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

Abstract

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 Å pore size stationary phase under aqueous conditions. Nonsize based interactions were minimal using this material, resulting in an effective calibration range of molecular masses 180 to 112 000 g?mol 21 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 ionexchanger 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 21 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 sizeexclusion 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.

Fluid dynamics in capillary and chip electrochromatography

“…1) demonstrate that a locally flat velocity profile for electrokinetic flow through the interparticle pore space of packed beds which are employed for EC separations in capillary and chip format does not exist, even in the thin-EDLlimit and with uniform surface properties throughout the packing. These results contradict relatively early anticipation [59][60][61] which still finds widespread acceptance [62][63][64][65][66][67][68][69].…”

Macrophage immunomodulatory activity of polysaccharides isolated from Juniperus scopolorum

“…Using this calibration, molecular weights of fractions J-I, J-II, J-III, J-IV, and J-V were estimated to be 680, 200, 100, 60 and 30 kDa, respectively. Furthermore, evaluation of the detector response as a function of polysaccharide concentration [28] indicated that the response was linear over the range of 0.25-2 mg/ml. Based on these results it appears that self-aggregation of the polysaccharides, which could falsely lead to high molecular weight determinations, did not occur with our assay.…”

Electrophoresis in gel channels

“…Electrophoresis without supporting gels has been exploited to develop separation methods, where the gels are used as functional elements of electrokinetic separation methods [10]. Examples are sol-gel based electrochromatography [11,12] and CIEF [13]. Additives of uncrosslinked polymers can be used to improve the separation of probe constituents in microchannels.…”