Reversed-phase HPLC uses a hydrophobic (non-polar) stationary phase paired with a polar mobile phase to separate compounds based on hydrophobicity.

Option Analysis

• A. A hydrophilic stationary phase is combined with a non-polar mobile phase: Incorrect. This describes normal-phase HPLC, where polar stationary phases (e.g., silica) retain polar analytes while non-polar solvents elute them.​

• B. A hydrophobic stationary phase is combined with a polar mobile phase: Correct. Reversed-phase features non-polar C18/C8 columns with aqueous-organic mobile phases (e.g., water-acetonitrile), retaining hydrophobic proteins/peptides longer.

• C. A hydrophobic stationary phase is combined with a non-polar mobile phase: Incorrect. Non-polar mobile phases suit normal-phase or hydrophobic interaction chromatography, not reversed-phase which requires polarity contrast for gradient elution.​

• D. A hydrophilic stationary phase is combined with a polar mobile phase: Incorrect. Both phases polar yields poor separation; hydrophilic stationary needs non-polar mobile for reversed polarity relative to normal-phase.

Correct Answer: B

Introduction to Reversed-Phase HPLC Phases

In reversed-phase HPLC, the hydrophobic stationary phase combined with polar mobile phase drives separations critical for GATE Life Sciences protein analysis. This “reversed” setup contrasts normal-phase, enabling gradient elution of peptides and small molecules by hydrophobicity.

Principles of Reversed-Phase HPLC

Non-polar stationary phases (C18 silica) retain hydrophobic analytes, while polar mobile phases (water-methanol) compete for elution. Gradients decrease polarity to release bound compounds, ideal for proteomics.​

Comparison of Phase Combinations

Hydrophobic stationary with polar mobile excels for high-resolution reversed-phase separations.​