CHROMATOGRAPHY

Lesson Plan
Basic principles of adsorption chromatography
Retention Theory
HETP
Selectivity
Parts of chromatograph instrument
Normal and reversed phase chromatography
Isocratic and gradient modes
Affinity chromatography
Gel permeation chromatography or size exclusion chromatography
Supercritical fluid chromatography
Countercurrent chromatography
Lipid separation by chromatography
Combination of two chromatographic methods
Antibiotic separation by TLC

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INTRODUCTION :
It is the most important and versatile technique for separation of a mixture and is used primarily
for analytical purposes.
The technique is based on differences in affinities of the compounds in a mixture towards a
suitable adsorbent.
Chromatography was first documented by Tsvet in 1903 – 06 when he was separating plant
extract using silica column. ‘Chroma’ means colour and ‘Graphy’ means ‘detection’. The separation
achieved is in molecular level.

History

It was the Russian botanist Mikhail Tsvet (Mikhail Semyonovich Tsvet) who invented the first
chromatography technique in 1901 during his research on chlorophyll. He used liquid-adsorption
columns to separate plant pigments. The method was described on December 30, 1901 at the XI
Congress of Naturalists and Doctors in St. Petersburg. The first printed description was in 1903, in the
Proceedings of the Warsaw Society of Naturalists, section of biology. He first used the term
chromatography in print in 1906 in his two papers about chlorophyll in the German botanical journal,
Berichte der Deutschen botanischen Geselschaft. In 1907 he demonstrated his chromatogaph for the
German Botanical Society. The phenomenon of precipitational separation was observed before Tsvet as
well. His contribution was turning the phenomenon into the method of scientific analysis.

The Greek word chroma in chromatography means color in English and refers both to Tsvet's name that
is literally translated from Russian as color and to the color of the plant pigments he was separating at
that time.

In 1952 Archer John Porter Martin and Richard Laurence Millington Synge were awarded the Chemistry
Nobel Prize "for their invention of partition chromatography". [1]

The technology of chromatography advanced rapidly throughout the 20th century. Researchers found
that the principles underlying Tsvet's chromatography could be applied in many different ways, giving
rise to the different varieties of chromatography. Simultaneously, advances continually improved the
technical performance of chromatography, allowing increasingly similar molecules to be resolved.

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VARIOUS USES OF CHROMATOGRAPHY
Chromatographic methods have been used for chemical analysis and for automated analysis of
process streams in process control (as process chromatography) since 1950/60’s. They also measure
variety of thermodynamic, kinetic and other physico-chemical properties.
Commercial separation processes involving chromatography is often called production or large
scale chromatography. Laboratory scale preparations are done by preparative chromatography.

Chromatography
Use of
Chromatography
[Detection (qualitative & quantitative)/ Production]

Chemical Analysis Physico-chemical measurement Separation Process

Process Chromatography Production Preparative

BASIC WORKING PRINCIPLE

The components of a mixture are separated as they pass through a column. The column contains a
‘stationary phase’ or adsorbent (solid or liquid impregnated solid) packed within it : The mixture is
carried through the column dissolved in a gas or liquid stream called ‘mobile phase’, ‘eluent’ or
‘carrier’. ‘Solute’ is the component in the feed mixture to be separated.
A discrete quantity of feed mixture is introduced into the column at the inlet. The mobile phase flow
causes the band of feed to migrate and split progressively into its component solute – bands or peaks.
1&2 Tiny iron balls + glass balls + oil

2 1 (mobile phase) are passed through

2 1 a column packed with magnetic

material.

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 Emergence of the bands at the column outlet is monitored by a suitable detector, and the
components are collected in sequence. A solute 1 will travel faster than solute 2 if it has lower affinity
to the stationary phase or higher affinity to the mobile phase than solute 2. The principle is one of
differential migration.
TYPES OF CHROMATOGRAPHIC METHODS
To separate compounds effectively requires appropriate choice of the chromatographic mobile
and stationary phases.
The principal types of chromatography involved in large-scale separations are as follows :
Gas Chromatography (GC)
 Gas – liquid (GLC)
 Gas – solid (GSC)
Liquid Chromatography (LC)
 Normal bonded phase (NP-BPC)
 Reverse bonded phase (RP-BPC)
 Liquid – solid (LSC)
 Ion Exchange (IEC)
 Size exclusion (gel permeation)(SEC/GPC)
 Affinity (AC)
 Hydrophobic interaction (HIC)

Super critical Fluid Chromatography (SFC)

Chiral Chromatography (GC, LC or SFC)
Gas Chromatography employs a gaseous mobile phase known as carrier gas. In liquid gas
Chromatography (GLC) the stationary phase is a liquid held on the surface and in the pores of a
normally inert solid support (typically diatomaceous silica). In gas-solid chromatography the stationary
phase is a solid adsorbent such as silica or alumina. These are cheap and long lasting. But there is
surface heterogeneity and irreproducibility. Porous polymer and molecular sieves are also used.
Typical surface area : 0.5 – 4 m2/g.
Liquid Chromatography employs stationary phase consisting of chemically bonded liquid
monolayer over a surface of solid support. The support is usually silica or alumina having particle size
of 5 – 25 nm and surface area of 200 – 800 m2/g. These are mechanically strong to withstand a pressure

