Theory of Affinity Chromatography

Theory of Affinity Chromatography describes selective binding of target molecules to ligands for precise separation.

1. Loading/Binding Phase: The sample is introduced into a column with immobilized ligand. The target molecule binds specifically, while non-target molecules pass through.
2. Washing Phase: Non-specifically bound molecules are removed using an appropriate buffer.
3. Elution Phase: The target molecule is released by disrupting the interaction, often by changing pH, ionic strength, or adding a competing ligand.
4. Regeneration Phase: The column is restored for reuse by washing away residual target molecules and elution agents.

Classification of Affinity Chromatography

1. Based on Type of Interaction:

   • Enzyme-Substrate/Inhibitor Chromatography: Enzymes attached to the resin separate substrates or inhibitors.
   • Antigen-Antibody Chromatography: Antibodies or antigens immobilized to capture their counterpart.
   • Receptor-Ligand Chromatography: Receptors immobilized to capture specific ligands.

2. Based on Elution Method:

   • Biospecific Elution: Uses agents that specifically interfere with target binding.
   • Non-specific Elution: Alters pH or ionic strength to disrupt all bound molecules.

3. Specialized Types:

   • Immunoaffinity Chromatography: Uses antibody-antigen interactions for high specificity.
   • Metal Chelate Chromatography: Captures proteins with affinity for certain metals.
   • Dye-Ligand Chromatography: Employs reactive dyes as ligands to bind specific proteins.

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