Kinetics of base deacetylation of chitin and chitosan as influenced by their crystallinity

Article abstract of DOI:10.1134/S1070427207100321

Structural properties of the initial and reprecipitated chitin and chitosan samples in dry and wet states were studied.

Full text of DOI:10.1134/S1070427207100321

ISSN 1070-4272, Russian Journal of Applied Chemistry, 2007, Vol. 80, No. 10, pp. 1753 1758.
Pleiades Publishing, Ltd., 2007.
Original Russian Text
pp. 1724 1729.
E.N. Chebotok, V.Yu. Novikov, I.N. Konovalova, 2007, published in Zhurnal Prikladnoi Khimii, 2007, Vol. 80, No. 10,
MACROMOLECULAR CHEMISTRY
AND POLYMERIC MATERIALS
Kinetics of Base Deacetylation of Chitin and Chitosan
as Influenced by Their Crystallinity
E. N. Chebotok, V. Yu. Novikov, and I. N. Konovalova
Murmansk State Technical University, Murmansk, Russia
Knipovich Polar Research Institute of Marine Fisheries and Oceanography, Murmansk, Russia
Received December 25, 2006; in final form, April 2007
Abstract Structural properties of the initial and reprecipitated chitin and chitosan samples in dry and
wet states were studied.
DOI: 10.1134/S1070427207100321
Base hydrolysis of chitin (CTN) and chitosan
(CSN) is one of the main procedures for deacetylation
of these natural polysaccharides [1].
solution with a 10% alkali concentration was filtered
through a Schott filter in a vacuum. Then, chitin was
precipitated by gradual neutralization of this solution
with HCl to pH 6.5. The precipitate was separated by
centrifugation, and a part of the wet reprecipitated
chitin was dried at 60 C.
The kinetics of deacetylation of chitin and chitosan
was analyzed in [2 4]. It was found that complete de-
acetylation of chitin and chitosan cannot be attained
in a single-stage treatment of these polysaccharides
with concentrated alkali. Several explanations were
suggested for the decrease in the rate of chitin de-
acetylation in the alkaline solutions.
Reprecipitation of chitin from acid solutions was
performed as follows. Chitin (10 g) was mixed with
cold concentrated HCl (200 ml), and the resulting
mixture was stored for a day with intermittent stir-
ring. Then, the chitin solution was filtered through
a Schott filter in a vacuum. The resulting solution
was diluted with cold distilled water (3 l) under stir-
ring. The resulting suspension was kept until the pre-
cipitation was complete, and then the chitin precipitate
was separated by centrifugatoin. The precipitate was
washed with distilled water to pH 6.5, and a part of
the resulting reprecipitated wet chitin was dried at
60 C.
This paper is devoted to elucidation of the mechan-
ism of chitin deacetylation on the basis of published
data and our experimental results.
EXPERIMENTAL
Chitin and chitosan were prepared in laboratory
by the known procedure [5] from shell-containing
wastes from king crab processing.
Chitosan was precipitated from an acid solution.
For this purpose, chitosan (2 g) was mixed with
a 0.1 M HCl solution (200 ml), and the resulting
mixture was kept for 18 h until the chitosan com-
pletely dissolved. The resulting solution was filtered
through a Schott filter under reduced pressure and
then was neutralized with stirring to pH 7.5 with
a 0.5 M NaOH solution. The chitosan precipitate was
separated by centrifugation and washed with distilled
water. A part of the wet chitosan was dried at 60 C.
Chitin and chitosan were deacetylated in a 50%
NaOH solution at 100 C. Samples with various de-
grees of deacetylation were prepared from chitin and
chitosan by repeated treatments with 50% NaOH so-
lution at 100 C.
Prior to reprecipitation from NaOH, chitin was
dissolved in alkali by the procedure described else-
where [6]. A suspension of chitin (3 g) in a 40%
aqueous solution of sodium hydroxide (75 g) was
stored for 3 h at 25 C under reduced pressure. The re-
sulting alkaline chitin was mixed with crushed ice
(225 g) at approximately 0 C. The resulting chitin
The degree of chitosan deacetylation was deter-
mined by potentiometric titration of the chitosan so-
1753

1754
CHEBOTOK et al.
weight of the acetylated monomeric unit of the poly-
saccharide; 42.0, difference between the molecular
weights of the acetylated and deacetylated monomeric
units; 1000, factor of conversion of milliliters to liters;
and 100, factor of conversion of DD to percents.
The degree of deacetylation of chitin and chitosan
was also determined by IR spectroscopy [7, 8].
The IR spectra were recorded on an IR-420 Shima-
1
dzu spectrophotometer (Japan) in the 4000 400 cm
range using KBr pellets. The pellets were prepared us-
ing samples ground in a TI-100 mill (C.M.T. Co,
Japan) for 10 min and dried to constant weight at 100 C.
