Characterisation of chitosan molecular weight distribution by multi-detection asymmetric flow-field flow fractionation (AF4) and SEC

A new method for the molecular weight (MW) determination of chitosans by asymmetric flow field flow fractionation (AF-FFF or AF4) coupled with multi-angle light scattering (MALS) and differential refractive index (RI) detectors is proposed. The method allows the separation of polymer from molecular aggregates typically found in chitosan solutions making possible the accurate determination of MW distribution of the polymer. The effect of different experimental conditions on the obtained results has been evaluated and compared with those obtained from SEC-MALS-RI (size exclusion chromatography-MALS-RI). The chitosans analysed were from different biological sources and of varying degree of acetylation. AF4-MALS-RI results revealed that although aggregates are minimised when using a good solvent, a fraction still remains, and it is only completely eliminated by filtration through small pore sizes (≤0.45 μm). This represents a limitation for the characterisation of high MW chitosan, where filtration can lead to considerable sample loss in the filter. The method described here has the advantage of not only allowing the identification and separation of the aggregates present, but also the accurate determination of the MW distribution for a wide range of chitosans of varying type where results are well correlated with those obtained by more conventional techniques.