Hydrophobic Interaction Chromatography

Hydrophobic Interaction Chromatography (HIC) is based on hydrophobic attraction between the stationary phase and the protein molecules. The stationary phase consists of small non-polar groups ( butyl, octyl or phenyl) attached to a hydrophilic polymer backbone (cross-linked dextran or agarose, for example). Separations by HIC are often designed using nearly opposite conditions to those used in ion exchange chromatography. The sample is loaded in a buffer containing a high concentration of a non-denaturing salt (frequently ammonium sulfate). The proteins are then eluted as the concentration of the salt in the buffer is decreased. Typical stationary phase ligands are shown here attached to a generic backbone.