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1. Studies on preparation of oligoglucosamine by oxidative
degradation under microwave irradiation
Jian Shaoa,
*, Yumin Yangb
, Qiqing Zhongc
a
Department of Chemistry, Nantong Medical College, Nantong 226001, China
b
Institute of Functional Biomolecule, School of Life Science, Nanjing University, Nanjing 210093, China
c
Department of Hygienic, Nantong Medical College, Nantong 226001, China
Received 4 February 2003; received in revised form 23 April 2003; accepted 11 May 2003
Abstract
Medicinal oligoglucosamine was prepared by oxidative degradation of chitosan with neutral hydrogen peroxide under microwave
irradiation. The effects of concentration of H2O2, volume of solution, amount of chitosan and irradiation time were investigated by
the orthogonal test, and the optimal conditions of degradation are described. The structure of the product was confirmed by FT-IR
spectrum analysis. The number average molecular weight of oligoglucosamine was determined by the method of end group analysis.
The experimental results show that oligoglucosamine can be conveniently and effectively obtained by oxidative degradation of
chitosan under microwave irradiation. The number average molecular weight of chitosan decreased to 900–1000. The changes in the
yield of oligoglucosamine are strongly dependent on the reaction time and the concentration of H2O2.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Chitosan; Oligoglucosamine; Degradation; Hydrogen peroxide; Microwave irradiation
1. Introduction
Chitosan, a copolymer of (1!4)-2-acetamido-2-
deoxy-b-d-glucan and (1!4)-2-amino-2-deoxy-b-d-glu-
can is a highly deacetylated derivative of chitin, one of
the most widespread polysaccharides in biomass. Con-
sequently, chitin and chitosan are necessarily biode-
gradable and bioresorbable. In addition to these
properties, common to every natural polymer, chitin
and chitosan are biocompatible and bioactive molecules
whether in their polymeric or oligomeric forms.
It has been reported that oligoglucosamine, a degra-
dation product of chitosan, has distinct physiologic
activities [1]. It was widely used in pharmaceutical,
functional foods, cosmetics and so on recently. Oli-
goglucosamine can be obtained either by chemical
hydrolysis or by enzymatic hydrolysis. More than 30
types of enzymes can be used for the degradation of
chitosan, but there are still some difficulties in large-
scale industrial processes [2].
Hydrochloric acid was used to hydrolyze chitosan,
and other attempts include the use of nitrous acid, con-
centrated sulfuric acid and hydrofluoric acid. We have
reported preparation of oligoglucosamine by treatment
chitosan with H2O2, because it is handy, easily available
and environmentally friendly [3].
Microwave irradiation using the commercial domestic
microwave oven has received increasing interest in
organic synthesis due to remarkable enhancements of
the rates of some organic reactions and significant
effects over conventional reaction [4].
In the present paper, we have hydrolyzed chitosan
with H2O2 under microwave irradiation in anticipation
of obtaining water-soluble oligoglucosamine.
2. Experimental
2.1. Materials and apparatus
Chitin was supplied by Nantong Water Products
Institute, Nantong, People’s Republic of China. Addi-
tionally it was ground through a granulating machine
and then sent through a 100-mesh granular membrane.
0141-3910/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0141-3910(03)00177-0
Polymer Degradation and Stability 82 (2003) 395–398
www.elsevier.com/locate/polydegstab
* Corresponding author.
E-mail address: ntyxyhx@yahoo.com.cn (J. Shao).

2. H2O2 and other reagents used were of analytical reagent
grade and used without further purification. All solu-
tions were prepared with distilled water.
Galanz Microwave Oven (WP700) was produced by
Shunde Galanz Electric Appliances Factory, Ltd; Nico-
let Impact 410 FT-IR spectrometer.
2.2. Preparation of chitosan
Chitin isolated from shrimp shell was pulverized and
treated with 40% aqueous sodium hydroxide at 110
C
for 4 h (three times) to give chitosan as an almost col-
orless powdery material. The degree of deacetylation
was 91.2% as determined by the titrimetric method, and
the viscosity average molecular weight was 2.2105
as
determined by a viscometric method using an Ostwald
viscometer at 25
C [2].
2.3. Preparation of water-soluble oligoglucosamine
Chitosan (2 g) was placed in a 250 ml Erlenmeyer
flask with 15% H2O2 (50 ml), 10-min later, the Erlen-
meyer flask (with a glass cover on it) was placed on the
center of the turntable of a microwave oven, the reac-
tion mixture was irradiated with microwave power
levels at 700 W for 4 min. After irradiation had been
stopped, allow the product to cool to room tempera-
ture, the mixture was filtered through a Buchner filter
under reduced pressure. The residue and the filter paper
were dried to constant weight in an infrared dryer. By
deducting the weight of the filter paper, the weight of
the unreacted chitosan can be obtained, and the amount
of water-soluble oligoglucosamine can be determined.
The filtrate was concentrated to about one-tenth with
a rotary evaporator under reduced pressure at 40
C.
