A

SEMINAR
ON
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

PROF. J.P. SHARMA (DIRECTOR) PRESENTED BY..

DR. R.K. RAO (PRINCIPAL) NIKITA DEWANGAN
GUIDED BY - HUMA NAZZ M.Sc.1st SEM
SIDDIQUI BIOTECHNOLOGY

G.D. RUNGTA COLLEGE OF SCIENCE & TECHNOLOGY

KOHKA-KURUD,BHILAI DURG (C.G.)
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

 INTRODUCTION
 VARIOUS ANALYTICAL TECHNIQUES
 PAPER CHROMATOGRAPHY
 ION-EXCHANGE CHROMATOGRAPHY
 GEL FILTRATION CHROMATOGRAPHY
S  AFFINITY CHROMATOGRAPHY
Y  HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
N  PAPER ELECTROPHORESIS
O  GEL ELECTROPHORESIS
P  ISOELECTRIC FOCCUSING
S  SPECTROPHOTOMETER
I  CENTRIFUGE
S  ENZYME LINKED IMMUNO SORBANT ASSAY
 CONCLUSION
 SUMMARY
 REFERENCES

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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

Introduction
I
N
In analytical biochemistry uses instruments and used to
T separate, identify and qualify biomolecules.
R
O
D
U
C Definition
T
I
An analytical techniques is a method that is used to
O
N
determine the concentration of a chemical compound.

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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

A
N
A
L  Chromatography
Y
T  Electrophoresis
I
C
A  Spectrophotometer
L
 Centrifugation
T
E
C  Enzyme Linked Immuno Sorbant Assay
H
N
I
Q
U
E
S
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

INTRODUCTION

Chromatography is an analytical technique dealing with the separation of

C closely related compound from a mixture. These include protein, peptides,
H amino acid, lipids, carbohydrates, vitamins and drugs.
R
O PRINCIPLE
M
 Chromatography (Greek ; chroma – colour, graphein – to write) usually
A
T consists of a mobile phase and a stationary phase.
O  The mobile phase refers to the mixture of substances( to be separate),
G dissolved in a liquid or a gas. The stationary phase is a porous solid matrix
R through which the sample contained in the mobile phase percolates.
A  The interaction between the mobile and stationary phases results in the
P
separation of the compounds from the mixture.
H
Y  TYPES OF CHROMATOGRAPHY - PAPER CHROMATOGRAPHY, ION
EXCHANGE CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY.
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

P INTRODUCTION
A
P Paper chromatography is useful for separating the mixture of amino acid,
E sugar, chemicals, lipid , urea and some drugs.
R
PRINCIPLE
C
H
R  The filter paper are used to support a stationary water phase while mobile
O organic phase moves down the suspended paper strip in a cylinder.
M
A  Separation is based on a liquid-liquid partition of the compounds.
T
O  Thus, this is essentially a form of partition chromatography between 2 liquid
G
R phases, although adsorption to the paper may also take place.
A
P
H
Y
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

P PROCEDURE –
A
P
E  The sample mixture is applied to a piece of
R filter paper the edge of the paper is
immersed in a solvent.
C  The aqueous component of the solvent
H system binds to the paper & forms a
R stationary phase.
O  The organic component that migrates on the
M paper is the mobile phase.
A  When the migration of the solvent is
T
upwards, it is referred to as ascending
O
chromatography.
G
R  In descending chromatography, the solvent
A moves downwards.
P  As the solvent flows, it takes along with it
H the unknown substances.
Y
Fig. 1; paper chromatography 6
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

P
A  The rate of migration of the molecules depends on the relative solubility in the
P
stationary phase (aqueous) & mobile phase (organic).
E
R

C  The paper (chromatography) is then removed, dried & developed

H
for the identification of the specific spots.
R
O
M
A  Identification of the compound :-
T
O The distance travelled by a particular component of a mixture (or solute)is used
G to identify it.
R
A
P
H Resolution Factor(RF) = Distance from origin run by the solute
Y Distance from origin run by the solvent 7
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

INTRODUCTION
Ion exchange chromatography is used for separation of amino acids, proteins,
nucleotides and charged molecule.
I
O PRINCIPLE
N Ion exchange chromatography involves the separation of molecules on the basis of
their electric charges. Ion exchange resins- cat ion exchanges & anion exchanges are
E used for this purpose.
X
C PROCEDURE- An anion exchanges (R⁺ A⁻) exchanges its anion (A⁻) with
H another anion (B⁻) in solution.
A R⁺ A⁻ + B⁻ = R⁺ B⁻ + A⁻
N
 A cation exchanges ( H⁺R⁻) exchanges its cat ion (H⁺) with another cat ion ( C⁺) in
G
E solution.
H⁺ R⁻ + C⁺ = C⁺ R⁻ + H⁺
 Thus, in ion exchange chromatography, ions in solutions are reversibly replaced by
ion-exchange resins.
 The binding abilities of ions bearing positive or negative charges are highly pH
dependent, since the net charge varies with pH .
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

