
Lyophilic colloids, 79
Lyophobic colloids, 79-80
Lysine-agarose, 404
M
Mass spectrometry, 504-505, 505(f)
Matrixless afl'mity separations, 402
Maxam-Gilbert DNA sequencing, 469-472
Membrane potential, 226
Membranes, 153154, 162

Metabolic turnover time, 524

Metal chelate affinity chromatography, 403-404
Method interference, 70
Micelles, 84-85
Microelectrophoresis, 426-427
Microns, 76
Miller indices, 296
Molar ellipticity, 274

Index
Molecular bending, 176
Molecular orientation, 160-171
in emulsions, 162-165
in membranes, 162
in monolayers, 165-168
Molecular weight determination, 207-208, 339342, 383-385, 447
by absorption spectrophotometry, 207-208
by approach to equilibrium, 342
by gel electrophoresis, 447
by gel permeation chromatography, 383-385
by osmotic pressure measurement, 114-116
by sedimentation equilibrium, 341-342
by sedimentation velocity, 339-340
by X-ray diffraction, 299-300
Monochromators, 194
Monolayers, 165-168
of steric acid, 168(i]
Mossbauer spectroscopy, 261-264
Moving boundary electrophoresis, 427-48,
427(0
N
Negatron emission, 481
Negatrons, 487
Nephelometry, 209{i]
Nernst equation, 53
Nernst glower, 193
Nitrate electrode, 71
Non-radioactive labeling, 506
Northern blotting, 425-426, 466
Nuclear magnetic resonance spectrometry,
247-258
applications of, 256-258
instrumentation for, 256(f)
Nuclear resonance, 249-251
Number average molecular weight, 115
O
Oncotic presure, 120
Optical density, 178
Optical quenching, 499
Optics/rotation, 271
Optical rotatory dislrsion, 271-283

instrumentation for, 282-283

ORD (see, optical rotatory dispersion)
Osmolarity, 114
hyperosmotic, 113
hyposmotic, 113
isosmotic, 113
Osmoreceptors, 117
Osmoregulation, 117-121
excretory systems and, 119-120
Osmosis, 109-121
definition, 109
osmotic behaviour of cells, 112-114
significance in biology, 116-121
Osmotic pressure
colloid osmotic pressure, 1 I0, 120
definition of, 109
electronic osmometer, 11
measurement of, 110-112
total osmotic pressure, I i0
Ouchterlony method, 461
Oxygen electrode, 72-73, 73(I)
Pair production, 488
Paper chromatography, 351-357
apparatus, 352-353
paper, development of, 352-5
ascending technique, 352-353
descending technique, 352-353
detection, 354-355
solvent, choice of, 353-354
Paper electrophoresis, 430-435, 431(f)
apparatus, 430
detection and quantitative assay, 432-34
elcctrophoretic run, 430-432
Partition chromatography, 346-349
Partition coefficient, 344-345
Passive diffusion, 100
Path-length, 177
PCR, (see polymerase chain reaction)
Peptizing colloids, (see protective
pH,
concept of, 14-17
clcctromctric determination of, 52-62
measurement of, 51-62
scale of, 15
pH meter, 62-64

pH paper, 64
Phosphorescence, 223, 497
Photodiodes, 198
Photoelectric absorption, 488
Photoemissive tubes, 197(t]
Photomultipliers, 197-198, 198(f)
Photovoltaic cells, 196-197, 197(f)
Physiological buffers, 35

602
quantitative analysis, 206
instrumentation for, 19--201
detection devices, 196-198
radiation sources, 193
sample containers, 195-196
wavelength selectors, 193-195
Vacuum ultraviolet region, 177(f}, 190
van Hofra equation, 114
Viscomctry, 136
applications of, 136-141
actin-myosin interaction, 138-139
association-dissociation studies, 138
chemical modifications, 138
DNA lincarity, 141
DNA polymerization, 140-141
Intercalation of dyes, 141
intrastrand disulphide bonds, detection of,
140
native structures of proteins, 140
protein denaturation, 137-138
size and shape of macromolecules, 136137
Viscornetcr, design of, 132-133
Biophysical Chemistry
Viscosity, 122-144
biological significance of, 141-143
blood flow and, 142-143
factors affecting, 128-131
hematocdt and, 143
measurement of, 131-136
of ellipsoids, 125
of random coils, 125-126
of spheres,. 124
Void volume, 377-378, 377(f}
Wall effects, 310, 311
Water regain value, 378
Wave number, 175
Wavelength, 175

Western blotting, 468-459

X-ray diffraction, 293-300
Z
Zeta potential, 88-92
measurement of, 90-92
Zonal centrifugation, 319
Zone electrophoresis, 428-430