Chromatographic techniques such as paper chromatography, thin layer chromatography, and column chromatography are used to separate mixtures based on differences in how components partition between a stationary and mobile phase. In paper chromatography, the stationary phase is water absorbed by a paper support and the mobile phase is a solvent such as ethanol or chloroform. Thin layer chromatography uses a finely divided substance coated on a glass plate as the stationary phase. Column chromatography packs the stationary phase into a glass or plastic column. Components separate as they migrate through the column at different rates based on their partitioning between the phases.

1. Chromatographic Techniques
Module IV
Bioanalytical Techniques
S.Y. Biotech

2. ● The term chromatography bunches together a family of closely related extremely powerful
separation methods.
● The first detailed description of chromatography is generally credited to Michael Tswett a Russian
biochemist who separated chlorophyll from a mixture of plant pigments in 1906.
● Chromatography is the method by which two or more compounds in a mixture are physically
separated by distributing between two phases.
● Two mutually immiscible phases are brought into contact with each other. One of these phases is
stationary, while the other is mobile.
● A stationary phase which can be a solid or liquid supported on a solid and a mobile phase, either
a gas or a liquid which flows continuously around the stationary phase.
● The mobile phase either moves over the surface or percolates through the interstices (intervening
space) of the stationary phase.
Introduction

4. ● The sample mixture introduced into the mobile phase undergoes repeated interactions
(partitions) between the stationary and mobile phases while being carried through the
system by the mobile phase.
● Different components of the sample mixture interact with the two phases
differentially on the basis of small differences in their physico-chemical
properties. Since these different rates of interactions govern the migration of the
sample components through the system. Each one of the components migrates at a
different rate.
● The compound which interacts more with the mobile phase and least with the
stationary phase migrates fast. The component showing least interaction with
the mobile phase while interacting strongly with the stationary phase migrates
slowly (retarded). This differential movement of the components is responsible for
their ultimate separation from each other.

5. General Principle of Chromatography
Partition Coefficient/Distribution Coefficient (K):
● Concept of partition coefficient is the basic principle of all chromatographic methods.
● Partition coefficient (also known as distribution coefficient) is a definitive term
normally used to describe the way in which a given compound distributes or
partitions itself between two immiscible phases: the stationary and the mobile
phase.
● The solute upon entering a chromatographic system immediately distributes itself
between the stationary and the mobile phase. If at a given time during
chromatography the mobile phase flow is stopped the compound will be in
equilibrium between the stationary phase and the stopped mobile phase.
● At this stage the concentration of the compound in each of the phases is described by
the partition coefficient, K. which is expressed as follows: K=Cs/Cm. Where Cs
and Cm are the concentrations of the compound in the stationary and the mobile
phases respectively.

6. Nature Of Partition Forces
● The distribution of a solute between the stationary and the mobile phase is a result of the
balance of forces between the solute molecules and the molecules of each phase.
● The partition coefficient, therefore, reflects the relative attraction or repulsion that
the molecules of the two phases show for the solute molecules and for themselves.
These attractive or repulsive interactions are accompanied by a release or consumption
of energy.
● The amount of interaction energy provides a measure of the strength of the interaction
and serves as a criterion to classify the interaction as physical or chemical in nature.
● It is the molecular constitution, which is fixed by the species of atoms present and by the
nature of bonds between them (metallic, ionic, covalent, co-ordination), which decides
the quality and the intensity of the physical interactions.
● Dispersion forces and electrostatic forces are the typical physical interactions which
contribute most to partitioning of the solute between two phases.

