This document discusses various chromatography techniques used to purify biomolecules, including affinity chromatography, ion exchange chromatography, size exclusion chromatography, and reversed phase chromatography. It provides details on how each technique separates molecules based on specific properties, such as biological recognition (affinity chromatography), charge (ion exchange chromatography), size (size exclusion chromatography), and hydrophobicity (reversed phase chromatography).

1. Purification
Techniques:Chromatography
Mr.Halavath Ramesh
Department of chemistry
Loyola College –Chennai
University of Madras
E-Mail: halavathramesh39@gmail.com

2. Introduction
Biomolecules are purified using purification techniques that separate according to differences in
specific properties .
Property Technique
1. Bio-recognition( Ligand specificity) …….Affinity Chromatography
2. Charge ……..Ion exchange chromatography
3. Size …..Gel filtration (some times called size
exclusion)
4. Hydrophobicity …… Hydrophobic interaction
chromatography, Reversed phase
chromatography

3. Affinity Chromatography
Affinity chromatography separates proteins on the basis of
a reversible interaction between a protein ( or group of
proteins) and a specific ligand coupled to a chromatography
matrix. Affinity chromatography is unique in purification
technology since it is the only technique that enables the
purification of a biomolecules on the basis of its biological
function or individual chemical structure. The technique can
be used to separate active biomolecules from denatured or
functionally different forms, to isolate pure substances present
at low concentration in large volumes of crude sample and also
to remove specific contaminants.

5. Ion exchange chromatography
Ion exchange chromatography is a techniques that is commonly used in
bio-molecule purification. It involves the separation of molecules on the
basis of their charge. This technique exploits the interaction between
charged molecules in a sample and oppositely charged moieties in the
stationery phase of the chromatography matrix. This type of separation is
difficult using other techniques as charge is easily manipulated by the pH
of buffer used.
Two types of ion exchange separation is possible - Cation
exchange and anion exchange. In anion exchange the stationary phase is
positively charged whilst in Cation exchange it is negatively charged. It
involves the four stages 1. Equilibrium 2. Sample application and wash 3.
Elution 4. Regeneration.
In ion exchange chromatography adsorption and desorption processes are
determined by the the properties of the three interaction entities:
1. The stationary phase 2. Mobile phase(Eluent) 3.The solute.

9. Size exclusion chromatography
Size exclusion chromatography (SEC) also called gel-filtration or gel-
permeation chromatography(GPC) uses porous particles to separate
molecules of different size. When an aqueous solutions is used to transport the
sample through the column the techniques is known as Gel filtration
chromatography. A mixture of molecules dissolved in liquid (the mobile phase
) is applied to a chromatography column which contains a solid support in the
form of microscopic spheres or beads( the stationary phase).
Buffer: The liquid used to dissolve the biomolecules to make the mobile phase
is usually called a Buffer.
Still, the use of the hydrodynamic volume, a size based on dynamical
properties, in the interpretation of SEC data is not fully understood. This is
because SEC is typically run under low flow rate conditions where
hydrodynamic factor should have little effect on the separation.

11. Reverse phase chromatography
Reversed –phase chromatography (also called hydrophobic
chromatography). Reversed-phase chromatography is a technique using alkyl chains
covalently bonded to the stationary phase particles in order to create a hydrophobic
stationary phase, which has a stronger affinity for hydrophobic or less polar compounds.
The use of a hydrophobic stationary phase is essentially the reverse of normal phase
chromatography, since the polarity of the mobile and stationary phases have been
inverted – hence the term reversed-phase chromatography. Reversed-phase
chromatography employs a polar (aqueous) mobile phase. As a result, hydrophobic
molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase,
and hydrophilic molecules in the mobile phase will pass through the column and are
eluted first.

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