Chromatography

CHROMATOGRAPHY
Chromatography
Principle; Retardation Factor; Retention times; Resolution;
Techniques – Thin Layer; Liquid chromatography- Normal
Phase (Adsorption); Reverse phase (Partition); Gel
Permeation (Size Exclusion); Ion Exchange; Affinity; High
Performance Liquid Chromatography (HPLC);
Instrumentation – HPLC; Applications (Molecular weight
determination of a protein using Gel Permeation; Separation
of proteins using Ion Exchange; Purification of mRNA from
Total RNA and Isolation of DNA binding proteins using
Affinity).
Chromatography
Chromatography is a physical method of separation in

which the components to be separated are
distributed between two phases one of which is
stationary (stationary phase) while the other (the
mobile phase) moves through it in a definite direction.
The chromatographic process occurs due to

differences in the distribution constant of the
individual sample components.
Terminology
Mobile phase is the extracting phase that moves in a definite direction.
It may be a liquid (LC), a gas (GC).
Ex: In the case of HPLC the mobile phase consists of a non-polar
solvent(s) such as hexane in normal phase or polar solvents in reverse
phase chromatography and the sample being separated. The mobile phase
moves through the chromatography column (the stationary phase) where
the sample interacts with the stationary phase and is separated.
Stationary phase is the substance fixed in place for the chromatography

procedure (or) the extracting phase that remains in a fixed position.
Ex: the silica (SiO2) layer or alumina (Al2O3) in thin layer
chromatography.
Terminology
Elution is the process of extracting one material from another by
washing with a solvent.
Eluent is the solvent that carries the analyte (or) the eluent or eluant is
the "carrier" portion of the mobile phase. It moves the analytes through
the chromatograph. In liquid chromatography, the eluent is the liquid
solvent; in gas chromatography, it is the carrier gas.
Eluate is the mobile phase leaving the column (or) the eluate is
the analyte material that emerges from the chromatograph. It
specifically includes both the analytes and solvent passing through the
column, while the eluent is only the carrier.
Terminology
Retention time is the characteristic time it takes for a particular analyte

to pass through the system (from the column inlet to the detector) under
set conditions (tr).
Retention volume is the volume of mobile phase needed to move a solute
from its point of injection to the detector (Vr).
Chromatogram is a plot of the detector’s signal as function of elution

time or volume.
Chromatography
Classification of chromatography according to

mobile phase:
1- Liquid chromatography: mobile phase is a
liquid.
2- Gas chromatography : mobile phase is a gas.
Chromatography
Classification according to the force (mechanism)

of separation:
1- Adsorption chromatography
2- Partition chromatography
3- Ion exchange chromatography
4- Gel permeation chromatography (GPC) or Size-
exclusion chromatography
5- Affinity chromatography
Chromatography
Classification according to the packing of the
stationary phase:
1- Paper chromatography (PC): the stationary

phase is a thin film of liquid supported on an
inert support.
2-Thin layer chromatography (TLC): the

stationary phase is a thin solid layer
supported on glass, plastic or aluminium
plates.
3-Column chromatography (CC): stationary

phase is packed in a glass column.
Techniques of Chromatography
There are two basic techniques of chromatography:
plane chromatography and
column chromatography
In plane chromatography the stationary phase is coated onto 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.
Chromatography
As opposed to plane chromatography, the stationary phase in column

chromatography is packed into a glass or plastic column. Each of these
techniques have their specific advantages, applications, and modes of
operation.
Paper Chromatography
• Separation of analytes in a mixture
It is carried out mainly by the flow of solvents on specially
designed filter paper.
There are two types of paper chromatography, they are:
1. PAPER ADSORPTION CHROMATOGRAPHY
Paper impregnated with silica or alumina acts as
adsorbent (stationary phase) and solvent as mobile
phase.
2. PAPER PARTITION CHROMATOGRAPHY

Moisture / Water present in the pores of cellulose
fibers present in filter paper acts as stationary phase &
another mobile phase is used as solvent
In general P.C – Paper Partition Chromatography

A method of plane chromatography using filter paper strips
as carrier or inert support.
The factor governing separation of mixtures of solutes on

filter paper is the partition between two immiscible
phases.
One is usually water adsorbed on cellulose fibres in the

paper (stationary phase).
The second is the organic solvent flows past the sample on

the paper (mobile phase).
Cellulose fibers in the paper hold moisture tightly through

formation of hydrogen bonds.
Partition occurs between the mobile phase
and the stationary aqueous phase bound by
the cellulose.
The separation depends on partition

coefficient of the solute.
c( stationary )
K
c(mobile)
A partition-coefficient or distribution-coefficient is the ratio
of concentrations of a compound in a mixture of
two immiscible phases at equilibrium. This ratio is therefore a
measure of the difference in solubility of the compound in these two
phases.
Nature of the Paper
 The paper commonly used consists of highly purified cellulose.

Cellulose, a polysaccharide of glucose, contains several thousand anhydro-
glucose units linked through oxygen atoms.
 Cellulose fibres in the paper hold moisture tightly through formation
of hydrogen bonds.
Cellulose
Types or Modes of Paper Chromatography
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 is good enough for simple chromatography.
The sample is applied to the paper as a small spot. This is done before
dipping the paper into the eluting solvent. A capillary tube is used to
transfer a small volume of sample for spotting.
There are two main techniques. which may be employed for the
development of paper chromatograms - ascending or descending
techniques.
In both cases the solvent is placed in the base of a sealed tank or glass jar
to allow the chamber to become saturated with the solvent vapour. After
equilibration of the chamber is achieved, the development of the
chromatogram may be started.
Ascending development
Descending development
If the development is to be performed by the ascending technique, the

paper is allowed to suspended so 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 above the surface of the solvent so that as the solvent
moves vertically up the paper by capillary action, separation of the
sample is achieved.
In the descending technique, the end of the paper near which the
samples are located is held in a trough at the top of the 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.
Firstly, the set up required for it is very simple.
Secondly, the resolution of sample by ascending technique is somewhat
better as compared to the descending technique. This is so because in
ascending chromatography, two forces are acting on the solute: the
capillary force, which makes it move up, and the gravitational 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 ascending technique is that it is very slow. The
descending technique, on the other hand is much faster than the
ascending technique. Based on the above advantages, one can choose
the technique which suits one's purpose most.
Radial Mode
A third, less used technique is the technique of radial development.
In this method the sample is spotted at the center of a circularly cut
disc of paper which is placed horizontally.
The center of the paper is connected with a wick to the solvent, which is
placed at the base of a jar.
The solvent rises up the wick and thence onto the paper through
capillary action.
The sample components now move outward radially forming concentric
circles of increasing diameters.
If the components to be separated are colored, the Chromatogram
developed by this method looks pleasing to the eye.
The resolution of components by this technique is sharper.
Two-dimensional chromatography
When large numbers of substances are to be separated on
a single chromatogram.
Development in a direction perpendicular to the first, and

with a solvent system different from that used initially
is often necessary.
The sample is applied on one corner of a square piece of

paper and after development with the first solvent, the
paper is dried, rotated 90o and developed in the second
direction.
Usually, different types of solvents systems are used in

each direction. It is essential that the first solvent be
completely volatile.
Two-dimensional chromatography
Choice of Solvent System
Usually in paper chromatography, the stationary phase is

water since it is very well adsorbed by cellulose. The mobile
phase, which is less polar flows over the polar stationary
phase.
The mobile phase is usually a mixture of various solvents

such as alcohols, acids, esters, ketones, phenols, amines, and
hydrocarbons etc.
Choice of Solvent System
Ideally, the solvent system should be chosen that the two
phases are immiscible. Moreover, the sample components
should have differing solubilities in the two phases. Such a
choice would lead to maximum separation.
Desirable characteristics that a solvent should possess are
(i) Partition coefficient of substances to be analyzed should

range from 1-100 in favour of the aqueous phase.
(ii) The solvent should be removable from the paper and to

this effect its boiling point should ideally be less than 200
°C.
(iii) The solvent should be stable. It should not become

oxidized when spread over the paper.
Detection
There are various methods of detection available. If the
sample components are colored, the analysis becomes simple
as the distinctive color itself identifies the component.
When the components are colorless, they can be imparted

color by spraying the paper with color producing reagents.
For example, in the detection of amino acids, Ninhydrin
reagent spread on the paper reacts with amines and amino
acids to form a blue or purple color.
Other methods of detection are (i) ultraviolet absorption, (ii)
fluorescence, and (iii) radioactivity.
Otherwise, the components may be extracted and chemical

and physical tests be performed on the extract.
Detection
Deep blue or Purple colored pigment

