Affinity chromatography is based on the exceptional ability of biologically active substances to bind specifically and reversibly other substances generally called ligands or affinity ligands or simply affinants. Affinity chromatography provide a high degree of protein purification due to the specificity of the binding step. Three most commonly used column chromatography methods are ion exchange chromatography, gel filtration and affinity chromatography. Ion exchange chromatography uses electrostatic interactions between the protein and the solid matrix to fractionate proteins.Gel filtration: It separates protein molecules by size. This method depends on special beads that permit small proteins to penetrate into their interior while excluding large proteins from this region.Ref: Affinity Chromatography By J. Turkova page 7, Principles of Molecular Biology: Genes to Proteins By Buon E. Trop.