UNIT-V

IONEXCHANGE CHROMATOGRAPHY

Pointsto be covered in this topic

Introduction

& Classification

lon exchange resins

’ Properties
* Mechanism of ion exchange resins

’* Factor affecting ion exchange

Methodology
’% Applications
 ION EXCHANGE CHROMATOGRAPHY

* INTRODUCTION
lon exchange chromatography can be defined as a reversible process in
which of similar charged are exchanged between solid and liquid.

The solid is known as an ion exchanger. It is an adsorption

chromatography, a useful and popular method for separation of a
mixture of similar charged substances into pure components. It is also
known as cation anion exchange chromatography.
DEFINATION
These are the substances capable of exchange of ions with the
electrolytic solution.
These are porous solid, swelling in water without dissolving in it. The most
common properties of all ion exchanger are as follows:
a) They are almost insoluble in water and organic solvents.
b) They are complex in nature (Polymeric in nature)
c) They have active or counter ions that will exchange reversibly with
other ions in a surrounding solution without any substantial change
in the mnaterial.
TYPE OF ION EXCHANGERS
There are three classes of ion exchangers, these includes
1. Resins
2. Gels

3.Inorganic exchangers
OResins
" Resins are amorphous particles of organic
CH=CH,
materials which arecomposed of polystyrene CH=CH2
and divinely benzene.
Polystyrene contains sites for exchangeable
functional groups. CH=CH2
" Divinyl benzene acts as across linking agents Styrene Divinyl benzene
"and offers adequate strength i. e., mechanicalstability.
> Classification of resins
L. According to their chemical nature, they can be classified
as:

a) Strong cation exchange resin: These types of resins are useful for the
chromatographicseparation of amino acids, rare earths and other
substances that contains sulphonic acid groups as the ionisable
groups.
b) Weak cation exchange resin: These resins are based on polymers of
methacrylic acid and possess carboxyl groups.
c) Strong anion exchange resin: These resins with positively charged
quaternary ammonium groups attached to cross linked polystyrene
frame work belong to this class. Trimethyl ammonium groups are used
for this resin.
d) Weak anion exchange resin: The tertiary amine resins and polyamine
resins having amixture of primary, secondary and tertiary amine
groupson the polystyrene net work are well known.
II. According to the source,they are classified as
1. Natural source: These are of two types
a) Cation: E.g: Zeolites, clay etc.
b) Anion: E.g: Dolomite
2. Synthetic source: These are of two types
a) Inorganic resins
b) Organic resins : Most widely used
III. Structural types of ion exchange resins
1. Pellicular type with ion exchange film: The particle size is ranging
from 30 -40 mm with 1- 2mm film thickness. These have verylow ion
exchange capacity to separate the ions. Their ion exchange efficiency
is 0.01- 0.1 meq/g of ionexchange capacity.
2. Porous resin coated with exchanger beads:The particle size ranges
 from 5 - 10m. They are totally porous in nature and highly efficient. Their
exchange capacities are from 0.5 -2meg/g of ion exchange resin.
3. Macro reticular resin bead: A reticular network of the resin is seen
superficially on the resin beads. They are not highly efficient and have
very low ion exchange capacities.
4. Surface sulfonated and thermostatically bonded with anion: It is less
efficient and also has less exchangeable capacity.
Ion exchange ilm Axion resin beads Macropores

Inertcore Acttve apacity

(4) (2) (3) (4)

Structural types of ion exchange resins.

