Kinetics and mechanism of degradation of chitosan by combining sonolysis with H2O2/ascorbic acid

Article abstract of DOI:10.1039/c6ra11197a

This study demonstrated the combined use of sonolysis with the H₂O₂/ascorbic acid (Vc) redox reaction to degrade chitosan (CS). The influence of operating parameters, such as the initial CS concentration (1-15 mg mL^-1), the input power level (15-45% of the total input power), the H₂O₂ and Vc concentration (10-70 mM) and the pH (3.0-5.0) of the reaction solution, on the molecular weight and degradation kinetics of CS were investigated and optimized. Based on the degradation kinetics, a synergetic effect of sonolysis with H₂O₂ and Vc on the degradation of CS was observed. Structural analysis by FT-IR and ¹H and ¹³C NMR indicated no significant difference between the chemical structure of chitosan before and after degradation. Moreover, intermolecular hydrogen bonds were broken, and the crystallinity of degraded chitosan decreased. Using the above analysis, CS degradation by the combination of sonolysis with the H₂O₂/Vc redox reaction was proposed to be due to mechanical effects along with HO attack on the β-1,4-glucoside linkages of glucosamine units. These results suggest that the sonolysis/H₂O₂/Vc combined technique is promisingly suitable for large-scale manufacture of chitooligosaccharide.

Full text of DOI:10.1039/c6ra11197a

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Kinetics and mechanism of degradation of chitosan
by combining sonolysis with H2O2/ascorbic acid
Cite this: RSC Adv., 2016, 6, 76280
Tiantian Wu,† Chunhua Wu,† Yingchun Xiang, Jiaqi Huang, Lanlan Luan, Shiguo Chen
*
and Yaqin Hu
This study demonstrated the combined use of sonolysis with the H2O2/ascorbic acid (Vc) redox reaction to
degrade chitosan (CS). The influence of operating parameters, such as the initial CS concentration (1–15 mg
mLꢀ1), the input power level (15–45% of the total input power), the H2O2 and Vc concentration (10–70 mM)
and the pH (3.0–5.0) of the reaction solution, on the molecular weight and degradation kinetics of CS were
investigated and optimized. Based on the degradation kinetics, a synergetic effect of sonolysis with H2O2
and Vc on the degradation of CS was observed. Structural analysis by FT-IR and 1H and 13C NMR
indicated no significant difference between the chemical structure of chitosan before and after
degradation. Moreover, intermolecular hydrogen bonds were broken, and the crystallinity of degraded
chitosan decreased. Using the above analysis, CS degradation by the combination of sonolysis with the
H2O2/Vc redox reaction was proposed to be due to mechanical effects along with HOc attack on the b-
1,4-glucoside linkages of glucosamine units. These results suggest that the sonolysis/H2O2/Vc combined
technique is promisingly suitable for large-scale manufacture of chitooligosaccharide.
Received 30th April 2016
Accepted 28th July 2016
DOI: 10.1039/c6ra11197a
www.rsc.org/advances
the most gentle in its preservation of chemical structure and
elimination of side reactions.14 Moreover, the separation and
1. Introduction
further purication that would be necessary in the case of
chemical treatment methods could be avoided.15 Over the last
several decades, most studies have focused on the use of low-
frequency sonolysis for the degradation of CS.8,13,14 The mech-
anism involved in the degradation of CS is generally accepted to
be due to the mechanical effects of shear force caused during
the collapse of cavitation bubbles.13 However, owing to the
specic structure of chitosan, its degradation efficiency by
sonolysis alone is still relatively low. From this starting point,
a simple free-radical degradation method was introduced to the
sonolysis system in this current study to produce lower molec-
ular weight CS.
