Size-exclusion capillary electrochromatographic separation of polysaccharides using polymeric stationary phases

We report the successful size-based separations of large, neutral polysaccharides using capillary electrochromatography (CEC). As the polysaccharides possessed little chromophore for photometric detection, two separate approaches were taken. In the first approach, indirect detection was combined with size-exclusion chromatography using a sulfonated polystyrene/divinylbenzene stationary phase. The separations were performed using a 300 A pore size stationary phase under aqueous conditions. Non-size based interactions were minimal using this material, resulting in an effective calibration range of molecular masses 180 to 112 000 g.mol(-1) for pullulans. In the second approach, the polysaccharides were derivatized with phenylisocyanate and were subsequently separated on columns made using a combination of high capacity ion-exchanger and a neutral polystyrene/divinylbenzene material of various pore sizes. The sulfonated ion-exchange phase provided the electroosmotic flow, while the mixed pore size material provided the extended calibration range. The linear range for this primarily nonaqueous system using tetrahydrofuran was determined to be from molecular masses 738 to 404 000 g.mol(-1) of the original, untagged pullulan. This approach overcame the limited solubility issue associated with analysis of some polysaccharides. Analysis of pullulan and amylose samples by CEC correlated well with results obtained by conventional high-performance liquid chromatography (HPLC). The size-exclusion electrochromatographic separations provide an alternative mode for determining the relative molecular weights of polysaccharides with reduced sample and solvent consumption, as well as analysis times.