11.24 : Affinity Chromatography

Affinity chromatography is a powerful technique extensively utilized for separating and purifying specific biomolecules from complex mixtures. It capitalizes on the highly selective binding between an analyte and its counterpart, such as antibody-antigen interactions. The counterpart is immobilized on the stationary phase, forming an affinity column. The stationary phase typically consists of solid support, such as agarose or porous glass beads, immobilizing the affinity ligand. The mobile phase serves a dual purpose – supporting strong binding between the analyte and ligand and subsequently weakening the interaction to elute the analyte. Only the analyte binds when the mixture containing the desired analyte is passed through the column, while other substances are washed through. The bound analyte can then be eluted by altering conditions like pH or ionic strength, which weakens its binding.

This method is particularly valuable in biochemistry for its specificity and application in isolating proteins, enzymes, substrates, antibodies, antigens, receptors, and hormones. Affinity chromatography can also be employed to isolate individual optical isomers of drugs, which may exhibit different therapeutic effects. This process involves using monoclonal antibodies specific to each stereoisomer.

Affinity chromatography's primary advantage is its exceptional specificity, which makes it ideal for the rapid isolation of biomolecules in preparative work.