1699

Characterization of deacetylated chitosan and

chitosan molecular weight review
Jonathan Z. Knaul, Mohammad R. Kasaai, V. Tam Bui,
and Katherine A.M. Creber

Abstract: Starting from a chitosan sample with a degree of deacetylation of 71%, three separate sample sets were
generated by successive deacetylation and reacetylation processes. The degree of deacetylation of samples was
determined by UV spectrometry supported by thermogravimetric analysis. The molecular weight of chitosan samples
was determined in a solvent system of 0.25 M CH3COOH/0.25 M CH3COONa, using viscometry and gel permeation
chromatography (GPC) with a TSK-gel column. The first set of samples had a similar degree of deacetylation (DDA)
but differing molecular weights. The second set of samples had a similar molecular weight but differing degrees of
deacetylation. The Mark–Houwink–Sakurada constants used for the determination of viscosity average molecular
weight and the universal calibration of the GPC system were K = 1.40 × 10–4 dL/g and a = 0.83. Results showed that
molecular weights determined from both techniques are in good accord only at lower degrees of deacetylation. This
may be attributed to the fact that the chemical structure of chitosan samples could have been largely altered with
increasing or decreasing degree of deacetylation. Nevertheless, the trend with which the molecular weights vary with
the deacetylation time is consistent over a limited DDA range. A literature review of molecular weight analysis of
chitosan is included.

Key words: chitosan, degree of deacetylation, gel permeation chromatography, molecular weight, viscometry.

Résumé : Utilisant comme produit de départ un échantillon de chitosane avec un degré de désacétylation de 71%, on a
préparé trois ensembles différents d’échantillon par des processus successifs de désacétylation et de réacétylation. On a
déterminé le degré de désacétylation des échantillons par spectrométrie UV associée à des analyses
thermogravimétriques. On a déterminé le poids moléculaire des échantillons de chitosane par viscosimétrie et par
chromatographie par perméation de gel (CPG) avec une colonne de gel de TSK, dans un système de solvant à 0,25 M
CH3COOH/0,25 M CH3COONa. Le premier ensemble d’échantillons présentait un degré de désacétylation (DDA)
semblable, mais des poids moléculaires différents. Le deuxième ensemble d’échantillons présentait des poids
moléculaires semblables, mais des degrés de désacétylation différents. Les constantes de Mark–Houwink–Sakurada
utilisées pour la détermination des poids moléculaires moyens par viscosité et pour la calibration universelle du
système de CPG sont les suivantes : K = 1,40 × 10–4 dL/mg et a = 0,83. Les résultats montrent que les poids
moléculaires déterminés par les deux techniques ne sont en bon accord qu’aux faibles degrés de désacétylation. On
peut attribuer cette observation au fait que la structure chimique des échantillons de chitosane peut avoir été fortement
modifiée par une augmentation ou une diminution du degré de désacétylation. Malgré tout, la tendance avec laquelle
les poids moléculaires varient avec le temps de désacétylation est consistent pour un degré limité de «DDA». On a
inclus une revue de la littérature relative à l’analyse des poids moléculaires des chitosanes.

Mots clés : chitosane, degré de désacétylation, chromatographie par perméation de gel, poids moléculaire, viscosi-
métrie.
Knaul et al. 1706

2-amino-2-deoxy- -D-glucan, is the N-deacetylated deriva-

Chitin, a (1–4)-linked 2-acetamido-2-deoxy- -D-glucan, is
a natural polysaccharide that occurs mainly in invertebrates,
fungi, and yeasts. It is the second most abundant natural
polymer after cellulose (1). Chitosan, a (1–4)-linked
tive of chitin, although 100% N-deacetylation is almost
never achieved. It is generally accepted that this biopolymer
is referred to as “chitosan” if chitin is N-deacetylated to
such a degree that it becomes completely soluble in dilute
aqueous acidic systems (1–5% by volume aqueous acetic

Received February 25, 1998.

