Gel-filtration (size-exclusion) chromatography: What is the principal basis of separation when using porous beads packed in a column?

Introduction / Context:
Gel-filtration (also called size-exclusion chromatography, SEC) separates macromolecules by size as they migrate through a matrix of porous beads.

Given Data / Assumptions:

• Stationary phase: beads with a defined pore size distribution.
• Mobile phase: buffer that does not promote adsorption.
• Analytes: proteins of varying hydrodynamic radius.

Concept / Approach:
Larger molecules are excluded from more pores and thus traverse a shorter path, eluting earlier. Smaller molecules enter more pores, experience a longer residence time, and elute later. While molecular shape may subtly affect hydrodynamic radius, the governing parameter is effective size.

Step-by-Step Solution:
Pack column with appropriate pore-size beads.Load sample under non-adsorbing conditions.Collect fractions; earlier fractions contain larger species.

Verification / Alternative check:
Calibration with standards of known molecular mass/size yields a linear relationship between elution volume and log molecular mass over a defined range.

Why Other Options Are Wrong:

• Shape only: Secondary effect; SEC is fundamentally size-based.
• Hydrophobicity/charge: Describe different chromatographies (HIC, ion exchange).

Common Pitfalls:
Using buffers that cause non-specific binding; overloading columns which degrades resolution.

Final Answer:
Size using porous beads packed in a column.