Then ethanol was added to obtain a white precipitate.
The precipitate was filtered off, reprecipitated with eth-
anol from distilled water, filtered off, and then washed
thoroughly with ethanol. The product was dried over-
night in vacuum at room temperature.
2.4. Determination of molecular weight of oligoglucosa-
mine
The number average molecular weight (M
n) of oli-
goglucosamine was determined by the method of end
group analysis [3].
Color-producing reagent: 0.5 g of potassium ferricya-
nide was accurately weighed and dissolved in 0.5 mol/l
sodium carbonate solution (1 l), kept the solution in
brown reagent bottle.
Standard solution: 1 g of d-glucosamine hydrochlo-
ride (GAH) was accurately weighed and dissolved in
distilled water. Then diluted it to 100 ml.
Several different volumes of standard solution and 2.0
ml of color-producing reagent were accurately added to
comparison tubes (10 ml) with a stopper, respectively.
The final volume was adjusted to 5 ml by adding dis-
tilled water. The tightly closed comparison tubes were
put into a boiling water bath for 15 min, and then they
were cooled to room temperature in cold water imme-
diately. After that, the absorbance values of the solution
in each of the comparison tubes were determined at 420
nm with distilled water as a blank system. The regressive
equation (absorbance value versus the amount of GAH)
was obtained.
A desired weight of dried oligoglucosamine were
accurately weighed and dissolved in 2.0 ml of color-pro-
ducing solution. The final volume of this solution was
adjusted to 5 ml by adding distilled water. The following
steps were as above.
The amount of GAH corresponding to this oligoglu-
cosamine could be consulted from the regressive equa-
tion, and the number average molecular weight (M
n) of
oligoglucosamine was then calculated by the following
equation:
M
n ¼
W1
W2
215:5
where W1 is weight of oligoglucosamine (g); W2 is the
amount of GAH corresponding the oligoglucosamine
from the regressive equation.
3. Results and discussion
No degradation method is better than treatment of
chitosan by H2O2, for the reaction needs no promoter
and the final product of H2O2 is H2O. Hydrogen per-
oxide (H2O2 ) is substantially more acidic than water,
with a pKa of 11.6. The perhydroxyl anion (HOO
) is
unstable. The decrease in stability of H2O2 is caused by
the instability of the HOO
. Heat and base will increase
the decomposition of H2O2. The perhydroxyl anion
reacts with H2O2 to form the highly reaction hydroxyl
redical (HO. ) and superoxide anion (O2
.).
H2O2 ! H+
+HOO
H2O2+HOO
! HO.+O2
.+H2O.
Both of them are much more powerful oxidants, and
will attack the glycosidic bond of chitosan to cause the
scission of the chitosan chain.
But the treatment of chitosan with H2O2 also leads to
the changes in the chemical structure. The changes such
as formation of carboxyl groups, deamination and ring-
opening are accompanied by the degradation of chit-
osan. The degree of these changes increases sharply with
the increasing treatment time [5].
The principle of microwave dielectric heating has been
reported by S.A.Galema [6]. Its basic viewpoint is that
microwave irradiation induces changed particles to
migrate or rotate, which results in polarization of polar
396 J. Shao et al. / Polymer Degradation and Stability 82 (2003) 395–398

3. particles, and the lag between this polarization and
rapid reversals of the microwave field creates friction
among molecules to generate heat. Because the prompt
change from the static to dynamic state is occurring
within the molecules, the microwave heating is occur-
ring within the molecules, the microwave heating is
described as ‘‘inside heating’’. Microwave irradiation
makes the rate of some organic reactions 1240 times
higher than that of classical methods. The energy level
of microwaves matches with the rotation energy level of
polar molecules, implying the energy of microwaves can
be absorbed rapidly by polar bonds. The absorbed
energy exchanging with the energy of translation makes
the depression of the activation energy and the increas-
ing of reaction activity within the polar bonds (such as
the C–O–C glycosidic bond).
3.1. Orthogonal test
In order to select the factor of reaction for the ortho-
gonal test, we have carried out several preliminary
experiments. The experimental results show that the size
of chitosan must be smaller than 100-mesh, and the
chitosan must be immersed in H2O2 solution for at least
10–20 min to make the chitosan swell completely.
In acidic or basic solution, the oxidizing power of
H2O2 is much higher than in neutral solution. The pro-
ducts prepared from acidic or basic solution are inso-
luble, and their FT-IR spectra shows that the carboxylic
group is formed.
The best condition for preparing oligoglucosamine
was studied by the orthogonal test. Four controllable
variables, volume of solution (ml), amount of chitosan
(g), concentration of H2O2 (%), and irradiation time
(min) were selected, each at three levels. The investi-
gated variables and their test levels are listed in Table 1.
Reference to the experimental design theory, the ortho-
gonal array L9(34
) was selected to arrange the test pro-
gram. The percentage of chitosan dissolved in water
(ws) was a criterion of each test. The test results are lis-
ted in Table 2.