I
O
N

E
X
C
H
A
N
G
E

C
H
R
O
M
A
T
E
FIG.:- 2 Ion exchange chromatography
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
A
F
F INTRODUCTION
I Affinity chromatography is a method of separating biochemical mixtures based on a
N highly specific interaction such as that between antigen and antibody, enzyme and
I substrate, Or receptor and ligand.
T PRINCIPLE
Y  The principle of affinity chromatography is based on the property of specific &
noncovalent binding of proteins to other molecules, referred to as ligands. For instance
C enzymes bind specifically to ligands such as substrates or cofactors.
H PROCEDURE
R
O  This technique involves the use of ligands covalently attached to an inert & porus
M in a column.
A  The immobilized ligands acts as molecular fish hooks to selectively pick up the
T
O desired protein while the remaining proteins pass through the column.
G  The desired protein captured by the ligands, can be eluted by using free ligand
R molecules.
A
 Alternatively, some reagents that can break protein- ligand interaction can also be
P
H employed for the separation.
Y 10
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

A
F
F
I
N
I
T
Y

C
H
R
O
M
A
T
O
G
R
A
P
H
Y 11
Fig:-3 Affinity chromatography
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

INTRODUCTION
 High Performance Liquid Chromatography (HPLC) is used to separate
components of a mixture by using a variety of chemical interactions between the
substances being analyzed & the chromatography column.

H
P
L
C

Fig; 4. HPLC 12
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

PRINCIPLE
 It may employ the principles of adsorption, partition, ion-exchange, exclusion
& affinity chromatography. This makes it an extremely versatile technique.
PROCEDURE
 The sample to be analyzed is introduced in a small volume to the stream of
H mobile phase.
P  It is retorted to specific chemical or physical interactions with the stationary
phase. It travels the length of the column.
L
 The amount of retardation depends on the nature of the analyte, stationary phase
C & mobile phase composition.
 At the time at which a specific analyte elutes is called the retention time.
 The use of pressure increases the linear velocity giving the components less
time to diffuse with in the column.

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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

E
L INTRODUCTION
E The movement of charged particles(ions) in an electric field resulting in
C their migration towards the oppositely charged electrode is known as
T electrophoresis.
R TYPES OF ELECTROPHORESIS – PAPER ELECTROPHORESIS,
O GEL ELECTROPHORESIS, ISOELECTRIC FOCUSSING.
P
PAPER ELECTROPHORESIS - Separation of charged particles is determined by
H differences in their migration rate which varies with electrical charge, size of
O particles & shape of particles.
R PROCEDURE -
 In this the sample is applied on a strip of filter paper wetted with dried buffer
E solution.
S  The end of the strip are dipped into the buffer reservoirs in which electrodes are
I placed. Then electric current is supplied allowing the molecules to migrate for
sufficient time.
S 14
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

P
A
 The paper is removed, dried & stained with a dye that specifically colors the
P substance to be detected which can be identified by comparing with a set of
E standards run simultaneously.
R For the separation of serum proteins whatman No.1 filter paper, veronal or tris
buffer at pH 8.6 & the stains amido black or bromophenol blue are employed.
E
L
E
C
T
R
O
P
H
O
R
E
S
I
S
15
Fig 5, PROCESS OF PAPER ELECTROPHORESIS
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

G
E INTRODUCTION
L
 It is used for the separation of proteins & nucleic acid.
E  This technique involves the separation of molecules based on their size, in
L addition to electric charges.
E
C PROCEDURE
T  In this technique gel is used as a stabilizing media.
R  Gel contains wells made with the help of comb.
O
P  Buffer is added in the apparatus.
H  In the well the sample is loaded.
O  The electric current is switched on.
R
E  The separated components can be identify either by labeling with a radio
S isotope or by UV- visible spectrophotometer.
I
S 16
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

G
E
L

E
L
E
C
T
R
O
P
H
O
R
E
S
I
S 17
FIG. :- 6 GEL GLECTROPHORESIS
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

I
INTRODUCTION
S
 It is used for purification of proteins.
O
E
 This technique is primarily based on the
L
immobilization of the molecules at isoelectric
E
pH during electrophoresis.
C
T
PROCEDURE
R
 Stable pH gradients are set up (usually in gel)
I
converting the pH range to include the
C
isoelectric points of the components in a
mixture.
F
O
 As the electrophoresis occurs, the molecules
C
migrate to positions corresponding to their
U
isoelectric points, gets immobilized & form
S
I sharp stationary bonds.
Fig -7 Isoelectric focusing
N
G  The gel blocks can be stained & identified. 18
 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

