A. Reverse-phase chromatography

B. Ion-exchange chromatography

C. Gel-filtration chromatography

D. Chromatofocusing

Proteins with identical molecular weight and isoelectric point (pI) but different amino acid compositions can be separated by reverse-phase chromatography, which exploits hydrophobicity differences arising from varied hydrophobic residue content.

Question Analysis

Same molecular weight eliminates size-based methods. Identical pI means same net charge at any pH, ruling out charge-based separation. Different amino acid composition creates hydrophobicity or polarity differences—key for specific techniques.

Option Analysis

A. Reverse-phase chromatography

Correct. Hydrophobicity varies despite same pI/mass due to different proportions of hydrophobic (Leu, Ile, Val, Phe) vs hydrophilic (Ser, Thr, Asp, Lys) residues. C18 columns bind hydrophobic regions stronger; elution order follows hydrophilicity.

B. Ion-exchange chromatography

Incorrect. Same pI = identical net charge at working pH. Both proteins bind (or don’t bind) resin equally, co-eluting regardless of amino acid differences.

C. Gel-filtration chromatography

Won’t work. Same molecular weight = identical hydrodynamic volume/stokes radius. Both proteins elute at same volume in isocratic size-exclusion.

D. Chromatofocusing

Fails. Creates internal pH gradient; proteins migrate to their pI. Identical pI = co-migration/co-elution.

Correct Answer

A. Reverse-phase chromatography exploits amino acid composition-driven hydrophobicity differences when size (MW) and charge (pI) match perfectly.

Technique Comparison Table

GATE Prep Insight

Amino acid composition affects three protein properties: hydrophobicity (RP-HPLC), charge distribution (potentially 2D properties), and conformational stability. Sequence isomers (same composition, different order) challenge even RP-HPLC, requiring MS or enzymatic digestion patterns.

Pro Tip: For GATE, remember RP-HPLC separates “look-alike” proteins when other properties match—classic scenario question pattern from previous chromatography MCQs in this series.