International Journal of Biological Macromolecules 31 (2002) 1 /8 www.elsevier.

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An optimised method to determine the degree of acetylation of chitin and chitosan by FTIR spectroscopy
M.L. Duarte a,*, M.C. Ferreira b, M.R. Marvao b, Joao Rocha c
b

Department of Chemistry and Biochemistry, CECUL, University of Lisbon, 1749-016 Lisbon, Portugal Department of Materials, National Institute of Engineering and Industrial Technology, Estrada do Paco do Lumiar, 1649-038 Lisbon, Portugal c Department of Chemistry, University of Aveiro, 3810 Aveiro, Portugal Received 12 November 2001; received in revised form 14 June 2002; accepted 14 June 2002

Abstract Linking up the user-friendly and low-priced FTIR with the more sophisticated and high-priced 13C CP/MAS NMR spectroscopies, an improved method to determine the degree of acetylation (DA) of chitins and chitosans was outlined. The method was established for the most complex polymorphic form (a-chitin) and for the most problematic range of DA values (most acetylated samples) and can easily be extended to the other polymorphic forms (b- and g-chitins) and to other ranges of DA values. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Chitin; Chitosan; Degree of acetylation

1. Introduction Chitin, poly-N -acetyl-D-glucosamine, is one of the most abundant, easily obtainable and renewable natural polymers, second only to cellulose. Depending on the sources, this polymer has been found in three polymorphic forms: a-, b- and g-chitin, which differ as regards the arrangement of the chains within the crystalline regions, implying different networks of hydrogen-bonds. The a-chitin is the most abundant and stable polymorphic form, exhibiting the most complex network of hydrogen-bonds. Chitosan, poly-D-glucosamine, is obtained by deacetylation of purified chitin with concentrated alkali and high temperature treatment, and frequently it is commercialised in powder or flake forms. While chitin is extremely insoluble in common solvents, chitosan is soluble in dilute acid solutions, being a much more tractable material with a large number and wide variety of applications, ranging from

* Corresponding author. Tel.: '/351-217-150-571; fax: '/351-217500-088 E-mail address: mlduarte@fc.ul.pt (M.L. Duarte).

biomedical to waste water treatment and fibre industries. Despite those specific chemical designations, the names chitin and chitosan actually correspond to a family of polymers varying in the acetyl content measured by the degree of N -acetylation (DA). The DA dictates the behaviour of these polymers, namely reactivity and solubility, its determination being one of the routine analyses performed for quality control on chitins and chitosans industrially produced. Furthermore, in the case of chitin, the DA determines the suitable conditions of deacetylation, a complex process still requiring investigation in order to assess the feasibility of quicker preparation of chitosan. Thus, the search for quick, user-friendly, low cost and accurate methods to determine the DA has been one of the major concerns over many decades. However, the inherent complexity of this particular system turns this apparently simple analytical problem into a very difficult one, which is well illustrated by the extensive number of different techniques, either destructive or non-destructive, yielding direct or non-direct and frequently non reproducible values [1 /5]. It is worth noting that nondestructive methods offer the advantage of avoiding manipulations of the polymers such as hydrolysis,

0141-8130/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 1 - 8 1 3 0 ( 0 2 ) 0 0 0 3 9 - 9