Detection
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. This ratio, which reflects the distribution coefficient of
the given solute, is known as the retardation factor (also known as
relative flow), Rf, and is constant for a given compound under standard
conditions.
Thin layer chromatography (TLC)
TLC is a method for identifying substances and

testing the purity of compounds.
TLC is a useful technique because it is relatively

quick and requires small quantities of material.
Separations in TLC involve distributing a mixture of two or more
substances between a stationary phase and a mobile phase.
The stationary phase:

is a thin layer of adsorbent (usually silica gel or alumina) coated on a
plate.
The mobile phase:
is a developing liquid which travels up the stationary phase, carrying the
samples with it.
Components of the samples will separate on the stationary
phase according to how much they adsorb on the stationary phase
versus how much they dissolve in the mobile phase.
TLC
In principle, the components will differ in solubility and in the

strength of their adsorption to the adsorbent and some components
will be carried farther up the plate than others.
Preparing the Chamber
To a jar with a tight-fitting lid add enough of the

appropriate developing liquid so that it is 0.5 to 1 cm deep
in the bottom of the jar.
Close the jar tightly, and let it stand for about 30 minutes so
that the atmosphere in the jar becomes saturated with
solvent.
Preparing the Plates for Development
With a pencil, etch two small notches into the adsorbent

about 2 cm from the bottom of the plate.
The notches should be on the edges of the plate, and each

notch should be the same distance up from the bottom
of the plate.
The notches must be farther from the bottom of the

plate than the depth of the solvent in the jar.
Using a drawn-out capillary tube, spot the samples on the

plate so that they line up with the notches you etched.
Developing the Plates
After preparing the development chamber and

spotting the samples, the plates are ready for
development.
Be careful to handle the plates only by their edges,

and try to leave the development chamber
uncovered for as little time as possible.
When the plates are removed from the chamber,

quickly trace the solvent front (the highest
solvent level on the plate) with a pencil.
Identifying the Spots (visualization)
If the spots can be seen,

outline them with a pencil.
If no spots are obvious, the
most common visualization
technique is to hold the plate
under a UV lamp.
Many organic compounds can be
seen using this technique, and
many commercially made
plates often contain a
substance which aids in the
visualization of compounds.
Visualizing Agents
Alkaloids- Dragendorff’s reagent
Sugars - Aniline phthalate
Amino acids - Ninhydrin
Iodine vapour - used extensively as a universal reagent for

organic compounds
KMnO4 - The most universal stain. Harsh oxidizer. Any

oxidizable compound such as alcohol, ether, ester, alkene,
alkyne, alkyl aromatic, ketone, carboxylic acid, amine, amide
and almost all the rest will show up as brown-yellow spots.
Interpreting the Data
The Rf (retention factor) value for each spot

should be calculated.
It is characteristic for any given compound on

the same stationary phase using the same
mobile phase for development of the plates.
Hence, known Rf values can be compared to

those of unknown substances to aid in their
identifications.
What does the Rf value tell you about

the solubility of a separated component
in the mobile Phase component?
Larger Rf value → more soluble
Smaller Rf value → less soluble

(Note: Rf values often depend on the temperature and the

solvent used in the TLC experiment.
the most effective way to identify a compound is to spot

known substances – authentic - next to unknown substances
on the same plate.)
In addition, the purity of a sample may be estimated from

the chromatogram.
An impure sample will often develop as two or more spots,

while a pure sample will show only one spot.
Advantages 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
complexity of a mixture.
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,
contaminants and adulterants.
It has also been widely used to resolve plant extracts and
many other biochemical preparations.
Column Chromatography
Stationary phase is
held in a narrow
tube through which
the mobile phase is
forced under
pressure or under
the effect of
gravity.
Open Column Chromatography
(Traditional column chromatography)
Traditional column chromatography is

characterized by addition of mobile phase
under atmospheric pressure and the
stationary phase is packed in a glass
column.
The stationary phase in column chromatography is a solid. The most
common stationary phase for column chromatography is silica gel
or alumina. Also possible are ion exchange chromatography, reversed-
phase chromatography (RP), affinity chromatography.
The stationary phases are usually finely ground powders or gels and/or
are microporous for an increased surface. There is an important ratio
between the stationary phase weight and the dry weight of the analyte
mixture that can be applied onto the column. For silica column
chromatography, this ratio lies within 20:1 to 100:1, depending on how
close to each other the analyte components are being eluted.
Columns
The columns are usually made up of glass or polyacrylate plastic.
Different columns differ in their dimensions. Laboratory columns usually
have a diameter of 2-70 mm and a length of 15-150 cm.
Choice of a column of a particular dimension is dictated by the amount of

sample; larger the sample volume larger the column chosen.
The ratio of the column diameter to its length also varies. Mostly, a ratio
of diameter to length between 1: 10 and 1: 100 can be chosen.
The commonly used glass columns have a sintered glass disc at the
bottom to support the stationary phase. A cheaper alternative is to use a
plug of glass wool with a small amount of quartz sand and glass beads.
Packing the Column
The selection of the method of packing depends mainly on the density
of the solid. Techniques used are the wet, dry & slurry methods. In all
cases avoid inclusion of air bubbles.
The column is fitted in the upright position and its bottom is
sealed with glass wool or such other supports.
The column is now filled to about one third its height with the
mobile phase.
A thick suspension, called slurry, of the stationary phase (gel,
adsorbent or resin) is gently poured into the column with its outlet
closed. The upper part of the column is stirred to ensure even packing
and to avoid air bubbles. The slurry is usually added till 3/4th of the
column is full.
Packing the Column
The outlet is now opened and the column is stabilized by

washing it with mobile phase. A filter paper disc or nylon
gauze or cotton is then placed on the surface of the column
to prevent disturbance during mobile phase/sample addition.
To prevent the column from drying, a layer of solvent is
always maintained above the column surface.
Column chromatography proceeds
by a series of steps
The mobile phase or eluent is either a pure solvent or a mixture of

different solvents. The eluent is chosen so that the different compounds
can be separated effectively. The eluent is optimized in small scale
pretests, often using thin layer chromatography (TLC) with the same
stationary phase.
There is an optimum flow rate for each particular separation. A
faster flow rate of the eluent minimizes the time required to run a
column and thereby minimizes diffusion, resulting in a better separation.
However, the maximum flow rate is limited because a finite time is
required for the analyte to equilibrate between the stationary phase and
mobile phase. A simple laboratory column runs by gravity flow.
The flow rate of such a column can be increased by extending the

fresh eluent filled column above the top of the stationary phase or
decreased by the tap controls. Faster flow rates can be achieved by
using a pump or by using compressed gas (e.g. air, nitrogen, or argon) to
push the solvent through the column (flash column chromatography).
The particle size of the stationary phase is generally finer in

flash column chromatography than in gravity column
chromatography. For example, one of the most widely used
silica gel grades in the former technique is mesh 230 – 400
(40 – 63 µm), while the latter technique typically requires
mesh 70 – 230 (63 – 200 µm) silica gel.
Introduction of Sample
It is necessary that the sample to be applied reaches the
surface of the column below the top layer of the solvent. The
sample is introduced in a concentrated solution and is allowed
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 5-10 cm. The
sample now sinks below the top layer of the solvent to the
surface of the column.
It is necessary to apply the sample in as less volume as

possible. This gives an initial tight band of material when the
separation begins and results in a sharper final separation.
Selection of the mobile phase
The liquid stationary & mobile phases should

have a considerable difference between their
solvent strength parameters.
Pure water > Methanol > Ethanol > Propanol >

