Elsevier

Talanta

Volume 45, Issue 4, February 1998, Pages 713-719
Talanta

The degree of deacetylation of chitosan: advocating the first derivative UV-spectrophotometry method of determination

https://doi.org/10.1016/S0039-9140(97)00288-9Get rights and content

Abstract

The degree of deacetylation (DD) is increasingly becoming an important property for chitosan, as it determines how the biopolymer can be applied. Therefore, a simple, rapid and reliable method of determining the DD for chitosan is essential. In this report, the DD of chitosan was determined by nuclear magnetic resonance (NMR), linear potentiometric titration (LPT), ninhydrin test and first derivative UV-spectrophotometry (1DUVS). The DD was calculated on a per mol basis instead of on a per mass basis. This is important as the molecular weights of N-acetyl-d-glucosamine and d-glucosamine are different. By converting the mass of N-acetyl-d-glucosamine and d-glucosamine into mols and calculating for the percentage of d-glucosamine present in the chitosan sample, a more accurate estimation of the DD can be obtained. Of the four methods, there is good correlation between 1DUVS and NMR. The concentration of chitosan solution for 1DUVS analysis was standardised as 0.1000 mg chitosan per ml of 0.0100 M acetic acid solution. The presence of d-glucosamine was corrected for by a reference curve for N-acetyl-d-glucosamine. 1DUVS is easy to perform, sensitive and the interference of other contaminants to the results is minimal compared with the other three methods. Therefore, we advocate 1DUVS to be used as the standard methods for routine determination of DD of chitosan.

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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