Ion-Exchange Chromatography: Basic Principles and Application

Ion-exchange chromatography (IEC) is a fractionation technique that allows for the separation of ionizable molecules on the basis of differences in their electrostatic properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, ability to perform simultaneous quantitation, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline basic laboratory protocols to partially purify a soluble serine peptidase from bovine whole brain tissue, covering crude tissue extract preparation through to partial purification of the target enzyme using a form of IEC, namely, anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described.