A. Ion exchange chromatography

B. Hydrophobic interaction chromatography

C. Chromatofocusing

D. Preparative chromatography

Answer: (B) Hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) requires samples with high ionic strength (e.g., ammonium sulfate saturation) to promote hydrophobic protein binding to the non-polar resin.[ from prior]

Option Analysis

(A) Ion Exchange Chromatography

Wrong. Ion exchange typically uses low ionic strength buffers (20-50 mM) for initial protein binding; high salt is used for elution, not loading.​

(B) Hydrophobic Interaction Chromatography

Correct. Proteins in high-salt conditions (2-4M ammonium sulfate) have reduced water solvation, exposing hydrophobic regions that bind to phenyl-Sepharose or octyl columns. Lowers salt elutes proteins.​

(C) Chromatofocusing

Incorrect. Uses low ionic strength polybuffer gradients (pH 11→4) on ion exchange resins for isoelectric focusing; high salt disrupts pH gradients.​

(D) Preparative Chromatography

Wrong. General term for large-scale purification; sample prep varies by method (no universal high ionic strength requirement).​

Hydrophobic interaction chromatography high ionic strength samples (2-4M ammonium sulfate) promote protein binding to hydrophobic resins, essential for protein purification in biochemistry.

HIC Sample Preparation Mechanism

High salt reduces water’s dielectric constant, dehydrating protein surfaces and exposing hydrophobic patches that bind resin ligands (phenyl, butyl groups).​
Samples typically start as (NH₄)₂SO₄ precipitates redissolved at 1.5-2M salt. Linear salt decrease (2M→0) elutes proteins by rehydration.​
Perfect after ammonium sulfate fractionation step.

Purification Method Sample Requirements

This clarifies why only hydrophobic interaction chromatography requires high ionic strength loading.​

GATE Exam Strategy

Tests understanding of sample prep before chromatography; HIC follows precipitation, ion exchange precedes it. Key pattern recognition question.