Obviously the order of influence of each variables on
the percent of chitosan dissolved in solution is
C BAD [7]. The variance of concentration of
H2O2 is the greatest, the optimum levels of each variables
is A2B2C3D2. Thus the optimum reaction condition was
obtained as follows: volume of H2O2 (ml): 50 ml, irra-
diation time : 4 min, concentration of H2O2:15%,
amount of chitosan: 2 g.
3.2. FT-IR spectra
FT-IR spectroscopy has been shown to be a powerful
tool for the study of the physicochemical properties of
polysaccharides. Figs. 1 and 2 give the IR spectra of
chitosan and oligoglucosamine. From Fig. 2, it can be
seen that no band is observed between 1650 and 1900
cm1
, which allows us to conclude that oxidated groups
such as the carboxylic or aldehyde groups do not exist
in oligoglucosamine. Meanwhile, the bands correspond
peroxidate, such as RCOOOCOR and RCOOOH, can-
not be found from Fig. 2. Moreover, the C–H binding
vibration of the b-pyranose is manifested through the
peak at 900 cm1
. All these indicate that the conditions
of this reaction system do not result in the ring-opening
oxidation of glucosamine repeating units.
The bands at 1608.4 and 660 cm1
, correspond to
binding vibration of –NH2, and the band at 1074.2
cm1
, corresponds to stretching vibration of C–N (the
Table 1
The investigated variables and their levels
Variables investigated Levels of each variable
1 2 3
A: volume of solution (ml) 25 50 100
B: irradiation time (min) 3 4 5
C: concentration of H2O2 (%) 5 10 15
D: amount of chitosan (g) 1 2 3
Fig. 1. FT-IR spectra of chitosan.
Table 2
Experimental arrangement and test result
Experiment number A B C D ws (%)
1 1 1 1 1 5.2
2 1 2 2 2 51.8
3 1 3 3 3 80.9
4 2 1 2 3 40.5
5 2 2 3 1 92.3
6 2 3 1 2 8.1
7 3 1 3 2 82.6
8 3 2 1 3 12.8
9 3 3 2 1 3.2
K1 137.9 128.3 26.1 100.7
K2 140.9 156.9 95.5 142.5
K3 98.6 92.2 255.8 134.2
Variance 42.3 64.7 229.7 41.8
J. Shao et al. / Polymer Degradation and Stability 82 (2003) 395–398 397

4. peak is too strong to correspond to stretching vibration
of C–C), indicate that the-NH2 group in chitosan do not
destroy or disappear by oxidation degradation with
H2O2 under microwave irradiation.
Thus, all the information obtained from IR spectra of
oligoglucosamine indicate that the breaking of the C–
O–C glycosidic bond leads to the chain scission. The
chemical reaction can be described as in Fig. 3.
3.3. Molecular weight of oligoglucosamine
Molecular weight of the chitosan samples can be esti-
mated by a viscometric method, but an accurate result
can be obtained only when the molecular weight of
chitosan is more than 104
. Molecular weight of water-
soluble oligoglucosamine is less than 1500 [3], so the
viscometric method cannot be used here.
The end group analysis method was used to determine
the number average molecular weight of oligoglucosa-
mine. As the hemiacetal hydroxyl of the reductive sugar
can color during the reaction with basic potassium fer-
ricyamide, the latter is used as a color-producing
reagent. Both GAH and oligoglucosamine have hemi-
acetal hydroxyl in their molecule, so they can be colored
by basic potassium ferricyamide.
The regressive equation which we obtained is:
A=97.6W0.133, where A is absorbance value; W is
the amount of GAH (g), and the correlation coefficient
(r) of this regressive equation is 0.998. Consulting from
this regressive equation, the amount of hemiacetal
hydroxyl in 1 g of oligoglucosamine is almost equal to
that of 1.1103
mol of GAH. The number average
molecular weight of oligoglucosamine is about 900–
1000. The molecular weight of glucosamine repeating
units is 160, so the number of glucosamine repeating
units is about 5–6. Meanwhile, we found no dependence
of the number average molecular weight of oligogluco-
samine on the reaction conditions shown in Table 2.
4. Conclusions
Preparation of oligoglucosamine by oxidative degrada-
tion of chitosan with hydrogen peroxide under microwave
irradiation is feasible and convenient. In this reaction
condition, hydrogen peroxide acts on the C–O–C glycosi-
dic bond and leading to the chain of chitosan scission.
The optimum reaction condition was as follows: volume
of H2O2 (ml): 50 ml; irradiation time: 4 min; concen-
tration of H2O2:15%; amount of chitosan: 2 g. The
structure of the product was confirmed by FT-IR spec-
trum. The number average molecular weight of oligoglu-
cosamine obtained by this method is about 900–1000.
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Fig. 2. FT-IR spectra of oligoglucosamine.
Fig. 3. The degradation of chitosan.
398 J. Shao et al. / Polymer Degradation and Stability 82 (2003) 395–398