> Propertiesof ion exchange resin

It must be chemically stable.
It should be insoluble incommon solvents.
The resin must be sufficiently hydrophilic.
It should have a sufficient degree of cross linking.
The swollen resin must be denser than water.
tcontain sufficient numbersof ion exchange groups.
2. Gels:
lon exchange gels are used for the separation of large molecules like
proteins, nucleicacids.
These are much softer than polystyrene resins
Dextron and its relatives are called as gels
Cellulose and dextran ionexchangers which are polymersof sugar glucose
possess large pore sizes and lower charge densities.
3.Inorganic exchangers
The combinations of hydrous oxides of highly charged ions, with
oxide more acidic than the other have been found to have ion exchanging
properties.
The amorphous precipitates have higher exchange capacities than the
crystalline compounds because of the greater surface area of the
former type of compounds.
E.g.: Titanium Arsenic has been used to absorb alkaloids, Hydrous
antimony peroxide has
been used to study exchange equilibrium of K and R, ions with
hydrogen and other ions.
MECHANISM OF IONEXCHANGE PROCESS
The mechanism of separation isby reversible exchange of ions between
the ions present in the solution and those present in the ion exchange
resin.
" lon exchange separations are mainly carried out in columns packed with
an ion exchanger.
There are two typesof ion exchanger, as follows:
a) Cationicexchangers:
It possesses negatively charged groups and these will attract positively
charged groups. These exchangers are also called acidic ion exch.ange
materials since their negative charges result from the proteolys1s ot
acidic groups.
Commonly used cation exchange resins are S-resin, sulfate deriv .Ilives:
andCM resins,carboxylate derived ions

Resin -CH2-SO Resin -0-CH:-C

(S-Cation exchanger ) (CM-Cation exchanger
b) Anionic exchangers:
It has positively charged groups, which will attract negatively charged
molecules.
This exchanger is termed as basic ion exchange materials since their
positive charges generally result from the association of protons with
basic groups.
Based upon the affinity of ions towards the matrix the ions like cation
and anion are separated. The ions that have less affinity towards
matrices will elute first and the ions that have more affinity towards
matrices it willelute later.
Commonly used anion exchange resins are Q-resin, a Quaternary amine;
and DEAE resin, DiEthyl Amino Ethane.

ÇH: ÇH:-CH
Resin -N CH3 Resin
CH3
-CH:-CH:NH,
CH:-CH,
(Q-Anion exchanger) DEAE-Anion exchanger)
The actual ion exchange mechanism is thought to be composed of five
distinct steps:
1. Diffusion of the ion to the exchanger surface. This occurs very quickly
in homogeneous solutions.
2. Diffusion of the ion through the matrix to the exchanger site. This is
dependent upon the degree of cross linkage of the exchanger and
the concentrationof the solution.
3. Exchange of ions at the exchange site occurs. This occur
instantaneously in an equilibrium process as follows:
Resin - SO;H + Na" Resins - S,Na + H*
Resin - N(CH;);OH +CI Resin -N(CH3);C1 + OH

4. Diffusion of the exchanged ion through the exchanger tothe surface

5.Selective desorption by the eluent and diffusion of the molecule into
 theexternal solution takes places
FACTORS AFFECTINGION EXCHANGE
The factor affectingionexchange are as follows:
a) Nature of ion exchange resin:
Cross linking & swelling is important factor which depends on the
proportion of cross linking agent (Divinyl benzene &polystyrene).
When more cross linking agent is present they are more rigid, but swells
less. When swelling is less , separation of ions of different sizes is
difficult as they nan not pass through the pores present & it becomes
selective to ions of different sizes. When less crosslinking agent is
present, they are less rigid but swells more.
When swelling is more separation will not be efficient as exchange of
functional groups does not take place due to wide pore hence
optimum quantity of cross linking agent should be added to the
polymericion exchange resins for the separation to be effective.
b) Nature of exchanging ions:
i) Valency of ions: At low concentration & at ordinary temperature
exchange increase with increase in valency.
Na< Ca2+< A|3+< Tht+
ii) Size of ions: For similar charged ions, exchange increases with decrease
the size of hydrated ion.
Li" <H* < Na < NHt
ii) Polarizability: Exchange is preferred for greater polarizable ion
|-< Br- < CI- <F
iv) Concentration of solution: In dilute solution, polyvalent anions are
generally adsorbed preferentially.
v)Concentration &charge of ions: If resin has higher +ve charge &solution
has lower tve charge, exchange is favoured at higher concentration. If the
resin has lower +ve charge and solution has high +ve charge, then exchange
is favoured at low concentration.
 METHODOLOGY
General components and methodology of an ion-exchange
chromatography are as follows
Elution column

