Ideal size-exclusion chromatography separates molecules primarily on the basis of hydrodynamic volume. This is achieved only when the chromatographic support is neutral and the polarity nearly equal to that of the mobile phase. When this is not the case, the support surface may begin to play a role in the separation process. As the magnitude of surface contributions becomes larger, the deviation from the ideal increases. Because the separation mechanism is different than that of ideal size-exclusion chromatography, selectivity could be increased in nonideal size-exclusion chromatography. This paper explores the use of size-exclusion chromatography columns with mobile phases that cause proteins to exhibit slight deviations from the ideal size-exclusion mechanism. Although there are many ways to initiate nonideal sizeexclusion behavior, the specific variable examined in this study is the influence of pH at low ionic strength. Individual proteins were chromatographed on SynChrom GPC-100, TSK-G2OOOSW, and TSKG3OOOSW columns at low ionic strength. It was found that a protein could be selectively adsorbed, ion excluded, or chromatographed in an ideal size-exclusion mode by varying mobile-phase pH relative to the isoelectric point of the protein. In extreme cases, molecules could be induced either to elute in the void volume or beyond the volume of total permeation. It is postulated that these effects are the result of electrostatic interactions between proteins and surface silanols on the support surface. Optimization of size-exclusion separations relative to protein isoelectric points is discussed.
Size-exclusion chromatography
(SEC)3 is that chromatographic process whereby molecules are separated on the basis of their hydrodynamic volume. In the absence of solute interaction with the support surface, the log of solute molecular weight is inversely proportional to the fractional pore volume available to a molecule between the limits of total exclusion and total permeation. It is well known that under certain conditions proteins may deviate from this pure size-exclusion retention mechanism (l-5). When mobilephase ionic strength is low, electrostatic in-