Simultaneous determination of molar degree of substitution and its distribution fraction, degree of acetylation in hydroxypropyl chitosan by 1H NMR spectroscopy

Under the assistance of 13C NMR and 1H-13C HSQC, we develop a novel 1H NMR assay for the substitution sites and degrees in hydroxypropyl chitosan (HPCS) by optimizing sample preparation and measurement method. We find that the chemical shift of HOD peak increases linearly with the increase of DCl concentration but declines with the rise of measurement temperature. According to the regression line, the HOD peak could be moved to a desired position of non-interference with other peaks by changing DCl concentration. Other DCl-responsive peaks are found and elucidated. Accordingly, the substitution fraction (NH2-substitution and OH-substitution) and the degree of acetylation are well discriminated and determined. The total molar degree of substitution (MS) obtained is basically consistent with those of elemental analysis and the existing NMR methods. This structural analysis is extendable to other amino-containing saccharides. The 1H NMR method could be used widely in acidsoluble polysaccharides and their derivatives.