J.Z. Knaul, V.T. Bui,1 and K.A.M. Creber. Department of Chemistry and Chemical Engineering, Royal Military College of
Canada/College Militaire Royal du Canada, P.O. Box 17 000, Stn Forces, Kingston, ON K7K 7B4, Canada.
M.R. Kasaai. Department of Food Science and Nutrition, Faculty of Agriculture, Pavillon Comtois, Laval University, Ste-Foy, QC
G1K 7P4, Canada.
1
Author to whom correspondence may be addressed. Telephone: (613) 541-6000. Fax: (613) 542-9489. E-mail: bui-v@rmc.ca

Can. J. Chem. 76: 1699–1706 (1998) © 1998 NRC Canada

1700
Fig. 1. Segments of cellulose, chitin, and chitosan polymers.
acid would be considered a dilute acidic medium). Structural
examples of chitin, chitosan, and the structurally similar
biopolymer cellulose can be found in Fig. 1. Chitosan is
well known as a nontoxic and degradable polymer. It is used
widely in the medical industry in products ranging from
burn dressings to drug delivery capsules.
Chitosan samples can be characterized for several parame-
ters including degree of deacetylation (DDA), turbidity, sol-
ubility, ash content, glass transition temperature, average
molecular weight, and molecular weight distribution. It is
known that viscometry and gel permeation chromatography
(GPC) are the two most commonly used secondary methods
for determining the molecular weight of polymers. They are
virtually related to each other through the use of the Mark–
Houwink–Sarakuda constants in both techniques (2, 3), and
the reliability of these constants for a given polymer–solvent
system determines the applicability of these secondary meth-
ods. As will be shown in the following section, the use of
these techniques is not always straightforward, especially in
the case of chitosan because of its complex hydrodynamic
behaviour, which greatly depends upon its degree of
deacetylation and the solvent system used.
In this study, one chitosan sample with a DDA of 71%
was deacetylated and then reacetylated, providing two series
of samples with either varying DDA or varying molecular
weight, and one series with constant DDA and molecular
weight. The samples were then characterized for the degree
of deacetylation by UV spectrometry supported by thermo-
gravimetric analysis, and for the molecular weights by vis-
cometry and gel permeation chromatography (GPC) using a
commonly used solvent system, 0.25 M CH3COOH/0.25 M
CH3COONa. The main objective of this work is first to de-
termine to what extent the viscometric and GPC data, based
on available values of the Mark–Houwink–Sarakuda con-
stants for the selected solvent system and for limited ranges
Can. J. Chem. Vol. 76, 1998
of DDA and molecular weight, could yield reliable
molecular weights of the above sets of chitosan samples, and
secondly, to determine the effects of the deacetylation and
reacetylation conditions, such as the reaction time and
amounts of reactants used, on the properties of the chitosan
samples obtained.
Viscometric studies on chitosan
The intrinsic viscosity, [h], of a polymer solution, includ-
ing nonelectrolytic and electrolytic polymers, is related to
the viscosity average molecular weight, Mv, of the polymer
through the Mark–Houwink–Sakurada equation (2, 3):
[1] [η] = K Mva
where K and a are constants depending on the polymer and
the solvent system used, as well as the temperature. Their
values are determined experimentally by evaluating the in-
trinsic viscosities of solutions of polymers for which the mo-
lecular weight has been determined by an independent
method. Several authors have reported the K and a values
for chitosan of varying DDA, in various solvent systems.
Table 1 summarizes data for Mv and related the constants K
and a, for different solvent systems and molecular weight
ranges of chitosan. The most commonly used solvent sys-
tems are buffer solutions formed with acetic acid/sodium ac-
etate at various concentrations. A significant result was
reported by Rinauldo et al. (10): for a narrow molecular
weight range, K and a were found to be nearly unchanged
for three samples of DDA of 98, 88.5, and 79%, respec-
tively. Normally, these constants have been found to vary
significantly when the DDA range of 69–100% is studied
over a large molecular weight range (9).
© 1998 NRC Canada
Knaul et al. 1701

Table 1. Published Mark–Houwink–Sakurada constants for chitosan.