Positive molecules
Negative molecules
High salt
Positively charged
gel beads of
charged DEAE
cellulose

Negatively charged Bound proteins are

proteins bind at low eluted at high salt
salt concentration concentration

i)Column:
Column used in the laboratories are made up of glass but those used in
industries are made up of either high quality stainless steel or polymer,
which are resistant to strongacids and alkalis.
Thegeometry of column depends completely on the separation factor. The
separation is improved by increasing the length of the column but the
length cannot be increased beyond a critical length.
" A Dimension of column is 20:1 to 100:1 for the higher effiiency can be
used.

ii) Packing of the column:

Inthis wet packing method is used.
The resins is mixed with the mobile phases and packed in the column
 The sample to be separated is dissolved in the mobile phases and
introduced all at once into the column.

iii) Application of the sample:

After packing the column,the solution to be analyzed is added to the top of
the column and allowed to pass through the bed of ion exchanger.
For thispurpose the syringe or pipette is utilized.
iv) Mobile phase:
The organic solvents are less useful so they are not used these days.
Only diferent strength of acids, alkalis and buffer are used as eluting
solvent. E.g.: 0.1 NHCI, 1N NaOH, phosphate buffers, acetate buffers,
borate buffers, phthalate buffers, etc.
v) Developments ofthe chromatogram and elution:
" After introduction of the sample, development of the chromatogram is
done by using different mobile phases.
The aqueous salt solution is adjusted to a constant ionic strength.
The choice of the mobile phase depends on the selectivity of the resin for
the solute ions. Following two types of elution techniques are used:
a) In Iso cratic elution technique, the same solvent composition is used
i.e., same strength of acids or alkalis or buffers are used.
b) In gradients elution techniques,initially less acidic or basic mobile
phase is used. Then, acidity or basicity is increased at regular
intervals. The different fraction of the elution is collected volume
wise or time wise and analyzed.
vi)Analysis of the elute or Detection:
Different fractions are collected with respect to the volume or time is
analyzed for their contents.
Several methods of analysis can be used which depends upon the nature
and quantity of the ionic species are:
nductometric method
Amperometric methods
Flame photometric method
 UV. Spectroscopy
Radiochemical methods using Geiger Muller counter, ionization chamber
method.
vii) Regeneration of ion exchange resin:
" The ion exchange resin after separation may not be useful for next
separationas exchange functional groups are lost. But due tothe cost
of ion exchange resins, they cannot be disposed off. Hence reactivation,
regeneration of the resins is mostimportant.
It refers to the replacement of the exchangeable cations or anions
present in the resin.
The charging of the column with strong acid like hydrochloric acid is used
for regeneration of the cation exchange resin while strong alkali like
sodium hydroxide or potassium hydroxide used for regeneration of
the cation exchange resin.
APPLICATIONS
1. Softening of hard water: Hardness of water is due to the presences of
Cal", Mg? and other divalent ions may be removed by passing the hard
water through the cation exchanger chargedwith Na ions.
2.Complete demineralization of water: This requires complete removal of
ions i.e., both cations and anions. For this, water is passed through an
acidic cation exchanger then metallic cations are exchanged with H*
ions.
3. Purification of organic compounds: Many natural products extracted in
water have been found to contain ions originally present in water. Those
ions can be removed by using ion exchange process.
4. Separation of amino acids: lon exchange methods can be used to separate
the complex mixture of 18 amino acids obtained by the acid hydrolysis
of proteins.
5. Purification and recovery of pharmaceuticals: The process is used for
purification and recovery of antibiotics, vitamins, alkaloids, hormones and
other chemicals of pharmaceutical importance during their
 manufacturing process.
6. Biochemical separations: Used for biochemical separations like some
drugs or metabolites from blood, urine or other biological fluids.
7. Other applications:
For the measurement of various active ingredients in medicinal
formulations.
For the measurement of drugs and their metabolites in serum and
urine, for residue analysis in food raw materials.
For the measurement of additives such as vitamins and preservatives
in food and beverages.
 UNIT-V
GEL CHROMATOGRAPHY

