An infrared investigation in relation with chitin and chitosan characterization J. Brugnerotto a , J. Lizardi b , F.M. Goycoolea b , W. Argu Èelles-Monal c , J. Desbrie Áres a , M. Rinaudo a, * a Centre de Recherches sur les Macromole Âcules Ve Âge Âtales, CERMAV-CNRS, af®liated with Joseph Fourier University, BP 53, 38041 Grenoble cedex 9, France b Centro de Investigacio Ân en Alimentacio Ân y Desarrollo, A.C., P.O. Box 1735, Hermosillo, Sonora 83000, Mexico c IMRE, Universidad de La Habana, La Habana 10400, Cuba Received 8 June 2000; received in revised form 1 September 2000; accepted 26 September 2000 Abstract The use of infrared spectroscopy for characterization of the composition of chitin and chitosan covering the entire range of degree of acetylation (DA) and a wide variety of raw materials is examined further. The ratio of absorbance bands selected was calibrated using 1 H liquid and 13 C CP-MAS solid-state NMR as absolute techniques. IR spectra of the structural units of these polymers validated the choice of baselines and characteristic bands. The bands at 1650 and 1320 cm 21 were chosen to measure the DA. As internal reference, the intensities at 3450 and 1420 cm 21 were evaluated. The absorption band ratios involving the reference at 3450 cm 21 had poorer ®t.. The absorption ratio A 1320 /A 1420 shows superior agreement between the absolute and estimated DA-values DA%  31:92A 1320 =A 1420 2 12:20; r  0:990: q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Chitin; Chitosan; Degree of acetylation 1. Introduction Chitin is the most important natural polysaccharide after cellulose found in crustaceous shells or in cell walls of fungi. However, it is not widely used for industrial applica- tions up to now because it is insoluble in many solvents, relatively dif®cult to isolate from natural sources in pure form and to prepare in a reproducible way under good economic conditions. That is why it is also dif®cult to char- acterize this polysaccharide. Its principal derivative is chitosan, obtained by deacety- lation of chitin. It is soluble in aqueous acidic medium due to the presence of amino groups. The name chitosan is reserved to partially or fully deacetylated chitin soluble in acidic aqueous conditions, it usually also means that the average degree of acetylation (DA) is around or lower than 0.5; in addition, the solubility is also controlled by the distribution of the acetyl groups remaining along the chain. For these different reasons, the characterization of chitin and chitosan is very delicate and has been largely discussed in the literature. Usually, a single technique cannot be adopted to cover the full range of DA, i.e. for chitin as well as for chitosan. For chitin, due to the lack of solubility, solid state NMR can be used [1±3], as well as infrared spectroscopy on ®lm or powder [3±8]; for samples in the pure form, elemental analysis can also be used but with lower accuracy [9]. For chitosan, which is soluble in aqueous medium, more methods are available and they have been also often discussed in the literature. The main techniques suggested are potentiometry [10,11], 1 H NMR [12±14], UV spectro- scopy [15±17] and infrared spectroscopy [3±8,18±26]. The most discussed technique is infrared spectroscopy because of its simplicity, but it needs a calibration versus an absolute technique. Many different calibration relations have been proposed, but they are still under discussion in the literature: some of the typical IR band ratios proposed will be discussed later. In this paper, we intend to improve the use of this technique as a way to characterize the degree of acetylation of chitin and/or chitosan. The aim of this inter-laboratory study is to discuss the application of infrared spectroscopy for DA determination whatever the degree of acetylation, the source, the salt form, the purity and the solubility of the polymer. It will allow us Polymer 42 (2001) 3569±3580 0032-3861/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0032-3861(00)00713-8 www.elsevier.nl/locate/polymer * Corresponding author. Tel.: 133-476-037-627; fax: 133-476-547-203. E-mail address: marguerite.rinaudo@cermav.cnrs.fr (M. Rinaudo).