Affinity Chromatography. principle, instrumentation

1. AFFINITY CHROMATOGRAPHY
Presented by : Dr. Vijaya U. Barge
(Vice Principal & Professor )
Pune District Education
Association’s Shankarrao Ursal
College of Pharmaceutical
Sciences & Research Centre.

2. Learning Objectives :
 After completing this sub-unit the students should be able:
• To study the principle involved in Affinity chromatography.
• To study the components of affinity medium.
• To study the steps involved in Affinity chromatography.
• To study the applications of Affinity chromatography.

3. Contents :-
• Introduction
• Principle
• Types of affinity chromatography
• Theory
• Advantages
• Disadvantages

4. Introduction :-
 Affinity chromatography :-
 It is a unique technique in the sense that it is based on the specific biological function of
the biomolecules of interest.
 This feature also makes affinity chromatography suitable for the selective separation of
active biomolecules and their isolation from the inactive or denatured forms .

5. Principle :-
 Affinity chromatography involves the covalent attachment of an immobilized
biochemical called affinity ligand to a solid support. When sample passed
through the column , only a solute that selectively binds to the complementary
ligand is retained ; other sample components elute without retention.
 The retained solutes can be eluted from the column by changing the mobile
phase condition
 By changing mobile phase composition means change in pH or ionic strength of
the molecules of interest can be isolated in pure form.

6. Diagram :-
 Fig. principal of affinity chromatography

7. Types of affinity chromatography :-
 Based on the nature of receptor present on matrix are as follows :-
 1. Bio-affinity chromatography.
 2. pseudo affinity chromatography.
a. Dye-affinity chromatography
b. Metal-affinity chromatography

8.  1.bio-affinity chromatography :- In this type of affinity chromatography ,
biomolecules used as receptor present on matrix and it has the biological
phenomeonon such as antibody – antizen enzyme substrate or enzyme inhibitor.
 2.pseudo –affinity chromatography :-
 a.Dye-affinity chromatography :- In this chromatography dyes are utilizes as
ligands in order to target protein.
 B.metal-affinity chromatography :- this is the most widely used method to purify
the proteins according

9. Theory :-
 By affinity chromatography highly selectivety separation of bio-molecules can be
achived throught their specific interaction.
 The separation exploit the “lock and key” binding that prevalent in bio logical system
 The stationary phase for affinity chromatography is solid such as agarose to which the
affinity ligand is immobilised.
 Agarose is very popular because of its porous meshwork and it permits the high
degree of ligand substitution and is more accessible to larger molecules.
 The ligands fixed to the stationary phase reversibly binds the desired biomolecule
present in the mobile phase. This material can be eluted from the column by changing
the composition of the mobile phase.

10. List of some of the commonly used interactions in affinity chromatography :-
Enzyme Substrate analogue or inhibitor
Antibody Antigen ( virus , cells )
Nucleic acid Complementary nucleic acid
Nucleic acid Histone or other nucleic acid binding
protein
Hormone Hormone receptor
Glutathione Glutathione S-transferase (GST) fusion
protein
Metal chelate His-tag fusion protein

11. Instrumentation :-
1.STATIONARY PHASE :-
 Instrumentation of affinity chromatography is same as per the high performance
liquid chromatography.
 Stationary Phase has variety of materials like as organic gels such as beaded agarose
, cellulose, dextran, combination of these polymers. The basic requirements of
stationary phase.
1. the stationary phase should be sufficiently hydrophilic to avoid
nonspecific binding of solutes and stable to most water compatible organic solvents.
2. a porous stationary phase permits a high degree of ligand substitution and is more
accessible to larger molecules.

12. 2. Affinity ligand :-
 The affinity ligands can be antibodies , enzyme inhibitors or other molecules that
reversibly and bioselective bind to complementary analyte molecules in the
sample.
 Affinity ligands are of two types :-
 1.specific ligand :- specific ligands bind only to one particular solute. The
advantage of specific ligand is high selectivity by using short columns to perform
seperations by step elution in less than 1 min.

13.  2.Group specific :- Group specific ligand bind to certain group of solutes. These
can be separated in one step as a group or separated from each other using
isocratic or gradient elution techniques. For better separation longer or more
efficient columns must be used.

14. 3. Sample preparation :-
 The sample must be clear solution free from solid particles. This can be achieved
by centrifugation or filtration. Protein solutions be centrifuged at least 10000 g.
cell lysates should be centrifuged at 40-50000 g.
 The factors affecting the interactions between the desired target protein and the
matrix- bound ligand ,should also be determined.
 Sample components interfering with the target protein and/or ligand should be
removed before loading onto the column.

15. Equilibration with a buffer facilitating the specific interaction :-
 The chromatography column is washed with 3-4 column volumes of the starting
(binding) buffer. The sample must also be equilibrated with this starting binding
buffer (if necessary , via solvent exchange or dialysis).

16. binding of the molecule of interest and Wash Out of the unbound material
:-
 During sample loading, considered the strength of the interaction. In case of
high- affinity samples, a high flow rate may be applied. In case of a weak
interaction and/or slow equilibration process, reduce the rate of sample loading.
After sample application, the column should be further washed with binding
buffer until all unbound components are removed.
 Regeneration :- After successful completion of the elution, the column can be
washed with several column volumes of binding buffer, and it can be then be
reused. For long term storage, one must ensure that the column is not exposed to
bacterial or fungal infection. The toxic compound sodium azide can be used to
prevent such infections.

17. Advantages :-
 High specificity , reproducible , simple
 Target molecules can be obtained in a highly pure state
 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.

18. Disadvantages :-
• Time consuming method
• More amounts of solvents are required hence becomes expensive.
• Intense labour requirement
• Non-specific adsorption cannot 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.

19. Applications:-
• Separation of mixture of compounds.
• Removal of impurities or in purification process.
• In enzyme assays
• Detection of substrates
• Investigation of binding sites of enzymes
• In in vitro antigen-antibody reactions
• Detection of single nucleotide polymorphisms and mutations in nucleic acids.

20. Referance :-
1. Gurudeep R. Chatwal , Sham K. Anand , instrumental methods of analysis.
2. Instrumentak method of Analysis Hobart H. Willard , John A. Dean , Frank A. Settle
, Jr.
3. Modern pharmaceutical Analytical Techniques by DR. Shashikant D. Barthate , DR.
MD. Rageeb MD. Usman , Poonam A. Salunke , Shital S. patil.