The aim of ion exchange chromatography (IEX) is to separate proteins based on their differences in net surface charge.

The pH at which the IEX will be performed depends on the pI of the protein of interest and the differences between the target and contaminant proteins. When the pH equals the pI, the protein has no net surface charge. At a pH above the pI, the protein will be negatively charged and bind to positively charged beads (ANION exchange). At a pH below the pI, the protein will be positively charged and bind to negatively charged beads (CATION exchange). After protein binding in low salt conditions, the column will be eluted in a gradient to high salt and each protein will elute at a characteristic salt concentration, allowing separation based on the net surface charges of the different proteins.

There are 2 different types of ion exchangers: strong ion exchangers and weak ion exchangers. Strong ion exchangers do not display a variation in IEX capacity upon changes in the pH. They remain fully charged over a broad pH range and maintain their binding capacity both at high and low pH. Weak ion exchangers do show a variation in IEX capacity based upon changes in the pH. They take up or lose protons upon pH changes and can provide a different selectivity when a strong ion exchanger doesn’t work for proper separation.