Free radical degradation is also used frequently to obtain low
molecular weight polysaccharides.16,17 For instance, the
hydroxyl radical (OHc) is a short-lived but powerful oxidant.18 It
can react with all sugars exceedingly fast.19 Generally, OHc can
be generated by the decomposition of hydrogen peroxide (H2O2)
in atmospheric air20 or via the Fenton reaction, which is cata-
lyzed by reduced transition metals, e.g., Fe2+ and Cu2+.16,21
Additionally, OHc can be formed in the presence of ascorbic
acid (Vc) without the need for transition metals because Vc
facilitates the formation of H2O2 from oxygen.22 Compared with
other OHc formation methods, the combination of H2O2 and Vc
in a redox reaction is a novel, eco-friendly procedure.23 Because
Vc can reduce H2O2 to OHc in the absence of trace metal,24
further separation and purication processes are simplied. To
date, the combination of H2O2 and Vc has been used for the
Chitosan (CS) is a cationic polysaccharide obtained by partial
deacetylation of chitin.1 It is a polymer composed of b-(1,4)-
linked glucosamine (GlcN) and N-acetylglucosamine (GlcNAc)
units in a random sequence.2 Due to its many desirable prop-
erties, such as biocompatibility, biodegradability, nontoxicity,
antioxidant activity, and antibacterial activity, CS has received
much attention as a functional biopolymer for diverse appli-
cations in biomedical, environmental, and agricultural
domains.3,4 However, CS always has a high molecular weight
and low solubility in aqueous solvents, which limit its applica-
tions, especially in the medical, cosmetic, and food industries.5
Additionally, several studies have shown that water-soluble CS
with low molecular weight has some special properties, such as
fat-binding and hypocholesterolemic and anti-tumor activities.6
Thus, to improve its solubility and properties, the preparation
of low molecular weight CS has become a hot topic for
researchers.7,8
A variety of techniques, including acid-4 and enzyme-
catalyzed hydrolysis,9,10 microwave,11 hydrodynamic cavita-
tion,5 and g irradiation,12 as well as sonolysis,8,13,14 have been
reported to prepare water-soluble chitosans with low molecular
weights. Among these methods, sonolysis is considered to be
College of Biosystems Engineering and Food Science, Fuli Institute of Food Science,
Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food
Technology and Equipment, Zhejiang University, Hangzhou, 310058, China. E-mail:
yqhu@zju.edu.cn
† These authors contributed equally to this work.
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degradation of hyaluronic acid,25 porphyran,26 and poly- mM), and the Vc concentration (0, 10, 30, 50 and 70 mM) and
saccharides from Athyrium multidentatum (Doll.) Ching27 and the pH (3.0, 3.5, 4.0, 4.5, and 5.0) of the reaction solution.
Enteromorpha linza.24 The degradation efficiency of these poly-
The intensity of the ultrasonic power that dissipated from
saccharides was signicantly enhanced in comparison to their the microtip with a radius of 10 mm was calculated according to
degradation by H2O2 or Vc individually at similar concentra- the report of our previously study.30 The input power levels were
tions. However, to the best of our knowledge, no report has been set at 15, 25, 35, and 45% of the total input power, which cor-
published on the application of this system for CS degradation. responded to ultrasonic powers of 182, 303, 424, and 545 W
Although many researchers have studied the coupling of the cmꢀ2, respectively.
OHc reaction with sonolysis for the efficient degradation of
Aer degraded, the treated chitosan solution was neutralized
hazardous materials,28 there have been few reports on its with 5% NH4OH solution to pH 7.5, then precipitated with
application to organic materials, including water-soluble poly- absolute ethanol. The chitosan precipitate was ltered, washed
saccharides.29 Furthermore, there has been no study on the with ethanol several times and lyophilization. The dried chito-
combination of sonolysis with the H2O2 and Vc redox system for san samples were collected for further GPC, FT-IR, XRD and
the degradation of CS.
NMR investigations.