Acetone > Ethyl acetate> Ether > Chloroform >
Dichloromethane >Benzene > Toluene > Carbon
tetrachloride > Cyclohexane > Hexane >
Pentane.
Techniques of Elution:
Column Development
Continuous passage of a suitable eluant (mobile phase)

through the packed column separates the components of the
sample. This process is known as column development. There
are two main techniques of elution:
(i) isocratic elution and (ii) gradient elution.
Elution Techniques
Technique Procedure
Isocratic elution Addition of solvent mixture of fixed composition

during the whole process.
Gradient elution Continuous or linear elution: in which there is

continuous change in the composition of the mobile
phase over a period of time (e.g. polarity, pH or ionic
strength).
Step wise or fractional elution: in which the change is

not continuous i.e. a sudden change in the
composition of the mobile phase is followed by a
period where the mobile phase is held constant.
Flow-Rate
Flow rate, Fc is expressed as the volume of mobile phase per unit time.
For a satisfactory resolution it is 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 eluant 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.
Column packing also influences flow rate. Unevenly packed column
leads to distortion of the flow leading to unsatisfactory resolution. More
densely packed columns retard the flow of the mobile phase and
decrease the flow rate. It is therefore very important to pack the
column optimally to obtain a good flow rate.
Analysis and Collecting of Eluent
The eluent, as it emerges from the column outlet is analysed. The

properties of a particular compound, viz., ultraviolet absorption, color or
fluorescence are exploited in its analysis.
To analyse the contents of the eluent from the column, it is
collected in small fractions. Fractions in small volumes may be collected in
different tubes in order to keep the column resolved 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.
Analysis and Collecting of Eluent
The modem approach is to continuously monitor the eluent coming out of

the column. The monitoring equipment is programmed to read the
inherent property of the desired compound such as the ultraviolet
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 absorption at 280nm.
A wide variety of monitoring equipments each exploiting a
different parameter are available. Some of these which may be cited as
examples are (i) fluorescence detectors (ii) polartmeters (iii)
voltameters (iv) refractometric detectors and (v) conductivity detectors.
etc.
Automated systems
Column chromatography is an extremely time consuming stage in any lab
and can quickly become the bottleneck for any process lab. Therefore,
several manufacturers have developed automated flash chromatography
systems (typically referred to as LPLC, low pressure liquid
chromatography, around 350–525 kPa or 50.8–76.1 psi) that minimize
human involvement in the purification process. Automated systems will
also include components normally found on more expensive high
performance liquid chromatography (HPLC) systems such as a gradient
pump, sample injection ports, a UV detector and a fraction collector to
collect the eluent. Typically these automated systems can separate
samples from a few milligrams up to an industrial many kilogram scale and
offer a much cheaper and quicker solution to doing multiple injections on
prep-HPLC systems.
Progress of a column chromatographic
separation showing the separation of two
solute bands.
Typical chromatogram of
Another view of the progress of a column detector response as a
chromatographic separation showing the function of retention time.
separation of two solute bands.
Measurement of the column’s void time, tm, and the retention
time, tr, and baseline width, w, for a solute.
Chromatogram
A chromatographic peak may be characterized in many ways, two of
which are shown in the above Figure.
The retention time, tr, is the elapsed time from the introduction of
the solute to the peak maximum.
The retention time also can be measured indirectly as the volume of

mobile phase eluting between the solute’s introduction and the
appearance of the solute’s peak maximum. This is known as the retention
volume, Vr.
Dividing the retention volume by the mobile phase’s flow rate, U,

gives the retention time, tr.
tr = V r / U
As shown in Figure, baseline width (w) is determined by the

intersection with the baseline of tangent lines drawn through the
inflection points on either side of the chromatographic peak.
Chromatogram
Besides the solute peak, Figure also shows a small peak

eluted soon after the sample is injected into the mobile
phase. This peak results from solutes that move through
the column at the same rate as the mobile phase. Since
these solutes do not interact with the stationary phase,
they are considered nonretained. The time (or) volume of
mobile phase, required to elute non-retained components is
called the column’s void time, tm or void volume, Vm.
Chromatographic Resolution
The goal of chromatography is to separate a sample into a series of
chromatographic peaks, each representing a single component of the sample.
Resolution is a quantitative measure of the degree of separation between two
chromatographic peaks, A and B, and is defined as
As shown in Figure, the degree of separation

between two chromatographic peaks improves
with an increase in R.
From equation, it is clear that resolution may

be improved either by increasing Δtr or by
decreasing wA or wB.
Gel Permeation Chromatography
Gel permeation chromatography is a separation method

dependent upon molecular size. The method is also known as
molecular sieve, gel filtration, or molecular exclusion or size
exclusion chromatography.
The advantages are: (i) gentleness of the technique

permitting separation of labile molecular species, (ii) almost
100% solute recovery, (iii) excellent reproducibility, and (iv)
comparatively short time and relatively inexpensive
equipment needed for its operation.
Principle
A column of gel beads or porous glass granules is allowed to attain
equilibrium with a solvent suitable for the molecules to be separated. If
the mixture of molecules of different size is placed on the top of such
an equilibrated column, the larger molecules pass through the interstitial
spaces between 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 with little resistance.
The small molecules, however, can enter the pores and are thereby
effectively removed from the stream of the eluting solvent. These
molecules are thus retarded.
The degree of retardation of a molecule is proportional to the time it

spends inside the gel pores, which is a function of the molecule's size and
the pore diameter. The molecules with Stokes' radii equal to or
exceeding pore diameter, do not enter the 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.
Diagramatic representation of solvent inside the gel bead (Vi), void

volume (V0) and total volume (Vt).
For a given type of gel, the distribution of a solute particle between
the inner and outer solvent (solvent within and outside the gel bead) is
defined by a distribution coefficient, Kd which is a function of its
molecular size.
The larger molecules which are usually excluded from the gel beads,
will have a Kd value of zero. Certain molecules which is smaller than the
pore size of the gel beads enters the pores in the gel matrix hence
they will have a Kd value of one. For molecules of intermediate size,
they will have Kd value between zero and one. This type of variation in
the Kd Values makes it possible to separate molecules in the narrow
molecular size range.
The volume of outer solvent i.e., the solvent surrounding the gel beads is
indicated as V0 the technical term for this is void volume.
The volume of solvent inside gel i.e., the inner solvent, is known as Vi.
The distribution coefficient as said above is symbolized by Kd.
The elution volume of solute, Ve, is dependent upon these three
variables. Thus,
Ve = V0 + Kd Vi
Kd = (Ve - Vo) / Vi or Kd = (Ve – Vo) / (Vt – Vo)
The volume of inner solvent, Vi can be calculated from the dry weight of
gel employed (a) and the water regain value (Wr) of the particular gel are
known. Thus,
Vi = a Wr
Wr is water regain value i.e., the amount of water it absorbs and swells.
If 2% agarose means 2g in 98g water.
So for 1 g, 49g water. So the Wr in this case is 49.
If 4% agarose means 4g in 96g water.
So for 1g, 24g water. So, the Wr in this case is 24.
If 6% agarose means, 6g in 94g water.
So for 1g, 15.6g water. So, Wr in this case is 15.6.
Therefore, if the water regain value increases, the pore size of the gel
increases, so that the exclusion limit increases.
The exclusion limit of the gel indicates the sizes of the molecules
that are excluded from the pores of the matrix and therefore elute
in the void volume.
The value of Kd is characteristic of a given molecule and does not vary
with the geometry of the gel bed.
However, the numerical value of Ve is dependent upon the size of the
column.
For two substances possessing different molecular weights and
therefore, different distribution coefficients (Kd1 and Kd2 ) the
difference in their elution volumes, Vs is given by
Vs = Ve1 - Ve2 = (V0 + Kd1 Vi) - (V0 + Kd2 Vi)
therefore
Vs = (Kd1 - Kd2)Vi
Thus, the sample volume applied for complete separation of

two substances should not exceed Vs.
The distribution coefficient and the elution volume of the

solute are both related to molecular weight of the molecules
being separated. One can therefore calculate molecular
weight of molecules if one determines the elution volume of
the solute by gel filtration experiment (see applications of
gel permeation chromatography).
Types of Gels
A gel filtration medium (stationary phase) should possess the
following characteristics:
(i) The gel material should be chemically inert.

