Fractionation and characterization of chitosan by analytical SEC and 1H NMR after semi-preparative SEC

Heterogeneity in molecular weight and degree of deacetylation (DDA) of chitosans from different sources and preparation methods were studied by fractionating chitosans, using semi-preparative SEC, and then determining molecular weight profiles of fractions by analytical SEC with multi-angle laser light scattering (SEC–MALLS), and degree of deacetylation (DDA) by 1H NMR. Fractionation of two high molecular weight chitosans from different manufacturers, produced fractions that spanned a wide range of molecular weight (number-average Mn), from 65 to 400 kDa in one case, that was not evident when unfractionated material was directly analyzed by SEC providing Mn = 188 kDa and PDI = Mw/Mn = 1.73. In a second case, fractions ranged from 20 to 600 kDa with unfractionated Mn = 145 kDa and PDI = 1.83. Fractionation of low molecular weight chitosans also showed a broad range of molecular weight in the original material, however, the fractions obtained with the TSKgel G4000W column in the Mn range of 5–100 kDa were essentially monodisperse with PDIs between 1.0 and 1.4. The DDA of one low molecular weight chitosan (10 kDa) produced by nitrous acid degradation was dependent on the Mn of the fraction. This semi-preparative fractionation procedure revealed important compositional heterogeneities of chitosans not evident in unfractionated material, and permitted the production of monodisperse low molecular weight chitosans with homogeneous properties.