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pyrolysis or derivatisation, the consequences of which are not always well known. Not only has infrared spectroscopy (IR) been the most widely used technique to determine the DA of those polymers, but also have the DA values obtained by this technique been frequently compared with DA values obtained by other techniques. The use of IR for that purpose requires the construction of a calibration line using ratios of absorbances (A ) of a probe band (PB), changing intensity with the DA, relative to a reference band (RB), not changing intensity with the DA, against standard DA values. The absorbances of PB and RB are determined by the baseline method (BL). But the construction of a reliable calibration line is not an easy task. On the one hand, the choice of the best PB, RB and BL combinations is not obvious, and this is well illustrated by the great number of APB(BL)/ARB(BL) ratios which have been used so far. On the other hand, the choice of the best standard technique among those described in the literature is not simple, since the variability of sources and isolation and preparation procedures of chitins and chitosans may entail that chemical reactions working for one particular sample may not work so well for another and again, this is well illustrated by a variety of methods which have been developed for this purpose [6 /17]. Recognising that (i) FTIR spectroscopy is a very attractive technique, as it is non- destructive, fast, sensitive, user-friendly, low-priced, suitable for both soluble and non-soluble samples; (ii) the above mentioned difficulties can be more easily and accurately solved by spectroscopists; (iii) this analytical problem is of immense importance for chitin and chitosan producers and researchers, we decided to optimise the FTIR technique to determine the DA of this fascinating system. In previous studies [17 /19] we chose the 13C cross polarisation magic angle spinning (CP/MAS) NMR spectroscopy as the standard technique to calibrate FTIR, since it is one of the most powerful, allowing direct determination of the DA, and, like FTIR, is suitable for soluble and non-soluble samples, non-destructive and requiring non-specific sample preparation. In addition, we carried out a comprehensive relaxation study in order to obtain accurate DA values of chitin and chitosan samples from 13C CP/MAS NMR spectra [19]. And, if it is true that currently, due to its high costs and some complexity, 13C solid state NMR will hardly be used in routine determinations, it is also true that producers and researchers can always order the determination of the DA of their samples to be used in the construction of the IR calibration lines from a specialised NMR laboratory. In fact, 13C solid state NMR together with FTIR are being increasingly used to

structural characterisation of solid chitin and chitosan derivatives [20]. In this paper, we perform a statistical evaluation of the ratios APB(BL)/ARB(BL) resulting from the combinations of all the PB, RB and BL used so far to calculate the DA of chitin and chitosan, using a-chitin and derived chitosans (most complex polymorphic form) covering the most problematic range of DA values (most acetylated samples), and outline an accurate and fast method to determine DA values of solid chitins and chitosans.

2. Experimental section 2.1. Materials Commercial chitin from crab shells (Sigma C-7170) was ground (grain size 5/100 mesh) and purified with 1 M HCl for 3 h at room temperature and refluxing 2 M NaOH for 48 h. The NaOH solution was replaced and the material washed with hot water every 6 h, until neutrality after the final 6 h treatment. Purified chitin had 0.32% ash and trace amounts of tyrosine, phenylalanine, lysine and histidine. The aminoacid analysis was carried out in an automatic analyser Biochrom 20 and required the addition of glucosamine hydrochloride to the standard aminoacids mixture in order to distinguish any aminoacid from the glucosamine resulting from sample hydrolysis. Five samples were obtained after deacetylation of purified chitin with 12 M NaOH at 110 8C under nitrogen atmosphere [21], for 2, 4, 6, 8 and 13 h, washing with hot water and replacing the solution after every 1 h treatment. 2.2. FTIR spectra FTIR spectra of the samples in KBr disc form were run in a Perkin /Elmer spectrometer 1725X with a resolution of 4 cm(1 and 32 accumulations. KBr discs were prepared in the usual way from very well dried mixtures of about 1 mg of the sample and 100 mg of KBr. The values of the different ratios APB(BL)/ARB(BL) obtained for each sample correspond to the average value of five spectra. 2.3.
13 13

C CP/MAS NMR spectra

C CP/MAS NMR spectra were recorded at 100.62 MHz on a Bruker MSL 400P spectrometer with a spinning rate of 5 kHz. The relevant parameters for signal acquisition were relaxation delay 0/5 s and contact time 0/1 ms as determined in [19]. The DA of the samples were determined dividing the intensity of the resonance of carbon from methyl group by the average intensities of resonances of carbons from

M.L. Duarte et al. / International Journal of Biological Macromolecules 31 (2002) 1 /8

the glucose ring: ICH3/(IC1'/IC2'/IC3'/IC4'/IC5'/IC6)/6 [24] and are 0.68, 0.50, 0.41, 0.29, 0.26, 0.16.