(ii) Gel material should provide a wide choice of pore and
particle sizes.
(iii) A given gel should have uniform particle and pore sizes.
(iv) The gel matrix should have high mechanical rigidity.
Types of Gels
No such material is available which will fulfil all the above

criteria satisfactorily. However, there are three types of
media that fulfil the criteria to quite some extent. They
include
(i) cross-linked dextrans (trade name Sephadex)

(ii) agarose (Sepharose, Bio-Gel A, Sagavac),
(iii) polyacrylamide (Bio-Gel P),
Types of Gels
Sephadex. For proteins and most of the bio-molecules,

Sephadex is by far the most popular of all the gels. It is
prepared by the microbial fermentation of sucrose, which
gives large polymers of glucose. These polymers, known as
dextrans, are used to prepare Sephadex. The agent used for
cross-linking dextran polymers is epichlorhydrin,
Cross linking Gl-Gl-Gl
Gl-Gl-Gl- O
OH CH2Cl
+ CH2
HOHC
H C OH
Sephadex
Gl-Gl-Gl- CH2Cl
CH2
OH
O
Gl-Gl-Gl n
Types of Gels
Types of Gels
Sephadex is obtained in different degrees depending on the

pore size.
High percentage of epichlorohydrin give high degree of

cross linking (small pore size). Lower percentage produce
sephadex with large pore size.
Characteristics of sephadex
1- highly stable gels

2- stable at pH 2-12
3- their particles are free from ions
4- insoluble in water and organic solvents
5- they swell in water and other hydrophillic solvents
Types of Gels
Agarose. Agarose gels are produced from agar. They are
linear polysaccharides of alternating residues of D-galactose
and 3,6-anhydro-L-galactose units. These gels are hydrophilic
and are almost completely free of charged groups.
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 up to a molecular weight of several million Daltons.
Agarose dissolves in H2O at 50 oC and on cooling forms gel

and insoluble below 40 oC. Freezing destroys the gel. The
agarose gels are compatible with all aqueous buffers and are
completely stable within the pH range of 4-10.
Types of Gels
Polyacrylamide. This very popular medium is produced by

polymerizing acrylamide into bead form.
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 300,000 Daltons. Polyacrylamide is insoluble in water and
common organic solvents and may be used in the pH range of
2-11.
Technique
Gel permeation chromatography can be performed by column
chromatographic technique.
Column preparation: The general principles of column preparation are
the same as described previously for column chromatographic
techniques. Prior to use, the gel must be converted to the swollen form.
This may be done by allowing a known weight of the gel to swell either in
water or in a weak salt solution. Although the initial swelling is fairly
rapid, the time taken to attain equilibrium may be longer. The greater
the porosity, the more will be the time required to reach equilibrium.
Thus, whereas overnight swelling is enough for Sephadex G-25, Sephadex
G-200 should be allowed to swell for three days. The swelling time can be
reduced drastically by heating the gel slurry on a boiling water bath for
1-5 hours.
Technique
The gel bed is supported in the column on a glass wool plug and the
previously swollen gel is added in the form of a slurry and allowed to
settle. Air bubbles must be removed by connecting the column to a
vacuum pump and the level of the liquid must never be allowed to go lower
than the top of the bed. Sample is applied in a manner indicated
previously.
The eluant is steadily added and the effluent collected in various
fractions to be analysed.
Detection. The common detection methods include collecting and

analysing fractions and continuous methods with flow cells in which
ultraviolet absorption, refractive index is measured.
Advantages of Gel Permeation
Gel permeation is the gentlest of the chromatographic techniques. It has
the following advantages not paralleled by any other chromatographic
technique.
1. Gel permeation depends only on the molecular sizes of the
macromolecules. The chromatography therefore can be conducted under
virtually any condition of the temperature, pH, ionic strength, and buffer
composition.
2. Apart from the above named conditions, macromolecules can be
damaged by adsorption too. Other chromatographic techniques do suffer
from some adsorption. Such macromolecules can be separated by gel
chromatography since adsorption here is almost nil.
3. there is less zone spreading in gel chromatography as compared to the
other techniques.
Applications
(i) As is evident, gel permeation chromatography is mainly used for the purpose
of separation of biological molecules leading to their ultimate purification.
Proteins, enzymes, hormones, antibodies, nucleic acids, polysaccharides have
been separated in various experiments which have used different types of
gels or glass granules. It is also the most satisfactory method for separating
DNA (from bacteria, usually Gram positive) from the invariable contaminants,
the teichoic acids.
(ii) 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.
(iii) Perhaps one of the most important applications of gel permeation
chromatography is in the determination of molecular weight of
macromolecules.
Molecular Weight Determination by
Gel Filtration
Gel chromatographic separations are achieved by means of differential

distribution of sample components between the stationary solvent within
the pores of a gel and the mobile eluting solvent outside the pores.
The distribution characteristics of a given macromolecule are a
function of the size and shape of the solute molecule and the size range
of the gel pores available.
Gel Filtration
In a gel filtration experiment, if one determines the elution volume of
the solute for a macromolecule, one can calculate its distribution
coefficient. If distribution coefficients of standard proteins of known
molecular weight are plotted against the log of their molecular
weights, the position of the distribution coefficient of the unknown
protein on the plot will lead to the determination of its molecular
weight.
Since guanidinium chloride makes the proteins behave like
randomly coiled linear homopolymers, it might be the ideal solvent for
molecular weight determination.
Gel Filtration
The solvent used is 6M guanidinium chloride in water, pH 6.
The sample is prepared by dissolving it at a concentration of about 1 % in 6M

guanidinium chloride and O.1M 2-mercaptoethanol (mercaptoethanol is used to
disrupt disulphide bonds between polypeptides).
The pH is adjusted to 8.6.
The sample is then incubated for about 8-10 hours.
Subsequently, the sample is carboxymethylated by addition of iodoacetic acid and

the pH re-adjusted to 8.6.
Chromatography is carried out and the elution volume of the desired

macromolecule is determined. This is used to calculate the distribution
coefficient of that molecule.
Once distribution coefficient of a number of polypeptide chains of known

molecular weights have been obtained, column calibration curves can be
constructed.
Gel Filtration
The most popular type of calibration curve is a plot of log molecular weight vs.
distribution coefficient. The position of distribution coefficient of unknown
polypeptide on such a plot then gives its molecular weight.
Alternatively, log molecular weight is plotted against elution volume.

Ion-exchange Chromatography
Ion-exchange chromatography (or ion

chromatography) is a process that allows the
separation of ions and polar molecules based on the
charge properties of the molecules.
Ion exchange may be defined as the reversible
exchange of ions in solution with ions
electrostatically bound to inert support medium.
The governing factor in ion exchange reactions is the electrostatic force
of attraction, which in turn depends mainly on the relative charge, the
radius of the hydrated ions and the degree of non-bonding interactions.
Ion exchange separations are carried out usually in columns 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 covalently bound to positive (anion exchanger) or negative (cation
exchanger) functional groups.
Ions bound electrostatically to the exchanger are referred to as the

counterions. This technique is extremely useful in the separation of
charged compounds.
Ion exchangers – Functional groups
Cation exchange chromatography
• Cation exchange chromatography

retains positively charged cations
because the stationary phase displays a
negatively charged functional group.
+ - _
- + + + -
R-X C +M B R-X M + C + B
Anion exchange chromatography
• Anion exchange chromatography retains

anions using positively charged
functional group:
+ _ + - + - + -
R-X A +M B R-X B + M + A
The basic process of ion exchange is illustrated in Figure. The exchanger is
prepared in a way that it is fully charged (Figure A). The sample containing 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 B):
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 similar to the counterion) in the

