A. Increased axial dispersion

B. Increase in number of theoretical plates

C. Increase in loading capacity

D. Increase in flow rate

Longer columns in size-exclusion chromatography should improve resolution for proteins with slight size differences, but fail when axial dispersion dominates band broadening. This limits separation gains despite increased length.

Question Analysis

Size-exclusion chromatography (SEC) separates by hydrodynamic volume. Longer columns increase theoretical plates (N), improving resolution (Rs ∝ √N). No improvement despite “partial flow” (some separation exists) indicates fundamental limitation.

Option Analysis

A. Increased axial dispersion

Correct. Longer columns amplify extra-column dispersion (system tubing, detector, injector) and diffusion effects. Peak broadening exceeds plate height gains, nullifying resolution. SEC’s narrow elution range makes it dispersion-sensitive.

B. Increase in number of theoretical plates

Incorrect. Longer columns increase N (N ∝ L), improving separation. This would enhance, not limit, resolution.

C. Increase in loading capacity

Wrong. Longer columns have proportionally higher capacity, but question specifies analytical separation of slight size differences—loading irrelevant.

D. Increase in flow rate

False. Flow rate typically held constant when testing column length. Higher flow decreases efficiency (higher HETP).

Correct Answer

A. Increased axial dispersion—SEC resolution plateaus when extra-column/system dispersion exceeds column-generated efficiency.

SEC Resolution Fundamentals

Resolution Rs = (√N/4) × (α-1) × (k/(1+k)), where N ∝ column length L. But total peak variance σ²_total = σ²_column + σ²_system. Long columns magnify σ²_system dominance.

GATE Prep Key Concept

SEC paradox: Optimal length ~30cm (not infinite). Beyond this, system dispersion limits returns. Use low-dw/dV UHPLC systems (V_extra-column <10% peak volume). for slight size differences, switch to narrower id columns or orthogonal methods (rp-hplc, iex).

Pro Tip: “Partial flow from each other” confirms SEC works but resolution-limited. Axial dispersion explains why “longer = no better” in practice. Classic chromatography efficiency question pattern.