Therefore, given the widespread use of these methods, the
combination of sonolysis with H2O2 and Vc was used to degrade
CS in the present study. The inuence of the initial CS, H2O2,
and Vc concentrations, the sonolysis intensities, and the solu-
tion pH, as well as the synergetic effect of sonolysis with H2O2
and Vc, on the molecular weight and degradation kinetics of CS
were investigated. The structural properties of the degraded
chitosan were characterized by FT-IR, 13C NMR, and XRD
spectral analyses. The mechanism of degraded CS is also
proposed. The current investigation may provide encourage-
ment for further exploration of the degradation of natural
polysaccharides.
2.3 Determination of the molecular weight of degraded CS
The average molecular weight (average Mw) and polydispersity
index (PDI) were determined by gel permeation chromatog-
raphy (GPC) according to our previously studies, with some
modications.30 The determination of average Mw was carried
out with a Waters 1525 HPLC system (Waters, Milford, MA, USA)
with a Waters Ultrahydrogel 250 and 2000 column. Twenty
microliters of the sample solution was injected and eluted with
0.2 M ammonium acetate (pH 4.5) at 40 ꢂC for 25 min at a ow
rate of 0.5 mL minꢀ1. The eluent was monitored with a Waters
2414 refractive index detector. The results were analyzed by the
GPC soware. Dextran standards (Sigma-Aldrich Chemical Co.,
St. Louis, MO, USA) with different molecular weights (from 0.5
to 670 kDa) were used to obtain calibration curves each time.
2. Material and methods
2.1 Materials
Chitosan was purchased from Qingdao Yunzhou Biochemistry
Co. Ltd. (Qingdao, China). The degree of deacetylation (DD) and
the molecular weight of the commercial chitosan used in this
study were determined experimentally and were found to be
92.21% and 209.45 kDa, respectively. Acetic acid (CH3COOH),
sodium acetate trihydrate (CH3COONa$3H2O), Vc, 5,5-dimethyl-
pyrroline-N-oxide (DMPO) and other reagents were purchased
from Sigma Chemical Co. (St. Louis, MO, USA).
2.4 Degradation kinetics models
The degradation behavior of CS was expressed according to the
model of rst-order reaction kinetics,7,31 and the degradation
rate constant (k) was determined by the following formula:
1
1
kt
m
1
¼
þ
¼
þ k0t
(1)
Mt
M0
M0
where Mt and M0 are the weight-average molecular weights of CS
samples at time t and time 0 (kDa), respectively. k (minꢀ1) and k0
(mol gꢀ1 minꢀ1) represent the rate constants; m is the molecular
weight of CS monosaccharides [m ¼ 161 + 42(1 ꢀ DD)], and t
(min) is the reaction time.
2.2 Experimental facility and batch experiments for the
demonstration of the synergetic effect of sonolysis-assisted
H2O2/Vc degradation CS
CS was completely dissolved in 1% acetic acid solution before
degradation. Sonication was performed with an ultrasonic
generator (Scientz-IID, Ningbo Scientz Biotechnology Co.,
2.5 Characterization of chitosan degradation products
Ningbo, Zhejiang, China) equipped with a 10 mm horn micro- The degree of deacetylation was determined by an acid–base
tip. The sample was added to 20 mL of nanopure water in a glass titration method from the literature.8 Chitosan samples (0.1 g)
tube (diameter ¼ 3 cm, height ¼ 10 cm), which was immersed in were dissolved in a known excess of 0.1 M HCl (10 mL). By
a water bath (DC-1006, Safe Corp., Ningbo, Zhejiang, China) to titrating this solution with a 0.1 M NaOH solution, a curve with
maintain a temperature of 25 ꢁ 1 ꢂC. The ultrasonic probe was two inection points was obtained. The amount of the acid
submerged to a depth of 1 cm from the surface of the liquid consumed between these two points was considered to corre-
layer.
spond to the amount of free amino groups in the solution. The
The effects of the following factors on the degradation of CS titration was performed using a pH meter (Mettler, Delta 340).
were investigated: the initial CS concentration (1, 2, 5, 10, and
The Fourier-transform infrared (FT-IR) spectra of the initial
15 mg mLꢀ1), the input power level (15, 25, 35, and 45% of the and degraded chitosan were recorded in powder form on KBr
total input power), the H2O2 concentration (0, 10, 30, 50 and 70 discs over the range of 400 to 4000 cmꢀ1 using an AVATAR 370
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spectrophotometer (Thermo Nicolet Corporation, Madison,
USA).
