Hydrophobic interaction chromatography (HIC) uniquely requires high initial ionic strength (1-2M ammonium sulfate) in the sample to promote protein binding through the salting-out effect.

Question Analysis

Identifies technique where sample buffer must contain high salt concentration at loading. Tests understanding of salt dependence across purification methods.

Option Analysis

A. Ion exchange chromatography

Incorrect. IEX uses low ionic strength (20-100 mM NaCl) for initial binding. High salt prevents protein-resin electrostatic interactions.

B. Hydrophobic interaction chromatography

Correct. HIC requires high salt (1-2 M ammonium sulfate) to dehydrate protein surfaces, exposing hydrophobic regions that bind resin. Elution decreases salt concentration.

C. Chromatofocusing

Wrong. Low ionic strength polybuffer systems. High salt disrupts pH gradient formation.

D. Gel filtration chromatography

No salt requirement. Physiological buffers (50-150 mM). Ionic strength irrelevant to size-based separation.

Correct Answer

B. Hydrophobic interaction chromatography

Salt Dependency Summary

HIC Mechanism

High salt → kosmotropic ions compete for water → protein surface dehydration → hydrophobic patches exposed → bind HIC resin (Phenyl, Butyl-Sepharose).

Typical protocol:

GATE Pattern Recognition

Q13 & Q16 identical question confirms HIC = high salt binding as core concept. Complements previous chromatography series:

Salt Rules:

• High salt start → HIC

• Low salt start → IEX

• High salt elution → IEX

• Low salt elution → HIC

Pro Tip: Ammonium sulfate precipitation (60-80%) often precedes HIC. Perfect orthogonal step after IEX (high salt eluate directly onto HIC). Never use high salt for IEX loading—classic mistake!

Note: This repeats Q13 exactly, reinforcing HIC as the definitive “high ionic strength” technique in GATE Life Sciences chromatography questions.