sample to be separated. This molecule can now exchange sites with the counterion
as shown in the above relationship.
The neutral and anionic molecules will not bind at all.
Once the exchange of counterion with the sample ion has been achieved, the rest
of the uncharged and like charged species can be washed out of the column.
Bound ions, X+ can now be eluted either by percolating the medium with increasing
concentrations of Y+ (this 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 species
(Figure C, D). Concentration of Y+ required to elute X+ will depend upon 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.
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 molecules, then, can bind to both, anion
and cation exchangers since they possess both types of charges.
The macromolecules can, however, be made to bear more negative

charges by increasing the pH (resulting in a stronger binding to anion
exchanger) or more positive charges by reducing the pH (resulting in a
stronger binding to cation exchanger).
The amphoteric nature of macromolecules, particularly proteins, can be

exploited for purification purposes by using ion exchange
chromatography.
If the pI of the amino acid is below the pH

of the buffer, then the amino acid will get
negative charge.
If the pI of the amino acid is above the pH

of the buffer, then the amino acid will get
positive charge.
Types of Ion Exchange Resins
Two main groups of materials (supports) are used to prepare ion
exchange resins: polystyrene, and cellulose.
Resins made from both of these materials differ in their flow properties,
ion accessibility, chemical and mechanical stability.
Polystyrene resins are prepared by polymerization reaction of styrene

and divinyl benzene. Acidic functional groups are easily introduced by
sulphonation in which a sulphonic acid group is attached to nearly every
aromatic nucleus. Sulphonic acids are strong acids with completely
dissociated protons. These protons, however, are not free to leave the
resin unless replaced by other positive ions. The total number of
equivalents of replaceable protons per unit volume of resin determines
the exchange capacity 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.
Cation exchange resin Anion exchange resin

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 quaternary ammonium group. Weak anionic exchanger can
be prepared with secondary amines, yielding a weakly basic tertiary
amine.
Polystyrene resins are very useful for separating small molecular weight
compounds.
The unsatisfactory separation of macromolecules on polystyrene type of

resins prompted development of the cellulose-based 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, softwood, and hardwood. Carboxymethylcellulose (CM-cellulose,
weakly acidic, cationic exchanger) where the CH20H group is converted
to -CH20CH2COOH, and DEAE cellulose CH20CH2CH2N(CH2CH3)2 (weakly
basic, anionic exchanger) are examples of main derivatives of practical
value.
Classification of ion exchange resins
• Strongly acidic cation exchanger ---sulphonic acid
groups attached to styrene and di vinyl benzene
copolymer.
• Weakly acidic cation exchanger---carboxylic acid

groups attached to acrylic and divinyl benzene co-
polymer
• Strongly basic anion exchanger-----quaternary

ammonium groups attached to styrene and divinyl
benzene co-polymer
• Weakly basic anion exchanger-----poly alkyl amine

groups attached to styrene and divinyl benzene co-
polymer
Preparation of the Exchange 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.
(i) Swelling the medium. This is also known as precycling. Swelling makes these
functional charged groups to become exposed. Cross-section of such a
swollen cation exchange bead is shown in Figure.
Swelling of anion exchangers is usually carried out by treating it first with an

acid (O.5N HCl) and then with a base (O.5N NaOH). Exactly the reverse is
the case with cationic exchangers.
Preparation of the Exchange Medium
If any, impurities like metal ions are present, the matrix can be treated
with a chelator such as EDTA.
(ii) Removal of very small particles of the exchanger (fines). To remove

fines, the exchanger is repeatedly suspended in a large volume of water
and after the larger polymers have settled down, the slow sedimenting
material (fines) is decanted.
(iii) Finally the exchanger has to be equilibrated with the suitable

counterions. This is accomplished by washing the exchanger with
different reagents depending upon the desired counterion to be
introduced (NaOH in case the counterion to be introduced is Na+; HCl if
H+ is the counterion, NaNO3 if NO3- is the counter ion; formic acid if
formate is the counterion, etc.).
Following conversion of the exchanger to the desired form, excess

counterions are removed by washing 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
compounds to be separated and the type of ion exchange being carried out
(anionic or cationic).
Anion 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 with anionic or cationic buffers respectively,
the buffer ions will indulge in ion exchange and hamper sample component
exchange.
The pKa of the buffer should be as near as possible to the pH at which
the system is buffered. The pH of the buffer should impart the same charge to
the sample ions as is present on the counterion.
Practical Procedure
Many biological components especially proteins, are stable only within a
fairly narrow pH range. Therefore, the exchanger selected should
operate within this range.
If the sample is more stable at a pH below its isoionic point, use of a

cation exchanger is advised. 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 of pH 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.
Practical Procedure
The columns used also have an effect on the resolution obtained. Columns
of a high diameter to height ratio usually give a better resolution.
The pH of the buffer used is usually maintained at about one pH unit

more or less 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 cation exchange chromatography is to be done, anionic buffers

such as acetate, barbiturate, 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.
Gradient elution is more commonly performed than isocratic elution.

Applications
(i) Perhaps the most stimulating use of ion exchange chromatography is in amino
acid analysis. In fact the amino acid "autoanalyser" is based on ion exchange
principle.
(ii) Ion exchange has been extensively used to determine the base composition of
nucleic acids. The mixture of nucleotides as a result of treatment with DNAses
and RNAses can be readily separated by ion exchange chromatography.
(iii) For many biological applications, ultrapure, metal ion free reagents are
needed. This is commercially performed by ion exchange chromatography.
(v) To find out the concentration of trace metals in biological samples which are
below the limit detected by atomic emission or atomic absorption spectrometry.
(vi) Apart from the above applications, ion exchange chromatography has been
used for the separations of many vitamins, other biological amines, and organic
acids and bases.
Amino Acid Analyser
To determine the amino acid composition of a protein, its hydrolysate is analysed
for different amino acids and the quantity of each of the amino acids present in
the protein.
The separation of amino acids from a protein hydrolysate is achieved using a

strong cation exchanger.
The protein hydrolysate is allowed to enter the ion exchange column at a pH of 1

to 3 and allowed to percolate slowly.
This low pH assures complete binding since all the amino acids will be positively
charged at this pH. Amino acids with largest positive charge (lysine, arginine and
histidine) at this pH will be bound to the resin most tenaciously.
The amino acids with least amount of positive charge (glutamic and aspartic acids)
will be bound less tightly.
Other amino acids (so called neutral amino acids such as glycine and alanine) which
possess positive charges intermediate between the two groups discussed above
will bind in an intermediate manner.
Amino Acid Analyser
When gradient elution is carried out by increasing the pH and ionic
strength of the eluting solvent, the acidic amino acids (glutamic and
aspartic acids) will be eluted first.
Next to elute out of the column will be the neutral amino acids
(glycine, alanine, valine, etc.).
The last to elute out will be the basic amino acids (lycine,
arginine, histidine) which will need a pH in excess of 10.
The above principles are made use of to make the entire process
automatic, so that elution, fraction collection, analysis of each fraction,
and recording of data are performed automatically in - a highly
integrated manner. Such an instrument is known as amino acid analyser.
Amino Acid Analyser
Most amino acid analysers use two columns, the second column being anion
exchange column which will separate the basic amino acids and ammonia
faster and more effectively.
The eluent emerging from the column(s) is mixed with ninhydrin color
reagent. The mixture is then heated to 105 °C to develop the color. The
mixture is then cooled.
The color intensity of the mixture is then determined by a colorimeter

