Practice question

Hydrophobic interaction chromatography separates proteins based on surface hydrophobicity differences under high salt conditions. Unlike reversed-phase that uses organic solvents, HIC preserves native structure. Stationary phase contains mildly hydrophobic ligands such as phenyl, octyl, or butyl groups attached to agarose backbone. In presence of high kosmotropic salt like ammonium sulfate, water structure around protein is enhanced, exposing hydrophobic patches that interact with matrix. Elution is achieved by decreasing salt concentration. Charged matrices belong to ion exchange, while polar solvents disrupt interactions. HIC is valuable for purifying membrane proteins and labile enzymes without denaturation.