ABSTRACT
This chapter deals with the optimization of high-performance affinity chromatography (HPAC) when peptides and proteins are used as the solute and ligand molecules. Affinity chromatography is a powerful technique for both investigating the molecular properties of protein-protein or peptide-protein interactions and for purifying proteins. The main advantage of HPAC over conventional affinity chromatography is the increased speed during the loading, elution, and regeneration steps of the separation. Ligands of small molecular weights, such as peptides, pose additional problems during ligand-support attachment. There are two classes of ligands that can be used in affinity chromatography: ligands can be specific for a particular molecule or may be multifunctional and bind a number of related molecules. There are numerous nonspecific desorption agents that disrupt the interaction between the ligand and solute molecule, including low pH or chaotropic ions. Non-specific desorbing agents, such as H+ ions or chaotropic salts, can be applied either as a step gradient or as a linear gradient.
