Polysaccharide characterization by hollow-fiber flow field-flow fractionation with on-line multi-angle static light scattering and differential refractometry

• First demonstration of multi-detector HF5 analysis of polysaccharides.
• Measured absolute molar masses of dextrans, pullulans, and larch arabinogalactan.
• Experimental results contrasted to SEC, off-line QELS, and simulations.
• Influence of analytical conditions on HF5 sample recovery studied.
• HF5 performed with commercially available (not in-house built) instrumentation.

Accurate characterization of the molar mass and size of polysaccharides is an ongoing challenge, oftentimes due to architectural diversity but also to the broad molar mass (M) range over which a single polysaccharide can exist and to the ultra-high M of many polysaccharides. Because of the latter, many of these biomacromolecules experience on-column, flow-induced degradation during analysis by size-exclusion and, even, hydrodynamic chromatography (SEC and HDC, respectively). The necessity for gentler fractionation methods has, to date, been addressed employing asymmetric flow field-flow fractionation (AF4). Here, we introduce the coupling of hollow-fiber flow field-flow fractionation (HF5) to multi-angle static light scattering (MALS) and differential refractometry (DRI) detection for the analysis of polysaccharides. In HF5, less stresses are placed on the macromolecules during separation than in SEC or HDC, and HF5 can offer a higher sensitivity, with less propensity for system overloading and analyte aggregation, than generally found in AF4. The coupling to MALS and DRI affords the determination of absolute, calibration-curve-independent molar mass averages and dispersities. Results from the present HF5/MALS/DRI experiments with dextrans, pullulans, and larch arabinogalactan were augmented with hydrodynamic radius (RH) measurements from off-line quasi-elastic light scattering (QELS) and by RH distribution calculations and fractogram simulations obtained via a finite element analysis implementation of field-flow fractionation theory by commercially available software. As part of this study, we have investigated analyte recovery in HF5 and also possible reasons for discrepancies between calculated and simulated results vis-à-vis experimentally determined data.