Points to be covered in this topic

& Introduction

Theory
Instrumentation

$ Application
 GEL CHROMATOGRAPHY

INTRODUCTION
Gel chromatography also called as Gel permeation chromatography,
Exclusionchromatography and Molecular sieve chromatography.
It is a simple and reliable method for separating molecules according to
their size.
It uses a porous material as the stationary phase &a liquid as a mobile
phase. The diameter of the pores of the porous material are of the order
50-300 A°, which is similar to the size of many molecules.
The molecules penetrate the pores according to their size. Small
molecules penetrate more rapidly than larger molecule.
The result is difference in the rate at which the molecule pass down the
column, the smaller molecules travelling faster than the larger
moleculeand become separated.
PRINCIPLE
A
mixture of molecules dissolved in liquid (the mobile phase) is appliedto a
chromatography column which contains a solid support in the form of
microscopic spheres, or "beads" (the stationary phase).
The mass of beads within the column is often referred to as the column bed.
The beads act as "traps" or "sieves" and function to filter small molecules
which become temporarily trapped within the pores. Larger molecules pass
around or are "excluded"from the beads.
Large sample molecules cannot or can only partially penetrate the pores,
whereas smaller molecules can access mostor allpores.
" Thus, large molecules elute first, smaller molecules elute later, while
molecules that can access allthe pores elute last from the column. Particles
of different sizes will elute (filter) through a stationary phase at different
rates.
 Gel Permeation Chromatography(GPC)
(A) (B) (C) (D) Solvent
flow
Small permeating
molecules
Sample
Large excluded mixture
molecules

Porous Gel
Bead
Large
molecules
elute first

CHROMATOGRAM

B C D
RETENTION TIME

Salt

-Large protein
Small protein
-Gel particle
 THEORY
Total volume of column packed with a gel that has been swelled by water
orother solvent is given by following equation:
V,= V,+ VI+ Vo

Where,V, = Total bed volume

V=Volume occupied by solidmatrix of gel
V,= Volume of solvent held in pores or interstices
V, = Free volume outside the gel particles
If conditions are assumed such that time taken for solute molecules to
diffuse into pore is less as compared totime spent by molecule near pore
and Separation process independent of diffusion process then under these
conditions:
V= V, + K¡. V,
V,=Volume of effluent flowing through column between point of sample
injection &sample emergence from column
K, =Distributioncoefficient
For large molecules: K, =0, V, = V,,
For molecules that can penetrate all the pores: Kd = 1, V, =V,+V,
INSTRUMENTATION
The important practical requirements for gel chromatography are as
follows: Chromatogram
Sample

