Hydrophobic Interaction

Hydrophobic interaction chromatography (HIC) is driven by non-polar interactions between a non-polar stationary phase and molecular hydrophobicity exposed by a high salt concentration mobile phase. Chaotropic salts in the mobile phase significantly alter hydration patterns around analytes in solution, forcing hydrophobic regions to reorganize and variably interact with a weakly non-polar stationary phase. Analytes generally elute in order of most hydrophilic to most hydrophobic, facilitated by decreasing salt concentration in the mobile phase. Low percentages of organic solvent in the mobile phase can be utilized to facilitate elution for strongly binding species.

HIC is generally considered a non-denaturing separation technique for biologics analysis, typically preserving tertiary and quaternary-structural interactions within proteins, antibodies, and nucleic acids. It is applied for protein separations, protein purifications, DNA and RNA isoform analyses, viral clearance for AAVs, analysis of monoclonal (mAbs) and bispecific antibodies (bsAbs), and analysis of Antibody-Drug Conjugates (ADCs).