Ion exchange chromatography is a powerful separation technique that separates components based on their charge. It involves using a stationary phase with charged groups that interact with the ionized groups on molecules in the mobile phase. The molecules are separated from a mixture as they compete for binding sites on the oppositely charged stationary phase. Common applications include purifying proteins, peptides, enzymes, and other biomolecules.

1. Mrs. Praveen Garg
VITS College, Satna
Ion Exchange Chromatography

2.  Chromatography is an important biophysical technique
that enables the separation, identification, and
purification of the components of a mixture for
qualitative and quantitative analysis.
 It is a powerful separation tool that is used in all
branches of science and is often the only means of
separating components from complex mixtures.
 Chromatographic separation is based on size, binding
affinities, charge, and other properties to separate
materials.
Introduction

3. Chromatography consist of two phases:
 Stationary phase: This phase is always composed
of a “solid” phase or “a layer of a liquid adsorbed on
the surface solid support”.
 Mobile phase: This phase is always composed of
“liquid” or a “gaseous component.”
 Molecule to be interact with the stationary and
mobile phase and separated according to their
interaction.
Chromatography Phase

4.  In this method, The electrostatic attraction between
oppositely charged ions.
 Many biological molecules like amino acid,
proteins, proteins have ionizable groups.
 They may carry a net positive or negative charge,
which can be utilized in separating mixture of
compounds.
 Ion exchange chromatography mainly carried out in
a column packed with a resin, which is act as
exchanger.
Ion Exchange Chromatography

5.  Ion exchange chromatography, adsorption based on
the properties of stationary phase, mobile phase and
solute molecule.
Stationary phase:
 Selection of a suitable ion-exchange matrix/resin is
the most important in ion exchange method.
 It is based on various factors: ion exchanger charge,
flow rate/sample volume and sample properties.
 Stationary phases consist of two structural elements-
 The charged groups: which are involved in the
exchange process.
 The matrix : on which the charged groups are fixed.

6. Ion exchange resin carries both the property of stationary
phase.
 Ion exchange resins are two types:
 Cation exchanger
 Anion exchanger
 Cation exchangers have negatively charge groups in the
resin and it attract positive charged molecules.
 Anion exchanger have positive charged groups in the
resin and it attract negatively charged molecules.
 Matrix or resin is a polymeric component, which can be
made up of cellulose, dextran, agarose, polyacrylamide,
silica etc.
 These polymer are suitable for purification of proteins.

8. Mobile phase (Eluent)
 Mobile phase or eluents consist of an aqueous solution
of a suitable salt or mixtures of salts with a small
percentage of an organic solvent are used in which
most of the ionic compounds are dissolved.
 Sodium chloride is most widely used for protein
separation due to has no important effect on protein
structure.
 The salt mixture can act as buffer or a separate buffer
can be added to the eluent if required.
 Nature and concentration of the competing ions and pH
of the eluent are the most important properties affecting
the elution characteristics of solute ions.

9.  Elution of the solute is influenced by the eluent
flow-rate and the temperature.
 Faster flow rates cause to lower elution volumes
because the solute ions have less opportunity to
interact with the fixed ions.
 Temperature has less impact, which can be change
according to ion exchange material type.
 Enhancement of the temperature increases the rate
of diffusion within column, leading to increased
interaction with the fixed ions and therefore larger
elution volumes.

10.  Additives which are protective agents found in the
mobile phase, generally used for maintain structure
and function of the proteins to be purified.
Commonly used eluent additives
 EDTA; Ethylenediamine tetraacetic acid
 Polyols; Glycerol, glucose, and saccharose
 Detergents;
 Urea and guanidinium chloride;
 Lipids;
 Organic solvents;
 Zwitterions;

11.  Anionic exchange Chromatography should be carried
out with cationic buffers.
 Cationic exchange Chromatography should be
carried out with anionic buffers.
 The pK of the buffer should be as near as possible to
the pH at which the system is buffered.
 This results in high buffer capacity, which can
withstand the local changes of pH in the column
easily.
Choice of buffer

12. Ion Exchange Chromatography:
 The most popular method for the purification of
proteins and other charged molecules is ion
exchange chromatography.
 Separation is based on electrical charges of the
molecles.
 In cation exchange chromatography positively
charged molecules are attracted to a negatively
charged solid support.
 In anion exchange chromatography, negatively
charged molecules are attracted to a positively
charged solid support.
Principle

13.  Ion exchange chromatography involves separation of
ionic and polar analytes using chromatographic
supports derivatized with ionic functional groups that
have charges opposite that of the analyte ions.
 The analyte ions and charged ions of the eluent
compete to bind to the oppositely charged ionic
functional group on the surface of the stationary
phase.
 Net surface charge of all molecules with ionizable
groups is highly pH dependent.
 Therefore, pH of the mobile phase should be selected
according to the net charge on a protein of interest
within a mixture.

15.  In ion-exchange chromatography separations have
been performed in the open-column mode.
 Column which is loosely packed with stationary phase
as small particles of resin made of 1-2 cm diameter
glass.
 The mobile phase or eluent contains the competing ion
and is passed continuously into the column and
percolates through it down.
 Sample mixture is applied to the top of the column and
allowed to pass into the bed of ion- exchange material.
 Eluent flow is then resumed and fractions of eluent are
collected at regular intervals from the column outlet.

18. General components of an ion-exchange chromatography
are presented as below:
 A high pressure pump with pressure and flow indicator,
to deliver the eluent
 An injector for introducing the sample into the eluent
stream and onto the column
 A column, to separate the sample mixture into the
individual components
 A detector, to measure the analyte peaks as eluent from
the column
 A data system for collecting and organizing the
chromatograms and data.

20.  Ion exchange chromatography can be applied for the
separation and purification of many charged or ionizable
molecules such as proteins, peptides, enzymes, nucleotides,
DNA, antibiotics, vitamins and etc.
 Ion exchange chromatography, which is also known as
adsorption chromatography, is a useful and popular method
due to its;
 high capacity,
 high resolving power,
 mild separation conditions,
 versatility and widespeared applicability,
 tendency to concentrate the sample
 relatively low cost
Application