Solvent Sample
delivery
pump
injecetorR Detector

GPC Column

Mobile phase
Instrumentation of Gelchromatography
1. Stationary Phase:
"Stationary Phase is Semi-permeable, porous beads with well defined
range of pore sizes.
" Beads are cross-linked polymers.
Smaller pore sizes are used for rapid desalting of proteins or for protein
purification. Intermediate pore sizes are used to separate relatively small
proteins. Very large pore sizes are used for purification of biological
complexes.
Stationary phase used for gel exclusion chromatography include
dextran, polyacrylamide and dextran poly acrylamide.
Properties of gel beads:
a) They should be chemically inert
b)They should be mechanically stable
c) They should have ideal and homogeneous porous structure (wide pore
size give low resolution).
d) They should have uniform particle and pore size.
e) The pore size of the gel must be carefully controlled.
2. Mobile phase
It composed of a liquid used to dissolve the biomolecules to make the
mobile phase permitting high detection response and wet the packing
surface.
The choice of mobile phase to be used in any separation will depend on
the type of separation to be achieved and component to be separated.
" The most common eluents in for polymers that dissolve at room
temperature. e.g. Tetrahydrofuran, Chloroform, Dimethyl formamide.
3. Column
"The column consist of astraight tube with abed support at the bottom.
The bed support allows only the liquid to pass through without disturbing
the bed material.
The simplest column consist of a straight tube containing some glass wool
into the bottom. Theglass woolis then covered with thin layer of quartz
" or glass beds.
Commercially Available Columns
Analytical column- 7.5-8mm diameters.
Preparative columns-22-25mmfor.
Usualcolumn lengths-25, 30, 50, and 60
Recently, narrow bore columns- 2-3 mm diameter have been introduced,
which save time and solve.
4. Pump
"A highly constant flow rate has to be maintained during the entire
chromatogram. Achange of the flow rate of only 0.1% can cause an error in
molar mnass of up to10%.
" Most pumps can only reproduce the flow rate to 0.2-0.3%. In-line filters
in the solvent reservoir may prevent particles from coming into the pump
heads, which might damage the check valves or the pump seals.
" Various pump used are: Syringe pumps, Reciprocating pumps.
5. Detectors:
"Various type of detector used are asfollows
A. Concentration sensitive detectors
" Bulk Property Detectors- Refractive Index (RI) Detector
"Solute Property Detectors- Ultraviolet (UV) Absorption Detector
Evaporative Detectors- Evaporative Light Scattering Detector (ELSD)
B.Molar mass sensitive detectors
a) Light Scattering Detectors
Low Angle Light Scattering (LALS) Detectors
" Multi-angle Light Scattering (MALS) detectors
b) Viscosity Detectors- Differential Viscometers
Other ; Flame lonization Detector (FID), A Mass Spectrometer or
Fourier Transform Infrared (FTIR) Spectrometer (FTIR) Spectrometer
 APPLICATION
Main application are as follows:
1. Purification: The application of gel chromatography is the
fractionation & purification of biological macromolecule. ie. protein,
polysaccharide, nucleic acid etc.
2. Molecular weight determination: When a polymer sample of mixed
molecular weight flows down the column there will be a separation of the
solute according to their molecular weight. The chromatogram results
from a separation based on the size of the sample molecule. It represents
distinct molecular weight distribution.
3. Protein-building studies: It is used to study the reversible binding of a
ligand to a macromolecule such as protein including receptor protein.
4. Solution concentration: Solution of high molecular weight substances
can be concentrated by addition of dry Sephadex G-25.
5. Desalting: By use of a column of, Sephadex G-25, solutions of high
molecular weight compound may be desalted. The separation of large
molecule of biologicalorigin from inorganic &ionisable species is termed as
desalting
 UNIT-V
AFFINITY CHROMATOGRAPHY

Points to be covered in this topic

Introduction

Theory
Instrumentation

$ Applications
 AFFINITY CHROMATOGRAPHY

INTRODUCTION
Affinity chromatography is a method of separating biochemical mixtures
based on a highly specific interaction such as that between antigen
andantibody, enzyme and substrate or receptor andligand.
Affinity chromatography, also known as bioselective adsorption, is
protein purification technique. It is widely used as a means of
separation andpurification with specificproperties.
Biological macromolecules, such as enzymes and other proteins, interact
with other molecules with high specificity through several different
types of bonds and interaction. Such interactions include hydrogen
bonding, ionic interaction, disulfide bridges, hydrophobic interaction, and
more.

The high selectivity of affinity chromatography is caused by allowing the

desired molecule to interact with the stationary phase and be bound within
the column in order to be separated from the undesired material
which will not interact and elute first.
Themolecules no longer needed are first washed away with a buffer
while the desired proteins are let go in the presence of the elutingsolvent
(of higher salt concentration).
PRINCIPLE
The principleof affinity chromatography is as follows:
The stationary phase is first loaded into a column with mobile phase
containing a variety of biomolecules from DNA to proteins (depending
on the purification experiment).
" Then, the two phases are allowed to bind.
Awash buffer is then poured through a column containing both bound
phases.
The wash buffer removes non-target biomolecules by disrupting their
weaker interactions with the stationary phase.
 Injection Adsorption Washing out Elute out
of sample of target impurities Of target
2

Adsorption Washing Elution

out

Gel

Spacer Ligand Target materials

Target biomolecules have a much higher affinity for the stationary phase,
and remain bound tothe stationary phase, not being washed away by
wash buffer.
An elution buffer is then poured through the column containing the
remaining target biomolecules.
The elution buffer disrupts interactions between the bound target
biomolecules with the stationary to a much greater extent than the wash
buffer, effectively removing the target biomolecules.
This purified solution contains elution buffer and target biomolecules
and is called elution.

