In Hydrophobic Interaction Chromatography the separation is based on protein hydrophobicity. The hydrophobic areas of a protein are attracted to the hydrophobic surface of the stationary phase under high aqueous salt concentrations (i.e at high surface tension). Therefore the protein binds to the stationary phase at high initial salt concentration. Elution is then achieved with a reverse ionic strength gradient. So by lowering the salt concentration in the mobile phase the surface tension decreases and the proteins desorb and elute. The differences in hydrophobicity lead to differences in elution.
The type of phases used in HIC have weak hydrophobicity such as short alkyl chains (i.e. C4, phenyl) and you would require a phase that you can bind the protein initially but will then allow desorption of the protein.
It uses aqueous mobile phase. No/limited organic mobile phase should be used. You can modify the salt used, although ammonium sulfate (1 or 2 M) or sodium chloride (3M) salts are most commonly used for HIC applications. The pH chosen is critical and must be kept constant as the pH will change interactions but pH 7 is a good starting point for method development. It is recommended to use the lowest concentration of salt possible for binding the protein to avoid protein precipitation or irreversible protein binding. Sample and binding buffer should also be the same molarity.¨