Reverse-phase chromatography separates proteins with identical molecular mass and isoelectric point by exploiting differences in amino acid composition that affect hydrophobicity.

Question Analysis

Same molecular mass eliminates size-based separation. Same isoelectric point (pI) eliminates charge-based separation at any pH. Remaining difference must be amino acid composition → hydrophobicity differences.

Option Analysis

A. Reverse-phase chromatography

Correct. RP-HPLC separates by hydrophobicity. Different amino acid compositions create varying proportions of hydrophobic (Leu, Ile, Val, Phe) vs hydrophilic residues, leading to different C18 column retention despite identical mass/pI.

B. Gel-filtration chromatography

Incorrect. Same molecular mass = identical hydrodynamic volume = co-elution. SEC cannot distinguish.

C. Ion-exchange chromatography

Wrong. Same pI = identical net charge at operating pH. Both proteins bind (or flow through) resin identically.

D. Chromatofocusing

Fails. Creates pH gradient; proteins stop at their pI. Identical pI = co-migration at same position.

Correct Answer

A. Reverse-phase chromatography

Technique Decision Matrix

Why RP-HPLC Succeeds

Two proteins can have identical MW/pI but different sequences:

X elutes later (more hydrophobic residues bind C18 stronger).

GATE Pattern Recognition

Identical to Question 4. This tests orthogonal separation principles:

• Q1-2: Charge/size (IEX, LCMS)

• Q3,11: Hydrophobicity (RP-HPLC)

• Q7-8: Size (SEC)

• Q6: Affinity (oligo dT)

• Q9-10: Technique fundamentals

• Q4,12: “Perfect match” scenario → RP-HPLC

Pro Tip: When all standard properties (size, charge) match, default to reverse-phase for amino acid composition differences. Classic “exception” question pattern.