7. Techniques Of Chromatography
Planar Chromatography
● The stationary phase is
coated onto a plane surface.
Column Chromatography
● The stationary phase is packed
into a glass or plastic column
Paper
Chromatography
Thin layer
Chromatography
● Ion Exchange chromatography
● Size Exclusion Chromatography
● Affinity Chromatography

8. Paper Chromatography

9. Nature of Paper
In paper chromatography support material consists of a layer of cellulose highly
saturated with water. Cellulose fibres in the paper hold moisture tightly through
formation of hydrogen bonds. The cellulose itself takes a negative charge in company of
water.
The paper exhibits weak ion exchange and adsorptive properties. Modified forms of paper
have been produced in which the paper has been impregnated with alumina, silica gel, ion
exchange resin.
Stationary Phase: Water molecules absorbed by paper
Mobile Phase: Ethanol, Methanol, Ether, Chloroform
Apparatus for 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.

10. Choice of solvent system
● The mobile phase may not be necessarily immiscible with water if water is being
used as the stationary phase. This is because the stationary phase water is very tightly
held by cellulose and will not mix with the mobile phase on this account.
● The mobile phase is usually a mixture of various solvents such as alcohols, acids,
esters, ketones, phenols, amines, and hydrocarbons. The solvents are selected in
such a way that the resolution of sample components is satisfactory.
● The components of the solvent system should be so chosen that the extent of
evaporation of each individual component is more or less similar.
● limit the number of components in the solvent system used to the barest minimum.
More components a solvent system contains the more difficult it will be to maintain a
saturated atmosphere in the chamber.
● The solvent system should be so 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. The time required will
also be short and the spreading of the separated zones will also be minimal.

11. Examples of solvent systems used in Paper Chromatography

12. The sample is applied to the paper as a small spot. This is done before dipping the paper
into the eluting solvent. Any device which can transfer a small volume of sample can be
used for spotting. Generally used devices are platinum loop, capillary tube or a
micropipette. Of these platinum wire is preferred because it can be reused with several
substances after heating on a flame. A micropipette can also be reused after its tip has been
disposed and a new tip applied. For some methods the sample may be applied as a narrow
streak at right angles to the flow of the solvent.
Sample Application

13. Paper Development
Techniques involved in paper development
(A)Descending (B) Ascending

14. ● In both cases of paper development 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.
● If the development is to be performed by the ascending technique the paper is
allowed to hang in 'or is suspended in a manner that the base of the paper is in contact
with the solvent at the base of the chamber. The sample spots should be in a position
just 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.

15. Radial Paper 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 then
onto the paper through capillary
action. The sample components
now move outward radially
forming concentric circles of
increasing diameters.

16. Paper Development
Technique
Advantages Disadvantages
Ascending ● Simple Setup
● High Resolution
● Slow
Descending ● Faster ● Less Resolution
● Difficult Setup
Radial ● Sharper Resolution
● Simple Apparatus
● Less samples get
investigated

17. Colored Samples: If the sample
components are colored, the analysis
becomes simple as the distinctive color
itself identifies the component.
Detection

18. Colorless samples: When the components are colorless (usually they are), they can be
imparted color by spraying the paper with color producing reagents. A case in point is
the detection of amino acids. Ninhydrin reagent spread on the paper reacts with amines
and amino acids to form a blue or purple color

19. Other methods of detection are (i) ultraviolet and infrared absorption, (ii) fluorescence,
and (iii) radioactivity.
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), and is constant for a given compound under standard conditions.

20. Two dimensional Paper Chromatography
● A plane surface is amenable to sequential development in two directions using two
different solvents. The paper, with the sample applied as a spot close to a corner, is
developed in the normal fashion by either ascending or descending procedure. The
development is continued until the faster moving component or solvent front
approaches the end of the paper.
● The paper is then removed and the solvent is allowed to evaporate. This paper is then
turned 90° and developed a second time with another solvent having totally different
eluting properties. Since the two solvents used have different eluting properties,
the distribution coefficients of individual components in them will also differ. Thus,
components which could not be separated using one solvent alone can be easily
separated by this procedure.This technique is known as two-dimensional
chromatography.

21. (A)First development in the direction indicated by the arrow does not
resolve B and C completely.
(B) Second development in a direction at right angles to the first using
a different solvent system resolves all components completely.