The crystallographic structures of the initial and degraded
chitosan were determined by a Bruker AXS D8 Advance X-ray
diffractometer (Bruker Inc., Germany) using Ni-ltered Cu Ka
radiation at 35 kV and 24 mA. The X-ray diffraction (XRD)
patterns of samples were recorded at a scanning rate of 10
minꢀ1 in the 2q range from 5 to 60ꢂ.
1
The H and 13C NMR spectra were recorded at 25 ꢂC with
samples dissolved in CD3COOD/D2O (1% acetic acid) using
a 600 MHz NMR spectrometer (Bruner, Switzerland).
Electron paramagnetic resonance (EPR) measurements were
conducted on a Bruker A300 EPR spectrometer (Bruner, Swit-
zerland), operating in 9.75 GHz microwave frequency with
a eld modulator frequency of 100 kHz. The magnetic eld was
scanned from 3300 G to 3500 G and the microwave power was
20 mW.
3. Results and discussion
3.1 Effects of reaction parameters on chitosan degradation
3.1.1. Effect of initial chitosan concentration. The effect of
solution concentration on the degradation of treated chitosan
was investigated, as shown in Fig. 1a and b. The kinetic rate
constants k and the determination coefficient of formula (1) R2
are presented in Table 1. The degradation efficiency decreased
as the concentration rose from 0.1 to 1.5 mg mLꢀ1, with lower
initial concentrations causing increased CS degradation. This is
consistent with published studies of CS degradation.5,7,13,14 The
kinetics study also revealed that k decreased with increasing CS
concentration (Fig. 1b; Table 1). This may be because the
increased polymer concentration led to a corresponding
increase in the solution viscosity, thereby reducing the extent of
the cavitation activity and hence the polymer scission rate. In
addition, as the concentration decreased, the entanglement
between the polysaccharide chains weakened.30 Thus, the
random coiled structure of the molecule became more extended
and therefore more vulnerable to attack from the shear force
and the hydroxyl radical, which improved the degradation.7
Therefore, lower solution concentrations improve CS
degradation.
Fig. 1 Effects of factors on CS degradation: (a) solution concentration
3.1.2. Effect of ultrasonic intensity. The inuence of (concentration, 1, 2, 5, 10 or 15 mg mLꢀ1; ultrasonic intensity, 424 W
cmꢀ2; H2O2 concentration, 50 mM; Vc concentration, 30 mM; pH 3.5).
ultrasound intensity on the degradation of CS is shown in
(b) Kinetics of CS degradation at different solution concentration; (c)
Fig. 1c and d. The kinetic rate constants k and the determina-
ultrasonic intensity (ultrasonic intensity, 182, 303, 424, or 545 W cmꢀ2
;
tion coefficient of formula (1) R2 are presented in Table 1. As
H2O2 concentration, 50 mM; Vc concentration, 30 mM; pH 3.5; and CS
concentration, 2 mg mLꢀ1); (d) kinetics of CS degradation at different
expected, the extent of degradation increased with an increase
in ultrasonic intensity. As the ultrasonic intensity increased ultrasonic intensity; (e) H2O2 concentration (concentration, 0, 10, 30,
50 or 70 mg mLꢀ1; ultrasonic intensity, 424 W cmꢀ2; CS concentration,
from 182 to 545 W cmꢀ2, the reaction rate constants (k) for CS
2 mg mLꢀ1; Vc concentration, 30 mM; pH 3.5); (f) kinetics of CS
increased from 4.494 ꢃ 10ꢀ7 to 1.245 ꢃ 10ꢀ6 mol gꢀ1 minꢀ1
.
