Ion Exchange Chromatography (IEX)
IEX is based on the reversible interaction between a charged protein and an oppositely charged chromatography medium. Biomolecules with even small differences in net surface charge can be separated, and very high resolution is obtained by choosing the optimal ion exchanger and separation conditions.
The net surface charge of a protein varies according to the surrounding pH. Typically, when the pH is above its isoelectric point (pI), a protein will bind to a positively charged anion exchanger. Below its pI, a protein will bind to a negatively charged cation exchanger.
Anion and cation exchangers are classified as strong or weak, depending on how much the ionization state of the functional groups vary with pH. A strong ion exchanger has the same charge density on its surface over a broad pH range, whereas the charge density of a weak ion exchanger changes with pH. The selectivity and the capacity of a weak ion exchanger are different at different pH values. We recommend to start with a strong ion exchanger (e.g., Q, S, or SP), and then to try a weak ion exchanger (e.g., DEAE or CM) if the selectivity is unsatisfactory.
In IEX, proteins bind as they are loaded onto the column at low ionic strength. The conditions are then altered so that bound substances are eluted differentially. Elution is usually performed by increasing salt concentration or changing pH in a gradient, or stepwise.
The most common salt is NaCl, but other salts can also be used.