set at 570 nm. There is second colorimeter which is set at 440 nm and
which reads the intensity of the color (yellow) produced by proline and
hydroxyproline.
Both the colorimeters are connected to the recorder which will record
the color intensity of each amino acid as it sequentially elutes out of the
column.
Each amino acid will produce a peak on the recorder and the area under
each peak will be proportional to the amount of each amino acid in the
mixture.
Affinity Chromatography
As opposed to all the chromatographic procedures described thus far
which exploited small physicochemical differences of molecules in a
mixture for their eventual separation, affinity chromatography exploits
the capacity of biomolecules for specific, non-covalent binding of other
molecules called ligands.
Biochemists are familiar with the concept that a given enzyme will bind
and react with only a group of substrates and will not react with
others. This amazing biospecificity is not limited only to enzymes;
a given hormone will bind to only a specific glycoprotein, known as
receptor, situated on the plasma membrane surface;
a given antibody will specifically bind to only a given antigen and not to
others.
Introduction
• Affinity Chromatography is a method of separating a
mixture of proteins or nucleic acids (molecules) by specific
interactions of those molecules with a component known as a
ligand, which is immobilized on a support. If a solution of,
say, a mixture of proteins is passed over (through) the
column, one of the proteins binds to the ligand on the basis
of specificity and high affinity (they fit together like a lock
and key).
• The other proteins in the solution wash through the column
because they were not able to bind to the ligand.
Principle
• Affinity chromatography is based on highly specific
biological interactions between two molecules such as
interactions between enzyme and substrate, receptor and
ligand, or antibody and antigen.
• These interactions which are typically reversible are used
for purification by placing one of the interacting molecules
referred to as affinity ligand onto a solid matrix to create a
stationary phase while a target molecule is in the mobile
phase. Many of the commonly used ligands coupled to
affinity matrices are now commercially available and are
ready to use.
Affinity Chromatography
The picture of affinity chromatography presented in the above
discussion would make one think that the desired macromolecule will be
totally purified in one single step.
However, in practice, a number of complications of this simple
idealized picture are always present.
Most such complications arise because of nonspecific adsorption
of sample components other than the desired one on to the matrix.
Usually ionic and hydrophobic interactions are involved in such non-
specific adsorption. This complication may be taken care of by judicious
choice of operating conditions (e.g., pH, temperature, or ionic strength).
Another type of complication arises when one uses ligands, which
interact with more than one kind of macromolecule present in a given
mixture.
Variables
Major variables, which need careful consideration, are
(i) The type of matrix used.

(ii) Selection of the ligand, its nature, and the means of covalently
binding it to the matrix,
(iii) The conditions exploited to bind and dissociate (elute) the
macromolecule from the column.
Supporting matrix
Characteristics desired of an ideal insoluble matrix for affinity
chromatography are
(i) The matrix should be inert to other molecules to minimize non-specific
adsorption.
(ii) It should possess good flow properties.
(iii) It should be chemically and mechanically stable at varying pH, ionic
strengths.
(iv) It should contain large numbers of suitable chemical groups for
ligand attachment.
(v) It should preferably be highly porous. High porosity provides a large
surface area for attachment of the ligand and allows better interaction
of the desired macromolecule with the immobilized ligand.
Immobilized Ligand
• The ligand can be selected only after the nature of the macromolecule
to be isolated is known.
• When a hormone receptor protein is to be purified by affinity

chromatography, the hormone itself is an ideal candidate for the
ligand.
• For antibody isolation an antigen or hapten may be used as ligand.
• If an enzyme is to be purified, a substrate analog, inhibitor, cofactor

or effector may be used as a the immobilized ligand.
Attachment of Ligand to Matrix
Several procedures have been developed for the covalent attachment of

the ligand to the stationary phase. All procedures for gel modification
proceed in two separate chemical steps:
1) Activation of the functional groups on the matrix and
2) Covalent attachment of the ligand to the functional group on

the matrix.
A wide variety of activated gels is now commercially available.
• CYANOGEN BROMIDE-ACTIVATED AGAROSE

This gel is especially versatile because all ligands containing primary amino
groups are easily attached to the agarose, since the gel is extremely reactive,
very gentle conditions may be used to attach the ligand.
• 6-AMINOHEXANOIC ACID(CH)-AGAROSE and 1,6-

DIAMINOHEXANE(AH)-AGAROSE
These activated gels overcome the steric interference problems by
positioning a six carbon spacer arm between the ligand and the matrix.
Ligands with free primary amino groups can be covalently attached to CH-
agarose, whereas ligands with free carboxyl groups can be coupled to AH-
agarose.
• CARBONYLDIMIDAZOLE(CDI)-ACTIVATED SUPPORTS
Reaction with CDI produces gels that contain uncharged N-alkylcarbamate
groups.
• EPOXY-ACTIVATED AGAROSE
This gel provides for the attachment of ligands containing hydroxyl, thiol or
amino groups.
The Arm
Need of a spacer arm. (A) Ligand without a spacer arm is closely held to the
matrix particle. The macromolecule cannot attach due to steric hindrance. (B)
ligand with a spacer arm. The macromolecule binds without any steric hindrance.
The Arm
Figure (A) shows that if a ligand is directly attached to activated groups

of the support, the macromolecules might encounter steric restrictions
due to which they might not become adsorbed to the matrix.
To avoid this difficulty, it is usual to introduce a spacer between
activated groups of the support and the ligand. This spacer is known as
the arm. Due to this, the ligand is projected at a distance from the
matrix and the desired macromolecule will bind to it without facing any
steric hindrance (Figure (B)).
A large variety of spacer arms have been investigated. Examples
include hexamethylenediamine, 3,3' - diaminodipropylamine, 1,6 -
diaminohexane, 6 - aminohexanoic acid, and 1,4-bis - (2,3-epoxypropoxy)
butane.
Selection of a Gel or Ligand
• Many type of matrix-ligand systems are
commercially available and cost are
reasonable so time can be saved by purchasing
preactivated gel for direct attachment of
ligand.
• Buffer
Buffer is used for formation of complex between a
matrix and ligand, as slight change in ionic
concentration weakens the interactions between
them.
• Affinity Elution
In this method a selective substance added to the
buffer causes selective elution of bound
macromolecule-ligand complex, resulting in elution
of desired macromolecule.
• Chaotropic Agents
If gentle and selective elution methods do not
release the bound macromolecule, then mild
denaturing agents can be added to the buffer,
the most powerful agents are urea, guanidine.
Experimental Procedure
• IS MATRIX LIGAND AVAILABLE
• NO YES
• SELECT GEL AND LIGAND SWELL GEL IN BUFFER
• COUPLE LIGAND
• PREPARE GEL FOR COLUMN
•
• PACK GEL IN GLASS COLUMN
• AND SET-UP COLUMN EQUIPMENT
•
• EQUILIBERATE COLUMN WITH BUFFER
•
• APPLY SAMPLE
•
• WASH COLUMN TO REMOVE
• UNBOUND MOLECULES
•
•
•
ELUTE BOUND MOLECULES
•
•
COLLECT AND ANALYZE ELUENT
• REGENERATE AND STORE GEL
Standard Matrix-Ligand Systems
Certain matrix-ligand systems have become standard for separating certain
types of macromolecules sharing a common structural/functional characteristic.
All of these are commercially available too. Some of them are as follows.
1. Heparin-agarose. Heparin is a natural anticoagulant found in the blood. It

interacts with a large number of blood components. However, when used as a
ligand. only a few components of the cell extract bind to it. Some substances
which have been purified using this system are as follows: DNA polymerase,
ribosomes, hepatitis B surface antigen, bone collagenase, uterine estradiol
receptor. etc.
2. Polynucleotide-agarose. oligo-dT columns are used for purification of mRNA.

Poly-U columns may also be used instead of oligo-dT. Similarly, Specific
polynucleotide sequences covalently linked to agarose may be used to purify
transcription factors and other DNA-binding proteins.
3. Lysine-agarose. Lysine-agarose does not bind to a large number of

substances. Among the substances that have been purified with this system are
ribosomal RNAs and the protein plasminogen.
Use of Affinity Chromatography in
Molecular Biology
Purification of mRNA from total RNA
In order to study gene expression, molecular biologists frequently have
to isolate mRNA from a total RNA preparation which contains other
types of RNA, viz., rRNA, and tRNA. Oligo dT (many units of
deoxythymidyllic 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 out. 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 immobilized on the matrix. Other RNA molecules do
not have such poly A tails. After other RNA molecules have eluted out,
mRNA molecules bound to the matrix are eluted by changing the eluting
conditions.
Isolation of DNA-binding Proteins
A transcription factor is a protein that recognizes and binds to a

specific DNA sequence lying in the regulatory region of a given gene.
This binding may promote the transcription of the given gene. To study
the mechanism of action of transcription factors, it is necessary to be
purify it. However, for quite a long time, scientists could not succeed in
purifying these proteins owing to the fact that these proteins are
present in extremely low concentrations in the cell (0.001% of total cell
protein). Conventional biochemical techniques cannot purity 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 1 and the sequence was called GC box owing to
the fact that it was formed entirely of the two nucleotides.
They also demonstrated that the binding of Sp 1 to the GC box

stimulated the transcription of the gene.
The problem was to purify this protein in order to study it.