4
about 6000 psia. The column is packed at this pressure, thus it is sometimes called high pressure liquid
chromatograph (HPLC).
This may be of two forms – normal phase and reversed phase. When silica is used as such, the
stationary phase is polar. Then the mobile phase must be non-polar (because the two phases must be of
opposite polarity). This is normal phase chromatography retaining polar compounds.
The reverse phase chromatography, the stationary phase is non-polar and the mobile phase is
polar. This retains non-polar molecules. As consumption of mobile phase dictates the economy of the
chromatographic process and reverse phase chromatography can employ water-based mobile phase
(polar), this is a comparatively cheaper process. The impregnated liquid is generally organic compound.
When people say C-18 column, it means it employs octa – decyl silane i.e. an organic compound
containing 18C atoms in a single molecular chain.
Ion Exchange Chromatography (IEC) is carried out with packing that contains charge bearing
functional groups. The ions exchangers are generally organic polymers. It is an important technique in
the purification of sensitive bio-molecules such as proteins.
Size exclusion or gel-permeation chromatography differs from, all other methods in separating
according to molecular sizes rather than the functional groups. The particulate packing has pores of well
defined size. Solute molecules too large to enter the pores are totally excluded from the interior and
elute first.
Affinity chromatography depends on the specific adsorption resulting from molecular
recognition. Bio-affinity chromatography uses the variety of such complexes e.g. ‘antibody-antigen,
enzyme – inhibitor, hormone – receptor etc.

Paper chromatography

In paper chromatography, chemical interactions with the paper make compounds travel at different rates.

A small spot of solution containing the sample is applied to a strip of chromatography paper about one
centimeter from the base. This sample is adsorbed onto the paper. This means that the sample will
contact the paper and may form interactions with it. Any substance that will react with (and thus bond
to) the paper cannot be measured using this technique. The paper is then dipped in to a suitable solvent
(such as ethanol or water) and placed in a sealed container. As the solvent rises through the paper it
meets the sample mixture which starts to travel up the paper with the solvent. Different compounds in

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the sample mixture travel different distances according to how strongly they interact with the paper.
Paper chromatography takes some time and the experiment is usually left to complete for some hours.

The final chromatogram can be compared with other known mixture chromatograms to identify sample
mixes. Two-way paper chromatography involves using two solvents and rotating the paper 90o in
between. This is useful for separating complex mixtures of similar compounds.

Thin layer chromatography (TLC)

In thin layer chromatography or TLC the stationary phase consists of a thin layer of adsorbent like silica
gel, alumina, or cellulose on a flat carrier like a glass plate, a thick aluminum foil, or a plastic sheet.

The process is similar to paper chromatography with the advantage of faster runs, better separations, and
the choice between different adsorbents. TLC is a standard laboratory method in organic chemistry.
Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the
qualitative analysis of reaction products.

TLC plates are made by mixing the adsorbent with a small amount of inert binder like calcium sulfate
(gypsum) and water, spreading the a thick slurry on the carrier, drying the plate, and activation of the
adsorbent by heating in an oven. The thickness of the adsorbent layer is typically around 0.1 - 0.25 mm
for analytical purposes and around 1 - 2 mm for preparative TLC.

Several methods exists to make colorless spots visible:

 Often a small amount of a fluorescent dye is added to the adsorbent that allows the visualization
of UV absorbing spots under a blacklight ("UV254").
 Iodine vapors are a general unspecific color reagent.
 Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the
plate.

Once visible, the spots can be quantified by way of calculating their Rf values. These values should be
the same regardless of the extent of travel of the solvent, and in theory are independant of a single
experimental run. They do depend on the solvent used, and the type of TLC plate.

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Gas-liquid chromatography

Gas-liquid chromatography is based on a partition equilibrium of analyte between a liquid stationary

phase and a mobile gas. It is useful for a wide range of non-polar analytes, but poor for thermally labile
molecules.

Ion exchange chromatography

Ion exchange chromatography is a method to separate molecules such as proteins by their charge in a
process of ion exchange. Ion exchange chromatography uses ion exchange resins as stationary phase.

Immobilized metal ion affinity chromatography

IMAC is a popular and powerful way to purify proteins. It is based on the specific coordinate covalent
binding between histidine or other unique amino acids (either naturally present on the surface of the
protein or grafted with recombinant DNA techniques) and various immobilized metal ions, such as
copper, nickel, zinc, or iron.

Salt concentration is increased to produce later fractions.

High performance liquid chromatography (HPLC)

High performance liquid chromatography, frequently referred to simply as HPLC, is a form of column
chromatography used frequently in biochemistry. The analyte is forced through a column by liquid at
high pressure, which decreases the time the separated components remain on the stationary phase and
thus the time they have to spread out within the column, leading to broader peaks. Less time on the
column then translates to narrower peaks in the resulting chromatogram and thence to better selectivity
(it's easier to differentiate one peak from another) and sensitivity (tall, narrow peaks can be easier to
discriminate from noise than shorter, broader peaks). Solvents used include any miscible combination of
water or various organic liquids (the most common are methanol or acetonitrile). Often, a gradient over
time in the solvent composition passing through the column is used to separate analyte mixtures, as a
function of how well the changing solvent composition differentially mobilizes the analyte. For instance,
using a water/methanol gradient, the more hydrophobic components will elute under conditions of
relatively high methanol, whereas the more hydrophilic will elute under conditions of relatively low

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methanol. Whether one starts with high methanol or low methanol depends on the nature of the
stationary phase.