KBr was recrystallized from an aqueous solution and
calcined at 600 C. The purity of KBr was checked
by IR spectroscopy. The weight ratio of the sample
and KBr in the pellet was 1 : 49. To prepare a pellet
with diameter and thickness of 13 and 0.42 mm, re-
spectively, the resulting mixture (120 mg) was com-
Fig. 1. IR absorption spectrum of a chitin sample. (I) Trans-
mission and ( ) wavenumber. Lines and points illustrate
the graphical processing of the data for DD calculation.
2
pacted at a 650-kg cm pressure for 1 min at room
temperature on an SSP-1 hand press (Shimadzu, Japan).
The procedure for DD determination was improved
for its use in a wide DD range. Using the samples
with DD varying from 0 to 98%, we found that the IR
1
absorption band at 2878 cm is the most appropriate
for measurements. This absorption band corresponds
to the stretching vibrations of the C H bond [8], and
its optical density obeys Beer’s law in the entire DD
range studied. The bending vibrations of the amino
Fig. 2. Absorption of chitin in the IR spectra vs. the degree
of deacetylation DD. (A
/A
) Ratio of the optical
1655 2878
1
densities of the bands at 1655 and 2878 cm
.
1
group (amide I) are observed at 1655 cm . The graph-
ical processing of the IR spectrum is illustrated in
Fig. 1.
lution with a 0.1 M HCl solution. The solution pH
was measured on an Anion 4151 pH-meter (Infraspak-
Analit Research and Production Enterprise, Russia).
The absolute error in determining the degree of de-
acetylation was 0.5%. The chitosan sample (0.2 g)
weighed with an accuracy of 0.0001g was dissolved
in a 0.1 M HCl solution (20 ml), and the resulting so-
lution was titrated with a 0.1 M NaOH solution with
the resulting potentiometric titration curve plotted,
pH = f (VNaOH). Then, the points of the maxima
of the first derivative, which correspond to the equi-
valence points in titration equivalence of excess HCl
(V1) and amino groups (V2), were determined.
The optical density was calculated as follows:
A2878 = log(AC/AB),
A1655 = log(DF/DE).
The degree of deacetylation was calculated from
the calibration dependence obtained for samples with
the known DD (Fig. 2). The resulting regression equa-
tion can be represented as
A1655 /A2878
=
0.04529DD + 4.4214, r2 = 0.9946,
The degree of deacetylation DD (%) was calculated
using the following expression:
where r is the linear-regression coefficient.
203.2
1000m
cNaOH(V2
The degree of crystallinity of the samples was de-
termined by the known procedures [10, 11].
DD =
100,
42.0 +
V1)
The structural properties of chitin and chitosan
samples were studied at the Tananaev Institute of
Chemistry and Technology of Rare Elements and
Mineral Raw Materials (Kola Scientific Center, Rus-
sian Academy of Sciences) on a DRON-2 diffractom-
where m is the content of chitosan in the sample, g;
cNaOH , concentration of the NaOH solution, M;
V2 V1, volume of the NaOH solution consumed in
titration of the amino groups, cm3; 203.2, molecular
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KINETICS OF BASE DEACETYLATION OF CHITIN AND CHITOSAN
1755
eter (Burevestnik Research and Production Associa-
tion; CuK , Ni filter, 24 kV, 10 mA) in the range
= 6 36 . The X-ray diffraction (XRD) patterns
2
were recorded from the initial wet samples and sam-
ples suspended in mineral oil deposited on sapphire
supports. The reflections were identified using JCPDS
tables [12] and published data [13].
The XRD patterns were mathematically processed
using the Graph Digitizer Ver. 1.9 (Nick’s Produc-
tion), PeakFit Ver. 4.12 (SeaSolve Software Inc.), and
Microsoft Excel 2002 program packages.
Kurita et al. [2] attributed the features of deacetyla-
tion to the crystal structure of natural chitin.
It is known that chitin is formed in nature in
various crystal modifications. In most cases, it has
a highly ordered structure, named -chitin, which
is typical of the majority of crustacea and insects
[14].
Other modifications, such as - and -chitin, are
also known [15]; they differ in the mutual orientation
of adjacent chitin macromolecules. Chitosan is also
characterized by high crystallinity, but its structure
differs from that of -chitin [11].
As shown in [1, 4, 11], strong intermolecular hy-
drogen bonds should affect the rate of chemical re-
actions of the functional groups of macromolecules
involved in formation of the crystal structure.
The degree of crystallinity, cr, is frequently de-
termined from the ratio of the integral intensity of
the peak of the crystalline regions in the diffraction
pattern to the total integral peak [10, 11].
To calculate the intensity of the peak originating
from the crystalline regions in the samples, the total
area under the diffraction curve Stot and the area of
the amorphous component Sam were determined
graphically.
