Size-exclusion chromatography (SEC), also known as gel filtration chromatography, is a separation technique used in biochemistry and analytical chemistry to separate molecules based on their size and shape. In this method, a porous gel or resin is used as the stationary phase, allowing smaller molecules to diffuse into the pores while larger molecules are excluded from entering them. This difference allows for the separation of components in a mixture as they pass through the column.
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Size-exclusion chromatography separates dissolved molecules by their hydrodynamic size in the chosen solvent. A larger species that cannot enter the packing's pores travels with the liquid between particles and elutes earlier. Smaller species explore more internal liquid and elute later. The column is not a sieve that permanently traps the large species: exclusion makes them leave first. “Gel filtration” is common for aqueous separations; “gel permeation chromatography” is common for organic-solvent analysis of polymers. SEC molar mass calibration translates size-dependent elution into a standard-relative estimate of molar mass, rather than a universal absolute measurement.
This makes SEC useful for separating proteins from small dissolved species, comparing protein aggregation, and characterizing dissolved polymer chains. The residual-group monitoring of polyurethane formation experiment already illustrates polymer calibration: its reported molar masses are relative to polystyrene standards. Size-exclusion characterization of chain polymers explains why an elution trace alone cannot count chains or establish their growth mechanism.