# DBT JRF 2022 — Question 49

Exam: DBT JRF 2022 · Section: A · Topic: Biochemistry › Chromatography Techniques · Difficulty: Medium

## Question

In Reverse Phase chromatography of a mixture of protein and sodium chloride:

## Options

- **A.** The salt will elute before the protein
- **B.** The salt and protein will co-elute
- **C.** The salt will elute after the protein
- **D.** The salt will elute but the protein will not

## Answer

Option A

## Explanation

Elution order in Reverse Phase chromatography: In Reverse Phase chromatography (RP-HPLC), the stationary phase is nonpolar (e.g., C18 alkyl chains), and the mobile phase is polar (aqueous/organic). Elution order is governed by hydrophobicity: • Polar/ionic molecules (low hydrophobicity) → elute FIRST (do not interact with stationary phase) • Nonpolar/hydrophobic molecules → elute LATER (retained by stationary phase) For a protein + NaCl mixture: • NaCl is ionic and highly polar → barely retained → elutes FIRST • Protein (especially if it has hydrophobic patches) → retained on C18 → elutes LATER Answer: Salt will elute before the protein (Option 1).

Source: https://mypyqbuddy.com/questions/in-reverse-phase-chromatography-of-a-mixture-of-6a104cd5920734408bc0c70e