Hydrophobic Interaction Chromatography

Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is based on non-polar interactions that are induced by high salt mobile phases. Stationary phases are similar to reversed phase chromatography (RPC) but the density of functional groups is lower. A weakly non-polar stationary phase is used with an aqueous mobile phase containing a high concentration of a chaotropic salt.

The technique is mainly applied to the separation of proteins, which are eluted by decreasing the salt concentration or by adding a low percentage of organic solvent. Although also based on hydrophobic interactions, selectivity in HIC separations is distinctly different from that in reversed phase chromatography. Despite the lower peak capacity in HIC compared to RPC, HIC has the advantage that the mobile phase conditions (primarily aqueous) do not usually disrupt higher-order protein structures.


HIC is used in the biopharmaceutical industry for the analysis of antibody drug conjugates (ADCs) or to determine the aggregate content of monoclonal antibodies.

TSKgel HIC Columns

TSKgel HIC columns are polymethacrylate-based with a choice of three ligands (butyl, ether and phenyl) with varied hydrophobicities from low to high, respectively. The TSKgel Phenyl-5PW and Ether-5PW columns are compatible with water-soluble organic solvents at concentrations below 50% while the TSKgel Butyl-NPR columns are compatible at concentrations below 20%.

TSKgel Column



Phenyl-5PW Most hydrophobic Applicable for the widest range of hydrophobicities; requires modest salt concentration to retain proteins
Ether-5PW Less hydrophobic Ideal for hydrophobic proteins such as membrane proteins
Butyl-NPR Least Hydrophobic Excellent choice for monoclonal antibody analysis and high speed applications; usually high recovery due to absence of pores