Carbohydrate Polymers 62 (2005) 316–320

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N-Deacetylation and depolymerization reactions of chitin/chitosan:

Influence of the source of chitin
Gemma Galed, Beatriz Miralles, Inés Paños, Alejandro Santiago, Ángeles Heras*
Instituto de Estudios Biofuncionales, Departamento de Quimica Fı́sica II, Facultad de Farmacia, Universidad Complutense de Madrid,
Pso. Juan XXIII, no 1. 28040 Madrid, Spain
Received 29 October 2004; revised 3 March 2005; accepted 15 March 2005
Available online 09 November 2005

Abstract
The deacetylation and depolymerization reactions of chitin/chitosan from three crustacean species (Paralomis granulosa, Lithodes
antarcticus and Palinurus vulgaris) were evaluated under the same conditions. The average molecular weight and the mole fraction of
N-acetylated units were the parameters studied in the resulting chitosans. During the N-deacetylation process P. granulosa,
L. antarcticus and P. vulgaris follow a pseudo-first order kinetics and their apparent rate constants are very similar. However, the
degradation rate of chitosan in the first 45 min of this process is higher for P. vulgaris. The depolymerization process follows a pseudo-
first order kinetics for the three species, but in the first 9 min P. vulgaris shows a slightly lower depolymerization rate. Hence, depending
on the ash contents, crystallinity and the physicochemical characteristics of chitin from these sources, the obtained chitosans show
different qualities.
q 2005 Elsevier Ltd. All rights reserved.

Keywords: Chitosan; Crustacean species; N-deacetylation; Degradation; Degree of depolymerization

1. Introduction a pseudo-first order reaction. However, the effect of the

biological source was not frequently mentioned.
Chitosan is a cationic biopolymer obtained from alkaline Chitosan is, like other polysaccharides, susceptible to a
N-deacetylation of chitinwhere the A-units GlcNAc [b(1/4) variety of degradation mechanisms, including oxidative–
2-acetamido-2 deoxy-b-D-glucopyranose] and D-units Glc reductive free radical depolymerization and acid, alkaline
[b(1/4) 2-amino-2-deoxy-b-D-glucopyranose] have been and enzymatic-catalysed hydrolysis. Degradation of
shown to be randomly distributed along the chains. polysaccharides occurs via cleavage of the glycosidic
Chitin has been found in a wide range of natural sources bonds. Controlling depolymerization of chitosan is useful
such as crustaceans, fungi, insects and some algae in order to adjust properties like viscosity, solubility and
(Tolaimate, Desbriéres, Rhazi, Alagui, Vincendon, 2000). biological activity (Rege & Block, 1999). Especially for
However, manufactured chitosan is usually obtained from biomedical applications high quality polymers of known
crustaceans (crab, krill and crayfish), especially because a molecular weight are mandatory as well as knowledge of
large amount of their exoskeleton is available as a by- the stability of chitosan over time in solution and in the
product of food processing (Methacanon, Prasitsilp, dry state (shelf life). Roberts (1992); Vårum; Ottøy and
Pitgsree & Pattaraarchachai, 2003) Smidsrod, (2001) have reviewed the results on the
The kinetics of heterogeneous alkaline deacetylation degradation of chitin and chitosan. The degradation of
of chitin has been studied by several authors as Chang, chitosan has been mostly described for chitosan solutions
Tsai; Lee and Fu; (1997) and it has been reported to be but the influence of the species in this reaction has not
been yet deeply studied.
Chitosans can be chemically depolymerized by different
* Corresponding author. Tel./fax: C34 91 394 32 84. mechanisms (acid hydrolysis, oxidative-reductive and
E-mail address: aheras@pluri.ucm.es (Á. Heras). nitrous acid depolymerization). The nitrous acid depoly-
0144-8617/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. merization reaction mechanism has been found to be
doi:10.1016/j.carbpol.2005.03.019 specific in the sense that HONO attacks the amino group
 G. Galed et al. / Carbohydrate Polymers 62 (2005) 316–320 317

