Which chromatography technique involves a pH gradient for separation?

Isoelectric focusing is a specialized technique that separates proteins based on their isoelectric point (pI), which is the pH at which a protein has no net charge. The method involves creating a pH gradient within a gel matrix. As an electric field is applied, proteins migrate through the gel until they reach the point where the pH matches their pI, at which point they will focus into discrete bands. This allows for a very precise separation of proteins according to their charge properties.

In contrast, size-exclusion chromatography separates molecules primarily based on their size; affinity chromatography relies on specific interactions between a target molecule and a binding partner; and ion-exchange chromatography separates proteins based on their net charge at a given pH, but it typically does not involve a pH gradient as part of the separation process itself. Therefore, the characteristic feature of using a pH gradient for separation is exclusive to isoelectric focusing.