A. Hydrophobic interaction chromatography

B. Reverse phase chromatography

C. Ion exchange chromatography

D. Immobilized metal anion chromatography

Reverse phase chromatography uses ion-pairing agents for elution of charged analytes on hydrophobic columns.

Correct Answer

The correct option is B. Reverse phase chromatography. Ion-pairing reagents like TFA or alkyl sulfonates enable separation of ions in reversed-phase (RP) HPLC, a key biochemistry purification technique.

Option Breakdown

• A. Hydrophobic interaction chromatography: Incorrect. HIC relies on high salt (e.g., ammonium sulfate) to promote hydrophobic binding; no ion-pairing agents involved.

• B. Reverse phase chromatography: Correct. Ion-pairing agents (e.g., heptanesulfonate for cations, TFA for anions) form neutral complexes with charged analytes, enhancing retention and elution control on C18 columns.

• C. Ion exchange chromatography: Incorrect. Uses charged resins directly for ionic binding; elution via salt gradients or pH shifts, not ion-pairing reagents.

• D. Immobilized metal anion chromatography: Incorrect. IMAC (e.g., Ni-NTA) targets histidine-tagged proteins via metal coordination; no ion-pairing needed.

Ion-pairing agents reverse phase chromatography enhances separation of charged molecules like nucleotides or peptides on nonpolar C18/C8 columns.

These agents (e.g., 5-10 mM tetrafluoroborate for anions) form lipophilic ion-pairs in the mobile phase, improving analyte retention. Gradient elution with increasing organic solvent (acetonitrile) releases them dynamically.

Technique Comparisons

This comparison aids GATE prep by showing RP’s unique ion-pair requirement.

Exam Applications

GATE tests mechanism knowledge: ion-pairing neutralizes charge for RP-HPLC of polar ions. Volatile agents like TFA suit MS detection.