Hydrophobic interaction chromatography relies on which protein property?

Hydrophobic interaction chromatography relies on which protein property?

• A. Net charge at physiological pH
• B. Overall molecular weight
• C. Hydrophobicity of amino acid side chains
• D. Specific ligand affinity

Answer: (C)

The key idea is that hydrophobic interaction chromatography separates proteins based on how hydrophobic their surface regions are. The column has a hydrophobic stationary phase, and under high salt conditions, water is structured in a way that strengthens hydrophobic interactions. Proteins with more exposed hydrophobic amino acid side chains bind more strongly to the column and thus elute later as the salt concentration is lowered. So the property being exploited is the hydrophobicity of the protein’s surface. Net charge at physiological pH would drive binding in ion-exchange chromatography, not HIC; overall molecular weight affects separation by size in size-exclusion chromatography; and specific ligand affinity governs affinity chromatography.

The key idea is that hydrophobic interaction chromatography separates proteins based on how hydrophobic their surface regions are. The column has a hydrophobic stationary phase, and under high salt conditions, water is structured in a way that strengthens hydrophobic interactions. Proteins with more exposed hydrophobic amino acid side chains bind more strongly to the column and thus elute later as the salt concentration is lowered. So the property being exploited is the hydrophobicity of the protein’s surface.

Net charge at physiological pH would drive binding in ion-exchange chromatography, not HIC; overall molecular weight affects separation by size in size-exclusion chromatography; and specific ligand affinity governs affinity chromatography.