Asymmetrical flow field-flow fractionation coupled to multiangle laser light scattering detector: optimization of crossflow rate, carrier characteristics, and injected mass in alginate separation

The coupling of the flow field-flow fractionation (FlFFF) to differential refractive index (DRI) and multiangle laser light scattering (LS) detectors is a powerful tool for characterizing charged polysaccharides such as alginate. However, the correct interpretation of the experimental results and extrapolation of meaningful molecular parameters by using an analytical tool with such a level of complexity requires improvement of the knowledge of the alginate behavior in the channel and careful optimization of the operating conditions. Therefore, the influence of the critical operating parameters, such as crossflow rate, carrier composition and concentration, and sample load, on the alginate retention was carefully evaluated. Combined information obtained simultaneously by DRI and LS detectors over the wide range of the crossflow rate, carrier liquid concentration, and injected amount, allowed to set the appropriate combination of optimal parameters. It was found that the crossflow rate of 0.25 mL/min, carrier solution containing 5x10(-2 )mol/L ammonium or sodium chloride, and 50-100 microg of injected sample mass were necessary to achieve complete separation and determination of the meaningful molecular characteristics. The values of the weight-average hydrodynamic radius (R(Hw)), radius of gyration (R(G)), and molar mass (M), obtained under the optimal conditions were in good agreement to those found for alginates in the literature.