Reversed-phase chromatography uses a non-polar stationary phase and a polar mobile phase, making option A correct.

This principle is key for biomolecule separation in exams like GATE Life Sciences.

Correct Answer

A. Stationary phase is less polar than the mobile phase

Option Analysis

Reversed-phase chromatography (RPC) separates biomolecules based on hydrophobicity using a hydrophobic column and aqueous-organic mobile phase.

• A. Stationary phase is less polar than the mobile phase: Correct. Stationary phase (C18/C8 silica) is non-polar/hydrophobic; mobile phase (water + acetonitrile/methanol) is polar. Hydrophobic biomolecules bind stronger to stationary phase.

• B. Stationary phase is more polar than the mobile phase: Incorrect—this describes normal-phase chromatography (polar silica, non-polar hexane mobile phase).

• C. Both the stationary and the mobile phase have the same polarity: Wrong—equal polarity eliminates separation mechanism (no partitioning gradient).

• D. Polarity of the mobile phase does not play any role: False—mobile phase polarity controls elution via gradient (e.g., increasing acetonitrile reduces retention).

Reversed-phase chromatography stationary phase polarity drives biomolecule separation in proteomics and pharmaceuticals, essential for GATE Life Sciences preparation.

Core Principle

In RPC, the stationary phase (non-polar C18 chains bonded to silica) has lower polarity than the polar mobile phase (water/acetonitrile). Hydrophobic molecules partition into stationary phase; polar ones elute first. Gradient elution increases organic solvent to elute retained analytes.

Normal vs Reversed-Phase Comparison

Reversed-phase dominates HPLC (90%+ applications) due to aqueous compatibility.

Why Stationary Phase Must Be Less Polar

Polar stationary phase fails RPC—biomolecules wouldn’t partition hydrophobically. Mobile phase polarity gradients (5-95% organic) precisely control retention time based on hydrophobicity.

GATE Exam Tip

This PYQ tests chromatography fundamentals. Remember: “Reversed” = reversed polarities vs normal phase. Practice with peptide maps where hydrophobic residues (Leu, Ile) elute later.