3. Results and discussion 3.1. FTIR spectroscopy

2.4. Statistical evaluation of the ratios APB(BL)/ARB(BL) [22,23] The statistical evaluation of all the calculated absorbance ratios was performed using the simple linear regression model. The regression model assumes that only y-direction random errors occur, hence to estimate the best line it is necessary to seek the line which minimises the deviations in the y -direction between the experimental and the estimated points (y-residuals). Since some of these residuals will be positive and others negative, it is reasonable to seek to minimise the sum of the squares of the residuals (least squares method). The line thus calculated indicates how y varies when x is set to chosen values and has the algebraic form given by Eq. (1): y0 bx' a (1)

where b is the estimated slope, a is the estimated intercept, y/ stands for the predicted APB/ARB ratios and x stands for the experimental DA 13C CP/MAS values. NMR To accurately choose the good regression lines among those we have studied, we need to work simultaneously with various criteria. The criterion commonly applied is the coefficient of determination, R2, which in the case of a straight line is equal to the square of the correlation coefficient, r2, and varies between 0 and 1, the latter representing a perfect fit of a line to a set of data points. Although correlation coefficient values are simple to calculate, they can easily be misinterpreted. So, in addition, we evaluated the significance of the model through the powerful statistical technique analysis of variance (ANOVA), which applies the one tailed F -test to calculate the F -value defined by the ratio of two variances: mean square due to regression and mean square due to residuals. The greater the F -value, the more significant is the model. The y-residuals are another criterion to provide valuable information on the success of the regression line. The residuals must be as small as possible if the line is to be a good fit to data points. In addition, we also calculated the 95% confidence limits for the slope and for the intercept, which must be as narrow as possible. To summarise, the calibration line chosen as the best fit to the experimental data is the one obeying the last mentioned criteria for the slope and intercept confidence limits and with R2 closer to one, the greatest F -value and the smallest residuals.

Figs. 1 and 2 depict the FTIR spectrum of a-chitin, the assignment of the relevant bands, and the BL, which have been used so far and other possible ones, to determine the absorbances of the PB and RB by the baseline method. The IR spectrum of a-chitin clearly shows the nonexistence of bands completely free of overlapping, and the consequent difficulty to choose the correct BL to determine the absorbances of PB and RB. In general, the complexity of the IR spectra of chitin and derived chitosans is due to the complicated and specific network of H-bonds in which the OH, C /O and NH groups are involved for each polymorphic form [25,26], and to the typical broadness of the IR bands of natural polymers. Further, when going from highly to less acetylated samples we might expect doublets in the NH bending region instead of a simple band. Thus, it is not surprising that researchers of chitin and chitosan field studying samples from different sources with different structures, submitted to different isolation and deacetylation processes should have used different ratios APB(BL)/ARB(BL) to determine the DA along these years (Table 1). From the point of view of a spectroscopist, it is obvious that it would be imperative to pursuit a detailed spectral analysis prior to the choice of the best PB and RB to solve this analytical problem. In what follows, we present a detailed analysis of the drawbacks associated with the PB and RB used so far to determine the DA of this system. 3.1.1. Probe bands The most commonly used PB have been the carbonyl stretching, nCO (amide I), and the NH bending, dNH (amide II), which clearly change their intensities with the DA. However, their positions and intensities are

Fig. 1. FTIR spectrum of a-chitin: PB and respective baselines.

M.L. Duarte et al. / International Journal of Biological Macromolecules 31 (2002) 1 /8

the decrease of the DA, probably due to the doublet expected for primary amines, may complicate its use as PB.

Fig. 2. FTIR spectrum of a-chitin: RB and respective baselines.

