Q.27 Match the entries in the Group I with the elution conditions in Group II.
Group I Group II
P. Ion-exchange chromatography 1. Isocratic solvent
Q. Hydrophobic column chromatography 2. Ampholytes
R. Gel filtration chromatography 3. Increasing gradient of salt
S. Chromatofocusing 4. Decreasing gradient of polarity
(A) P-4,Q-1,R-2,S-3 (B) P-4,Q-3,R-1,S-2
(C) P-3,Q-4,R-1,S-2 (D) P-3,Q-4,R-2,S-1
Correct Answer: (C) P-3, Q-4, R-1, S-2. Ion-exchange chromatography uses an increasing salt gradient, hydrophobic interaction chromatography employs a decreasing polarity gradient, gel filtration runs isocratically, and chromatofocusing relies on ampholytes for pH gradients.
Technique Principles
These purification methods separate biomolecules based on charge, hydrophobicity, size, or isoelectric point. Elution conditions optimize resolution by modulating interactions between analytes and stationary phases during protein or nucleic acid isolation.
Correct Matching Breakdown
P. Ion-exchange chromatography → 3. Increasing gradient of salt. Proteins bind via electrostatic interactions; elution applies a NaCl gradient (0-1 M) to disrupt ionic bonds progressively, eluting less tightly bound species first.
Q. Hydrophobic column chromatography → 4. Decreasing gradient of polarity. Hydrophobic proteins bind in high-salt conditions (e.g., 1.5 M ammonium sulfate); elution decreases salt or polarity to weaken hydrophobic interactions.
R. Gel filtration chromatography → 1. Isocratic solvent. Size-exclusion separates by molecular weight using a constant buffer; larger molecules elute first without gradient, relying on porous matrix exclusion.
S. Chromatofocusing → 2. Ampholytes. Polyampholytes create a stable pH gradient (e.g., pH 8-4); proteins focus and elute at their pI where net charge is zero.
Option Explanations
Option (A) P-4, Q-1, R-2, S-3 incorrectly pairs ion-exchange with polarity gradient (charge-based, not hydrophobic) and gel filtration with ampholytes (size-based, not pI).
Option (B) P-4, Q-3, R-1, S-2 mismatches ion-exchange to polarity (ignores salt disruption) and hydrophobic to salt gradient (reverses decreasing polarity requirement).
Option (D) P-3, Q-4, R-2, S-1 wrongly assigns gel filtration to ampholytes (isocratic by nature) and chromatofocusing to isocratic solvent (pH gradient essential).
Bioprocess Applications
In biotechnology downstream processing, matching elution to technique enhances purity and yield—ion-exchange for charge variants, hydrophobic for aggregates, gel filtration for desalting, chromatofocusing for isoforms. Gradients prevent co-elution in complex mixtures like fermentation broths.
