Introduction Principle Procedure Elution Types Application Limitation Conclusion

Chromatography:Chromatography is a collective term for a set of laboratory

techniques for the separation of mixtures.Affinity chromatography: Discovered by Pedro Cuatrecasas 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.

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)

KINETICS IN AFFINITY CHROMATOGRAPHY:

.

COMPONENTS OF AFFINITY PHASE:-

Matrix: for ligand attachment. Matrix should be

chemically and physically inert.

Spacer arm: used to improve binding between ligand

and target molecule by overcoming any effects of

steric hindrance. Ligand: molecule

that binds reversibly to a specific target molecule or

group of target molecules.

The matrix is an inert support to which a ligand can be directly or indirectly coupled.

It has some peculiar qualities like:-a. Does not itself adsorb molecules to a significant

amount.b. Ligand must be coupled without altering its binding

properties.c. 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 .

SUPPORT MATERIALS TRADE NAME

Agarose Sepharose 2B,4B,6B

Cross-linked dextran Sephadex

Polyacrylamide Bio-gel-P

Cross linked cellulose Matrex Cellufine

Agarose/Polyacrylamide Ultrogel

Silica Hypersil WP 300, Ultrasphere, Zorbax

Methacrylate Eupergit

Polystyrene Poros-50

Dextran/Polyacrylamide Sephacryl

• To prevent the attachment of the ligand to the matrix interfering with its ability to bind the macromolecules.

• The optimum length~ 6-10 carbon atoms or their equivalent.

• More commonly used for small immobilized ligands.

• Example:-1,6-diamino hexane and 6-amino hexanoic acid.

COUPLING AGENT NAME OF MATRIX LIGANDCyanogen-bromide Agarose enzymes, coenzymes,

antigens, antibodies, nucleic acid , proteins etc.

6-aminohexanoic acid;1,6-diaminohexane

Agarose Ligands having a free amino or carboxyl group

1,4-bis-(2,3-epoxypropoxy)butane

Agarose Sugars , carbohydrates, ligands with hydroxyl ,amino or thiol group.

2-thiopyridyl Agarose Thiol compound or sulfur containing proteins

Carboyldiimidazole Agarose N-nucleophiles

Aminoethyl ; hydrazide Polyacrylamide Carboxyl and amino ligands

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:

1. ligand must exhibit specific and reversible binding affinity for the target substance(s)

2. It must have chemically modifiable groups that allow it to be attached to the matrix without

destroying binding activity.

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, growth factors, serine protease inhibiter

Proteins A and G Immunoglobins

Poly (A) RNA containing poly (U) sequences , some RNA specific proteins

Lysine r RNA

Cibacron Blue F3G-A Nucleotide-requiring enzymes , coagulation factors

Fatty acids Fatty-acid binding proteins

Nucleotides:5’-AMP 2’,5’ -ADP

NAD+ -dependent dehydrogenases , some kinasesNADP+ -dependent dehydrogenases

Acriflavin Nucleotides

Addition of free ligands Introducing another protein

Lectin Affinity ChromatographyImmuno Affinity chromatographyMetal Chelate ChromatographyDye Ligand ChromatographyCovalent 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.

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 protirn G.

Special form of chromatography in which an immobilised metal ions such as Cu2+ , 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 agarose.

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.

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.

Purification of substances from biological mixture.

Separation of native from the denatured form of protein.

Purify and concentrate an enzyme in a solution. Purification of immunoglobulin. Purification of small amount of biological

materials from high levels of contaminating substances

Single step purification. The matrix can be reused rapidly. The matrix is a solid, can be easily washed and

dried. Give purified product with high yield. Affinity chromatography can also be used to

remove specific contaminants, such as proteases.

Non-specific adsorption can not be totally eliminated, it can only be minimized.

Limited availability and high cost of immobilized ligands.

Proteins get denatured if required pH is not adjusted.

Affinity chromatography is one of the most reliable chromatographic techniques.

This technique offers high selectivity, hence high resolution & usually high capacity for proteins of interest.

It is theoretically capable of giving absolute purifications even from complex mixtures in a single process.

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