69. Which one of the following purification steps always requires a high initial ionic strength in the sample?
A. Ion exchange chromatography
B. Hydrophobic interaction chromatography
C. Chromatofocusing
D. Gel filtration chromatography
Hydrophobic interaction chromatography (HIC) requires high initial ionic strength in the sample to promote protein binding to the hydrophobic resin.
Correct Answer
The correct option is B. Hydrophobic interaction chromatography. This is a standard concept in protein purification protocols tested in exams like GATE Life Sciences.
Option Breakdown
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A. Ion exchange chromatography: Incorrect. It uses low ionic strength (20-50 mM salt) for initial loading to allow protein binding; high salt elutes proteins.
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B. Hydrophobic interaction chromatography: Correct. High salt (e.g., 2-4 M ammonium sulfate) reduces water solvation, exposing protein hydrophobic regions for resin binding; salt gradients elute.
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C. Chromatofocusing: Incorrect. Employs low ionic strength polybuffer pH gradients on ion exchangers for isoelectric separation; high salt disrupts gradients.
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D. Gel filtration chromatography: Incorrect. Size-exclusion method unaffected by ionic strength; separates by molecular size in any aqueous buffer.
Hydrophobic interaction chromatography high ionic strength is essential for effective protein separation in biochemistry labs.
High salt concentrations like ammonium sulfate (1.5-4 M) in the sample promote “salting out,” forcing hydrophobic protein patches to interact with the resin (e.g., phenyl-Sepharose). Decreasing salt elutes bound proteins by rehydration.
Technique Comparisons
Other methods start with low or neutral salt:
| Technique | Initial Ionic Strength | Binding Principle | Sample Prep Example |
|---|---|---|---|
| HIC | High (2-4 M) | Hydrophobic exposure | Ammonium sulfate precipitation |
| Ion Exchange | Low (20-50 mM) | Charge-based | Dialysis to low salt |
| Chromatofocusing | Low (polybuffers) | pI focusing | pH-adjusted low salt |
| Gel Filtration | Any/neutral | Size exclusion | Direct load |
This table highlights why only HIC demands high ionic strength loading for GATE revision.
Exam Strategy
GATE questions test protocol order: ammonium sulfate precipitation often precedes HIC. Remember: high salt loads HIC, elutes ion exchange.