The degree of crystallinity, cr, was calculated by
the following expression:
Fig. 3. XRD patterns of (1) initial chitin and (2) dry and
Stot
Sam
=
100,
(3) wet chitin reprecipitated from solutions of (a) NaOH
and (b) HCl. (I) Peak intensity and (2 ) diffraction angle;
the same for Fig. 4.
cr
Stot
where Stot is the total area under the diffraction curve
in the angle range 2 = 6 36 , and Sam, the area of
the amorphous component in the same range.
The results listed in the table show that reprecipi-
tation virtually completely restores the crystal struc-
tures of chitin and chitosan. The diffraction peaks of
the restored structures of the chitin samples recrys-
tallized from solutions of NaOH (Fig. 3a) and HCl
(Fig. 3b) are broader as compared to the initial chitin,
but their positions in the XRD patterns are similar.
The degrees of crystallinity evaluated by the above
procedure are listed in the table.
It should be noted that the XRD patterns exhibit
a series of peaks typical of the crystal structure of
-chitin, described in [12, 13].
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1756
CHEBOTOK et al.
As for chitosan samples, their crystal structure was
also restored during reprecipitation (Fig. 4), with
certain differences from the initial chitin structure.
Two main peaks in the XRD pattern of chitosan are
shifted as compared to -chitin. In the XRD patterns
of chitin with DD less than 71%, two peaks at 2 9.2
and 19.1 are observed. In the case of chitosan with
DD 95%, these two peaks are shifted to 2 11.2 and
20.1 20.4 , respectively. These experimental data do
not contradict those reported in [2, 11, 16 18].
Partial restoration of the -chitin structure after
recrystallization and drying of the samples of - and
-chitin was also confirmed by NMR, IR, and Raman
spectroscopy [19].
On the whole, the above data suggest that the crys-
tal structure of chitin and chitosan is restored after
recrystallization of the initial sample from alkali and
acid and their drying (see table).
The XRD patters of the wet chitin and chitosan
samples show that they are virtually amorphous
(Figs. 3 and 4, curves 3).
Fig. 4. XRD patterns of (1) initial chitosan and (2) dry
and (3) wet chitosan reprecipitated from HCl solu-
tions.
It should be noted that the weak peaks observed in
the XRD patterns of the wet recrystallized chitin
(Figs. 3a, 3b) are absent in the XRD patterns of
the wet recrystallized chitosan (Fig. 4).
To compare the crystal structures of the chitin
samples reprecipitated from acid and alkali, their
XRD patterns were normalized as in [16]. It was
found that dry chitin samples reprecipitated from
NaOH and HCl exhibit similar XRD patterns, which
suggests similarity of their structures.
It has been suggested previously [2, 20] that de-
acetylation of crystalline chitin proceeds more slowly
than that of the amorphous compound.
Ottoy et al. [21] studied the DD distribution of
various fractions of partly deacetylated chitosan ex-
hibiting different solubilities in acid. Their exper-
imental results suggest that deacetylation proceeds
by the diffusion mechanism in which the deacetyla-
tion rate is controlled by the rate of alkali diffusion
into the solid particles, rather than by the ratio of
the crystalline and amorphous regions in the polysac-
charide structure.
Our results agree with experimental data reported
in [2, 16].
It is assumed [2] that, in the course of recrystal-
lization, the -chitin structure is restored only partly,
simultaneously with the formation of other crystal
forms and amorphization.
Degree of crystallinity, cr , of chitin and chitosan samples
Chang et al. [22] showed that the deacetylation rate
depends on the alkali concentration: as the NaOH con-
centration increases, the reaction rate constant be-
comes higher. Chang et al. suggested that the rate of
heterogeneous deacetylation can be controlled by both
the chemical reaction and the diffusion of the reactant
and reaction products in the polysaccharide particle.
Sample
cr , %
61.1
Initial dry chitin
Chitin reprecipitated from NaOH, dry:
from NaOH, dry
55.5
60.7
8.8
from HCl, dry
from NaOH, wet
from HCl, wet
8.5
35.8
Thus, our experimental results and published data
suggest that the kinetics of deacetylation of dry re-
precipitated chitin and chitosan should be similar to
that of the initial samples, whereas the rate of de-
acetylation of the wet recrystallized samples should
be higher. However, our experimental kinetic de-
Initial dry chitosan, DD 82%
Chitosan reprecipitated:
from HCl, DD 82%, dry
from HCl, DD 82%, wet
37.5
3.8
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KINETICS OF BASE DEACETYLATION OF CHITIN AND CHITOSAN
pendences of deacetylation do not fit in the model
1757
analyzing the effect of the degree of crystallinity of
chitin on the degree of its deacetylation.