Comparison of Gas and Liquid Chromatography :

1) GC is best suited to separate materials of molecular weight below 300. LC is the preferred
method for heat sensitive materials and to separate liquid having higher boiling point than in GC,
though there is some overlap in the range of molecular weight.
2) In GC, the mobile phase is gas and in LC, the mobile phase is liquid.
3) LC requires no vaporizer to inject material into the mobile phase. Low energy consumption is
advanced as a virtue in LC.

Theory of Chromatography :

Chromatography is a separation method that exploits the differences in partitioning behavior between a
mobile phase and a stationary phase to separate the components in a mixture. Components of a mixture
may be interacting with the stationary phase based on charge, relative solubility or adsorption. There are
two theories of chromatography, the plate and rate theories.

Retention

The retention is a measure of the speed at which a substance moves in a chromatographic system. In
continuous development systems like HPLC or GC, where the compounds are eluted with the eluent, the
retention is usually measured as the retention time Rt or tR, the time between injection and detection. In
interrupted development systems like TLC the retention is measured as the retention factor Rf, the run
length of the compound divided by the run length of the eluent front:

The retention of a compound often differs considerably between experiments and laboratories due to
variations of the eluent, the stationary phase, temperature, and the setup. It is therefore important to
compare the retention of the test compound to that of one or more standard compounds under absolutely
identical conditions.

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Plate theory

The plate theory of chromotography was developed by Archer John Porter Martin and Richard Laurence
Millington Synge. The plate theory describes the chromotography system, the mobile and stationary
phases, as being in equilibrium. The partition coefficient K is based on this equilibrium, and is defined
by the following equation:

K is assumed to be independent of concentration, and can change if experimental conditions are

changed, for example temperature is increased or decreased. As K increases, it takes longer for solutes
to separate. For a column of fixed length and flow, the retention time (tR) and retention volume (Vr) can
be measured and used to calculate K.

Retention theory :
This theory allows calculation of the time for which the solute is retained in the column between
injection and elution.
The retention time, t R is defined in terms of solute – concentration vs. time plot registered by a
detector at the column outlet.
An average molecule of a given solute moves repeatedly in and out of the stationary phase
during its passage through the column. The molecule spends some of its time being ‘mobile’ with the
mobile phase and some of its time being ‘stationary’ at the stationary phase. If R is the ratio of the total
time spent by this average molecule in the mobile phase to the total time spent in the column, then,
tM uM
R = =
tR u
where u M : velocity at which the solute band moves along the column, and u is the velocity of the
mobile phase. tR is the time taken by a solute to pass through the column; tM is the mobile phase hold-
up and is measured as the retention time of a non-sorbed solute. tR’ is the adjusted retention time, that is
total time spent by the solute in the stationary phase, tR-tM
Superficial velocity, ν
u =
Є
where Є : voidage, fractional volume of column occupied by mobile phase.
ν : volumetric flow rate / area.

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 Now, the average molecule is representative of a large number of similar molecules. Thus this
time ratio may be looked as the fraction of the total molecules, which are in the mobile phase at
equilibrium.

tM nM
R = =
tR n M+ n S
1 nS
= 1 + = 1 + K’
R nM
1
R =
1 + K’
where, K’ is the capacity factor or mass distribution co-efficient, n S / n M . n S → no. of molecules in
the static phase and n M → no. of molecules in the mobile phase.

tM uM 1
R = = =
tR u 1 + K’
u
uR =
1 + K’
and tR = t M (1 + K’)
If q → conc. of solute in stationary phase and
c → conc. of solute in mobile phase then,

nS 1–Є
= K
nM Є
where, K = q/c = distribution co-efficient of the solute between two phases.
(assumed that isotherm is linear)
1–Є
and tR = tM 1 + K
Є
using this equation t R can be calculated if K and Є are known.
Change in column type, flow rate and type of mobile phase affects retention time.

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Separation efficiency and band broadening :
The equation mentioned above relates only to the average molecule and so describe only the
mean retention of a band. The mean retention is that of the peak of the band if the band is symmetrical.
In practice, there are several causes of band broadening. The broadening makes a
chromatographic process “inefficient”.
To understand the efficiency and inefficiency, we have to understand the following :
 Plate height or HETP
 Particle size of packing
 Resolution,
 Selectivity.

Plate Height or HETP :

The efficiency of a chromatographic column is often measured in terms of HETP. Martin and
Synge defined HETP as the unit of column length sufficient to bring the solute eluting out with the
mobile phase from it into equilibrium with the solute in the stationary phase in the unit. Thus HETP H
of a column of length L and no. of plates N is related as :
H = L/N
To maximize separation efficiency, small H and large N is required. Generally, 75,000 to
100,000 numbers of plates are there per meter of chromatographic columns.
Normally a peak is a bell – shaped or Gaussian Curve,
Number of plates N = C ( V / W )2
= C ( tR / tW )2
w band width, C = 25 (by 5 T method)
16 (tangent method) and
5.54 (1/2 peak height method).

Tangent method converts the actual peak to a Gaussian one.

From Curve : tR

Conc.

Time tW

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 Therefore greater is the ratio tR / tW , more is the value of N. It is required that the stationary and
the mobile phase is well dispersed. This may be achieved by packing the column with very fine and
porous particles with large surface area. tW is the width of a solute band at the baseline.

Particle size and band broadening :

There are several band broadening processes operating in a chromatographic column. Each
contributes to a term towards plate height that is efficiency / inefficiency of a column.
H = A + B/u + CS u + Cm u .

