Chromatography Hydrophobic Interaction HIC

Hydrophobic interaction chromatography (HIC) is a bioanalytical separation method for proteins in which they retain their native form – and thus also their biological activity. The separation is based on interactions of nonpolar surface regions of a protein with a hydrophobic stationary phase. As in the case of salt precipitation, these interactions only occur when the salt concentration of the solution is increased. An increased salt concentration leads to an increase in surface tension, which results in a partial removal of the hydrate shell and thus to an exposure of hydrophobic regions of the protein. These hydrophobic surface areas of an analyte molecule now in turn interact with the hydrophobic residues of the stationary phase. It should be noted that as the hydrophobicity of a protein increases, the salt concentration required for binding to the stationary phase decreases. Therefore, to elute the proteins, chromatography is performed with a decreasing concentration gradient. This is probably also the most significant difference to reversed-phase chromatography.