Ion Exchange Chromatography

Some ion exchangers are regarded as weak, that is functioning best over a comparatively narrow pH range, while others are strong, that is functioning over a wider pH range. Exchangers with quaternary amino or sulfonic acid groups behave as strong anion and cation exchangers, respectively, while aromatic/aliphatic amino and carboxylic acid groups are weak anion and cation exchangers, respectively. Choice of ion exchange stationary phase is heavily influenced by knowledge of the pI of the protein of interest.

In an ion exchange chromatography experiment (Figure), sample is applied to a stationary phase which has been charged with the ion to be exchanged; the counterion (e.g. Cl−). The protein (and any other species of like charge in the sample) may exchange with this counterion, binding to the ion exchanger. It is important that the sample is free of components of like charge since these are usually present in large molar excess relative to the concentration of protein. The ion exchanger will not distinguish between proteins possessing a single positive charge and, for example, a Na+ ion also present in the sample.

Desalting is carried out before ion exchange either by gel filtration chromatography, by dialysis or by centrifugal filtration. Elution of bound proteins is achieved by reversing the process of binding and, again, exchanging a counterion for protein. This is usually carried out by applying a large excess of a salt (e.g. NaCl) containing the counterion in the mobile phase. Because proteins have different net charge, they may bind to an ion exchanger at a given pH with a variety of strengths, that is some proteins may bind strongly whilst others bind weakly or not at all. We can take advantage of this to separate proteins by applying salt in a continuous gradient. Weakly bound proteins will elute first from such a system while strongly bound proteins elute later.

Selection of an Ion Exchanger

Below the isoelectric pH the protein has a positive charge and binds to a cation exchanger. Above the isoelectric pH the protein has a negative charge and binds to an anion exchanger.

Choice of Buffer Cationic buffer should be used with anion exchanger. Anionic buffer should be used with cation exchanger.

Ion exchangers are commonly used in column form.