Size exclusion chromatography elutes proteins in decreasing order of molecular weight—largest proteins exit first, unable to enter column pores, while smallest enter pores and elute last.

Protein Molecular Weights

• Myosin: 524 kDa (largest)

• Catalase: 221 kDa

• Serum albumin: 68 kDa

• Myoglobin: 16 kDa

• Cytochrome c: 13 kDa (smallest)

All fall within 5-400 kDa fractionation range, so size-based separation applies.

Option Analysis

A. Myoglobin, catalase, cytochrome c, myosin, serum albumin

Wrong. Myoglobin (16 kDa) cannot elute first—much smaller than myosin (524 kDa).

B. Serum albumin, myoglobin, cytochrome c, catalase, myosin

Incorrect. Serum albumin (68 kDa) elutes before smaller proteins; catalase/myosin reversed.

C. Myosin, catalase, serum albumin, myoglobin, cytochrome c

Correct. Perfect decreasing MW order: 524 → 221 → 68 → 16 → 13 kDa.

D. Cytochrome c, myosin, myoglobin, catalase, serum albumin

Wrong. Smallest cytochrome c (13 kDa) cannot elute first.

Correct Answer

C. Myosin, catalase, serum albumin, myoglobin and cytochrome c

SEC Elution Principle

Large proteins excluded from pores → travel void volume → elute first. Small proteins permeate pores → longer path → elute last.

GATE Prep Strategy

Memorize standard protein MWs:

• Myosin: 500+ kDa (muscle)

• Catalase: ~220 kDa (H₂O₂ enzyme)

• BSA/serum albumin: 66-68 kDa (lab standard)

• Myoglobin: 16-17 kDa (oxygen storage)

• Cytochrome c: 12-13 kDa (electron transport)

Golden Rule: SEC = BIG first, small last. Opposite of gel electrophoresis. fractionation range confirms all proteins separable (no total exclusion or permeation).