22. Applications of Paper chromatography
● To control of purity of pharmaceuticals
● The detection of adulterants and contaminants in foods and drinks
● The study of ripening and fermentation
● The detection of drugs and dopes in animals and humans
● The analyses of cosmetics
● The analyses of the reaction mixtures in biochemical labs are all performed
routinely with paper chromatography technique.

23. Thin layer Chromatography
● In this technique, a thin layer of a finely
divided substance is deposited on to a flat
glass plate.
● The sample to be separated is spotted at one
end.
● The plate is dipped into the solvent in a
glass jar and the development carried out by
the ascending technique.
● After the development, the layer can be
dried and the components detected by
various methods available.
● Thin layer chromatography may be carried
out by either adsorption principle or
partition principle.

24. ● The glass plate on which the thin layer is prepared should be even and is thoroughly
washed and dried before layer application.
● The material of which the thin layer is to be made is usually mixed with water in such
a proportion that a thick suspension, known as slurry results. This slurry is applied
to a plate surface as a uniform thin layer by means of a plate" spreader' starting at one
end of the plate and moving to the other in an unbroken uniform motion.
● The nature of the desired chromatographic separation dictates the thickness of the
slurry layer used. Thus, for analytical separations the thickness of the layer is
usually 0.25 mm, while for preparative separations the thickness of the layer might
be about 5 mm. Although thin layer technique can be used for many different types
of chromatographic separations such as adsorption.
● The plates are dried after application of slurry.
Preparation of layer

26. Sample application: This is absolutely similar to that described for paper chromatography.
except that care should be taken not to scrape the thin layer while applying the sample.
Plate Development:
● The choice of solvents and the methods of elution are much the same as for paper
chromatography.
● The procedure must of course be conducted in a closed chamber to prevent
evaporation of the solvent and the technique used is ascending out of necessity.
Two dimensional chromatography may also be carried out much in the same way as
described for paper chromatography.
One of the greatest advantages of TLC is the speed at which the separation is achieved.
Generally 10-30 minutes are sufficient. However with certain compounds about 90
minutes may be required.

27. Detection
Several detection methods are available.
Many of these have already been named in the section on paper chromatography for
example: ultraviolet absorption, fluorescence, autoradiography if the components are
radio-labeled or production of colors by chemical treatment.
Those specific for TLC are:
(i) spraying the plate with 25-50% sulphuric acid in ethanol and heating. This results
in charring of most of the compounds which show up as brown spots.
(ii) Iodine vapour is used extensively as a universal reagent for organic compounds.
The iodine spot disappears rapidly but can be made more permanent by spraying with
0.5% benzidine solution in absolute ethanol. Iodine vapour is seen concentrated in the
form of a cloud over the region where the components have separated. These spots can
then be scraped out eluted and analysed quantitatively.

28. Quantification by densitometer:
On plate quantification of the separated
components might be achieved by employing a
densitometer which not only measures the
ultraviolet or visible absorption of the separated
components but also gives a complete
absorption spectrum of the compound for
identification purposes. Precision made
densitometers are now commercially available.
In case the substance has been radiolabeled.
radio-chromatogram scanning might be
employed to quantitate the separated
components.

29. 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.
● It may otherwise be used as an aid in order to find out the best conditions for large-
scale chromatography.
● Because of its speed and simplicity it is often used to follow the course of
reactions.
● Thin layer technique has often been used to identify drugs, contaminants and
adulterants.
● It has also been widely used to resolve plant extracts and many other biochemical
preparations.