T K Mv range Ref.
Solvent (°C) % DDA (mL/g) a Method (× 104) (year)
0.2 M CH3COOH/ 8.93 × 10–2 0.71 SD 1.38 4 (1974)
0.1 M NaCl/
0.4 M Urea
0.1 M CH3COOH/ 25 Derived 3.04 × 10–5 1.26 Absorbance 5 (1982)
0.2 M NaCl chitosan
0.17 M CH3COOH/ 25 90–100 1.115 0.147 SD 1.5–0.16 6 (1980)
0.47 M NaCl
2% CH3COOH/ 25 82–88 1.38 × 10–2 0.85 SD 1.5–0.16 7 (1985)
0.2 M CH3COOH
+ dichloroacetic acid
0.33 M CH3COOH/ 21 80–82 3.41 × 10–3 1.02 Diffusion 5.2–13.3 8 (1986)
0.3 M NaCI SD
0.1 M CH3COOH/ 25 ≈80 1.81 × 10–3 0.93 63–0.48 5, 9 (1988)
0.2 M NaCl
0.2 M CH3COOH/ 30 69 0.104 × 10–3 1.12 LS 25.1–1.94 9 (1991)
0.1 M CH3COONa 84 1.424 × 10–3 0.96
91 6.589 × 10–3 0.88 1.94
100 16.80 × 10–3 0.81
0.3 M CH3COOH/ 25 98 0.082 0.76 GPC/MALL 20.3–29.0 10 (1993)
0.2 M CH3COONa 88.5 0.076 0.76 LS/LALLS
79 0.074 0.76
0.02 M CH3COOH/ 20 100 Log K0.1 = a0.1 = 0.6169 OM 1.5–31.0 11 (1993)
0.1 M CH3COONa 85 –0.427 – + 0.759FA
and chitosan chloride 40 3.821FA
0.2 M CH3COOH/ 25 42 1.14 SD 12 (1993)
0.2 M CH3COONa 89 0.521
1% CH3COOH 30 0.0474 0.723 LS 20.46–65.74 13 (1993)
0.5 M CH3COOH/ 25 69.8 0.119 0.59 GPC/LS 274–11.5 14 (1993)
0.5 M CH3COONa
0.3 M CH3COOH/ 25 Plots but no values SEC3, LS 15 (1993)
0.2 M CH3COONa provided

Gel permeation chromatography (GPC) studies on
chitosan
The first study using a GPC system (16) was reported in
1976, using a long column packed with glass beads, and 2%
aqueous acetic acid as the mobile phase. A strong absorb-
ance peak was observed at 254 nm, which would appear to
be an unusual result, as chitosan is known to absorb between
200 and 204 nm (17). The chitosan sample studied was
found to have an Mn of 9.36 × 105 and an Mw of 2.05 × 106.
The GPC system was calibrated based on dextran standards,
but a universal calibration was not done. In a later study
(18) by the same authors, it was assumed that the differ-
ence in solution behavior between chitosan and the dextran
standards was negligible given their similarity in molecular
structure.
The molecular weight of chitosan samples varying in de-
gree of deacetylation between 78 and 99% was analyzed
using an in-line GPC/LALLS method (19), using a solvent
system of 0.2 M acetic acid/0.l M sodium acetate at 30°C.
The molecular weights were found to be between 8.5 × 105
and 4.9 × 105. These values decreased with a rise in treat-
ment temperature and with an increase in degree of
deacetylation. This is in accordance with results obtained us-
ing an off-line laser light scattering method (20). Nine
chitosan samples of different sources and varying molecular
weights were analysed using a GPC/MALLS system (21)
with dextran standards for the calibration. A dissimilarity
between the hydrodynamic behaviour of dextran standards
and chitosan samples was suggested as the probable cause of
the overestimation of the molecular weights of chitosan samples.
In a recent study (22) using a size exclusion chromatogra-
phy (SEC) system with different solvents ranging from a
0.3 M acetic acid solution to a 0.3 M acetic acid/0.2 M
sodium acetate buffer, polystyrene standards were used for
the calibration of the SEC system. A universal calibration
was not mentioned.
© 1998 NRC Canada
1702 Can. J. Chem. Vol. 76, 1998

Table 2. Deacetylated and reacetylated chitosan sample information.

Sample code % DDA (±3.4%) % Water + impurities (±1.5%) Deacetylation/reacetylation conditions

P59a 70.8 15.4 As received from NovaChem Ltd.
Deacetylated samples:
P59b9 87.3 12.5 P59a, 1 h, 50% NaOH (aq), 100°C, N2
P59c9i 94.5 4.8 P59b9, 1 h, 50% NaOH (aq), 100°C, air
P59c9ii 94.9 5.9 P59b9, 2 h
P59c9iii 95.0 6.2 P59b9, 3 h,
P59c9iv 95.1 5.9 P59b9, 4 h
P59c9v 95.3 5.1 P59b9, 5 h
P59c9vi 95.3 5.3 P59b9, 5.4 h
P59c9vii 95.3 3.8 Same conditions as for P59c9ii
P59c9viii 95.1 3.2
P59c9ix 95.4 3.0
P59d9i 96 3 P59c9ix, 0.5 h, 50% NaOH (aq), 100°C, air
Reacetylated samples:
B 90.1 4.3 P59d9i with 0.1004 g acetic anhydride
C 87.7 4.0 P59d9i withO.1236 g acetic anhydride
D 87.8 3.6 P59d9i withO.1445 g acetic anhydride
E 79.2 5.9 P59d9i withO.2845 g acetic anhydride
F 64.3 12.6 P59d9i with 0.5202 g acetic anhydride

