ABSTRACT
Both hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC) rely on hydrophobic interactions with the stationary phase to effect separation. The special utility of HIC lies in its ability to separate proteins on the basis of their surface accessible hydrophobicity without, in general, causing denaturation and loss of enzymatic or other activity. HIC can be used to accomplish this because it differs from RPC in three areas: mobile phase solvents utilized; ligand density on the column support; and hydrophobicity of the column matrix. The primary effect of increasing ligand density in HIC is enhanced protein retention. The selectivity and retention of a HIC column can be modified by adjusting mobile-phase variables such as salt type, salt concentration, pH, temperature, and mobile-phase modifiers. Preparative-scale versions of analytical HIC columns are available from a number of manufacturers. Another area where HIC can supplement RPC is purification of hydrophilic peptides.