Term Mechanism
A = 2 λ dp Inequalities in patterns of flow in column packing.
B/u = 2 γ Dm/u Axial diffusion in mobile phase.
/ 2 Lack of equilibrium between solute in the two phases due to
2 k df
CS u 

3 1  k / DS
u
 slow mass transfer in stationary phase film.

Slow mass transfer of solute in mobile phase during

d p 2
Cmu  u passage of solute to and from interface with stationary
Dm
phase.

Dm = Diffusivity of solute in mobile phase

Ds = Diffusivity of solute in stationary phase
df = Effective thickness of stationary liquid film
dP = diameter of packing particles
k’ = capacity factor
u = average mobile phase velocity
ω = packing geometry factor
λ = eddy diffusion constant in a packed bed
γ = obstruction (labyrinth) factor for diffusion through packed bed

Smaller (3 – 8 μm) sized particles are used in HPLC than GC (120 – 300 μm). Smaller particles entail
much higher pressure drops (40 – 400 bar). Large scale LC employs larger particles (10 – 70 μm) than
analytical HPLC. Not only particle size, but also particle size distribution is important for good

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performance. A narrow distribution leads to a good efficiency. Pore size distribution is also another
important factor.

RESOLUTION :
The resolution of two neighbouring peaks is defined as

t R 2  t r1 Difference in retention time

R/ 
 
t w1  t w2 / 2
=
Average of the peak width as the base

The resolution may be related to the selectivity ( α ) capacity factor ( K’) and the number of theoretical
plates (N) as follows :

 α  1  k  k1/  k 2/
/
R    N k/ 
/
where,
 4  1  k
/
 2

A ‘capacity factor’ in a chromatographic separation is defined as –

Total amount of solute in the stationary phase
K’ =
Total amount of solute in the mobile phase
This may, intern, be represented in terms of retention times :
tR–tM
K’ =
tM

SELECTIVITY :
The ‘Selectivity’ of compound 2 over 1 is defined as :

tR2  tm k 2/
α 21   /
t R1  t m k1

This is a ratio of mass distribution co-efficient. It is therefore a thermodynamic rather than

kinetic factor. It is analogous to the relative volatility in distillation.
The value of α depends mainly on the nature of the two solutes, on the stationary and mobile
phase (LC).

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TAILING :
If the mobile phase is in plug flow and axial dispersion effects are small, a sharp peak is obtained
for a compound in the sample. Dispersion effects make a peak broader and reduce its height. If too
much peak broadening occurs, adjacent peaks may partially over lap. The operating conditions of the
instrument have to be so adjusted that it gives a sharp and nearly. Gaussian Plak. In many causes the
rear end of the peak forms a “tail”. This is called ‘tailing’. Tailing reduces plate number and hence
resolution.

Gas Chromatography :
A gas chromatograph consists essentially of the following parts :
Flow meter
Sample injection

Chart recorder (Data station)

Brain
Gas Column
Cylinder (heart)

Detector (eye)

1) Supply of carrier gas from high-pressure cylinder :

The carrier gas used is either helium, nitrogen, hydrogen or argon, the choice of gas depending
on factors such as availability, purity required, consumption and type of detector employed. Helium is
preferred when thermal conductivity detectors are employed, because of its high thermal conductivity
relative to that of the vapours of most organic compounds. The operating efficiency of the GC is largely
dependent on maintenance of a constant flow of carrier gas.
2) Sample Injection System :
Liquid samples are injected by a micro-syringe with hypodermic needle. The latter in inserted
through a self-sealing silicone rubber septum and the sample injected smoothly into a heated metal block
at the head of the column. The temperature of the sample port should be such that the liquid is rapidly
vaporized without decomposing or fractionating. As a rule of thumb the sample port temperature should
be the b.p. of the most volatile compound in the sample. The sample size should be minimum consistent
with the detector sensitivity.

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3) The Column :
The actual separation of sample components depends mainly upon the inert solid support, type
and amount of liquid phase, method of packing, length and temperature. Analytical columns are usually
prepared with 2 – 6 mm internal diameter glass tubing or 3 – 10 mm outer diameter metal tubing, which
is normally coiled for compactness. Glass columns are used for the sample components decomposing
by metal – contact.
The HETP is proportional to the average particle diameter so that theoretically the smallest
possible particles should be preferred in terms of column efficiency. But decrease in particle size will
increase the necessary gas pressure. For effective packing, the internal diameter of column should be at
least eight times the particle diameter.
The relative amount of stationary liquid phase in the column packing is usually expressed on the
basis of the percent by weight of liquid phase present. 15 percent loading means 100 g column packing
contains 15 g of liquid phase on 85 g inert support.
4) The Detector :
The function of the detector is to sense and to measure the separated components. The choice of
detector will depend on factor such as nature and concentrations of the components.
Thermal conductivity,
Flame ionization
Electron capture.

Thermal Conductivity Detector :

This is the most common type of detector used in gas chromatography. These detectors employ
a heated metal filament or a thermistor to sense changes in the thermal conductivity of the carrier gas
stream.
Two pairs of matched filaments are arranged in a wheat stone bridge circuit. Two filaments in
opposite arms of the bridge are surrounded by the carrier gas only while the other two filaments are
surrounded by the effluent from the chromatographic column. When pure carrier gas passes over both
the reference and sample filaments the bridge is balanced, but when a vapour emerges from the column,
the bridge becomes unbalanced. The extent of this imbalance is proportional to the concentration of the
vapour. This signal is fed to the recorder. The differential technique is based on the measurement of the
thermal conductivity between the carrier gas and the carrier gas / sample mixture.