Table 1 Absorbance ratios used so far and found in the present work to determine the DA of chitin and chitosan APB(BL)/ARB(BL) A1550(BL1)/A2878(BL4) A1655(BL3)/A2867(BL5) A1655(BL1)/A3450(BL4) A1554(BL2)/A897(BL7) A1655(BL3)/A3450(BL4) (A1655(BL3)'A1630(BL8))/ A3450(BL4) (A1655(BL3)'A1630(BL8))/ A1070(BL6) (A1655(BL3)'A1630(BL8))/ A1030(BL6) A1560(BL2)/A1070(BL6) A1560(BL2)/A1030(BL6) A1626(BL2)/A2877(BL5) (A1663(BL2)'A1626(BL2))/ A2877(BL5) A1561(BL2)/A1074(BL6) A1561(BL2)/A1025(BL6) Year of proposal Sannan et al., 1978 [6] Miya et al., 1980 [7] Moore et al., 1980 [8], Domszy et al., 1985 [9] Miya et al., 1985 [10] Baxter et al. , 1992 [11], Roberts, 1997 [3] Shigemasa et al., 1996 [12] Shigemasa et al., 1996 [12] Shigemasa et al., 1996 [12] Shigemasa et al., 1996 [12] Shigemasa et al., 1996 [12] Present work

affected by remaining proteins and calcium binding to the chitin and chitosan matrix [25]. Further, a great number of BL can be used to determine their absorbances. The doublet at 1663 and 1626 cm (1 is assigned to nCO and arises from two types of H-bonds in which the C /O groups are involved in a-chitin [26]. Both bands may suffer interference from OH bending, dOH, of water at approximately 1640 cm (1 if the samples are not well dried. Surprisingly, until 1996, only the higher frequency component of the nCO doublet at 1663 cm (1 had been used as PB (Table 1). The dNH at 1561 cm (1 band has not been considered a good PB for highly deacetylated samples [27 /29] but it has been considered a reasonably good one for the other samples. However, a band appearing at 1597 cm (1 with

3.1.2. Reference bands Until today, the most commonly used RB had been OH and CH stretchings, respectively, nOH and nCH, at 3448 and 2877 cm (1. Although for nOH a change of intensity with DA is not expected, this band may suffer interferences from a second nOH at approximately 3480 cm (1, due to another type of H-bonds in which the CH2OH groups are involved in a-chitin [26], and from the intense NH stretching, nNH, at 3269 cm (1, the intensity of which varies with the DA, of course, besides nOH from water if the samples are not conveniently dried. These interferences become particularly relevant when BL4 is used to determine the absorbance of the nOH. The nCH at 2877 cm (1 lies in a complex spectral region where at least five bands have been observed due to symmetric and asymmetric stretchings of CH from the ring and from CH2OH and CH3 groups [25], only this one changing its intensity with the DA. Although these bands are not unequivocally assigned, in our FTIR spectra the intensity of the band at 2877 cm (1 did not change with the DA. Three CO stretchings, nCO, at 1159, 1074 and 1025 cm (1 have been analysed [10,12] and only the last two have been proposed as RB [12]. The intensity of the band at 1159 cm (1, assigned to the asymmetric nCO of bridge oxygen [25], may change if depolymerisation occurs during the deacetylation treatment. In the region of the bands at 1074 and 1025 cm (1, at least four bands have been observed due to the nCO of the ring COH and of the COC and CH2OH groups, the intensity of which are not expected to change with the DA. Finally, the band at 895 cm (1 besides not clearly assigned is relatively weak.

3.2. Statistical evaluation of the APB(RB)/ARB(BL) ratios Since we were aware of the spectral peculiarities of these samples, it became equally imperative for us to perform a statistical evaluation of the APB(BL)/ARB(BL) ratios resulting from the combinations of all the PB, RB and BL previously used and some new possible ones, as well as to develop an improved method to evaluate those ratios. Using the simple regression model, we constructed 120 calibration lines APB(BL)/ARB(BL) versus standard DA values obtained from the optimised 13C CP/MAS NMR spectra, which were evaluated according to the criteria mentioned before. Table 2 shows the R2 and F values for all the studied ratios.

