Chapter 16 Chromatography of proteins

Abstract

Publisher Summary This chapter focuses on various techniques used for the chromatography of proteins, such as conjoint liquid chromatography (LC), perfusion chromatography, hydrophobic-interaction chromatography (HIC), reversed-phase liquid chromatography (RP-LC), ion-exchange chromatography (IEC), and size-exclusion chromatography. Chromatography performed with media of different separation principles, packed into a single chromatographic column, is named “conjoint liquid chromatography.” This technique allows two-dimensional chromatography to be carried out in a single step without column switching. HIC is the method of choice for maintaining the native conformation and biological activity of proteins. Owing to its high resolving power, RP-LC is widely used as an analytical method for peptides and proteins, and it is also applied on an industrial scale. The most common support material for RP-LC is silica. Characteristics of the bonded phase—such as ligand density, surface area, and porosity—depend on the base matrix. IEC exploits the fact that every protein has a different composition of acidic and basic amino acids. Reversible binding occurs to oppositely charged groups immobilized on an ion-exchange resin. Elution is effected by increasing the ionic strength and the concentration of salt ions. Salt ions compete with protein molecules for opposite charges on the resin, and depending on the strength of interactions, proteins are removed from the sorbent at a certain salt concentration.