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Flame Ionization Detector :
The basis of this detector is that the effluent from the column is mixed with hydrogen and burned
in air to produce a flame which has sufficient energy to ionize solute molecules having low ionization
potentials. The ions produced are collected at electrodes and the resulting ion current measured.
Electron Capture Detector :
Most ionization detectors are based on measurement of the increase in current, which occurs
when a more readily ionized molecule appears in the gas stream. The electron capture detector differs
from other ionization detectors in that it exploits the recombination phenomenon. It is based on the
electron capture by compounds having an affinity for free electrons – the detector thus measures a
decrease rather than increase in current.
A β – ray source is used to generate ‘slow’ electrons by ionization of the carrier gas flowing
through detector. When an electron – capturing gas emerges form the column and reacts with an
electron, the net result is the replacement of an electron by a negative ion of greater mass with a
corresponding reduction in current flow.
LIQUID CHROMATOGRAPHY (HPLC)
Gas Chromatograph fails in case of compounds having low vapour pressure or being unstable at
elevated temperature. Such compounds have to be separated by liquid chromatography using solid
adsorbents of small sized particles (5 – 10 μm) at an elevated pressure (300 – 3000 psi). This technique
is called high performance (or high pressure) liquid chromatography (HPLC).
Rotary Micro-syringe
injection valve Column Detector Recorder
Pump

Solvent

Filter

The solvent is taken up through filter by pump fed to rotary injection value, fitted with sample
loop, filled from micro-syringe and then to column, detector and waste. The output is displayed on
recorder. The pump supplies the solvent at a constant pressure upto 4500 psi and flow rates of few
mL.min-1. The sample size is of the order of 1 – 20 μL. The column is made of thick-walled strain.

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As steel tubing, 10 or 20 cm long with 2 –3 mm bore and packed with particles 5 – 10 μm which may be
:
 Porous solids (Al2O3, Silica gel) for use with low – polarity solvents.
 Bonded-phase packings which are suitable for sue with polar solvents such as aqueous ethanol.
 Ion change resins, useful for the separation of amino acids or inorganic anions.

Normal and Reverse Phase Chromatography :

In normal phase, mobile phase is non-polar. Therefore the least polar part of the solute comes out first.
Typical stationary phase is silica gel, alumina etc. Typical mobile phase is isooctane –
chloroform. The disadvantages of normal phase chromatography lie in the fact that it is limited to
compounds soluble in relatively non-polar solvents. The regeneration of the stationary phase is difficult
and it adsorbs moisture.

In reverse phase, stationary phase is non-polar and mobile phase is polar. Therefore the most
polar component comes out first. Common solvents here are acetonitrile, methanol, tetra-hydrofuran or
isopropanol. Reversed phase (RP) liquid chromatography

Traditionally HPLC stationary phases are polar, whereas so-called "reverse" phase (RP-HPLC)
stationary phases are hydrophobic. On an RP-HPLC column, then, hydrophobic analytes would tend to
be retained on the column, eluting more readily as the proportion of the hydrophobic component of the
mobile phase is increased. RP-HPLC has lower resolution than GC.

Isocratic and Gradient methods in liquid chromatography :

In liquid chromatography, a solvent gradient is often used. A mixed solvent, with its
composition changing with time in a predetermined, fashion, done by automatically adjusting the flow
rates of the individual solvent, is used as the mobile phase so that the eluting environment can be
controlled to cause elution of the component in a controlled manner. In isocratic system the
composition of the mobile phase remains constant over time.
The detector used in HPLC measure a property of the solution or solutes. The solution property
may be either refractive index or conductance whereas the solute properties may be measured by
spectrophotometer or spectrofluorimeter. R.I. or refractive index detector is the universal detector

17
whereas UV – vis detector is the most commonly used one. With RI detector, it is not permissible to
change the composition of the mobile phase.

COMPARISON WITH OTHER SEPARATION PROCESSES

The most obvious virtue of chromatographic methods is their great separating power. In
choosing suitable methods to separate a given mixture of compounds, the general characteristics
favouring a chromatographic method are :
 It can achieve difficult separations
 It can meet high product purity specification,
 It can separate heat sensitive compound because of the small residence time, absence of reflux
and low operating temp. (LC & SFC).
 It has relatively low energy consumption,
 It can split an n – component mixture into n – pure components in one column instead of (n – 1)
columns as in distillation and other counter current processes.

But throughputs from chromatographic columns, specially GC, are much less than from a
distillation column of same diameter, unless the separation is a ‘difficult’ one. For the ‘difficult’
separation processes with α < 1.2, GC is preferred to distillation whereas in case of α > 1.6, distillation
is preferred. Chromatographic methods are in general advantageous for difficult separation where high
product purities are specified.
The same advantages are exhibited by LC compared with liquid extraction, ultra filtration and
adsorption.

Affinity chromatography

It is a chromatographic method of separating biochemical mixtures, based on a highly specific biological

interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.
Affinity chromatography combines the size fractionation capability of gel permeation chromatography
with the ability to design a stationary phase that reversibly binds to a known subset of molecules.

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Uses

Affinity chromatography can be used to:

 Purify and concentrate a molecule from a mixture into a buffering solution

 Reduce the amount of a molecule in a mixture
 Discern what biological compounds bind to a particular molecule, such as drugs

Principle

Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell
lysate, growth medium or blood serum. The molecule of interest will have a well known and defined
property which can be exploited during the affinity purification process. The process itself can be
thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or
medium. The other molecules in solution will not become trapped as they do not possess this property.
The solid medium can then be removed from the mixture, washed and the target molecule released from
the entrapment in a process known as elution.

