Practice question

Size exclusion chromatography separates strictly by hydrodynamic volume, not by charge or affinity. Porous inert beads allow small molecules to diffuse into pores, increasing path length and retention, while large molecules unable to enter are restricted to void space between beads. Consequently, large molecules travel shortest distance and emerge first, followed by progressively smaller species. This elution order opposite to many other methods enables molecular weight estimation, desalting, and removal of aggregates. Understanding inverse relationship between size and retention is crucial for interpreting elution profiles and selecting column with appropriate fractionation range matching analyte sizes.