Permeation of small molecules in aqueous size-exclusion chromatography vis-a-vis models for separation

K-SEC, the chromatographic partition coefficient for size-exclusion chromatography, is defined as the fraction of the column pore volume into which a solute can permeate. The column pore volume is commonly measured as the elution volume of a small molecule, e.g., D2O in the case of aqueous separations. We found that the elution volumes of a number of small molecules vary inversely with molecular size, so that the choice of any single small molecule is arbitrary. For a large number of small molecules and oligomers, we found two distinct regions in the dependence of K-SEC(1/2) on solute size R, with a discontinuity at R congruent to 0.6 nm. These results are explained in terms of two sets of pores: (a) those accessible to all solutes, which behave to a first approximation as cylindrical cavities, and (b) micropores, accessible only to solutes smaller than 0.6 nm, If K-SEC is calculated by using an adjusted pore volume, reduced by the 20% attributable to micropores, the data for the larger solutes are found to conform to the cylindrical pore model: K-SEC = (1 - R/r(p))(2).