Binding to the solid phase may be achieved by column chromatography, whereby the solid medium is
packed onto a chromatography column, the initial mixture run through the column to allow binding, a
wash buffer run through the column and the elution buffer subsequently applied to the column and
collected. These steps are usually done at ambient pressure (as opposed to HPLC or FPLC).

Alternatively binding may be achieved using a batch treatment, by adding the initial mixture to the solid
phase in a vessel, mixing, separating the solid phase (by centrifugation for example), removing the
liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the
eluate.

Sometimes a hybrid method is employed, the binding is done by the batch method, then the solid phase
with the target molecule bound is packed onto a column and washing and elution are done on the
column.

A third method, expanded bed adsorption, which combines the advantages of the two methods
mentioned above, has also been developed. The solid phase particles are placed in a column where

19
liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensure that
the solid phase does not exit the column with the liquid phase.

Specific uses

Affinity chromatography can be used in a number of applications, including nucleic acid purification,
protein purification from cell free extracts and antibody purification from blood serum.

Antibody affinity also known as Immunoaffinity Chromatography

Another use for the procedure is the affinity purification of antibodies from blood serum. If serum is
known to contain antibodies against a specific antigen (for example if the serum comes from an
organism immunized against the antigen concerned) then it can be used for the affinity purification of
that antigen. For example if an organism is immunized against a GST-fusion protein it will produce
antibodies against the fusion-protein, and possibly antibodies against the GST tag as well. The protein
can then be covalently coupled to a solid support such as agarose.

For thoroughness the GST protein and the GST-fusion protein can each be coupled separately. The
serum is initially allowed to bind to the GST affinity matrix. This will remove antibodies against the
GST part of the fusion protein. The serum is then separated from the solid support and allowed to bind
to the GST-fusion protein matrix. This allows any antibodies that recognize the antigen to be captured
on the solid support. Elution of the antibodies of interest is most often achieved using a low pH buffer
such as glycine pH 2.8. The eluate is collected into a neutral tris or phosphate buffer, to neutralize the
low pH elution buffer and halt any degradation of the antibody's activity. This is a nice example as
affinity purification is used to purify the initial GST-fusion protein, to remove the undesirable anti-GST
antibodies from the serum and to purify the target antibody.

Immobilized metal ion affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent
binding of amino acids to metals, particularly histidine. This technique works by allowing proteins with
an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt,
nickel, copper, zinc, or iron ions. Many naturally occurring proteins do not have an affinity for metal
ions, therefore recombinant DNA techniques can be used to introduce this property into a protein of

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interest. Methods used to elute the protein of interest include changing the pH, or adding a competitive
molecule, such as imidazole.

Recombinant proteins

Possibly the most common use of affinity chromatography is for the purification of recombinant
proteins. Proteins with a known affinity are tagged in order to aid their purification. The protein may
have been genetically modified so as to allow it to be selected for affinity binding, this is known as a
fusion protein. Tags include His-tags and GST (glutathione-S-transferase) tags. His6-tags have an
affinity for nickel or cobalt ions which are coordinated with a chelator for the purposes of solid medium
entrapment. For elution, an excess amount of a compound able to act as a metal ion ligand, such as
imidazole, is used. GST has an affinity for glutathione - commercially available immobilized as
glutathione agarose.

Supercritical fluid chromatography

Supercritical fluid chromatography (SFC) is a normal phase chromatographic technique in which mobile
phase is a supercritical fluid.
Supercritical fluid is a state of matter that is intermediate between a gas and a liquid in its properties.
This state is reached when a gas or liquid solvent is subjected to temperature and pressure condition
exceeding a particular critical point. Beyond this point the compound will neither be a gas nor be a
liquid, but will possess properties of both gas and liquid. Whether the supercritical fluid would behave
more as a gas or as a liquid, will depend on the pressure and temperature.
SFC is important for separation and determination of a group of compounds that are not conveniently
handled by GC or HPLC because either of non-volatility or thermal lability or of having no functional
groups that could be detected by spectroscopic or electrochemical techniques of HPLC.
It was first proposed by J Lovelock in 1958. Thermally labile porphyrins were separated by Klespar et
al in 1962.

Advantages of SFC
(a) Supercritical fluid has density and viscosity lower than that of liquid. This results in large
diffusion co-efficients for solutes and also higher optimum velocity in SFC than in LC.
Efficiency is more and plate height (H= A+B/u+Cu) is less.

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 (b) SFs have higher density than gas. Mobile phase has higher chance of interaction than gas in GC.
(c) Detectors for both GC and LC can be used for SFC. So the process is flexible.

Drawbacks
High pressure operating conditions.
Difficult to maintain supercritical conditions.
Difficult to separate and collect prorducts

Applications
Separation of natural products, drugs, explosives and propellants.

Instrument
Both temperature and pressure should be controlled to maintain the fluid in supercritical conditions.
Generally CO2 is used as supercritical fluid. Pump head and input CO2 must be cold. Since CO2 is too
non-polar, co-solvents are methanol, ethanol or isopropanol.

