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

www.elsevier.com/locate/ijbiomac

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. Marvão b, João Rocha c
a
Department of Chemistry and Biochemistry, CECUL, University of Lisbon, 1749-016 Lisbon, Portugal
b
Department of Materials, National Institute of Engineering and Industrial Technology, Estrada do Paço 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 biomedical to waste water treatment and fibre indus-

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 poly-
morphic 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 deace-
tylation of purified chitin with concentrated alkali and
high temperature treatment, and frequently it is com-
mercialised 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: /351-217-
500-088
E-mail address: mlduarte@fc.ul.pt (M.L. Duarte).
tries.
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 appar-
ently simple analytical problem into a very difficult one,
which is well illustrated by the extensive number of
different techniques, either destructive or non-destruc-
tive, yielding direct or non-direct and frequently non
reproducible values [1
/5]. It is worth noting that non-
destructive 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
2 M.L. Duarte et al. / International Journal of Biological Macromolecules 31 (2002) 1
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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 construc-
tion 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 absor-
bances 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 men-
tioned difficulties can be more easily and accurately
solved by spectroscopists; (iii) this analytical problem is
of immense importance for chitin and chitosan produ-
cers 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 cali-
brate 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 pre-
paration.
In addition, we carried out a comprehensive relaxa-
tion 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 deter-
mination of the DA of their samples to be used in the
construction of the IR calibration lines from a specia-
lised 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 combina-
tions 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, phenyla-
lanine, 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 cm1 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
C CP/MAS NMR spectra
13
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 /5 s and
contact time /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 3

the glucose ring: ICH3/(IC1/IC2/IC3/IC4/IC5/IC6)/6 3. Results and discussion

[24] and are 0.68, 0.50, 0.41, 0.29, 0.26, 0.16.
3.1. FTIR spectroscopy

Figs. 1 and 2 depict the FTIR spectrum of a-chitin,

2.4. Statistical evaluation of the ratios APB(BL)/ARB(BL)
the assignment of the relevant bands, and the BL, which
[22,23]
have been used so far and other possible ones, to
determine the absorbances of the PB and RB by the
The statistical evaluation of all the calculated absor-
baseline method.
bance ratios was performed using the simple linear
The IR spectrum of a-chitin clearly shows the non-
regression model.
existence of bands completely free of overlapping, and
The regression model assumes that only y-direction
the consequent difficulty to choose the correct BL to
random errors occur, hence to estimate the ‘best’ line it
determine the absorbances of PB and RB.
is necessary to seek the line which minimises the
In general, the complexity of the IR spectra of chitin
deviations in the y -direction between the experimental
and derived chitosans is due to the complicated and
and the estimated points (y-residuals). Since some of
specific network of H-bonds in which the OH, C /O and
these residuals will be positive and others negative, it is
NH groups are involved for each polymorphic form
reasonable to seek to minimise the sum of the squares of
[25,26], and to the typical broadness of the IR bands of
the residuals (least squares method). The line thus
natural polymers. Further, when going from highly to
calculated indicates how y varies when x is set to
less acetylated samples we might expect doublets in the
chosen values and has the algebraic form given by Eq.
NH bending region instead of a simple band. Thus, it is
(1):
not surprising that researchers of chitin and chitosan
field studying samples from different sources with
ŷ b̂x â (1)
¯ different structures, submitted to different isolation
and deacetylation processes should have used different
where b̂ is the estimated slope, â is the estimated ratios APB(BL)/ARB(BL) to determine the DA along these
intercept, ŷ/ stands for the predicted APB/ARB ratios years (Table 1).
and x stands ¯ for the experimental DA 13C CP/MAS From the point of view of a spectroscopist, it is
¯
NMR values. obvious that it would be imperative to pursuit a detailed
To accurately choose the good regression lines among spectral analysis prior to the choice of the best PB and
those we have studied, we need to work simultaneously RB to solve this analytical problem.
with various criteria. In what follows, we present a detailed analysis of the
The criterion commonly applied is the coefficient of drawbacks associated with the PB and RB used so far to
determination, R2, which in the case of a straight line is determine the DA of this system.
equal to the square of the correlation coefficient, r2, and
varies between 0 and 1, the latter representing a perfect 3.1.1. Probe bands
fit of a line to a set of data points. Although correlation The most commonly used PB have been the carbonyl
coefficient values are simple to calculate, they can easily stretching, nCO (amide I), and the NH bending, dNH
be misinterpreted. So, in addition, we evaluated the (amide II), which clearly change their intensities with the
significance of the model through the powerful statis- DA. However, their positions and intensities are
tical 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. Fig. 1. FTIR spectrum of a-chitin: PB and respective baselines.
4 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.