M.L. Duarte et al. / International Journal of Biological Macromolecules 31 (2002) 1 /8 Table 2 Coefcients of determination, R2, and F -values of the signicance tests for the ratios APB(BL)/ARB(BL) ARB(BL) A3448(BL4) [3,9,11] A2877(BL4) [6] A2877(BL5) [7] A1159(BL6) [10,12] A1074(BL6) [12] A1025(BL6) [12]
1 A895(BL6)

A895(BL7) [10]

APB (BL ) A1663(BL1) [8,9] 1 A1663(BL2) A1663(BL3) [3,7,10,11] 1 A1663(BL9) A1 1626(BL1) 1 A1626(BL2) A1 1626(BL3) A1626(BL8) [12] A1561(BL1) [6] A1561(BL2) [10,12] A1663(BL1)'A1 1626(BL1) 1 A1663(BL2)'A1626(BL2) A1663(BL3)'A1 1626(BL3) A1663(BL3)'A1626(BL8) [12] 1 A1663(BL9)'A1626(BL8) *Present study.

0.9790, 0.9745, 0.9800, 0.9528, 0.9811, 0.9705, 0.9773, 0.9586, 0.9789, 0.9781, 0.9801, 0.9736, 0.9804, 0.9796,

186 153 196 81 207 131 172 92 186 178 197 148 200 192

0.9793, 0.9806, 0.9819, 0.9742, 0.9825, 0.9856, 0.9830, 0.9722, 0.9773, 0.9784, 0.9809, 0.9832, 0.9834, 0.9833,

189 202 217 151 224 273 231 140 172 181 206 233 237 235

0.9907, 0.9949, 0.9855, 0.9433, 0.9922, 0.9972, 0.9746, 0.9422, 0.9837, 0.9910, 0.9918, 0.9970, 0.9819, 0.9786,

426 782 272 66 506 1436 154 65 241 442 486 1317 217 183

0.9878, 0.9921, 0.9863, 0.9514, 0.9897, 0.9948, 0.9747, 0.9426, 0.9808, 0.9888, 0.9891, 0.9943, 0.9827, 0.9802,

325 503 288 78 384 759 154 66 204 354 362 694 227 198

0.9949, 0.9926, 0.9933, 0.9669, 0.9944, 0.9807, 0.9824, 0.9548, 0.9921, 0.9969, 0.9953, 0.9896, 0.9900, 0.9885,

778 537 593 117 712 203 223 84 500 1279 843 381 396 344

0.9952, 0.9952, 0.9899, 0.9508, 0.9955, 0.9856, 0.9760, 0.9423, 0.9893, 0.9965, 0.9959, 0.9934, 0.9852, 0.9826,

821 827 392 77 884 274 163 65 369 1150 978 597 266 225

0.9623, 0.9613, 0.9750, 0.9031, 0.9462, 0.9357, 0.9675, 0.9292, 0.9722, 0.9799, 0.9558, 0.9518, 0.9727, 0.9664,

102 99 156 37 70 58 119 52 40 194 86 79 142 115

0.9579, 0.9536, 0.9433, 0.7943, 0.9325, 0.8872, 0.9181, 0.8451, 0.9488, 0.9565, 0.9488, 0.9334, 0.9325, 0.9194,

91 82 66 15 55 31 45 22 74 88 74 56 55 46

0.9622, 102

0.9759, 162

0.9472, 72

0.9529, 81

0.9666, 116

0.9527, 81

0.9235, 48

0.8310, 20

The detailed analysis of the statistical results sequentially followed the four steps listed below. The first two steps are: i) identification of the R2 /0.9900 values for all PB(BL) combinations, ii) identification of the R2 /0.9900 values for all the RB(BL) combinations. The radar representations of the R2 values of all PB(BL) and of all RB(BL) combinations shown in Figs. 3 and 4 proved to be very helpful for the choice of the best combinations. As expected, according to our spectral analysis we did not find any plausible reasons to use as PB the 1663 cm (1 band of the nCO doublet and to exclude the other band, or not to use the sum of absorbances of both bands, while it remained some doubt about the quality of the dNH band as PB. The main problem associated with the PB was the correct choice of the respective BL and of the RB(BL) to combine with them. It was easily concluded from Fig. 3 that the best PB(BL) combinations are: all PB(BL1), all PB(BL2) and 1663(BL3). As far as RB are concerned, Fig. 4 clearly shows that the best combinations are 2877(BL5), 1159(BL6), 1074(BL6) and 1025(BL6), again in accordance with our predictions based on the spectral analysis. The nOH band with BL4, so far extensively used as RB, either with dNH, or with the higher frequency band of the nCO doublet did not prove to be a reasonably good RB, since the R2 values for all the calibration lines APB(BL)/A3448(BL4) vary from 0.9811