Finally, four chitosan samples with degrees of deacetyl-
ation (23) varying between 91 and 92% were characterized
using a GPC system with a series of TSK-gel columns and a
mobile phase consisting of 0.3 M acetic acid/0.l M ammonium
acetate buffer. This study showed that a more accurate anal-
ysis of the molecular weight distribution of chitosan samples
can be made through GPC by application of the polydis-
persity index and the statistical skewing factor “g.” This factor
is a statistical measure of skewness of the chromatographic
peaks. When g = 0, the peak of the sample is symmetrical.
When g > 0, the peak has a long tail at high elution volumes
and thus is skewed toward low molecular weights. When g <
0, the peak is skewed toward higher molecular weights.
Thus g gives an indication of whether the polymer contains
more high or low molecular weight chains. However, the
chromatographic peaks are, in effect, influenced by both mo-
lecular separation and by axial dispersion as well as adsorption
effects. Thus, for example, a skewed peak can result from ad-
sorption.
The above GPC work can be summarized in the following
manner. Buffer solutions of acetic acid are suitable for the
mobile phase, dextran standards are commonly used for the
calibration of the GPC columns, and the dissimilarity in hydro-
dynamic behaviour between chitosans and calibration standards
should be taken into account. However, a large difficulty fre-
quently encountered in the calibration of GPC systems arises
from the fact that the hydrodynamic behaviour of chitosan in
a given solvent system, indirectly represented by the Mark–
Houwink–Sakurada constants, varies with its degree of
deacetylation as well as with its molecular weight range.
Materials
One chitosan sample with a degree of deacetylation of
71%, lot no. P59a, was obtained in the form of flake from
Novachem Limited of Dartmouth, Nova Scotia, Canada.
This is a shrimp-based product from the Gulf of St. Lawrence.
The viscosity of a solution of 1% chitosan (by weight) in 1%
(by volume) aqueous acetic acid was provided as 740 cP.
The following chemicals were all obtained and used as re-
agent grade from Sigma–Aldrich Canada Ltd: sodium hy-
droxide, acetic acid, acetic anhydride, methanol, and sodium
acetate. Chemicals used in the GPC experimentation were
HPLC grade.
One set of six samples having similar degrees of
deacetylation but differing molecular weights, and another
set of six samples with differing degrees of deacetylation,
but similar molecular weights, were prepared from the origi-
nal chitosan sample according to the following deacetylation
and reacetylation processes. In addition, three samples of the
same DDA and molecular weight were also prepared.
Deacetylation and fragmentation
Chitosan identification codes and the particulars of de-
acetylation are noted in Table 2. Deacetylations were carried
out by heating solutions of chitosans in aqueous NaOH solution,
50% by weight, at 100°C, for the times noted in Table 2,
under a nitrogen atmosphere. The reaction product was sub-
sequently washed in distilled water until reaching a neutral
pH, and then dried in an oven at 50°C for 16 h. In the first
experiment, the original chitosan, code P59a, was deacetyl-
ated for 1 h; the reaction product was code-named P59b9.
All subsequent deacetylation reactions were conducted under
air in order to aid in the breakup of chitosan molecules.
After washing and drying, this sample, P59b9, was further
reacted over the course of 6 h and one sample was removed
every hour, yielding a set of chitosan samples with similar
degrees of deacetylation but decreasing molecular weight.
The products from this experiment were code-named P59c9(i–vi).
In a second experiment, three separate samples of P59b9
were further deacetylated for 2 h under the same conditions
in order to arrive at three samples with similar DDA values
and molecular weights. Their code names are P59c9(vii–ix).
The final DDA values, also listed in Table 2, were deter-
mined by UV spectrometry, following a reported method (17).
© 1998 NRC Canada
Knaul et al.
Reacetylation
To obtain a series of chitosan samples with similar molec-
ular weights but varied degrees of deacetylation, a method
previously described (24) was followed. The experiment
used a sample of P59c9(ix) and further deacetylated it for
30 min. After washing and drying the product, it was code
named P59d9(I). Five samples (2 g) of P59d9(i) were each
placed into a flask containing 100 mL distilled water and
stirred for 1 h before adding 100 mL of 2% aqueous acetic
acid, then continuously stirred for one more hour. Methanol
(200 mL) was added to each flask and stirred for a further
10 min. The final addition to each solution included 100 mL
of methanol containing acetic anhydride, ranging in amount
from 6.86 × 10–4 mol for sample B to 5.10 × 10–3 mol for
sample F.
The solutions were then stirred for a further 16.5 h at
room temperature. At the end of this period, methanolic am-
monia was added to each solution in order to precipitate the
chitosan. The precipitants were recovered and washed with
distilled water using a sintered glass funnel under vacuum.
