ion exchange chromatography, principle and application

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