degradation at different H2O2 concentration; (g) Vc concentration
(concentration, 0, 10, 30, 50 or 70 mg mLꢀ1; ultrasonic intensity, 424
W cmꢀ2; CS concentration, 2 mg mLꢀ1; H2O2 concentration, 50 mM;
This nding could be attributed to the enhancement of cavita-
tion energy.32 Similar observations were made by others for the
degradation of CS,13 sea cucumber fucoidan,30 dextran,33 and pH 3.5); (h) kinetics of CS degradation at different Vc concentration; (i)
pH value (3.0, 3.5, 4.0, 4.5 or 5; ultrasonic intensity, 424 W cmꢀ2; CS
a polysaccharide from Phellinus linteus.32 Higher ultrasonic
intensity can effectively increase the number of cavitation
concentration, 2 mg mLꢀ1; H2O2 concentration, 50 mM; Vc concen-
tration, 30 mM; pH 3.5); (j) kinetics of CS degradation at different pH
value.
events and result in more opportunities for enhanced degra-
dation.14 In addition, increased ultrasonic intensity accelerated
the generation of OHc radicals from H2O2 or H2O2/Vc,34 which
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Table 1 Degradation rate coefficients and determination coefficient
of formula (1) R2 of chitosan degradation
3.1.4. Effect of Vc concentration. The effect of the Vc
concentration on CS degradation is shown in Fig. 1g and h. A
degradation trend similar to that seen for H2O2 was observed
for Vc. It can be seen that addition of Vc (10–50 mM) can greatly
Process
k0 (mol gꢀ1 minꢀ1
)
R2
Solution concentration (mg mLꢀ1
)
1
2
5
1.333 ꢃ 10ꢀ6
1.138 ꢃ 10ꢀ6
4.380 ꢃ 10ꢀ7
1.435 ꢃ 10ꢀ7
8.260 ꢃ 10ꢀ8
4.494 ꢃ 10ꢀ7
7.741 ꢃ 10ꢀ7
1.138 ꢃ 10ꢀ6
1.245 ꢃ 10ꢀ6
1.141 ꢃ 10ꢀ7
2.696 ꢃ 10ꢀ7
1.138 ꢃ 10ꢀ6
1.442 ꢃ 10ꢀ6
1.560 ꢃ 10ꢀ6
2.835 ꢃ 10ꢀ7
4.747 ꢃ 10ꢀ7
1.138 ꢃ 10ꢀ6
1.593 ꢃ 10ꢀ6
1.517 ꢃ 10ꢀ6
0.977 reduce the required dosage of H2O2 and increase the degrada-
0.983
tion efficiency. In addition, excess Vc provides no advantage for
CS degradation. From the kinetic study (Table 1), it can be seen
that the most degradation efficiency was found at c(H2O2)/c(Vc)
¼ 1. Based on these results, it was concluded that the molar
0.994
0.934
0.977
0.988
10
15
182
303
424
545
0
10
30
50
70
0
Ultrasonic intensity (W cmꢀ2
H2O2 concentration (mM)
)
0.996 ratios of the two reagents should be kept at 1 to maximize
0.986
0.974
0.973
efficiency in obtaining lower Mw CS.
3.1.5. Effect of initial pH. The pH of the solution plays an
important role in CS degradation with H2O2/Vc redox reactions.