The solids were finally dried in a convection oven at 50°C
for 72 h.
Characterization
Thermogravimetric analysis
The water content of the chitosan samples, listed in Ta-
ble 2, was determined using a Texas Instruments 2050 TGA
thermogravimetric analyser. The sample was heated from
room temperature to 300°C at 10°C per min under nitrogen.
The loss in weight of a chitosan sample up to 180°C was
taken to be the result of evaporation of water and volatile
impurities.
Viscometry measurements
The viscosities of the chitosan samples were measured in
a solvent of 0.25 M CH3COOH/ 0.25 M CH3COONa at
24.95 ± 0.05°C using an Ubbelohde capillary viscometer,
model Cannon no. 100 E482, which yielded an average flow
time of 62.29 s for the solvent system. The uncertainty in
flow time was ±0.5 s, with at least three measurements for
each solution. Six dilutions with the solvent were used for
each chitosan sample. The flow time data were used to cal-
culate the relative viscosity, the reduced viscosity, and then
the intrinsic viscosity, [η] (by a linear regression of the
reduced viscosity versus concentration). The overall error in
the intrinsic viscosity, resulting mainly from the flow time
and concentration uncertainties, and the temperature fluctua-
tion, was estimated as being ±0.6 dL/g, which led to a maxi-
mum error of ±5% in the values of viscometric average
molecular weight. The correlation coefficients were better
than 0.950 for all chitosan samples.
Gel permeation chromatography (GPC)
GPC was carried out using the same instrumentation and
method as previously described by Kasaai et al. (25). The
solvent used was the same as that used in the viscosity mea-
surements. The GPC system incorporated a Hewlett–Packard
1050 series pump. The flow rate was maintained at
0.40 mL/min under a pressure of 1.041 ± 0.021 MPa with
zero attenuation. The system also included an HP 1050 se-
ries autoinjector and an HP 1047A refractive index detector.
1703
The column used was a TSK-gel GMPWXL column pro-
vided by Phenomenex with dimensions of 300 mm ×
7.8 mm. The temperature of the column was maintained at
35°C. The standards used to calibrate the column were
pullulan. All data provided by the GPC system were col-
lected and analysed using the Hewlett–Packard ChemStation
software package, B.02.06, 1994.
Degree of deacetylation and thermogravimetric analysis
Table 2 summarizes all relevant information concerning
the deacetylated and reacetylated chitosan samples obtained
from the original sample, P59a. Knowledge of the water
content as well as traces of volatile impurities in the chitosan
samples was essential since these amounts would affect,
first, the determination of their degree of deacetylation,
DDA, presented in Table 2 (using data from UV spectrome-
try), and, secondly, the calculation of the concentration C in
viscosity measurements. A typical thermogravimetric analy-
sis (TGA) curve for chitosan samples shows a significant
weight loss up to about 150°C followed by a plateau until
about 250°C, and then a marked decrease in the weight of
sample. It is believed that beyond 180°C all sample weight
loss should be due to the thermal degradation of short and
long molecular chains of chitosan. Repeated TGA runs on
the same chitosan samples showed an uncertainty in water
and impurities content of about 1.5%. This was the largest
contributing factor to the error in the DDA determination,
which was estimated as being ±3.4%. This error refers to
the maximum single standard deviation after several DDA
determinations performed on the same chitosan sample. The
TGA results for the temperature range up to 180°C are in
good agreement with those obtained by weighing chitosan
samples before and after being placed in an oven at 50°C for
24 h. This TGA method for determining the water and vola-
tile impurities content has also been successfully used by
Qin (26) for the same purpose.
The deacetylation of the original chitosan sample, P59a,
for 1 h in a nitrogen atmosphere increased the DDA from
70.8 to 87.3% and then up to 94.5% with one more hour of
deacetylation in air. For further deacetylation times, the DDA
remained nearly constant at about 95.0% while the molecu-
lar weight of samples decreased only slightly; this is shown
in Table 3 and will be discussed later. It is hard to draw firm
conclusions about the oxidative effect caused by oxygen in
air on the deacetylation of chitosan, but it is clear that longer
reaction times contribute to the chain degradation, or possibly
to some branching formation of chitosan polymer molecules.
Values of DDA for the reacetylation series, B–F, showed an
appreciable decrease in DDA with increasing amount of
acetic anhydride, which confirms the success of the
reacetylation method used.
Intrinsic viscosity, [η], and viscosity average molecular
weight, Mv
Reduced viscosity values for typical chitosan samples
are plotted in Fig. 2 as a function of concentration. The lin-
earity of all traces is attributed to the presence of a suitable
amount of sodium acetate in the acetic acid solution. With-
out this salt, one would expect to observe a large depend-
© 1998 NRC Canada
1704 Can. J. Chem. Vol. 76, 1998