30. Column Chromatography

31. Column Chromatography
● Column chromatography is an often used and routinely carried out technique which
is adaptable to all the major types of chromatography.
● The stationary phase in column chromatography is packed into a glass or plastic
column
● Important factors in column chromatography
a. Selection of column
b. Packing of column
c. Introduction of sample
d. Column Development
e. Elution
f. Analysis

32. Selection of column
● Material: Glass and Polyacrylate Plastic
● Laboratory Columns:
1. Diameter: 2-70 mm
2. Length: 15-150 cm
3. Ratio of diameter: length is between
1: 10 and 1: 100.
● larger the sample volume larger the column
chosen
● Columns have a sintered glass disc at the
bottom to support the stationary phase.
● Thermostat jacket is used to control temperature
fluctuations

33. Packing Of Column

34. 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 degassed stationary phase is gently poured into the column with its outlet closed.The slurry is
added upto ¾ th of column
The outlet is now opened and the column is stabilized by washing it with mobile phase.
A filter paper disc or nylon gauze is then placed on the surface of the column to prevent disturbance during
mobile phase/sample addition
Many commercial columns provide an opening at the top. which can be fitted with capillary tubing through
which solvent drips into the column. This provision decreases the disturbance of the column surface
considerably. To prevent the column from drying. a layer of solvent is always maintained above the column
surface.

35. Introduction of Sample
Remove the top layer of the solvent and then carefully add the sample on
column by using pipette.
Allow the sample to run into the column
Add the solvent in the column to a height of 5-10 cm.Connect the column
with 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.

36. Important factors to consider during sample application
● An alternative and better method for sample application is to mix the sample with
sucrose or ficoll to a concentration of about 1% to increase sample density. The
sample now sinks below the top layer of the solvent to the surface of the column.
● The dye bromophenol blue can be used in place of sucrose.
● Alternatively it is possible to reach the column surface directly with the help of a
syringe or capillary tubing.
● It is necessary to apply the sample in as less a volume as possible. This gives an
initial tight band of material when the separation begins and results in a sharper final
separation.
● An additional precaution is to desalt the sample to avoid anomalous adsorption
effects.

37. Column Development
Continuous passage of a suitable eluent (mobile phase) through the packed column
separates the components of the sample applied to the column.
Techniques Of Elution
Gradient Elution
Isocratic Elution
● When a single solvent is used as
an eluant during development.
● pH, ionic strength or polarity of
the eluant is changed with
respect to time.
● Composition of the mobile phase is
changed giving rise to a gradient
● Two solvents of differing
compositions have to be mixed in
correct proportions before entering
the column.

38. Flow Rate (Fc)
● Flow rate, Fc is expressed as the volume of mobile phase per unit time. This is a
very important criterion for suitable column development.
● For a satisfactory resolution it is absolutely necessary that the eluant flow should be
maintained at a stable rate.
● The easiest way to maintain a stable flow rate is to use a peristaltic pump to force
the eluant on to or out of the column.
● An increase in the flow rate of the mobile phase through the column leads to
shortening of the time necessary for separation. An undue increase in the flow rate
decreases the efficiency of resolution and time economy.
● The flow rates used differ with respect to the nature of the sample and the nature of
the stationary phase. However. commonly used flow rates fall between 30-120
ml/hour cm2

39. Fraction Collection
● Collecting effluent fractions manually can be both boring and time consuming.
● A range of automatic fraction collectors are available commercially. They are designed to
collect a definite volume of the effluent in each tube before a new tube is placed in
position automatically.
● Different fraction collectors are programmed to operate in different ways. Some fraction
collectors are fitted with electronic device to measure the number of drops falling in a tube.
This number can be predetermined so that after a set number of drops have fallen into a
given tube, a new tube comes into position.
● Other fraction collectors allow the effluent to enter each tube for a fixed interval of time.
● A better method of fraction collection is by programming the fraction collector for
collection of definite volume per tube. The fractions collected are subsequently analysed.

40. Analysis of Effluent
The effluent as it emerges from the column outlet is analysed. The properties of a
particular compound i.e. ultraviolet absorption, color or fluorescence are exploited in its
analysis. Alternatively the compound may be labeled before application of the sample to
the column and its radioactivity exploited for its analysis.
Approaches to analyse Solutes in Effluent
Classical Approach Modern Approach
Collect the effluent in equal fractions
and to subsequently analyse each
fraction for the presence and content
of the solute.
continuously monitor the effluent 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.

