HYDROPHOBIC INTERACTION CHROMATOGRAPHY : Principles and Methods

Hydrophobic Interaction Chromatography (HIC) is a purification technique used to separate proteins and other biomolecules based on their hydrophobic properties. The method involves a high-salt buffer that promotes binding of hydrophobic regions of molecules to a hydrophobic stationary phase, typically a hydrophilic matrix with attached hydrophobic groups (like butyl, octyl, or phenyl). During the process, molecules are loaded onto the column and eluted by gradually decreasing the salt concentration, which weakens hydrophobic interactions and allows for the selective elution of bound molecules. HIC is widely used in biopharmaceuticals for protein purification, particularly for proteins with exposed hydrophobic regions and as a polishing step in downstream processing.