Table 3. Average molecular weight values for chitosan samples.

Sample % DDA Mv × 10–3

code (±3.4%) [η] (dL g–1) (±5%) Mn × 10–3 Mw × 10–3 Mw/Mn
P59a 70.8 12.03 881 855 1619 1.89
P59b9 87.3 11.22 809 427 842 1.97
P59c9i 94.5 10.22 724 119 302 1.52
P59c9ii 94.9 9.84 691 223 333 1.49
P59c9iii 95.0 9.60 671 200 310 1.55
P59c9iv 95.1 9.75 684 221 318 1.44
P59c9v 95.3 9.40 654 183 269 1.47
P59c9vi 95.3 9.2 637 184 265 1.44
P59c9vii 95.3 9.93 699 208 307 1.48
P59c9viii 95.1 9.79 687 233 341 1.46
P59c9ix 95.4 9.77 685 223 319 1.43
P59d9i 96 9.00 621 179 252 1.41
B 90.1 8.58 586 247 457 1.85
C 87.7 8.57 585 260 502 1.93
D 87.8 8.51 580 297 608 2.05
E 79.2 7.90 531 270 550 2.04
F 64.3 7.52 500 335 699 2.09

Fig. 2. Reduced viscosity versus concentration for some chitosan samples.

ence of the reduced viscosity on the concentration, most
importantly in the low concentration range. This is typical
for a polyelectrolyte whose molecular chains become
extended as they progressively lose their charge due to the
escape of some counterions from the polymer backbones,
leaving the latter oppositely charged. The effect of an ex-
tended polymer molecule on the viscosity of the solution is
greater than that of a random polymer chain. Consequently,
if the polyelectrolyte solutions contain a sufficiently large
amount of simple electrolyte, such as sodium chloride or so-
dium acetate, their reduced viscosity is expected to change
linearly with concentration similar to that of an uncharged
polymer solution.
The intrinsic viscosity values, [η], and the limited value of
reduced viscosity at C = 0, are presented in Table 3. These
values allowed the calculation of the viscosity average mo-
lecular weight, Mv, also reported in Table 3, according to the
following equation:
[2] [η] = 1.40 × 10–4 Mv0.83