0.985
The pH value of the solution was adjusted by 1 M acetic acid or 1
0.986
0.990 M sodium hydroxide. As Fig. 1i and j show, the highest CS
0.960
0.996
0.985
degradation efficiencies were found at pH 4.0. A similar trend
was found for the reaction rate constants (Table 1). This
phenomenon might be related to the stability of H2O2 and the
Vc concentration (mM)
10
30
50
70
0.986
protonation of the CS molecular chain at various pH
0.982
0.969 values.20,25,35 Studies have shown that the stability of H2O2
pH
3.0 4.705 ꢃ 10ꢀ7
0.992
decreases with increasing pH and that it quickly decomposes in
3.5 1.138 ꢃ 10ꢀ6
0.986
0.997
0.994
0.983
alkaline conditions.25 Thus, its ability to generate free radicals
declined, and the greater the pH, the poorer the degradation
effect. On the other hand, the protonation of NH3+ groups in the
4
1.568 ꢃ 10ꢀ6
4.5 9.784 ꢃ 10ꢀ7
5
6.202 ꢃ 10ꢀ7
3.172 ꢃ 10ꢀ7
9.261 ꢃ 10ꢀ7
2.126 ꢃ 10ꢀ6
Sonolysis
H2O2/Vc
Sonolysis assisted H2O2/Vc
0.953 CS chain under acidic condition generates electrostatic inter-
0.975
0.989
actions between molecules and makes the chain easy to stretch
and break, which is convenient for degradation. However, it is
also noted that, when increasing the concentration of H+, the
concentration of anions in the acid will increase as well, making
also improved the degradation efficiency of CS. Consequently,
the degradation efficiency increased with the ultrasonic inten-
sity. It is worth noting that the Mw of CS dropped rapidly in the
initial 30–40 min and then decreased slowly and slightly over
the remaining 20–30 min of ultrasonic treatment, regardless of
ultrasonic intensity. This may be attributed to the preferential
breaking of longer polymer chains over shorter chains by shear
force and the OHc radical.13,18,24,30
3.1.3. Effect of H2O2 concentration. As Fig. 1e illustrates,
the degradation rate of CS was enhanced with the increased
H2O2 concentration. The kinetic rate constants k increased
obviously with H2O2 concentration from 10 to 50 mM;
however, at higher H2O2 concentrations, the change in k was
insignicant (Fig. 1f and Table 1). This indicates that when
a certain amount of H2O2 was added, a reasonable amount of
Vc could increase the production of OHc, thus contributing to
the degradation of CS. This result was in accordance with
previous studies on the degradation of others polymer by
OHc.17,24,27 Additionally, as discussed above, small molecules
with shorter chains are not sensitive to OHc radical attack,
which decreases the efficiency of degradation. It should be
noted that the decrease in the Mw of CS was strongly depen-
dent on the concentration of H2O2 when c(H2O2)/c(Vc) < 1.
Nevertheless, higher concentrations of H2O2 (>50 mM) were
not effective on the reaction, as measured by degradation
efficiency (Fig. 1f), resulting in a waste of resources. According
to these results, it can be concluded that the optimal
more salt linkage in CS molecules and thus increasing steric
hindrance to glycosidic bond cleavage and reducing the rate of
degradation of the molecules.7,36 Therefore, the optimum pH in
the experiment was found to be 4.0.
3.1.6. Comparison of sonolysis-assisted H2O2/Vc degrada-
tion, sonolysis alone and H2O2/Vc alone. To investigate the
synergetic effects of sonolysis with H2O2/Vc on the degradation
of chitosan, 50 mL CS solution (2 mg mLꢀ1) was treated with
sonolysis alone, H2O2/Vc alone, and H2O2/Vc in the presence of
sonolysis at 25 ꢁ 1 ꢂC for 60 min. The resulting Mw was plotted
as a function of time. The kinetic rate constants (k) and the
determination coefficient of formula (1) R2 are presented in
Table 1. As illustrates in Fig. 2a and b, the degradation efficiency
of CS using a combination of sonolysis with H2O2/Vc was
signicantly higher than that for sonolysis or H2O2/Vc alone.
The degradation efficiency of CS by H2O2/Vc system is
Fig. 2 Effect of the degraded methods on the degradation efficiency
concentration of H2O2 for degradation experiments is 50 mM. of CS.
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signicant higher than that of sonolysis alone. Interestingly,
sonolysis and H2O2 appear to have a synergistic effect in CS
degradation, because the degradation rate constants of the
combined process (ksonolysis+H O +Vc) are greater than the sum of
2
2
the rate constants of the individual processes (ksonolysis
+
kH O +Vc). Compared with other degradation methods, sonolysis-
2
2
assisted H2O2/Vc degradation showed remarkable advantages,
such as lower required concentration of H2O2, shorter reaction
time and lower molecular weight obtained. To conrm the
properties and the mechanism of degraded CS, these three
samples were selected for further characterizations.
