Size exclusion chromatography elutes largest proteins first as they cannot enter column pores and travel the shortest path through void volume.

Molecular Weights Ranked

• RNA polymerase: 450 kDa (largest)

• Serum albumin: 138.5 kDa

• Helicase: 68 kDa

• Proteinase inhibitor: 23 kDa

• Cytochrome-c: 13.7 kDa (smallest)

Option Analysis

A. Cytochrome-c (13.7 kDa)

Incorrect. Smallest protein enters all pores, takes longest path, elutes last.

B. Proteinase inhibitor (23 kDa)

Wrong. Second smallest—elutes second-to-last.

C. RNA polymerase (450 kDa)

Correct. Largest protein completely excluded from pores, elutes first at void volume.

D. Serum albumin (138.5 kDa)

No. Second largest—elutes second.

Correct Answer

C. RNA polymerase elutes first as largest protein (450 kDa).

Complete Elution Order

1. RNA polymerase (450 kDa) – 1st

2. Serum albumin (138.5 kDa) – 2nd

3. Helicase (68 kDa) – 3rd

4. Proteinase inhibitor (23 kDa) – 4th

5. Cytochrome-c (13.7 kDa) – 5th (last)

GATE Pattern Recognition

This follows Question 7 pattern (myosin→catalase→albumin→myoglobin→cytochrome c). SEC Golden Rule: LARGEST elutes FIRST, smallest LAST. All proteins within typical fractionation range.

Memorize standard MWs:

• RNA polymerase: 400-450 kDa

• Serum albumin: ~66-140 kDa (context-dependent)

• Cytochrome c: ~12-14 kDa (classic smallest standard)