of D-units, with the subsequent cleavage of the following 2.3. Depolymerization

glycosidic linkage (Allan and Peyron, 1995).
The physicochemical properties of chitosan, as well as its The chitosan products obtained from the N-deacetylation
functionality, depend on two fundamental parameters: the reaction above mentioned were dissolved (0.68%, wt%) in
degree of deacetylation and the degree of polymerization. HCl 0.1 M, stirred for 12 h and filtered through 5 mm TMTP
These parameters can be influenced by the species and by membrane. After that, 1.5 ml of 31.36 mM KNO2 were
the conditions used in both reactions. added to the solutions and separated in eight different
The purpose of this study was to evaluate the influence recipients for each selected time (3, 6, 9, 15, 30, 60, 90 and
of the crustacean source on the deacetylation and 120 min). The depolymerization was carried out by stirring
depolymerization reactions of chitin and the physicochem- at 35 8C and was stopped by precipitating the depolymer-
ical properties of the resultant macromolecules. To this ized chitosans with 80% (v%) acetone in distilled water,
end, the hydrolysis of the N-acetyl linkage (N-deacetyla- washing later with acetone 90% (v%) and pure acetone
tion) of chitins and the O-glycosidic linkages (depolymer- (Sannan, Kurita, & Iwakura, 1976). All the experiments
ization) of partially N-acetylated chitosans were studied at were done in triplicate.
fixed conditions for three different crustacean species
(Lithodes antarcticus, Paralomis granulosa and Palinurus
2.4. Characterization of samples
vulgaris). The kinetics of N-deacetylation and depolymer-
ization reactions were investigated. The mole fraction of
 w) Chitins from the three sources were characterized by
N-acetylated units (FA) and average molecular weight (M 13
0 C-CP-MAS NMR experiments in solid-state (Heux,
of the by-products and the apparent rate constants (k ) for
Brugnerotto, Desbrières, Versali, & Rinaudo, 2000). The
these reactions were calculated.
NMR experiments were performed on a Bruker WB
DPX-400 spectrometer using the magic angle spinning
(MAS) technique. The contact time was 1 ms, the sweep
2. Materials and methods width 40 kHz, and the recycle delay 3 s for the 13C. A
typical number of 800 scans were acquired for each
spectrum. The chemical shifts were externally referenced
2.1. Materials to those of adamantane (38.3–29.2 ppm). The spinning
speed was set at 7 kHz for all samples. Moisture and ash
Chitins from the species L. antarcticus, P. granulosa and contents analysis were carried out by gravimetric measures.
P. vulgaris were purchased from IDEBIO S.L. (Salamanca, Protein contents were determined according to the nitrogen
Spain). Samples were milled and sieved. The particle size content determined by elemental analysis, allowing for the
was between 0.5 and 1 mm. NaOH, KNO2, acetone and HCl acetylation degree of chitin and the chitin content of the
were analytical grade reagents from Panreac Quı́mica S.A. sample (Lizardi et al., 2002).
(Barcelona, Spain). Chitosans were characterized by measuring their FA by
first derivative UV-spectrophotometric method proposed
2.2. N-Deacetylation by Muzzarelli; Rocchetti; Stanic and Weckx (1997). The
M w was calculated from the intrinsic viscosity values using
Chitins from the three different species were treated the Mark-Houwink equation with the constants aZ0.93
identically with NaOH 50% (wt-%) (10 ml per g of chitin) at and kZ1.81!10K3 cm3/g (Roberts, 1992). Data of
110 8C for 4 h under a N2 atmosphere (Joensen, 1999) in a intrinsic viscosity were measured in an Ubbelohde
batch reactor designed in our laboratory. Samples were viscometer (purchased from Schott-Gerate, Germany) at
taken out of the reactor every 15 min for 4 h (plus a sample 25 8C using 0.1 M HOAc-0.2 M NaCl as solvent (Roberts,
at 360 min) and washed with distilled water until pH 7. All 1992). Two different viscosimeters were used for the
the experiments were done in triplicate. characterization of the chitosan hydrolyzates obtained from

Table 1
 w of the chitins and chitosans from the three biological species
FA, Moisture, ash and protein contents and M