41. 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 280 nm.
Diagrammatic representation of a typical
elution profile. The profile is prepared by
plotting a measurable property of the sample
components (absorbance in this
diagram)against the eluted volume as it
emerges from the column. The peaks in such a
pattern represent individual zones of sample
components (proteins in this case since the
absorbance is measured at 280 nm. separated
from each other during column development.
It is possible to identify each component in the
pattern on the basis of its elution volume i.e
the volume of effluent collected which
corresponds to the apex of the peak. To
quantitate each component its peak height at
the apex (a) is multiplied with peak width at
half height (b).

42. Size exclusion chromatography
● Also known as molecular sieve, gel filtration, gel permeation or molecular
exclusion chromatography.
● Separation is dependent on molecular size.
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.

43. Illustration of principle of molecular sieving. (A) Schematic representation of
exclusion of large molecule. (B) Effect of particle size on their elution rates.

44. Characteristics of gels
A gel filtration medium should possess the following characteristics:
(i) The gel material should be chemically inert.
(ii) It should preferably contain vanishingly small number of ionic groups.
(iii) Gel material should provide a wide choice of pore and particle sizes.
(iv) A given gel should have uniform particle and pore sizes.
(v) The gel matrix should have high mechanical rigidity.

45. Types of gels
i) Cross-linked dextrans (trade name Sephadex)
ii) Agarose (Sepharose, Bio-Gel A, sagavac)
(iii) Polyacrylamide (Bio-Gel P)
(iv)Porous glass and silica granules Bio-Glas Porasil
(v) polystyrene (Styragel, Bio-Beads)
Vi) Other gels have been used for gel filtration, include macroreticular polyvinyl acetate
(Merck-a-Gel OR), microparticulate aluminas and silica's (Spherisorb) and cellulose
packings in bead form (bead celluloses have not been developed specially for gel
filtration but are known to have molecular sieving properties).

46. Different types of Sephadex gels

47. Commonly used Polyacrylamide gels

48. Commonly used Agarose gels

49. Commonly used porous glass beads

50. Solute behaviour on molecular sieve gels
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.
Kd = 0:when the solute molecule is large
and completely excluded from the inner
solvent.
Kd = 1: if the solute molecule is small
enough to penetrate the gel pores and
diffuse into the inner solvent
Volume of
solvent inside
gel
Volume of
solvent
surrounding
the gel
Total Volume

51. Sample volume(Vs)
For two substances possessing different molecular weights and therefore different
distribution coefficients (Kd1 and Kd2) the difference in their effluent volumes. Vs, is
given by
Thus. the sample volume applied for complete separation of two substances should not
exceed Vs. The distribution coefficient and the effluent volume are both related to molecular
weight of the molecules being separated. One can therefore calculate molecular weight of
molecules if one determines the effluent volume by gel filtration experiment

52. Technique
● Gel permeation chromatography can be performed either by column or thin layer
chromatographic techniques.
● Packing of column: The gel bed is supported in the column on a glass wool plug or
nylon net 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 (see section on column chromatography).
● Application of sample: The volume of sample that should be applied varies as per the
column size and the type of the gel used.

53. ● Elution: The eluent is steadily added and the effluent collected in various fractions to
be analysed. A knowledge of the effluent volume of a particular compound is useful
for the calculation of its distribution coefficient, which might be useful for molecular
weight determination.
● Detection: The common detection methods include collecting and analysing fractions
and continuous methods with flow cells in which ultraviolet absorption, refractive
index, or radioactivity is measured.