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Countercurrent Chromatography (CCC)

Countercurrent chromatography is a form of liquid chromatography that uses a liquid stationary phase
that is immobilized only by a centrifugal force without a solid support. The mobile phase is also a liquid
(immiscible with the stationary liquid phase) that is pumped to the c column in a countercurrent mode.
There are two modes by which the stationary phase is retained by centrifugal force – hydrostatic and
hydrodynamic.

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In hydrostatic method the column is rotated about a central axis. Hydrostatic instruments are sometimes
called centrifugal partition chromatography. High performance or high speed CCC can be achieved by
using a helical coil based on Archimedes screw force to retain the stationary phase in the column.
Hydrodynamic CCC uses ‘planetary centrifuge’ where a closed helical tube was rotated on a planetary
axis as it turned on a sun-axis.

Applications
Separation of natural products, DNA, active enzyme, vitamins.
Advantages
All components in the sample solution can be separated.
Irreversible adsorption and contamination of samples can be avoided
Crude sample can directly be injected – no sample preparation is required.
Versatile process
Simple mechanism
High throughput
Preservation of biological characteristics

Gel permeation (filtration) chromatography or Size exclusion chromatography (SEC)

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SEC is the separation of mixtures based on their hydrodynamic volume (loosely said molecular size) of
components.
A column of gel particles or porous matrix is in equilibrium with a mobile phase suitable for the
separation of molecules. Gel filtration resin can thus be thought of as beads containing pores of a
definite size range. Large molecules are completely excluded from the pores and will pass through the
space in between the gel beads and will come first in the effluent because the volume of the column
appears smaller to the larger molecules. Smaller particles which can enter the pores of the beads

Chromatographic methods for separation of lipids (long chain mono- and poly-unsaturated fatty
acids)

Long chain fatty acids are organic compounds in which the hydrocarbon chain length may vary from 10-
30 carbon. The hydrocarbon chain can be saturated or unsaturated (one or more double bonds). Based
on the number of double bonds, unsaturated fatty acids are classified as:
 Mono-unsaturated fatty acids (MUFA)
 Poly-unsaturated fatty acids (PUFA)
 Eicosanoids (derived from PUFA)
MUFA and PUFA can be separated and identified by any of the following techniques:

TLC (thin layer chromatography) for lipids

Many types of stationary phases classified as normal and reverse phases are used for TLC of MUFA and
PUFA. The most popular normal stationary phase layers are silica gel, alumina, cellulose, starch,
polyamides and kiesel guhr. Of the mentioned stationary phases, the best one is silica gel which can be
additionally modified by impregnation with different agents like Ag, Cu, Co, Zn.
Small amount of acetone, diethyl ether, ethanol can be added to the mobile phase (generally hexane) in a
volume ratio of 9:1 for better separation.
Advantage of this process is simplicity and the TLC plate can be home made. Sometimes it is combined
with mass spectroscopy.

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High performance liquid chromatography (HPLC) for lipids
HPLC is often used for separation of lipids. Following aspects are worth noting:
 Reverse phase is more efficient and universal.
 Influence of geometric isomerization ie cis-vs-trans is very important. It is reported that cis-
isomers are eluted before the trans-ones.
 Those with short C-chains are eluted first in comparison with long C-chains
 FID or ED enhances the applicability
 For accurate detection upto very small amount derivatization is done. Use of UV-vis detector is
facilitated by derivatization by phenacyl bromide, p-bromo-phenacyl bromide etc.
 Fluorescence detector has higher sensitivity.
 Mobile phase may be 25:35:75:0.4 volume ratio of tetrahydrofuran: acetonitrile: water: acetic
acid.
 Photodiode array detector may also be used in 190-240 nm range.
 Column is generally Supelcosil C18

Gas Chromatography (GC) for lipids

Following are the important aspects of using GC for separation of lipids
 Derivatization to suitable volatile derivatives facilitate separation. Alkyl derivatives are
preferred. H+ catalyzed methylation or OH- catalyzed esterification are often done.
 Polar columns are the most suitable
 Most popular detector is FID
 Carrier gas may be N2, H2, He
 Sometimes combined with MS.

Supercritical fluid chromatography (SFC) for lipids

 Following are the important aspects
High flow rate and low temperature process
 Derivatization is not required
 Nowadays can be operated at subcritical conditions
 CO2 is the mobile phase and that can be modified by organic solvents

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  Open tubular, packed capillary columns may be used.

Combination of two chromatographic techniques-2D chromatography

Multi-dimensional chromatography is nowadays becoming popular. It combines high resolution, large
peak capacity and flexibility for separation of difficult mixtures that could not be separated by
conventional single dimensional chromatographic techniques.
In 2D chromatography, two different chromatographic columns are connected in sequence, and the
effluent from the first system is transferred onto the second column. Application of 2D LC (liquid
chromatograph) is suggested when very complex mixtures have to be separated. In a typical HPLC
experiment, the average peak width is 30 s while the chromatogram is about 1 h long, so at most 120
compounds can be separated. This peak capacity can be substantially improved when the effluent of the
first column is collected in fractions and is further analyzed by a separate chromatographic run, usually
based on a different separation mechanism. This can be implemented in both offline and online modes.
A typical online experiment for 2D HPLC is used for proteomics applications where a complex mixture
of digested proteins has to be analyzed (often thousands of peptides are present in the sample). The
digested sample is first injected onto a cation-exchange column, as the commonly used trypsin yields
basic peptides. First, the neutral peptides elute from the column, and these are washed onto the next,
very short octadecyl silica column. This column binds (and therefore concentrates) the first fraction of
peptides. After changing the solvent composition (switching to a different solvent mixture) the peptide
fraction is washed onto a longer, analytical octadecyl silica column, where the peptides are separated on
the basis of their polarity (a typical RP-HPLC application). In the next step the cation-exchange column
is washed with an eluent containing low salt concentration, which elutes the weakly retained peptides.
These are trapped, washed, and analyzed on the octadecyl silica column similarly to the first fraction. In
repeated steps the cation-exchange column is washed with eluents of higher and higher salt content and
thus peptides with higher and higher basicity are eluted from the column. These fractions are trapped
and analyzed on the C18 column as described earlier. In summary, the peptides are fractioned according
to their basicity on the first column (first dimension) and the obtained fractions are further separated on
the basis of their apolar character on the second column (second dimension). This protocol reduces
coelution and thus enhances the confidence of identification for unknown proteins.