It was decided to exploit the high affinity of this protein to the GC box
sequence GGGCGG.
They synthesized oligonucleotides containing multiple repeats of the
above sequence and coupled them to solid beads.
A crude nuclear extract was passed through a column consisting of these
beads coupled to the oligonucleotide sequence.
The column was then washed to remove non-specifically binding proteins.
Finally the beads were washed with high salt buffer which disrupted the
Sp 1 binding to DNA. This resulted in the elution of the Sp 1.
Different Types of chromatography
Mode or type Stationary phase Mobile phase Mechanism
Adsorption Solid that attracts Liquid or gas Solutes move at different rates
Chromatography the solutes according to the forces of attraction
to the stationary phase.
Partition Thin film of liquid Liquid or gas Solutes equilibrate between the 2
Chromatography formed on the phases according to their partition
surface of a solid coefficients
inert support
Ion Exchange Solid resin that Liquid Solute ions of charge opposite to the
Chromatography carries fixed ions containing fixed ions are attracted to the resin
& mobile electrolytes by electrostatic forces & replace the
couterions of mobile counterions.
opposite charge
attached by
covalent bonds
Molecular Exclusion Porous gel with no Liquid Molecules separate according to their
Chromatography attractive action size:
on solute 1.Smaller molecules enter the pores
molecules of the gel, and need a larger volume
of eluent.
2.Larger molecules pass through the
column at a faster rate.
Affinity Solid on which Liquid or gas Special kind of solute molecules

Chromatography specific molecules interact with those immobilized on
are immobilized the stationary phase
High Performance Liquid Chromatography
High performance liquid chromatography is a product of the Scientific

effort towards optimization of the conventional column
chromatography. Ordinarily, a conventional chromatographic
experiment takes long time. To reduce the time of experiment, one can
increase the flow rate thereby reducing the retention time of the
solute components. Although this reduces the time of experiment, it
also reduces the efficiency of the column because the solute
components undergo lesser number of equilibration, i.e., the theoritical
plate number is reduced. To circumvent this, if one uses a longer
column, the diffusion effects increase and the peak is broadened.
HPLC
To increase the flow rate, the pressure has to be increased. The

conventional supports can tolerate pressure only up to a particular
limit; at higher pressures their structure is affected thereby causing
flow rate anomalies.
All these factors were efficiently resolved with the development of

high performance liquid chromatography (HPLC). This method uses an
extremely high pressure (up to 8000 psi). The flow rate therefore is
high and the experimental time is shortened considerably.
HPLC
There is no loss in efficiency also because the supports that are used
here are different and the support particles are very small and more or
less, uniform in size. The lateral diffusion is less because of two factors,
the smaller particle size and the enormous pressure, which reduces the
time that a solute spends in the column. Due to this, band broadening is
minimized. HPLC is therefore highly efficient and has a very fast speed
of resolution.
The technique may be used with very small amounts of sample (pico or
even femto gram).
HPLC
Block Diagram of HPLC
Components of HPLC
Six major components needed to perform HPLC are

(i) A solvent reservoir to store the mobile phase.
(ii) High pressure pump to push the mobile phase through the column.
(iii) A device to inject the sample into the mobile phase.
(iv) 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 chromatogram.
Solvent Reservoir and the Solvents
The solvent reservoir should meet the following criteria:

(i) it must contain volume enough for repetitive analysis;
(ii) it must have a provision for degassing the solvents;
(iii) it must be inert to the solvent.
Solvent degassing within the reservoir is performed usually by heating
or by application of vacuum or by treating it with ultra sonic sounds.
The type of separation desired dictates the choice of the mobile phase.
Isocratic separations can be carried out with a single solvent or a fixed
proportion mixture of two solvents. For gradient elution, however, the
development solvent composition changes continuously. This is achieved
by a gradient programmer usually attached to the HPLC assembly.
Pumping Systems
Pumping system can said to be the heart of HPLC.
A good pump should have the following qualities:

(i) A pulseless stable flow. Absence of pulsations minimizes detector
noise.
(ii) A suitable pump should provide solvent flow-rates of 0 .5-10 ml/min,
which is compatible with most HPLC modes.
(iii) A constant volume delivery.
(iv) Amenable to high pressures of up to 6000 psi.
(v) The pump should be adaptable to gradient operation.
Pumping systems available for HPLC are:
(i) Liquid displacement by compressed gases (holding coil)

(ii) Pneumatic amplifier
(iii) Piston/diaphragm driven by a moving fluid
(iv) Reciprocating piston
(v) Syringe pumps
Holding coil
This unit is usually available with less expensive HPLC systems. A
large holding coil made up of stainless steel tubing 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
applied. These pumps can provide pressures up to 1500 psi., and cannot be
used for 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 resolutions.
Pneumatic amplifier
This pump also uses compressed gas for pressure. The pump has a
piston driven by the compressed gas. The pump uses gas at
comparatively 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 pulseless
flow and is ideal for quantitative purposes.
Moving fluid 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
gradient elution.
Reciprocating piston
These pumps use a piston that is in direct contact with the solvent.
The piston may be driven with motors and gears. 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 forward move. The outlet to the columns close during the
backward move to maintain the pressure in the column.
Syringe pump
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
Two methods are available to introduce the sample.
(i) The first method employs a micro syringe designed to withstand high
pressures. With the help of this micro syringe, the sample is introduced
either directly onto the column. Preferably the sample is injected when
the pressure has dropped to almost one atmosphere after switching the
pump off. This technique is known as stop flow injection. Alternatively,
the sample can be injected while the system is under high pressure.
(ii) The second method employs a small volume metal loop which can be
filled with the sample. An appropriate valve then channels the eluant
from the pump through the loop directly onto the column. The sample is
thus carried spontaneously with the eluant to the column.
The Column
The columns for HPLC are usually made up of stainless steel, aluminium,
copper or PTFE. Stainless steel columns are preferred since they can
withstand pressures up to 8000 psi. Straight columns of between 20-50
cm in length are generally used. Short columns are required for liquid
adsorbent and liquid-liquid chromatography, whereas for other modes
longer columns are necessary. The internal diameter of the columns is
usually 1-4 mm. The columns usually possess an internal mirror finish,
which allows efficient packing. The packing material is supported by a
porous stainless steel or teflon plug/disc at the end of the column.
Column Packings
Schematic representation of the structure of three types of supports