3.2 Properties of the degradation products
3.2.1. DD and molecular distribution of degraded CS. The
DD and molecular distribution of the native and degraded
chitosan samples are listed in Table 2. The DD of degraded
chitosan samples did not appear to change with decreasing
molecular weight, suggesting that the N-acetyl-glucosamine is
stable enough during degradation and that glycosidic bonds
might be randomly cleaved. Table 2 also suggests that the oli-
gomerized CS samples have low polydispersity (Mw/Mn), with
a similar molecular weight distribution as the initial CS.
3.2.2. FT-IR spectra of degraded CS. The structures of the
degraded CS as well as the initial CS were investigated by FT-IR
spectrometry, and the results are shown in Fig. 3. The charac-
teristic absorption peaks of untreated CS are assigned as
follows: major absorption bands approximately 3380 cmꢀ1 were
attributed to O–H stretching; the signal at 2870 cmꢀ1 was due to
C–H stretching of CH2 groups; the feature absorption peaks at
1640 cmꢀ1 and 1560 cmꢀ1 were attributed to the bending
vibration of amide I and amide II groups, respectively; the
signal at 1153 cmꢀ1 and 1075 cmꢀ1 corresponded to the
deforming vibrations of C–O–C and C–O groups; and the band
at 898 cmꢀ1 was attributed to the b-(1,4)-glycosidic bond in the
CS chain.2,35 As can be seen, the FT-IR spectra of all the
degraded CS are similar to that of native CS though some
differences exist, indicating that the main chain structure of
degraded CS still remained during degradation process.
Compared to sonolysis degraded CS, the intensity of the amide I
and amide II groups H2O2/Vc treated CS (c and d) showed
a lower than that of native CS, suggesting the degradation of CS
by H2O2/Vc system were more acutely than sonolysis alone. This
was in agreement with the molecular weight data of degraded
CS samples, as discussed above. Additionally, no new band
Fig. 3 FT-IR spectra of the degraded CS as well as initial CS. (a) Initial
chitosan; (b) sonolysis alone; (c) only with H2O2/Vc; (d) sonolysis in the
presence of H2O2/Vc.
approximately 1730–1740 cmꢀ1, assigned to –COOH groups,
was seen, indicating that the degradation mainly involved
cleavage of b-glycosidic linkages and that no –COOH was
formed. Moreover, there was no obvious change in the charac-
teristic peaks at 1640 cmꢀ1 caused by amide I group stretching
vibrations, indicating no obvious deacetylation during the
degradation process. Similar results have been found aer the
degradation of CS by ozone,8 sonolysis,8 microwave,11 and
hydrodynamic cavitation.5
3.2.3. NMR spectra of degraded CS. The detailed structure
of CS before and aer degradation was further investigated by
1H and 13C NMR, and the results are shown in Fig. 4. As shown
in Fig. 4, native CS exhibited a typical 1H NMR spectra that
a single peak at d 1.95 ppm (the N-acetyl protons), at d 2.9 ppm
(H-2) and multiple peaks at d 3.3–3.7 ppm (H-3, H-4, H-5 and H-
6).37,38 As similar to FT-IR analysis, the 1H NMR spectrum of
degraded CS has similar to that of the original CS, which
coincided with the reported data.38 For 13C NMR of CS samples,
there was also almost no change in the CS spectrum before and
aer degradation, except that the absorption peak of the C-1
carbon downshied. This strongly suggested that, aer degra-
dation, the electron density around the C-1 and C-4 carbon
changed;39 therefore, the breakage and formation of chemical
bonds most likely occurred at or near the C-1 or C-4 carbon.