© 1998 NRC Canada

Knaul et al. 1705

This equation for the solvent system of 0.25 M acetic Fig. 3. Size exclusion chromatograrns of initial chitosan
acid/0.25 M sodium acetate has been developed by Kasaai et al. (A) P59a, and three deacetylated samples: (B) P59b9,
(25), using combined viscometry–GPC methods performed (C) P59c9i, (D) P59c9vi, respectively.
on relatively monodisperse chitosan samples, with pullulan
standards used for the calibration of the GPC system where
the discrepancy between the hydrodynamic behaviour of
chitosan and pullulan standards was taken into account by a
universal calibration method. The value of a = 0 means that
chitosan molecules behave like random polymer chains in
the given solvent system. This is supported by the results of
other authors (7, 9, 10), Table 1, for similar solvent systems.
As an approximation, the hydrodynamic behaviour of
chitosan in the given solvent system is assumed to be nearly
the same within a limited DDA range (e.g., between 71 and
95%). It is thus found that the intrinsic viscosity and then Mv
largely decrease with increasing DDA, up to 2 h of reaction
time, and then only slightly diminish with further reaction
times. Chain scission might have occurred during the
deacetylation process, and this may, to a certain degree “out-
weigh,” but not occlude, the effect of coil expansion due to
electrostatic repulsion, which closely depends on the DDA
of samples. The above results do not agree with those
reported by Roberts and co-workers (5, 24), where an oppo-
site trend was observed (e.g., the intrinsic viscosity and
molecular weight increased with increasing DDA). This
trend was attributed (5, 24) to the fact that the cationic effect
of the positively charged amine groups on the chitosan back-
bone plays a greater role in coil expansion than do the acetyl P59c9ix may lend support to the reproducibility of the
groups present on a more highly acetylated chitosan solu- deacetylation process used as well as the reliability of the
tion. However, the reported trend has been observed with the DDA determination and the viscosity measurements.
use of a 0.1 M acetic acid/0.2 M sodium chloride solvent
system (5), which did not have the same buffer effect as the Gel permeation chromatography
acetic acid/sodium acetate solvent systems. Consequently, it The typical GPC chromatograms of some samples are pre-
is suggested in this study that, as a result of their suitable sented in Fig. 3, from which the number and weight average
buffer effect, the acetic acid/sodium acetate solvent systems molecular weights, Mn, Mw, and the polydispersity index
could well minimize the difference in electrolytic effects of were deduced, and are listed in Table 3. The original chitosan
amine groups and acetyl groups. This, at least for a limited sample P59a has a relatively narrow molecular weight distri-
DDA range, leads to a lesser dependence of K and a on bution with a polydispersity index of 1.89 and, interestingly,
DDA. Furthermore, it has been proposed (9) that, with in- all deacetylated samples have even narrower molecular
creasing DDA, chitosan chain stiffness decreases due to a weight distributions with a polydispersity index of as low as
decreased presence of hydrogen bonds between the mono- 1.41. This likely resulted from the washing operation per-
mer units of the chitin chain. In the more acetylated chitosan formed on deacetylated samples where relatively short
chain, two types of intramolecular H-bonds are available to chitosan chains could have been advantageously drained out.
restrict coiling of the chains: first, between the CH2OH In most cases, except for samples P59a and P59b9, Mw and
group and the O atom of the acetyl group of two neighbour- Mn are smaller than Mv obtained from viscosity measure-
ing repeat units and, secondly, between the OH group and ments. This is typical of most polymers when applying a sta-
the b-deoxy of two neighbouring segments. Thus, by in- tistical analysis of the molecular weight distribution (25).
creasing the DDA, the former type of H-bonds will be re- On the other hand, results for the reacetylated samples, B–F,
duced, leading to an increase in the coiling of chitosan do not exhibit this trend, e.g., Mw becomes larger than Mv
chains. This H-bond effect may be a significant factor con- when the DDA of the samples decreases. This is likely due
tributing to the dependence of K and a constants on the to the uncertainty in the use of the K and a values for the
DDA of chitosan. Regarding the reacetylated sample series, calibration of the GPC system over such a large DDA range
the B–F samples, their intrinsic viscosity and molecular within which the differences in the molecular conformation of
weight show only a slight decrease as the DDA greatly de- chitosan such as discussed above should become over-
creases from 96.0 to 64.3%. This may result from combined whelming. In addition, there may be another cause, such as a
effects of, first, a possible chain scission following the greater tendency for the samples, with increasing DDA, to
reacetylation process and, secondly, changes in the confor- adhere to the column bed, thus resulting in longer elution
mation of chitosan chains due to differences in their electro- times. Column bed – sample interaction is typically associ-
static repulsion, H-bonds, and branching extent, over a large ated with an electrostatic attraction. The buffer effect of the
DDA range. Finally, the almost unchanged values of the in- solvent used would have suitably attenuated this interaction
trinsic viscosity and Mv of the samples from P59c9vii to within a small DDA range but not completely eliminated it

