SizeExclusion

Size exclusion refers to the separation or filtering of particles according to their effective size, typically their hydrodynamic radius, rather than their chemical identity. In size-exclusion chromatography (SEC), a sample is passed through a column packed with porous beads. The pores create a network of restricted access; large solutes cannot enter most pores and therefore travel through the column mainly via the spaces between beads, eluting first. Small solutes can enter pores to varying extents, increasing their path length and delaying elution. Thus the elution order reflects size relative to the pore structure rather than specific chemical interactions with the stationary phase.

The technique relies on a calibration between elution volume and a size descriptor such as the molecular weight, hydrodynamic radius, or Stokes radius, using standards of known size. The resulting curve allows estimation of the size distribution of unknown samples. Size exclusion is commonly referred to as gel filtration or gel permeation chromatography, depending on the context and industry.

Applications of size exclusion include characterization and purification of polymers, proteins, and other biomolecules, as well as desalting and buffer exchange. It is particularly useful for separating or estimating monomers, oligomers, and aggregates, as well as for determining molecular size distributions in solution. However, size exclusion has limitations: it measures apparent size in the mobile phase, so molecular shape and interactions with the stationary phase can influence results; sample compatibility and pH or buffer conditions must be controlled; and resolution depends on the pore size distribution of the column and the range of sizes in the sample.