Advantages

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The theory relating to the relative peak capacities of 1D and 2D is well-documented and provides a basis
for the advantages in separation of the 2D technique. Peak capacity determines the number of peaks of a
finite width that can be separated within a specified time window.
On coupling 2 columns together, in sequence, there is a considerable increase in the theoretical and
experimental peak capacity because of the enhanced separation achieved in the second column, with
peaks from the first column being separated with a greater efficiency within a particular time window,
which eliminate or minimize peak overlap.
The peak capacity-n, for 2DGC or LC is the product of the peak capacities for column 1 and 2, n11D and
n21D respectively, and not the sum. In an ideal state the increase is an order of magnitude greater.
Thus, n2D = n11D × n21D (1)
Another consideration for the application of the 2D mode is the analysis time. For 2DGC the total
analysis time is simply the sum of the time to elute the components of interest from the first column plus
the time for elution and detection of all of the selected components from the second column. If heart
cutting is not to be applied, the total analysis time would be the sum of the elution times for all analytes
from the first and second columns to the point of final detection.

Instrumental augmentation
The coupling of two capillary columns in sequence originally was designed for heart-cut analysis of a
selected analyte group eluted from the first column using a splitter to divert from or allow analyte
transfer to the second column. Over the past several decades with the added capability of modulators
and when coupled with mass spectrometry, the applications of the techniques of 2D chromatography
have grown, immensely.
The introduction of a modulator for the transfer of analytes from column 1 to column 2 enables a greater
separation and efficiency. Basically the introduction of a modulator has several requirements involving
electronic control of the interface, transfer of analytes, and computer software for generation of the 1D
and 2D chromatograms and, frequently 3D profiles by signal processing. A number of designs of
modulator (Cryogenic modulator; Flow modulator; Heart-cutting; In-line valve system; Longitudinally-
modulated cryogenic system; Out-line valve system; Pneumatic modulator; Reinjection. The
introduction of a modulator for the transfer of analytes from column 1 to column 2 enables a greater
separation and efficiency. Basically the introduction of a modulator has several requirements involving
electronic control of the interface, transfer of analytes, and computer software for generation of the 1D

28
and 2D chromatograms and, frequently 3D profiles by signal processing (see section 4.3). A number of
designs of modulator (Cryogenic modulator; Flow modulator; Heart-cutting; In-line valve system;
Longitudinally-modulated cryogenic system; Out-line valve system; Pneumatic modulator; Reinjection )
Thermal modulator (for a further description refer to have been employed for a range of applications.
The modulator is a piece of hardware that transfers effluent from the exit of the primary column to the
head of the secondary column as a repetitive series of pulses. The commercially available units have
primarily employed a thermal modulation strategy that required the consumption of large amounts of
liquid cryogen coolant and gaseous working fluid. Thermal modulation produces optimal resolution but
also sacrifices some of the simplicity of conventional GC. Valve-based modulation has received less
attention than thermal modulation. However, valve based modulators use straightforward, low-cost
designs and do not require additional consumables. Two main classes of valve-based modulators have
been developed: sub- sampling and differential flow. Sub-sampling modulators generate pulses by
briefly diverting small amounts of primary column effluent to the head of the secondary column. While
easy to put into practice, sub-sampling modulators lead to decreased sensitivity and must employ short
modulation periods. Differential flow modulators sample all of the primary column effluent, which
means that sensitivity is not sacrificed and larger modulation periods can be used. However, these
devices also use high secondary flows that lead to elevated column pressures and limit the use of direct
mass spectrometric detection.

Modulator incorporates a proprietary deactivated flow channel and integrated gas supply connections

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providing unprecedented robustness that is easy to use and maintenance free. CFT (Capillary flow
technology) Modulator is not limited by sample volatility providing efficient collection and injection of
solutes across the volatility range.
The CFT has advantages as follows:
1. No cryogenic-cooling needed to re-focus, providing significant savings to the lab
2. Collects the material from the first column, dividing a peak into multiple cuts
3. Focuses the material collected from each cut into a narrow band
4. Introduces the bands sequentially into the second column

Separation of antibiotics by TLC

Gabriel Hancu et al (Advanced Pharmaceutical Bulletin, 2013, 3(2), 367-371) separated beta-lactams
present in penicilins and cephalosporins by TLC.

Compounds

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Stationary phase

10x20 and 20x20 cm pre-coated silicagel GF254 HPTLC glass plates (Merck, Germany).

Mobile phase

All beta-lactams can be detected in UV light at 254 nm (green fluorescence) and 366 nm (blue
fluorescence). Applying reagents such as ninhydrin or exposing the chromatoplate to iodine vapor can
diminish the detection limit.

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