to 0.9528. The third and fourth steps are: iii) identification of the R2 /0.9900 values for APB(BL)/ ARB(BL) resulting from PB(BL) and RB(BL) combinations chosen in (i) and (ii) (Table 2). iv) choice of the best APB(BL)/ARB(BL) ratios based on the strict criteria: R2 /0.9900, F /1000, smallest residuals and narrowest confidence limits. Finally, we found that the best absorbance ratios to accurately determine the DA by FTIR spectroscopy are: A1626(BL2)/A2877(BL5), (A1663(BL2)'/A1626(BL2))/A2877(BL5), A1561(BL2)/A1074(BL6), A1561(BL2)/A1025(BL6). It is worth noting that amongst the great number of ratios used so far, only the last two are in accordance with previously but recently proposed ones [12]. However, the two best ratios we found do not agree with any previously proposed ones, although these have been extensively used until today [3]. These remarks mean, that along the years, the DA values of chitin and chitosan samples have not been accurately determined by IR spectroscopy. Furthermore, they are liable to invalidate the DA values obtained by other techniques to the extent that they were compared with the DA values determined by IR values.

4. Conclusions To conclude, we are in a position now to propose a fast and accurate method to determine the DA of chitins and chitosans, linking up FTIR with 13C CP/MAS NMR spectroscopies and involving the following steps:

M.L. Duarte et al. / International Journal of Biological Macromolecules 31 (2002) 1 /8

Fig. 3. Radar representation to identify PB(BL) combinations associated with R2 /0.9900.

1) Preparation of standard and unknown samples carefully dried, the more demineralised and deproteinised the better and completely free of paramagnetic impurities. All of these requirements must be confirmed by appropriate tests. The standard DA values must cover the range of DA values of unknown samples. 2) Determination of the DA of the purified samples to be used as standards by 13C CP/MAS NMR spectroscopy with the optimised parameters: relaxation delay of 5 s and contact time of 1 ms and using Ottys method [24]. The producers and the researchers of chitins and chitosans can always order this determination from any specialised NMR laboratory. 3) Determination of the ratios: A1626(BL2)/A2877(BL5), (A1663(BL2)'/A1626(BL2))/A2877(BL5), A1561(BL2)/ A1074(BL6), A1561(BL2)/A1025(BL6) from FTIR spectra of the purified samples, using the baseline method. 4) Construction of the calibration lines using the absorbance ratios determined in (3) and the standard DA values determined in (2).

5) Evaluation of the calibration lines, following the method described above. 6) If the calibration lines did not accurately fit the experimental data, a detailed analysis of the FTIR spectra of the samples must be carried out in order to choose other possible ratios to be submitted to the same statistical evaluation. 7) The construction of specific calibration lines for each particular isolation and deacetylation procedures to which the samples are submitted is required for reliable values of DA to be obtained by FTIR technique. The producers and the researchers of chitins and chitosans can equally order the construction of the specific calibration lines from specialised laboratories. Once the best calibration line is established and validated, the accurate determination of the DA of unknown samples can be obtained by FTIR spectroscopy in relatively short time. Finally, the proposed method to accurately determine the DA of chitins and chitosans by FTIR spectroscopy can easily be extended to other range of DA values and

M.L. Duarte et al. / International Journal of Biological Macromolecules 31 (2002) 1 /8

Fig. 4. Radar representation to identify RB(BL) combinations associated with R2 /0.9900.

to the other polymorphic forms, b- and g-chitins and derived chitosans, provided a previous detailed IR spectra analysis is performed.

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