Size-exclusion (gel filtration) chromatography — Predict elution order and bead access for proteins of different sizes

Introduction:
Gel filtration separates macromolecules by hydrodynamic size. The stationary phase contains porous beads; accessibility to pore volume determines retention and elution order.

Given Data / Assumptions:

• Beads have defined fractionation ranges.
• Large proteins are excluded from more pores than small proteins.
• Mobile phase carries solutes through the packed bed.

Concept / Approach:
Exclusion from pores reduces path length, so very large proteins traverse mainly the interstitial volume and elute early. Smaller proteins take longer paths by entering pores, eluting later. This principle enables estimation of native molecular weight using standards.

Step-by-Step Solution:

Verification / Alternative check:
Plotting elution volume vs log(MW) with standards yields a calibration curve used to infer native size for unknowns.

Why Other Options Are Wrong:

Common Pitfalls:
Ignoring shape effects (elongated proteins behave larger); operating outside the bead fractionation range; interpreting nonspecific interactions as size effects.

Final Answer:
Both (a) and (b).