It is known that, as DD increases, the crystal-
linity of chitin decreases and, when DD reaches 90
95%, a new crystal structure typical of chitosan starts
to form [2, 17]. In this case, the rate and degree of
chitin deacetylation should increase with the initial
DD owing to the rise in the content of amorphous
regions. Previously, Novikov et al. have shown [23]
that the rate of deacetylation of chitin and chitosan
are independent of the initial degree of deacetylation
of the corresponding polysaccharide. The shapes of
the kinetic curves for chitin and chitosan with various
initial DD (up to 95%) were similar [23, 24].
Fig. 5. Kinetic curves of deacetylation of (1) dry chitin
and (2) wet chitin reprecipitated from 50% NaOH solution
at 100 C. (DD) Degree of deacetylation and ( ) time.
tions of pseudo-first order: (1) deacetylation and (2)
formation of chitin complex with hydroxide ions.
Yaghobi and Mirzadeh [25] explained the increase
in the degree of deacetylation in the course of repeated
alkaline treatments as follows. They suggested that
the initial structure of chitin hinders penetration of
NaOH molecules to all the polysaccharide molecules.
When the alkaline treatment is performed continuous-
ly, the rate of deacetylation decreases at DD 75%.
The authors assume that water-washing of chitin or
partly deacetylated chitin after the first stage of de-
acetylation is of particular importance. Washing
changes the chitin morphology, causing its swelling,
and makes chitin macromolecules more accessible to
NaOH molecules. At the same time, the authors at-
tribute the decrease in the rate of continuous de-
acetylation to a decrease in the concentration of
NaOH molecules from 50 to 43%, i.e., to a concen-
tration at which, as they believe, deacetylation is
terminated.
Figure 5 shows the kinetic dependences of de-
acetylation of dry chitin (curve 1) and wet chitin
reprecipitated from NaOH solution (curve 2). As can
be seen, the rate of deacetylation of wet reprecipitated
chitin ( 4.0%) is lower than that of the dry sample
cr
(
59.0%). This fact suggests solvation (hydra-
cr
tion) of polysaccharide molecules in NaOH solution.
The water dipoles are located in a certain order near
the acetamide and glycoside bonds of the chitin
macromolecules. In accordance with the mechanism
of deacetylation in the alkaline medium [29], hy-
drolysis of the acetamide bonds starts with a nucle-
ophilic attack of the carbonyl carbon atom of the acet-
amide group by OH ions. The hydrate shell probably
hinders this nucleophilic attack, causing a decrease
in the deacetylation rate.
However, this explanation contradicts the ex-
perimental data. Novikov et al. have shown [26] that
the NaOH concentration decreases insignificantly
(from 50 to 49.4%). If the decrease in the rate of
deacetylation were due to a decrease in the alkali
concentration below some limit, then its increase
would accelerate deacetylation. However, the rate
of deacetylation does not increase in the presence of
excess NaOH [26]. Probably, the deacetylation rate is
determined by formation of solvated structures owing
to interaction of polysaccharide macromolecules and
low-molecular-weight electrolyte with the solvent.
The above results suggest that the influence exerted
by the degree of crystallinity of the samples studied
on the deacetylation rate is significantly weaker than
that exerted by the hydrate shell probably formed
around the polysaccharide macromolecules, which
hinders nucleophilic attack of the hydroxide ions and
decreases the deacetylation rate.
CONCLUSIONS
(1) The IR spectrophotometric procedure for eval-
uating the degree of deacetylation of chitin and
chitosan samples was improved.
For example, Percot et al. [27] suggested formation
of NaOH hydrates, which are less active in chitin de-
acetylation than nonhydrated alkali molecules.
(2) The possibility of formation of a hydrate shell
around the macromolecules and its effect on the de-
acetylation rate were suggested.
Previously, we suggested in [23, 24, 28] that OH
complexes with chitin are formed and showed that de-
acetylation can be described by two parallel reac-
(3) The influence exerted by the degree of crystal-
linity of the samples on the deacetylation rate is
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 80 No. 10 2007

1758
CHEBOTOK et al.
weaker thanthat exerted by the hydrate shell formed
around the polysaccharide macromolecules. This shell
hinders nucleophilic attack of the hydroxide ions and
decreases the deacetylation rate.
13. Paralikar, K.M. and Balasubramanya, R.H., J. Polym.
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ACKNOWLEDGMENTS
15. Cho, Y.-W., Jang, J., Park, C.R., and Ko, S.-W.,
Biomacromolecules, 2000, vol. 1, no. 4, pp. 609 614.
The authors are grateful to staff members of
the Tananaev Institute of Chemistry and Technology
of Rare Elements and Mineral Raw Materials (Kola
Scientific Center, Russian Academy of Sciences)
L.D. Rogachev for X-ray diffraction analysis and
to O.A. Zalkind for assistance in interpretation of
the IR data.
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