Affinity chromatography

A decade and a half after its introduction, affinity chromatography, a method of purification based on biological recognition, has become a major means for the purification of biologically active molecules. Despite the enormous expansion of the field and the variety of biologically active materials that have been purified, the general approach has not changed markedly. Basically the same carriers (mainly agarose) and the same activating reagents (mainly CNBr) remain the predominant means for applying the technique. It is not surprising, therefore, that the same concepts and difficulties continue to prevail. The major problems involve the lack of effective procedures for efficient elution, particularly with high‐affinity interacting systems. Certain non‐specific interactions are also inherent in CNBr systems due to ion exchange and hydrophobic interactions. In addition, there is often leakage of ligand from the carrier due to the unstable isourea linkage. In view of these difficulties, alternative means for activation have been considered. In this chapter, the chemistry of activation and coupling of ligands are described, together with newer and more effective methods for preparation and assay of affinity matrices.