Samples FA Moisture content (%) Ash content (%) Protein content (%)  w (kDa)
M
L. antarcticus (chitin) 0.82 1.32 6.25 0 –
P. granulosa (chitin) 0.87 5.94 5.25 5.52 –
P. vulgaris (chitin) 0.77 5.25 0.19 0 –
L. antarcticus (chitosan) 0.16 10.99 0.64 0 490.7
P. granulosa (chitosan) 0.16 9.02 1.64 0 518.0
P. vulgaris (chitosan) 0.14 8.96 0.05 0 423.7
318 G. Galed et al. / Carbohydrate Polymers 62 (2005) 316–320

(a) 0.35

0.3

0.25

Fa
0.2

0.15

0.1
0 50 100 150 200 250 300 350 400
(b) Reaction time (min)

Fig. 2. Plot of the determined FA versus deacetylation time for the three
species (%L. antarcticus; & P. granulosa; :P. vulgaris). Deacetylation
conditions: NaOH 50% wt%; 110 8C.

the reaction. The plots between the determined FA and the

deacetylation time are shown on Fig. 2. The apparent rate
(c)
constants (k 0 ) for the deacetylation and correlation
coefficients (R) from the regression analysis are shown in
Table 2. All species present similar deacetylation rates and
in the initial period the deacetylation reaction occurs at a
very fast speed under the conditions studied in agreement
with previous studies (Focher, Beltrame, Naggi, & Torri,
1990).
200 150 100 50 0
Some authors report that the influence of the species is a
13
Fig. 1. Solid state C-CP-MAS NMR spectra of the chitins obtained from factor that increases the standard deviation of the rate constant.
(a) Lithodes antarcticus, (b) Paralomis granulosa and (c) Palinurus However, differences in crustacean species and the chitin
vulgaris. isolation procedure have shown to cause a variation in
morphology or composition between chitin samples, leading
the depolymerization reactions varying the diameter of to different behaviour during deacetylation (Chang, Viton, &
their capillary (Nm. 525.20II and Nm. 525.13 Ic). Domard, 1997; Lamarque, Viton, Domard, 2004).
The degradation of the different chitosans along the
deacetylation process was studied. Fig. 3 shows the time-
3. Results and discussion course decrease of M  w versus deacetylation time. In the
range 15–45 min a higher rate of degradation can be
3.1. Characterization of chitins and chitosans observed for P. vulgaris compared to L. antarcticus and
P. granulosa, which present a similar trend. From this time a
Chitins isolated from the three crustacean species under stabilisation of the relative decrease occurs in all cases. The
study were characterized (Table 1). Fig. 1 shows the 13C difference among species can be confirmed by the plots of
CP-MAS NMR spectra of the different chitins analysed. ln M w0 =M
 wt versus deacetylation time up to 45 min.
Signals in all cases were characteristic of chitins with high P. vulgaris presents an apparent rate constant of one order
acetylation degrees. Single lines could be assigned in each of magnitude higher than those for L. antarcticus and
spectrum to six of eight chemically distinct carbon atoms of P. granulosa (Table 3).
the 2-deoxy-2-acetamide-D-glucose repeat unit. Spectra In the presence of alkali, polysaccharide chains are found
showed that the C3 and C5 carbon atoms give two partially to undergo degradation because of the high concentration of
resolved peaks for a-chitin (Tanner, Chanzy, Vincendon, reagents and prolonged reaction times required to obtain a
Roux, & Gaill, 1990). Final chitosans obtained from these complete deacetylation (Tolaimate et al., 2000). It is well
chitins by N-deacetylation were also characterized
(Table 1). Table 2
Parameters of the N-deacetylation reaction for the three species according
to pseudo-first order kinetics
3.2. N-Deacetylation
Species k 0 (minK1) R

Experimental data of the N-deacetylation kinetics were L. antarcticus 4.46!10 K2

0.8717
studied between 0 and 360 min. Data were modelled via P. granulosa 3.89!10K2 0.8950
P. vulgaris 4.45!10K2 0.9004
first- and second-order kinetic models and were best
adjusted to a first order equation for the first stages of k 0 ; Apparent kinetic constant and R; correlation coefficient.
 G. Galed et al. / Carbohydrate Polymers 62 (2005) 316–320 319

