Characterization of chitosan using triple detection size-exclusion chromatography and 13C-NMR spectroscopy

Abstract

The biopolymer chitosan has shown a great potential for a tremendous number of applications despite the fact that typical chitosan preparations are always mixtures of different chemical entities, natural impurities and process-induced impurities. Chitosan preparations described in the literature or offered on the market are analytically highly undefined which prevents a detailed structure-activity-relationship (SAR) analysis. The aim of this thesis is to accomplish the prerequisites necessary for a sophisticated study of the polymers chitin/chitosan. Within this thesis a SAR guided multi-dimensional analysis is proposed including a physicochemical description (molecular weight (MW), polydispersity (MW/MN), fraction of acetylation (FA), and determination of the purity content combined to biological effects on two different bacteria (Escherichia coli, Vibrio fischeri ). Results are obtained by methods such as triple detection size-exclusion chromatography (refractive index, light scattering (90° and 7°), viscometry), inhibition assays and transmission electron microscopy (TEM). Furthermore, an improved method to determine the pattern of acetylation PA from heterogeneously as well as homogeneously prepared chitosan is shown. No link between the different production processes and the formation of a specific pattern of acetylation was found. For all investigated sample a random-dominated pattern was found. The obtained PA values include also calculated inter-day and inter-analyst variations of the carbon nuclear magnetic resonance technique (13C-NMR). The conformational analysis of chitosan, again achieved with the triple detection SEC, revealed an interesting behavior. Using different theoretical models (Benoit-Doty, Odijk-Houwart, Bohdanecky) different Kuhn segment lengths (lK) were found for the two different samples. While the first approached linear chain behavior, the second showed similarity to dextran, a branched polysaccharide. Dissolution studies on chitin and chitosan showed positive results in 18 cases (chitosan) and 6 cases (chitin), respectively, by testing 58 different ionic liquid samples in total. In case of chitin, ionic liquids may be promising candidates for future dissolution studies of this biopolymer. However, chitin behaves completely different in dissolution assays than its relative cellulose which may based on three different dissolution mechanism each for chitosan, chitin and cellulose, respectively.