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
Fig. 2. FTIR spectrum of a-chitin: RB and respective baselines.
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
Table 1 varies with the DA, of course, besides nOH from water if
Absorbance ratios used so far and found in the present work to the samples are not conveniently dried. These interfer-
determine the DA of chitin and chitosan ences become particularly relevant when BL4 is used to
APB(BL)/ARB(BL) Year of proposal determine the absorbance of the nOH.
The nCH at 2877 cm 1 lies in a complex spectral
A1550(BL1)/A2878(BL4) Sannan et al., 1978 [6] region where at least five bands have been observed due
A1655(BL3)/A2867(BL5) Miya et al., 1980 [7]
A1655(BL1)/A3450(BL4) Moore et al., 1980 [8], Domszy et al.,
to symmetric and asymmetric stretchings of CH from
1985 [9] the ring and from CH2OH and CH3 groups [25], only
A1554(BL2)/A897(BL7) Miya et al., 1985 [10] this one changing its intensity with the DA. Although
A1655(BL3)/A3450(BL4) Baxter et al. , 1992 [11], Roberts, 1997 these bands are not unequivocally assigned, in our FTIR
[3] spectra the intensity of the band at 2877 cm 1 did not
(A1655(BL3)A1630(BL8))/ Shigemasa et al., 1996 [12]
A3450(BL4)
change with the DA.
(A1655(BL3)A1630(BL8))/ Shigemasa et al., 1996 [12] Three CO stretchings, nCO, at 1159, 1074 and 1025
A1070(BL6) cm 1 have been analysed [10,12] and only the last two
(A1655(BL3)A1630(BL8))/ Shigemasa et al., 1996 [12] have been proposed as RB [12]. The intensity of the
A1030(BL6) band at 1159 cm 1, assigned to the asymmetric nCO of
A1560(BL2)/A1070(BL6) Shigemasa et al., 1996 [12]
A1560(BL2)/A1030(BL6) Shigemasa et al., 1996 [12]
bridge oxygen [25], may change if depolymerisation
A1626(BL2)/A2877(BL5) Present work occurs during the deacetylation treatment. In the region
(A1663(BL2)A1626(BL2))/ of the bands at 1074 and 1025 cm 1, at least four bands
A2877(BL5) have been observed due to the nCO of the ring COH and
A1561(BL2)/A1074(BL6) of the COC and CH2OH groups, the intensity of which
A1561(BL2)/A1025(BL6)
are not expected to change with the DA.
Finally, the band at 895 cm 1 besides not clearly
assigned is relatively weak.
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 absor-
3.2. Statistical evaluation of the APB(RB)/ARB(BL) ratios
bances.
The doublet at 1663 and 1626 cm 1 is assigned to Since we were aware of the spectral peculiarities of
nCO and arises from two types of H-bonds in which the these samples, it became equally imperative for us to
C /O groups are involved in a-chitin [26]. Both bands perform a statistical evaluation of the APB(BL)/ARB(BL)
may suffer interference from OH bending, dOH, of water ratios resulting from the combinations of all the PB, RB
at approximately 1640 cm 1 if the samples are not well and BL previously used and some new possible ones, as
dried. well as to develop an improved method to evaluate those
Surprisingly, until 1996, only the higher frequency ratios.
component of the nCO doublet at 1663 cm 1 had been Using the simple regression model, we constructed
used as PB (Table 1). 120 calibration lines APB(BL)/ARB(BL) versus standard
The dNH at 1561 cm 1 band has not been considered DA values obtained from the optimised 13C CP/MAS
a good PB for highly deacetylated samples [27
/29] but it NMR spectra, which were evaluated according to the
has been considered a reasonably good one for the other criteria mentioned before. Table 2 shows the R2 and F -
samples. However, a band appearing at 1597 cm 1 with values for all the studied ratios.
 M.L. Duarte et al. / International Journal of Biological Macromolecules 31 (2002) 1
/8 5