54. Thin layer gel filtration chromatography
● For clinical use thin layer gel filtration (TLG) is ideal since very small sample
volume is required for this technique.
● In this technique a layer of hydrated gel is applied to the plate. There is no need of a
fixative to adhere the gel beads. The plate is not dried at all and is placed in an
airtight container at an angle of 20°.
● The plate is connected to reservoirs at either end by means of filter paper bridges.
Equilibrium must be carried out for at least 12 hours. Such equilibrium serves to
normalize the ratio between the stationary and mobile phase volumes.
● The sample may be applied either as a spot or as a band. The plate may then be
developed for a suitable time and the separated components detected by suitable
means.

55. ● TLG is used mainly for the study of hydrophilic substances which require mild
conditions (proteins, peptides and nucleic acids).
● TLG has been used to study certain enzymes such as adenosine deaminase
(separation of high and low molecular weight forms), collagenase from human
granulocytes, glyoxylate reductase, lactate dehydrogenase and RNA synthetase (all of
these for molecular weight determination) and polyphenoloxidase for size
heterogeneity studies.
● TLG has found numerous applications in clinical immunology and immunochemistry.
● The technique has been used as screening procedure for several immunopathological
conditions involving altered immunoglobulin levels.

56. Advantages
1. Gel permeation depends only on the molecular sizes of the macromolecules. The
chromatography therefore these macromolecules can be separated under the
conditions where they are stable. (pH, Ionic strength and buffer composition)
2. Other chromatographic techniques do suffer from some adsorption. Such
macromolecules can be separated by gel chromatography since adsorption here is
almost nil.
3. The macromolecules having renaturing capacity can be separated from the similar
sized molecular by destabilizing them. Such instability causes large scale changes in
their sizes making them migrate differently from other molecules which may have the
same molecular weights. After purification the molecules are again renatures.

57. Applications
● Separation of biological molecules leading to their ultimate purification. Proteins,
enzymes, hormones, antibodies, nucleic acids, polysaccharides and even viruses have
been separated in various experiments which have used different types of gels or
glass granules.
● It the most satisfactory method for separating DNA (from bacteria. usually Gram
positive) from the invariable contaminants the teichoic acids.
● Removal of salts and small molecules from macromolecules.
● Dilute solutions of macromolecules with molecular weights higher than the exclusion
limit may be readily concentrated by utilizing the hygroscopic nature of the dry
gel. Sephadex G-200 absorbs 20 times its weight of water. although G-25 is preferred
for its rapid action. This treatment leaves the macromolecular solution concentrated
but at the same time unaltered in pH or ionic strength.
● Determination of molecular weight of macromolecules

58. Ion Exchange Chromatography
● Ion exchange may be defined as the reversible exchange of ions in solution with
ions electrostatically bound to inert support medium.
● This technique is extremely useful in the separation of charged compounds (even
uncharged molecules can be "charged" by variance of pH as we will see later).
● 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.

59. Types of Ion Exchangers
Cation
Exchanger
Anion
Exchanger
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.

60. Isoelectric Point: The isoelectric point (pI) is the pH at which a particular molecule
carries no net electrical charge.
If the pI of molecule is less than the pH of surrounding solution the molecule will
possess a negative charge
pI< pH= Negative charge on molecule
If the pI of molecule is greater than the pH of surrounding solution the molecule will
possess a positive charge
pI > pH = Positive charge on molecule

61. Materials used to prepare Ion exchange resins
Two main groups of materials are used to prepare ion exchange resins: polystyrene, and
cellulose. Resins made from both of these materials differ in their flow properties, ion
accessibility, and chemical and mechanical stability. Selection of one or the other type of
resin is done on the basis of compounds being separated.

62. Choice of Buffers

63. Cation Exchange Chromatography

64. Anion Exchange Chromatography

65. Example

68. Applications
● Ion exchange chromatography has been used for the separations of many vitamins,
other biological amines, and organic acids and bases.
● Amino acid analysis
● 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.
● Fast and effective method of water purification.
● Determining the concentration of trace metals.