Reverse phase chromatography on C18 columns separates proteins by hydrophobicity, with less hydrophobic proteins eluting first in water:acetonitrile mobile phases. Proteins A, B, C have decreasing average hydrophobicity (A most hydrophobic, C least) but same molecular mass, so elution follows hydrophilicity order.

Question Analysis

C18 reverse phase uses non-polar stationary phase and polar mobile phase (20% water:80% acetonitrile). Hydrophobic proteins bind stronger to C18, requiring more organic solvent to elute. Decreasing hydrophobicity means A binds tightest, C weakest.

Option Analysis

A. A will elute first followed by B and then C

Incorrect. Most hydrophobic A binds strongest to C18, elutes last as it needs highest acetonitrile % to desorb.

B. A will elute first followed by C and then A

Invalid sequence (A elutes twice). Least hydrophobic should elute first, not A.

C. C will elute first followed by A and then B

Wrong order. After C (least hydrophobic), B elutes before A (more hydrophobic than B).

D. C will elute first followed by B and then A

Correct. Elution: C (least hydrophobic) → B → A (most hydrophobic). Least hydrophobic has weakest C18 binding, elutes first in 20:80 water:acetonitrile.

Reverse Phase Principles

Hydrophobic proteins partition more into non-polar C18; hydrophilic stay in polar mobile phase. 80% acetonitrile weakens hydrophobic interactions progressively.

GATE Prep Tips

Same mass eliminates size-exclusion effects. Amino acid composition drives hydrophobicity differences. Gradient elution (increasing acetonitrile) sharpens peaks: 5-95% over 30-60 min typical. For intact proteins, use wide-pore C18 (300Å); peptides use 120Å.