INSTRUMENTATION
" The important practical requirements for affinity chromatography are as
follows:

1.Matrix
The matrix is an inert support to which a ligand can be directly or
indirectly coupled.
The most useful matrix materials are agarose and polyacrylamide.
The matrix to be effective it must have certain characters which are as
follows:
a) Matrix should be chemically and physically inert.
b) It must be insoluble in solvents and buffers employed in the process.
c) It must be chemically and mechanically stable.
d) It must be easily coupled to a ligand or spacer arm onto which the ligand
can be attached.
e) It must exhibit good flow properties and have a relatively large surface
area for attachment.

2.Ligand
"It refers to the molecule that binds reversibly to a specific target
molecule.
" 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 tobe purified, a substrate analog, inhibitor, cofactor, or
effector may be used as a the immobilized ligand.
3.Solvents
The primary buffer in affinity chromatography is the one in which the
resides. This buffer should not degrade the matrix in any way. The buffer
should also have a reliable effect on the sample. The ideal buffer minimizes
nonspecific interactions while maximizing the specific interaction between
the sample and the ligand.
The other major solvent to consider in affinity chromatography is the
elution buffer.
The purpose of theelution buffer is to wash away unbound proteins initially
and at higher concentration release the desired protein from the ligand.
4.Spacer arms
"It is used to improve binding between ligand and target molecule by
overcoming any effects of steric hindrance.
 "Since the success of affinity chromatography resides in its ability to bind an
active site to its corresponding ligand, if the protein binding region cannot
join with the immobilized ligand the technique is effectively useless.
" Steps involving in affinity chromatography

Mal.
1. Loading of affinity column 2. Proteins sieve through matrix of
affinity beads

4. Wash off proteins that do not bind.

3. Proteins interact with affiuty
ligand with some binding loosels
and others tightly

6. Elute proteins that bind tightlv to

5. Wash off proteins that bind loosely ligand and Collect purified protein of
interest
 APPILICATIONS
1. Proteinpurifying
" Affinity chromatography has a large range of protein purifying
applications. Extra cellular and other receptor proteins can also be
purified by affinity chromatography.
It allows protein purification in arelatively short amount of time with a
high yield.
2. Isolation of enzyme
Enzymes can be isolated by a host at different ligands fit for bioselective
adsorption. For example, adenosine monophosphate (AMP) can be
immobilized and used to bind those proteins exhibiting an affinity for
AMP, ADPor ATP.
3. Immobilized Metal Affinity Chromatography In Proteomics
" It has been proved that the progress of proteomics is mostly determined by
the development of advanced and sensitive protein separation
technologies.
Immobilized metal affinity chromatography (IMAC) is a powerfulprotein
fractionation method used to enrich metal-associated proteins and
peptides
4. Affinity Chromatography To The Study Of Drug-Melanin
Binding Interactions
" Affinity chromatography using chromatographic stationary phases based on
physically adsorbed or chemically bonded melanin provides a useful
tool for studying the interactions of small molecules and metal ions with
melanin
5. Purification of lectins by Biospecific affinity
chromatography
Biospecific adsorbents which can be used for the purification of lectins are
easily prepared by a one-step reaction between Epoxy-activated
Sepharose 6 Band Lectin-specificsugars.
6. Purification of plasma proteins for therapeuticuse
Affinity chromatography is apowerful technique for the purification of
many proteins in human plasma. It is being used in the production of various
licensed therapeutic plasma products, such as : Factor VIII, Factor IX, Von
Willbrand Factor, Protein C, Antithrombin III, and Factor XI.