1000 Table 4
Results of the depolymerization reaction of chitosans obtained from the
900
three species according to the first order kinetics, k 0 ;kinetic constant for the
800
Mw (kDa)

first 9 min, R; correlation coefficient

700
600 Species k 0 (minK1) R
500
L. antarcticus 0.1506 0.9023
400
P. granulosa 0.1556 0.8916
300 P. vulgaris 0.1343 0.8901
0 50 100 150 200 250 300 350 400
Reaction time (min)

 w decrease versus N-deacetylation time for the three

Fig. 3. Plot of the M depolymerization reactivity as the degree of deacetylation
species (%L. antarcticus; & P. granulosa; : P. vulgaris). Deacetylation of the polymer increases but in the case of our chitosans the
conditions: NaOH 50% wt%; 110 8C.
initial FA was similar. These authors reported that the
known that chitin degradation occurs inevitably during depolymerization rate is independent of the size of the
N-deacetylation although the molecular mechanism has not chitosan substrate. This point should be further investigated
been elucidated. Our reaction is carried out in a nitrogen focusing on other parameters as cristallinity indexes.
atmosphere to reduce the molecular weight modification FA of the depolymerized samples showed relative
caused by oxidative mechanisms, as some authors report standard deviations below 1.5%, so no changes in FA were
(Mima, Miya, Iwamoto, & Yoshikawa, 1983). found throughout depolymerization (results not shown).
The different degradation rates could be due to the different In conclusion, chitin from P. vulgaris has similar rate of
ash content and the cristallinity of samples. In our case, chitins N-deacetylation to the other two species while its
with higher ash contents (Table 1, L. antarcticus and degradation rate is one order of magnitude higher.
P. granulosa) presented slower degradation rates during Differences in the degradation rate during the
deacetylation. The higher ash content can reduce the N-deacetylation reaction can be attributed to the higher ash
accessibility of the reagents to the raw chitin for both species. content of the chitins from P. granulosa and L. antarcticus
Some authors consider that the heterogeneous N-deacetylation compared to that of P. vulgaris. This is in accordance to the
might be controlled by either the reaction on solid surface, or fact that the former belong to the same crustacean family
the diffusion of reactant from bulk fluid to the solid surface (Lithodidae).
influencing the rate of the reaction (Levenspiel, 1972). On the other hand, the rate of depolymerization of
P. vulgaris is lower than that of L. antarcticus and
3.3. Depolymerization P. granulosa. Differences in the depolymerization reaction
remain unexplained although they could be related to the
The kinetics of the depolymerization reaction for the biological source of the initial chitosan samples.
chitosans obtained from the three species by nitrous acid Under the conditions studied, chitins and chitosans from
was studied between 0 and 120 min. In the plots of M w L. antarcticus and P. granulosa presented analogous
decrease versus depolymerisation time two domains were behaviour. These results show that the biological source of
observed: a rapid decrease occurred up to 9 min and from chitin is a key factor in the N-deacetylation and depolymer-
 w leveled off (plots not shown).
this point the M ization rates and properties of the resulting chitosans.
Plots of ln M w0 =M
  wt versus depolymerization time in the
range 0-9 min were adjusted to a first order equation
behaviour (Table 4). For P. vulgaris the depolymerisation Acknowledgements
rate was lower than those of L. antarcticus and P. granulosa.
The differences in the ash content of the initial chitosans Financial support from Project MAT2000-0037-P4-03
cannot explain in this case the different reactivity with (Spanish Ministry of Science and Technology) and Idebio
regard to the depolymerization reaction. Allan and Peyron S.L. is acknowledged. B. Miralles was the recipient of a
(1995) under similar depolymerization conditions have post-doctoral fellowship from the Spanish Ministry of
described that chitosan exhibits significantly decreased Education.

Table 3
Results of the N-deacetylation reaction for the three species studied, k 0
apparent kinetic constant for degradation up to 45 min, R: correlation
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