SPECTROPHOTOMETER – Spectrophotometer is a method to measure how much a

S chemical absorbs light, by measuring the intensity of light as a beam of light passes
P through sample solution.
E PRINCIPLE OF SPECTROPHOTOMETER - The principle of spectrophotometer is
C based on Lambert’s Beer’s law. :-
T
1. Lambert’s law (Bouguer’s law) - this law states that the amount of light absorbed is
R directly propotional to the length or thickness of the solution under analysis.Thus,
O
P I / I0 = e -kb
H  I = the intensity of the transmitted light, I0 = the intensity of the incident light, b = the
absorbing thickness, better known by the term path-length, k = the linear absorption
O coefficient of the absorbing material,
T  the power term in the above relationship can be removed by converting to the logarithmic
form, thus,
O In I /I0 = - kb or In I0 /I = kb,
M 
2.
Changing the common logarithms we get
E 2.303 Log10 I0/I = kb
T
2. Beer’s law - this law states that the amount of light absorbed is directly proportional
E
to the concentration of the solute in solution. Thus,
R
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

2.303log10 I0/I = kʹC

S  Where kʹ = absorptivity constant , C = the concentration of the
P absorbing material.
E
C  The beer- lambert’s law then becomes:
T
R Log10 I0/ I = abC
O
P
H
O
T
O
M
E
T
E
R

Fig - 8 spectrophotometer
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

 CENTRIFUGE is device for separating particles from a solution according

to there size, shape, density, viscosity of the medium.
 PRINCIPLE : - An object moving in a circle at a steady angular velocity will
C experience a force F directed outwards. This is the basis of centrifugation. Angular
E
velocity ω and the radius of rotation ‘r’ in cm collectively determine the magnitude
N
T of the force F,
R  F = ω2r
I
F  If F is expressed in the form of gravitational force it is divided by 980.
U  RCF = ω2r / 980
G
 RCF = relative centrifugal force
E
 But ω = 2π radian = 2π/60 r/min
 RCF = 4π2( r/m)2r/ 3600× 980
 RCF = 1.118 × 15-5 rpm2r
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

ENZYME LINKED
IMMUNOSORBANT
ASSAY
E
 ELISA is based on affinity.
L  An ELISA test can
detect both current and
past infections. As well as
I
antibodies, an ELISA test
can also be used to detect
S antigens (substances that
provoke an immune
A response, such as bacteria
or proteins) or enzymes.

Fig ; 9ELISA
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

 Analytical techniques in biochemistry provides an opportunity for

researchers and scientists to explore the advanced and latest research
developments in the field of biochemistry .

C  Biochemistry deals with the structures, functions and interactions of

O biological macromolecules such as proteins, nucleic acids, carbohydrates and
N lipids which provide the structure of cells and perform many functions
C associated with life.
L
U  It can be used both for separation & identification of macromolecules
S & micro molecules.
I
 In biochemistry, the term macromolecule is applied to the four
O conventional biopolymers ( nucleic acids, proteins , carbohydrates & lipids )
N as well as non polymeric molecules with large molecular mass.

 The constituent molecules from which macromolecules are assembled are

called monomers, dimers or tetramers.
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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

 An analytical technique is a method that is used to determine the concentration of

a chemical compound.
 CHROMATOGRAPHY is used to separation of closely related compound from a
S mixture.
U  Chromatography types – PAPER CHROMATOGRAPHY, ION- EXCHANGE
M CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY, HPLC.
 ELECTROPHORESIS – The movement of charged particle an electric field
M resulting in their migration towards the oppositely charged electrode .
A  Electrophoresis types – PAPER ELECTROPHORESIS, GEL
R ELECTROPHORESIS, ISOELECTRIC FOCUSING.
 Spectrophotometer is the quantitative measurement of the reflection or
Y transmission properties of a material as a function of wavelength.
 CENTRIFUGE - it is a device for separating particles from a solution according to
their size, shape, density and velocity of medium.
 ELISA - An enzyme-linked immunoassay (ELISA) is a test that detects
antibodies in the blood.

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 ANALYTICAL TECHNIQUES IN BIOCHEMISTRY

U. SATYANARAYANA 2010 BIOTECHNOLOGY

R
E
F
J.L JAIN SIXTH EDITION 2010 FUNDAMENTAL
E
OF
R
BIOCHEMISTRY
E
N
C
E
UPADHAYAY FOURTH EDITION2007 BIOPHYSICAL
S
AND
BIOCHEMISTRY

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THANK

YOU