The most popular method for the purification of proteins and other charged molecules is ion exchange chromatography.  In cation exchange chromatography positively charged molecules are attracted to a negatively charged solid support. Conversely, in anion exchange chromatography, negatively charged molecules are attracted to a positively charged solid support. To optimize binding of all charged molecules, the mobile phase is generally a low to medium conductivity (salt concentration) solution. The adsorption of the molecules to the solid support is driven by the ionic interaction between the two moieties and binding capacities are generally quite high. The strength of the interaction is determined by the number and location of the charges on the molecule and solid support. By increasing the salt concentration (generally a linear salt gradient) the molecules with the weakest ionic interactions are disrupted first and elute earlier in the salt gradient. Those molecules that have a very strong ionic interaction require a higher salt concentration and elute later in the gradient.

The pH of the mobile phase buffer must be between the pI or pKa of the charged molecule and the pKa of the charged groups on the solid support. For example, a molecule with a pI of 8.2 is run in a mobile phase buffer at pH 6.0 with the solid support pKa at 1.2 in cation exchange chromatography. In anion exchange chromatography a molecule with a pI of 6.8 is run in a mobile phase buffer at pH 8.0 with the solid support pKa at 10.3. Typically, this rule is immutable but there are cases where there is an exception.

An alternative to the use of a linear gradient is to use step gradients. This requires less complicated equipment and can be very effective if the appropriate concentrations of salt are known, usually from linear gradient experiments.

Many chromatographers also use changes in pH to affect a separation.  In cation exchange chromatography, raising the pH of the mobile phase buffer will cause the molecule to become less protonated and hence less positively charged. The result is that the protein no longer has the capability to form a strong ionic interaction with the negatively charged solid support which causes the molecule to elute from the chromatography column. In anion exchange chromatography, lowering the pH of the mobile phase buffer will cause the molecule to become more protonated and hence more positively charged. The result is that the protein no longer has the capability to form a strong ionic interaction with the positively charged solid support which causes the molecule to elute from the chromatography column.