13. Initial high ionic strength is needed among the following purification techniques in sample?
A. Ion-exchange chromatography
B. Hydrophobic interaction chromatography
C. Chromatofocusing
D. Preparative chromatography
Hydrophobic interaction chromatography (HIC) requires high initial ionic strength (1-2M ammonium sulfate) to promote protein binding to hydrophobic resin through enhanced hydrophobic interactions.
Question Analysis
“Initial high ionic strength” refers to sample preparation buffer before loading onto column. Tests understanding of salt dependence for different chromatographic modes.
Option Analysis
A. Ion-exchange chromatography
Incorrect. IEX uses low ionic strength start (20-50mM NaCl). High salt competes with protein binding, used for elution (0.1-1M gradient), not loading.
B. Hydrophobic interaction chromatography
Correct. HIC exploits salting-out effect: High salt (1.5M (NH₄)₂SO₄) dehydrates protein surface, exposing hydrophobic patches that bind resin. Elution decreases salt.
C. Chromatofocusing
Wrong. Uses low salt polybuffer pH gradients. High ionic strength disrupts pH gradient formation.
D. Preparative chromatography
Vague/incorrect. Not specific technique. Preparative refers to scale, not mechanism.
Correct Answer
B. Hydrophobic interaction chromatography
Salt Role Comparison
| Technique | Initial Salt | Binding Mechanism | Elution |
|---|---|---|---|
| Ion Exchange | Low (20mM) | Electrostatic | Increase salt |
| HIC | High (1-2M) | Hydrophobic | Decrease salt |
| Chromatofocusing | Low | pH gradient | pH change |
| Affinity | Low/variable | Specific binding | Competitor |
HIC Protocol
1. Ammonium sulfate precipitation (60-80% saturation)
2. Load on Phenyl-Sepharose / Butyl-Sepharose in 1.5M (NH₄)₂SO₄
3. Wash unbound proteins
4. Elute: 1.5M → 0M salt gradient
GATE Prep Essential
Classic salt dependence question pattern:
High salt binding → HIC
Low salt binding → IEX
High salt elution → IEX
Low salt elution → HIC
Mnemonic: “HIC loves high salt to stick, IEX loves low salt to stick”
Pro Tip: Ammonium sulfate > NaCl for HIC (better salting-out). Often follows precipitation step (Q13 links to practical workflow). Perfect orthogonal complement to IEX/RP from previous questions.
1 Comment
Vanshika Sharma
January 29, 2026Hydrophobic interaction chromatography