11. A mixture of three peptides A, B and C is loaded in a C18 reverse-phase chromatography column.
A mixture of water and acetonitrile is used as the solvent with increasing concentration of the latter in a gradient mode.
If the hydrophobicities of the peptides are in the order A < B < C, the order in which they will elute from the column would be:
A. A followed by B followed by C
B. B followed by C followed by A
C. C followed by B followed by A
D. A followed by C followed by B
In C18 reverse-phase chromatography with increasing acetonitrile gradient, peptides elute from least hydrophobic to most hydrophobic as organic solvent strength progressively disrupts hydrophobic interactions with the stationary phase.
Question Analysis
Hydrophobicity order: A < B < C (A least hydrophobic, C most hydrophobic). C18 column (octadecyl, very non-polar). Mobile phase starts aqueous (water-rich), becomes increasingly organic (acetonitrile). Gradient elution increases acetonitrile % over time.
Reverse Phase Principle
-
Hydrophilic/less hydrophobic peptides have weak C18 binding → elute early (low acetonitrile %)
-
Hydrophobic peptides bind strongly → require high acetonitrile % to elute later
Option Analysis
A. A followed by B followed by C
Correct. Least → intermediate → most hydrophobic matches RP elution: A (weakest binding) elutes first, then B, then C (strongest binding, needs highest acetonitrile).
B. B followed by C followed by A
Wrong order. Most hydrophobic C cannot elute before intermediate B.
C. C followed by B followed by A
Opposite of RP principle. Most hydrophobic C elutes last, not first.
D. A followed by C followed by B
Incorrect sequence. C (most hydrophobic) cannot elute before B (less hydrophobic).
Correct Answer
A. A followed by B followed by C
RP-HPLC Gradient Elution Visualized
| Time → | Early (low ACN) | Middle | Late (high ACN) |
|---|---|---|---|
| A (least hydrophobic) | Elutes 1st | – | – |
| B (intermediate) | Binds | Elutes 2nd | – |
| C (most hydrophobic) | Binds | Binds | Elutes 3rd |
Typical gradient: 5% → 60% acetonitrile over 30-60 min. A elutes ~10% ACN, B ~30% ACN, C ~50% ACN.
GATE Pattern Recognition
Identical to Question 3 (proteins A>B>C hydrophobicity → C, B, A elution). RP-HPLC Golden Rule: LEAST HYDROPHOBIC FIRST. Opposite of ion-exchange (charge-based). Complements previous chromatography questions (SEC, IEX, affinity).
Pro Tip: C18 = standard peptide/protein RP column. Acetonitrile = universal organic modifier (vs methanol, slower). TFA (0.1%) typically added for ion-pairing/sharp peaks. For GATE: Hydrophobicity ∝ retention time in RP-HPLC.
1 Comment
Vanshika Sharma
January 29, 2026Option A is correct answer