1. S.GOKULAKRISHNAN
M.Pharm (Pharmaceutics) – I Year,
Department of Pharmaceutics,
College of Pharmacy,
Mother Theresa Post Graduate and Research Institute of Health Sciences,
(A Government of Puducherry Institution)
Puducherry.
AFFINITY CHROMATOGRAPHY
GOKULAKRISHNAN CHROMATOGRAPHY 1

2. HISTORY OF AFFINITY
CHROMATOGRAPHY
• 1930s, first developed by A.Wilhelm Tiselius-a swedish
biochemist, won the Nobel Prize in 1948.
• Used to study enzymes and other proteins.
• Relies on the affinity of various biochemical compounds with
specific properties.
GOKULAKRISHNAN CHROMATOGRAPHY 2

3. AFFINITY CHROMATOGRAPHY:
Discovered by Pedro and Meir Wilcheck.
Affinity chromatography is a type of chromatography
that makes use of a specific affinity between a substance
to be isolated and a molecule that it can specifically
bind.
GOKULAKRISHNAN CHROMATOGRAPHY 3

4. PRINCIPLE
Based on specific affinity between substance to be isolated and a
molecule that it can specifically bind(a ligand).
M + L ML
Macromolecule Ligand (attached Complex to matrices)
GOKULAKRISHNAN CHROMATOGRAPHY 4

5. EXAMPLES
 Antigen -Antibody
 Antibody- Antigen
 Substrate -Enzyme
 DNA- Histon
 Hormone -Binding Protein/Receptor
GOKULAKRISHNAN CHROMATOGRAPHY 5

6. SPECIFICITY OF AFFINITY
CHROMATOGRAPHY
Specificity is based on three aspect of affinity:
Matrix: for ligand attachment.
Spacer arm: used to bind ligand to matrix.
Ligand: molecule that binds reversibly to a specific
target molecule(site of interaction).
GOKULAKRISHNAN CHROMATOGRAPHY 6

7. PROCEDURE
 The Sample is injected into the equilibrated affinity
chromatography column.
 Only the substance with affinity for the ligand are
retained on the column.
 The substance with no affinity to the ligand will elute
off.
 The substances retained in the column can be eluted
off by changing the pH of salt or organic solvent
concentration of the eluent.GOKULAKRISHNAN CHROMATOGRAPHY 7

8. GOKULAKRISHNAN CHROMATOGRAPHY 8

9. MATRIX
The matrix is an inert support to which a ligand can be directly
or indirectly coupled.
It has some peculiar qualities like:-
 Does not itself adsorb molecules to a significant amount.
 Ligand must be coupled without altering its binding
properties.
 Stability under a wide range of experimental conditions
such as high and low pH, detergent and dissociating
conditions.
 The most useful matrix materials are agarose and
polyacrylamide GOKULAKRISHNAN CHROMATOGRAPHY 9

10. SUPPORT MATERIALS USED IN
AFFINITY CHROMATOGRAPHY
GOKULAKRISHNAN CHROMATOGRAPHY 10
SUPPORT MATERIALS TRADE NAME
Agarose Sepharose 2B,4B,6B
Cross-linked dextran Sephadex
Polyacrylamide Bio-gel-P
Cross linked cellulose Matrex Cellufine

11. LIGAND:
The ligand is the molecule that binds reversibly to a
specific molecule or group of molecules ,enabling purification
by affinity chromatography.
The selection of the ligand for affinity chromatography is
influenced by two factors:
Ligand must exhibit specific and reversible binding affinity
for the target substance(s)
It must have chemically modifiable groups that allow it to be
attached to the matrix without destroying binding activity.
GOKULAKRISHNAN CHROMATOGRAPHY 11

12. COMMENLY USED LIGANDS
GOKULAKRISHNAN CHROMATOGRAPHY 12
LIGAND AFFINITY
Concanavalin A Glycoproteins and polysaccharides
Calmodulin Calmodulin binding enzymes
Avidin Biotin containing enzyme
Heparin
Lipoproteins, lipases , coagulation factors,
DNA
polymerases , steroid receptor proteins
serine protease inhibiter
Proteins A and G Immunoglobins

13. TYPES
 Lectin Affinity Chromatography
 Immuno Affinity chromatography
 Metal Chelate Chromatography
 Dye Ligand Chromatography
 Covalent chromatography
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14. LECTIN AFFINITY CHROMATOGRAPHY
 Used for purification of glycoproteins particularly membrane receptor
proteins.
 Lectins are a group of proteins produced by plants & animals, which have the
ability to bind carbohydrates and glycoproteins.
 Used to separate mixtures of cells by taking advantage of the saccharide
components of their outer membrane.
 Commonly used lectins are:
ConcanavalinA,
Soyabean lectin,etc.
GOKULAKRISHNAN CHROMATOGRAPHY 14

15. IMMUNO AFFINITY CHROMATOGRAPHY
 Exploited in the isolation & purification of a range of proteins including
antigens, membrane proteins of viral origin.
 Used for purification of antibodies. Ligands used is Protein A and protein B.
METAL CHELATE CHROMATOGRAPHY
 Special form of chromatography in which an immobilised metal ions such as
Cu 2 + ,Zn2 +,Mn2 +,Ni2 + etc. are used.
 Used for purification of proteins containing imidazole groups or indole
groups.
 Commonly metal ions are immobilised by attachment to an imino-diacetate or
tris(carboxymethyl)ethylenediamine substituted.
GOKULAKRISHNAN CHROMATOGRAPHY 15

16. DYE LIGAND CHROMATOGRAPHY
 Uses a number of triazine dyes as ligands. Most widely used dye is Cibracron
Blue.
F3G-A.
Used for purification of lipoproteins, interferons, coagulation factors etc.
COVALENT CHROMATOGRAPHY
 Developed specifically to separate thiol containing proteins.
 Most commonly used ligand is a disulphide 2’-pyridyl group.
 Used for purification of a number of proteins but its use is limited by its cost
and rather difficult regeneration stage.
GOKULAKRISHNAN CHROMATOGRAPHY 16

17. AFFINITY CHROMATOGRAPHY
Can be used,
 Purify and concentrate a substance from a mixture into a buffering
solution.
 Reduce the amount of a substance in a mixture.
 Discern what biological compounds bind to a particular substance,
such as drugs.
 Purify and concentrate an enzyme solution.
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18. APPLICATIONS
Used in Genetic Engineeringnucleic acid purification
Production of Vaccinesantibody purification from
blood serum
Basic Metabolic Researchprotein or enzyme
purification from cell free extracts.
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19. ADVANTAGES OF AFFINITY
CHROMATOGRAPHY
 Extremely high specificity.
 High degrees of purity can be obtained.
 The process is very reproducible.
 The binding sites of biological molecules can be simply
investigated.
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20. DISADVANTAGES OF AFFINITY
CHROMATOGRAPHY
 Expensive ligands
 Leakage of ligand
 Degradation of the solid support
 Limited lifetime
 Non-specific adsorption
 Relatively low productivityGOKULAKRISHNAN CHROMATOGRAPHY 20

21. REFERENCE
 Hand book of Affinity chromatography, principles and
Method from GE Healthcare
 Practical Biochemistry, Principles and techniques by Keith
Wilson and John Walker, Cambridge University Press
 Affinity Chromatography: A Review ,Journal of Pharmacy
Research;May2011, Vol. 4 Issue 5, p1567
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