commonly used for HPLC. (A) microporous, (B) pellicular and (C) bonded
phase.
To minimize the band broadening due to coarser particles, stationary supports
for HPLC have been designed 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 small particles
should also be able to withstand the increased pressure that they will be
subjected to.
Three forms of column packing material are available based on a rigid solid
rather than a gel structure:
(i) microporous supports where micropores ramify through the particles. These
particles are generally 5-10 mm in diameter.
(ii) pellicular supports consist of a solid inert core onto which are coated several
porous particles. These supports are therefore superficially porous.
(iii) bonded phases where the stationary phase is chemically bonded to an inert
support .
Column Packing Procedure
The packing has to be uniform without any cracks or channels for
obtaining optimum separation. Rigid solids and hard gels should be
packed as densely as possible. Care should, however, be taken to not to
fracture the particles. Usually a method known as high pressure slurry
technique is used for packing the column. A dense suspension of the
packing material is prepared in a suitable solvent. The column is sealed
with a porous plug at the bottom. The slurry is now pumped into the
column at high pressure. The column so packed is then equilibrated for a
long time by passing the developing solvent through it. 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 before chromatography is not necessary. Thus, organic or
biological materials can be applied to the column without any
pretreatment. This, however, clogs the column after a few applications as
the column during separation retains many undesirable components of the
biological samples. To circumvent this problem, a short column (2-10 cm)
precedes the main column. This short column is known as guard column
and its function is to retain those biological components which would
otherwise clog the main column. The guard column has the same diameter
and the same packing as the main column.
Detectors
Since the quantity of the sample applied to the analytical column is small, it is
imperative that the sensitivity of the detector is sufficiently high and stable.
UV/VIS photometers can be used for HPLC. These detectors are inexpensive,
sensitive, insensitive to normal flow and temperature fluctuations and well suited
for gradient elution. They are however, sample selective, as only those sample
molecules which absorb at 254 nm or 280 nm can be detected. Thus, lipids,
carbohydrates, fatty acids, hydrocarbons etc., are undetectable by these
detectors.
UV/VIS spectrophotometers with wavelength selection range of 200-800 nm are
very popular HPLC detectors. These can be either recording spectrophotometers
or manual wavelength selection spectrophotometers. The advantage with these is
that a range of biological substances can be detected by selection of appropriate
wavelengths. Apart from the above two, refractive index monitors and
fluorescence detectors are also available with HPLC systems.
Preparative HPLC
If the study involves characterization and further studies on the

separated components. it is imperative that a larger quantity of the
pure components is needed. For this preparative HPLC may be
performed. Preparative HPLC can be carried out using the same
packing materials and solvents. The only difference being that the
columns is of a larger internal diameter (10-20 mm) so that a larger
volume of the sample can be processed. Repeated application of the
sample and collection of effluent fractions containing the desired
components can give a fair yield of the component of interest.
Specialized Techniques:
Reverse Phase Chromatography
As opposed to the usual polar stationary phase and a less polar or non-
polar mobile phase, the stationary phase in reverse phase
chromatography is hydrophobic (hydrophobic bonded phase usually
possessing C18 or C8 functional groups) and the mobile phase is polar
(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 increases, their retention times will also
increase since they will interact more with the non-polar stationary
phase. The reverse phase system is therefore very useful for
separation of non-polar solutes. Water, an extremely polar solvent
becomes the weakest eluent here. Methanol and acetonitrile are
stronger eluents than water. Solvents of intermediate eluting strength
can be obtained by mixing one of these solvents with water.
It is usual to separate compounds soluble in organic solvents by either
partition or adsorption chromatography.
Compounds possessing different functional groups are ideally separated
by adsorption chromatography on silica with a non-polar solvent as the
mobile phase.
Weakly ionic or non-ionic substances which are water soluble yield to
separation by reverse phase partition mode.
On the other hand strongly ionic solutes soluble in water should be
separated by the ion-exchange mode. However, reverse phase partition
mode can also be applied to these substances. This technique is known
as ion-pairing.
Types of HPLC
Based on Mode of Separation
1.Normal phase chromatography - stationary phase is polar
(hydrophilic) and mobile face is non-polar (hydrophobic).
2.Reverse phase chromatography- stationary face is non-polar

(hydrophobic) and mobile face is polar (hydrophilic).
•Polar-Polar bonds and Non Polar-Non Polar bonds have more affinity than
Polar-Non Polar bonds.
•Reverse phase chromatography is more commonly used as drugs are

usually hydrophilic
Based on Principle of Separation
1. Gel permeation or Size exclusion

2. Ion exchange
3. Affinity
Based on Elution Technique
Isocratic elution
•A separation in which the mobile phase composition remains constant

throughout the procedure is termed isocratic elution.
• In isocratic elution, peak width increases with retention time linearly

with the number of theoretical plates. This leads to the disadvantage
that late-eluting peaks get very flat and broad.
• Best for simple separations
• Often used in quality control applications that support and are in close
proximity to a manufacturing process.
Based on Elution Technique
Gradient elution
•A separation in which the mobile phase composition is changed during
the separation process is described as a gradient elution.
•Gradient elution decreases the retention of the later-eluting

components so that they elute faster, giving narrower peaks . This also
improves the peak shape and the peak height.
Best for the analysis of complex samples

• Often used in method development for unknown mixtures
• Linear gradients are most popular
Based on Scale of Operation
1.Analytical HPLC
No recovery of individual components of substance
2.Preparative HPLC
Individual components of substance can be recovered

Based on Type of Analysis
1.Qualitative analysis
Analysis of a substance in order to ascertain the nature of
its chemical constituents.
We can separate individual components but cannot assess the

quantity in this analysis.
2.Quantitaive analysis
Determining the amounts and proportions of its chemical

constituents.
Quantity of the impurity and individual components can be

assessed.
Applications of HPLC
In recent times HPLC has emerged as a method of choice for analytical
purposes. The biggest advantage that it has over other techniques is
the speed of analysis which is many times more than other techniques
except, perhaps, for GLC. The sample requirement is also very low for
this technique and as less as a few femtograms of the sample will be
analysed satisfactorily. On top of all this the detectors that are
employed in HPLC are non-destructive in 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 their metabolites, animal and plant hormones and complex lipids.

Chromatography

Caricato da

Informazioni sul documento

Titolo originale

Copyright

Formati disponibili

Condividi questo documento

Condividi o incorpora il documento

Opzioni di condivisione

Hai trovato utile questo documento?

Questo contenuto è inappropriato?

Copyright:

Formati disponibili

Chromatography

Caricato da

Copyright:

Formati disponibili

CHROMATOGRAPHY

Chromatography is a physical method of separation in

The chromatographic process occurs due to

Stationary phase is the substance fixed in place for the chromatography

Retention time is the characteristic time it takes for a particular analyte

Chromatogram is a plot of the detector’s signal as function of elution

Classification of chromatography according to

Classification according to the force (mechanism)

1- Paper chromatography (PC): the stationary

2-Thin layer chromatography (TLC): the

3-Column chromatography (CC): stationary

In plane chromatography the stationary phase is coated onto a plane

As opposed to plane chromatography, the stationary phase in column

2. PAPER PARTITION CHROMATOGRAPHY

In general P.C – Paper Partition Chromatography

The factor governing separation of mixtures of solutes on

One is usually water adsorbed on cellulose fibres in the

The second is the organic solvent flows past the sample on

Cellulose fibers in the paper hold moisture tightly through

The separation depends on partition

 The paper commonly used consists of highly purified cellulose.

If the development is to be performed by the ascending technique, the

Development in a direction perpendicular to the first, and

The sample is applied on one corner of a square piece of

Usually, different types of solvents systems are used in

Usually in paper chromatography, the stationary phase is

The mobile phase is usually a mixture of various solvents

Desirable characteristics that a solvent should possess are

(i) Partition coefficient of substances to be analyzed should

(ii) The solvent should be removable from the paper and to

(iii) The solvent should be stable. It should not become

When the components are colorless, they can be imparted

Otherwise, the components may be extracted and chemical

Deep blue or Purple colored pigment

TLC is a method for identifying substances and

TLC is a useful technique because it is relatively

The stationary phase:

In principle, the components will differ in solubility and in the

To a jar with a tight-fitting lid add enough of the

With a pencil, etch two small notches into the adsorbent

The notches should be on the edges of the plate, and each

The notches must be farther from the bottom of the

Using a drawn-out capillary tube, spot the samples on the

After preparing the development chamber and

Be careful to handle the plates only by their edges,

When the plates are removed from the chamber,

If the spots can be seen,

Alkaloids- Dragendorff’s reagent

Sugars - Aniline phthalate

Amino acids - Ninhydrin

Iodine vapour - used extensively as a universal reagent for

KMnO4 - The most universal stain. Harsh oxidizer. Any

The Rf (retention factor) value for each spot

It is characteristic for any given compound on

Hence, known Rf values can be compared to

What does the Rf value tell you about

Larger Rf value → more soluble

Smaller Rf value → less soluble

(Note: Rf values often depend on the temperature and the

the most effective way to identify a compound is to spot

In addition, the purity of a sample may be estimated from

An impure sample will often develop as two or more spots,

Traditional column chromatography is