A mixture contains three similarly sized peptides P, Q and R. The peptide P is positively
charged, Q is weakly negative and R is strongly negative. If this mixture is passed through
an ion-exchange chromatography column containing an anionic resin, their order of elution
will be
Options:
(A) P, Q, R
(B) R, Q, P
(C) Q, R, P
(D) P, Q and R elute together
Official Answer Key:
B
Step-by-Step Solution & Explanation
### **Official Solution & Step-by-Step Derivation (GATE BT 2018 Q.18)**
**1. Governing Concept & Question Classification**:
- **Section**: BT
- **Question Type**: **MCQ** (1 Mark)
- **Topic**: Downstream Processing / Chromatography (Ion Exchange & Hydrophobic Interaction Chromatography)
**2. Detailed Step-by-Step Analysis**:
- The peptide mixture consists of similarly sized peptides:
- Peptide **P**: Positively charged.
- Peptide **Q**: Negatively charged.
- Peptide **R**: Electrically neutral, highly hydrophobic.
- **Anion-exchange chromatography (AEC)**:
- The stationary phase has a **positively charged matrix** (e.g., DEAE).
- Positively charged peptide **P** is repelled by the cationic matrix and elutes immediately in the flow-through/unbound fraction.
- Neutral peptide **R** does not bind via ionic interactions and elutes without binding.
- Negatively charged peptide **Q** binds strongly to the positively charged matrix via electrostatic interactions and is retained until eluted with high salt (e.g., NaCl gradient).
- Therefore, AEC successfully binds and purifies peptide **Q**, separating it from P and R.
- **Hydrophobic interaction chromatography (HIC)**:
- Relies on hydrophobic interactions between nonpolar patches on proteins and hydrophobic ligands (e.g., phenyl, octyl, or butyl groups) on the matrix at high salt concentrations (e.g., $(NH_4)_2SO_4$).
- Peptide **R** is highly hydrophobic, so it binds tightly to the HIC matrix, whereas hydrophilic/charged peptides P and Q have minimal hydrophobic affinity and elute earlier.
- Eluting with a decreasing salt gradient releases peptide **R** in pure form.
- Thus, Anion-exchange followed by Hydrophobic interaction chromatography (or vice versa) uniquely resolves all three peptides.
- **Option-by-Option Evaluation**:
- **Option (A) [INCORRECT]**: Cation exchange binds P, but reverse phase would not cleanly resolve Q from R without severe denaturing conditions compared to HIC.
- **Option (B) [CORRECT]**: Anion-exchange separates negatively charged Q, and Hydrophobic interaction chromatography isolates neutral hydrophobic peptide R.
- **Option (C) [INCORRECT]**: Size-exclusion chromatography (gel filtration) cannot separate similarly sized peptides.
- **Option (D) [INCORRECT]**: Cation exchange and size exclusion fails because size exclusion cannot resolve similarly sized peptides.
**3. Final Answer / Verification Summary**:
- **Official GATE Answer Key**: **B**
- **Scoring Criteria**: +1.0 Mark (Negative marking: -0.33 marks).
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