Table 2
Coefficients of determination, R2, and F -values of the significance tests for the ratios APB(BL)/ARB(BL)

ARB(BL) A3448(BL4) A2877(BL4) A2877(BL5) [7] A1159(BL6) A1074(BL6) A1025(BL6) 

A895(BL6) A895(BL7)
[3,9,11] [6] [10,12] [12] [12] [10]

APB (BL )
A1663(BL1) [8,9] 0.9790, 186 0.9793, 189 0.9907, 426 0.9878, 325 0.9949, 778 0.9952, 821 0.9623, 102 0.9579, 91

A1663(BL2) 0.9745, 153 0.9806, 202 0.9949, 782 0.9921, 503 0.9926, 537 0.9952, 827 0.9613, 99 0.9536, 82
A1663(BL3) [3,7,10,11] 0.9800, 196 0.9819, 217 0.9855, 272 0.9863, 288 0.9933, 593 0.9899, 392 0.9750, 156 0.9433, 66

A1663(BL9) 0.9528, 81 0.9742, 151 0.9433, 66 0.9514, 78 0.9669, 117 0.9508, 77 0.9031, 37 0.7943, 15
A1626(BL1) 0.9811, 207 0.9825, 224 0.9922, 506 0.9897, 384 0.9944, 712 0.9955, 884 0.9462, 70 0.9325, 55

A1626(BL2) 0.9705, 131 0.9856, 273 0.9972, 1436 0.9948, 759 0.9807, 203 0.9856, 274 0.9357, 58 0.8872, 31
A1626(BL3) 0.9773, 172 0.9830, 231 0.9746, 154 0.9747, 154 0.9824, 223 0.9760, 163 0.9675, 119 0.9181, 45
A1626(BL8) [12] 0.9586, 92 0.9722, 140 0.9422, 65 0.9426, 66 0.9548, 84 0.9423, 65 0.9292, 52 0.8451, 22
A1561(BL1) [6] 0.9789, 186 0.9773, 172 0.9837, 241 0.9808, 204 0.9921, 500 0.9893, 369 0.9722, 40 0.9488, 74
A1561(BL2) [10,12] 0.9781, 178 0.9784, 181 0.9910, 442 0.9888, 354 0.9969, 1279 0.9965, 1150 0.9799, 194 0.9565, 88
A1663(BL1)A1626(BL1) 0.9801, 197 0.9809, 206 0.9918, 486 0.9891, 362 0.9953, 843 0.9959, 978 0.9558, 86 0.9488, 74

A1663(BL2)A1626(BL2) 0.9736, 148 0.9832, 233 0.9970, 1317 0.9943, 694 0.9896, 381 0.9934, 597 0.9518, 79 0.9334, 56
A1663(BL3)A1626(BL3) 0.9804, 200 0.9834, 237 0.9819, 217 0.9827, 227 0.9900, 396 0.9852, 266 0.9727, 142 0.9325, 55
A1663(BL3)A1626(BL8) 0.9796, 192 0.9833, 235 0.9786, 183 0.9802, 198 0.9885, 344 0.9826, 225 0.9664, 115 0.9194, 46
[12]

A1663(BL9)A1626(BL8) 0.9622, 102 0.9759, 162 0.9472, 72 0.9529, 81 0.9666, 116 0.9527, 81 0.9235, 48 0.8310, 20

*Present study.

The detailed analysis of the statistical results sequen- to 0.9528.

tially 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 absor-
bances 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
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) combi-
nations 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]. How-
ever, 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. Further-
more, 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:
6 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 depro-
teinised the better and completely free of paramag-
netic 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: relaxa-
tion delay of 5 s and contact time of 1 ms and using
Ottøy’s method [24]. The producers and the re-
searchers 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 stan-
dard 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 proce-
dures 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 con-
struction 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 spectro-
scopy 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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