The degree of deacetylation of chitosan: advocating the first derivative UV-spectrophotometry method of determination
Introduction
Over the past 30 years, chitosan, the (β-1,4)-linked d-glucosamine derivative of the polysaccharide chitin, has been extensively advanced as a promising renewable polymeric material. Wide ranging applications in many areas for chitosan include wastewater treatment, food, agriculture, cosmetic/personal care and biotechnological and pharmaceutical industries [1]. While chitosan has lived up to some of these expectations, significant barriers to its broader usage exist. These include supply cost, variability in quality and poor methods of characterisation of its properties [2]. One of the more important property is the degree of deacetylation (DD), which determines whether the biopolymer is chitin or chitosan. The arbitrary DD of ≥40 defining chitosan [3], plays an important role in defining the use of chitosan as DD influences the physical, chemical and biological properties of chitosan such as the tensile strength of films [4], ability to chelate metal ions [5]and immunoadjuvant activity 6, 7.
Many methods have been used to determine the DD of chitosan. These include infrared spectroscopy (IR) 8, 9, 10, near infrared spectroscopy (NIR) [11], UV-spectrophotometry [12], first derivative UV-spectrophotometry (1DUVS) [13], colloidal titration [14], linear potentiometric titration (LPT) [15], enzymatic determination [16], nuclear magnetic resonance (NMR) [17], ninhydrin test [18]and circular dichroism measurements [19]. The choice of method appears to be arbitrary and often does not correlate well with others [12]. A standard method used to determine the DD of chitosan that satisfies the producers and end users is essential, if the wider exploitation of chitosan is to be realised. A standard method has to be simple, rapid, cost effective and reliable yet tolerate the presence of impurities, especially protein, a common contaminant. From our survey, the use of the 1DUVS as proposed by Muzzarelli and Rocchetti is the simplest and most convenient among all the presently available methods. The 1DUVS method requires only very small amounts of sample and relies on simple reagents and instrumentation. In addition, the results obtained from 1DUVS are reasonably independent of protein contamination. Therefore, we wish to advocate the use of 1DUVS as the standard method for determining the DD of chitosan.
In this paper, we have evaluated and refined the 1DUVS method. The DD of commercial and purified commercial chitosan were determined and the results compared with those of NMR, LPT and ninhydrin test.
Section snippets
Materials and methods
Chitosan was obtained from Fluka (Switzerland) and Tokyo Kasei (Japan). d-Glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) were obtained from Sigma (USA). All other chemicals were of reagent grade.
Results and discussion
In the estimation of the DD, methods which assess the amine or acetyl amine groups on the glycoside unit of chitosan directly would be preferred. Methods such as circular dichroism, NMR, GPC and thermogravimetry are not suitable for routine purposes because of the cost, specialist considerations and sophistication which renders them more appropriate for research purposes. Elemental analysis has long been the common method to ascertain the DD of chitosan. The method requires very pure samples
Acknowledgements
The authors acknowledge the National University of Singapore for financial sponsorship of this work (RP 920633) and the award of a research scholarship for S.C. Tan.
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