Answer: (B) Reverse phase chromatography

Ion-pairing agents (e.g., TFA, TEA, heptanesulfonate) are used in reverse phase chromatography (RPC) to improve retention and peak shape of charged analytes on hydrophobic C18/C8 columns by forming neutral ion-pairs.

Option Analysis

(A) Hydrophobic Interaction Chromatography

Wrong. HIC uses high salt (ammonium sulfate) for binding to mild hydrophobic ligands; no ion-pairing agents involved.[ prior]

(B) Reverse Phase Chromatography

Correct. Ion-pair reagents in mobile phase (5-20 mM) pair with charged analytes (e.g., peptides, nucleotides), making them more hydrophobic for C18 retention. Gradient elution with organic solvent (acetonitrile).

(C) Ion Exchange Chromatography

Incorrect. Uses charged resins directly; salt gradients elute—no ion-pairing needed or used.​

(D) Immobilized Metal Anion Chromatography (IMAC)

Wrong. IMAC uses metal chelates (Ni-NTA) for His-tagged proteins; histidine coordinates metal—no ion-pairing.[ prior]

Reverse phase chromatography ion pairing agents enable separation of charged molecules like peptides and nucleotides on hydrophobic columns, essential for analytical biochemistry.

Ion Pairing Mechanism in RPC

Ion-pairing agents (alkyl sulfonates, quaternary amines) form neutral complexes with charged analytes in aqueous mobile phase: R-SO₃⁻ + R’NH₃⁺ → [R-SO₃⁻…R’NH₃⁺].​
This lipophilic ion-pair partitions into C18 stationary phase; gradient (%B acetonitrile) elutes by disrupting pairs.
TFA (0.1%) most common for peptides; heptafluorobutyric acid (HFBA) for maximum retention.

Chromatography Ion Pairing Comparison

This clarifies why only reverse phase chromatography requires ion-pairing agents.​

GATE Exam Applications

Tests understanding of mobile phase additives; pairs with prior chromatography questions. Remember: ion-pairing = reverse phase only.