© 1998 NRC Canada

1706 Can. J. Chem. Vol. 76, 1998

over such a large DDA range. Furthermore, due to the 7. A.I. Gamzazade, V.M. Slimak, A.M. Skljar, E.V. Stykova,
branching conformation of chitosan (27)2, neither the sec- S.S.A. Pavlova, and S.V. Rogozin. Acta Polym. 8, 420 (1985).
ondary viscometry nor the GPC methods used can provide 8. N.V. Pogodina, G.M. Pavlov, S.V. Bushin, A.B. Mel’nikov, et
accurate values of molecular weights compared to an abso- al. Polym. Sci. USSR, 28, 251 (1986).
lute method (for example, off-line laser light scattering). 9. W. Wang, B. Shuqin, L. Shuqing, and W. Qin. Int. J. Biol.
Regarding the series P59c9, it was expected at the begin- Macromol. 13, 281 (1991).
ning that with increasing deacetylation time chitosan would 10. M. Rinaudo, M. Milas, and P.L. Dung. Int. J. Biol. Macromol.,
reach a plateau in its DDA (27), while the molecular weight 15, 281 (1993).
11. M.W. Anthonsen, K.M. Varum, and O. Smidsrod. Carbohydr.
would lower significantly. This is not the case since the mo-
Polym. 22, 193 (1993).
lecular weights change just slightly, and it is shown that 6 h
12. N. Errington, S.E. Harding, K.M. Varum, and L. Illum. Int. J.
of deacetylation caused virtually no reduction in molecular Biol. Macromol. 15, 113 (1993).
weight. 13. D.G. Rao. J. Food Sci. Technol. 30, 66 (l993).
14. G.A.F. Roberts. Chitin chemistry. Macmillian Press, London.
1992. p. 64.
15. G.A.F. Roberts and W. Wang. Adv. Chitin Sci. 1, 279 (1996).
In this study, starting from one mother chitosan sample, 16. A.C.M. Wu and W.A. Bough. J. Chromatogr. 128, 87 (1976).
three series of deacetylated and reacetylated samples were 17. R.A.A. Muzzarelli, C. Jeuniaux, and G.W. Gooday. Chitin in
successfully produced: one with similar DDA values of 95% nature and technology. Plenum, New York. 1986. p. 385.
but molecular weight varying slightly from 724 000 to 18. A.C.M. Wu. Methods Enzymol. 161, 447 (1988).
637 000, another with DDA values decreasing from 90% to 19. M. Miya, R. Iwamoto, S. Yoshikawa, and S. Mima. Kobunshi
61% and molecular weight varying only slightly from Ronbunshu, 43, 83 (1986).
586 000 to 500 000, and the last one with both DDA and 20. R.A.A. Muzzarelli, C. Lough, and M. Emanuelli. Carbohydr.
molecular weight constant. The DDA of samples was deter- Res. 164, 433 (1987).
mined using UV spectrometry with the help of 21. R.G. Beri, J. Walker, E.T. Reese, and J.E. Rollings. Carbohydr.
thermogravimetric analysis, which provided the water and Res. 238, 11 (1993).
volatile impurities content of the samples. The reduced vis- 22. I. Hall, D. Gillespie, K. Hammons, and J. Li. Adv. Chitin Sci.
cosity as well as molecular weights of samples were deter- 1, 361 (1996).
mined by viscometry and GPC methods using the solvent 23. A. Denuziere, N. Yagoubi, A. Baillet, and D. Ferrier. S.T.P.
system of 0.25 M acetic acid/0.25 M sodium acetate and Pharma. Sci. 5, 481 (1995).
pullulan standards for the universal calibration of the GPC 24. G.G. Maghami and G.A.F. Roberts. Makromol. Chem. 189,
system. The difficulty with which accurate values of molec- 195 (1988).
ular weights could be deduced from the above methods is re- 25. M.R. Kasaai, J.A. Arul, and G. Charlet. Proc. 7th Int. Conf. on
lated to the dependence of the constants K and a, for a given Chitin and Chitosan. Lyon, France. 1997. p. 421.
26. Y. Qin. The production of fibres from chitosan. Ph.D. Thesis
solvent system, on the DDA and molecular weight ranges of
12085, University of Leeds, England. 1991. p. 70.
the chitosan samples. Nevertheless, the results obtained did
27. C. Yomota, T. Miyazaki, and S. Okada. Colloid Polym. Sci.
show consistent trends in variation of the intrinsic viscosity 271, 76 (1993).
and molecular weights with the deacetylation time and with
the amount of acetic anhydride used in the reacetylation pro-
cess. It remains unclear whether there may be a possible oxi-
dative effect on the deacetylation performance caused by
oxygen in the air. DDA: degree of deacetylation
GPC: gel permeation chromatography
HPLC: high-performance liquid chromatography
LS: light scattering
1. F. Shahidi. Canadian Chemical News. 47(8), 25 (1995).
LALLS: low-angle laser light scattering
2. P.J. Flory. Principles of polymer chemistry. Cornell University
Press, Ithaca, N.Y. 1953.
MALLS: multiple angle laser light scattering
3. P.J. Flory. J. Am. Chem. Soc. 65, 372 (1943). OM: osmometry
4. V.F. Lee. University Microfilms (Ann Arbor) 74/29446 (1974). SD: sedimentation and diffusion
5. G.A.F. Roberts and J.G. Domszy. Int. J. Biol. Macromol. 4, SEC3: size exclusion chromatography with three
374 (1982). detectors (refractive index, in-line viscosity,
6. L.A. Berkovich, G.I. Timofeyera, M.P. Tsyurupa, and V.A. and light scattering)
Davankov. Vysokomol. Soedin. Ser. A. 22, 1834 (1980). VIS: viscometry

2 Dr. W.S. Wong. Viscotek Corporation, Houston, Texas. Private communication, January, 1998.

© 1998 NRC Canada