According to previous reports on the degradation of CS,4,39,40 it is
reasonable to believe that the degradation of CS in the current
study also occurred via attack on the C-1 or C-4 carbon and then
breakage of the adjacent C–O–C glycosidic bond in the main
chain. Moreover, no new signals were obvious at approximately
175 ppm, assigned to the –COOH group, which proved that no
–COOH group had formed. These data further conrmed that
the main backbone structure of CS did not change during the
degradation process.
Table 2 Degree of deacetylation (DD) and molecular distribution of
the native and degraded CSa
CS samples DD (%)
Mw (kDa)
Mn (kDa)
Mw/Mn
a
b
c
92.13 ꢁ 1.64 209.49 ꢁ 4.13 120.40 ꢁ 2.37 1.74 ꢁ 0.12
93.12 ꢁ 2.25 60.62 ꢁ 3.81 42.70 ꢁ 3.46 1.42 ꢁ 0.10
91.42 ꢁ 1.81 27.25 ꢁ 2.94 18.41 ꢁ 2.59 1.48 ꢁ 0.16
3.2.4. X-ray diffraction patterns of degraded CS. Fig. 5
shows the X-ray diffraction patterns of the original CS and the
degraded CS. The original chitosan exhibited two characteristic
peaks at 2q ¼ 11.31 and 20.18ꢂ, in agreement with the ndings
d
90.67 ꢁ 2.23
7.67 ꢁ 1.37
5.28 ꢁ 2.25 1.45 ꢁ 0.14
a
a: Native chitosan; b: sonolysis alone; c: only H2O2/Vc with; d:
sonolysis in the presence of H2O2/Vc.
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Fig. 5 X-ray diffraction patterns of the degraded CS as well as initial
CS. (a) Initial chitosan; (b) sonolysis alone; (c) only with H2O2/Vc; (d)
sonolysis in the presence of H2O2/Vc.
AH2 + H2O2 / HOc + H2O + DKG
where AH2 is Vc and DKG represents 2,3-diketogu-lonic acid.
To conrm the generation routes of OHc and it role in
degradation process, the electron paramagnetic resonance
(EPR) were introduced to detect free radicals and DMPO was
used as the spin trap. As shown in Fig. 6, the H2O2 + Vc + DMPO
system show a strong EPR signal, and the presence of CS in
reduced the EPR signal, however, no EPR signal were observed
for the CS + DMPO and Vc + DMPO only. These data suggested
that the mixing of H2O2 and Vc leads to a production of cOH and
furthermore to the degradation of CS, resulting in a decrease of
the molecular weight of CS. Similar phenomenon were reported
by others.43,44
The OHc radical possess an unpaired electron and is a very
powerful oxidant. Its reactions are thought to be non-selective.20
According to previous free-radical degradation studies, OHc can
quickly abstract the H atom from C-1, C-2, C-3, C-4, or C-5 of
pyranose rings in a (1 / 4)-linked CS chain39,45 and form CS
macro radicals ((GlcNc)m ꢀ (GlcN)n, Rc):
Fig. 4 1H and 13C NMR spectra of the degraded CS as well as initial CS.
(a) Initial chitosan; (b) sonolysis alone; (c) only with H2O2/Vc; (d)
sonolysis in the presence of H2O2/Vc.
from previously studies.41 Compared with the original chitosan,
the degraded CS had reduced peak intensities in its X-ray diffrac-
tion patterns. The crystallinity of products formed from sonolysis
combined with H2O2/Vc had the lowest intensity compared with
the other two samples. These results demonstrated that the CS
degraded by synergetic sonolysis with H2O2/Vc caused a signicant
loss of the crystal structure. Therefore, the solubility of degraded
CS also increased substantially. This decrease in crystallinity is also
seen in CS degraded by other methods.5,7,38,40,41
3.3 Mechanisms of the synergetic effects of sonolysis-
assisted H2O2/Vc for the degradation of chitosan
Sonolysis-assisted H2O2/Vc is a mild, effective, and environ-
mentally friendly method to degrade polysaccharides. It is
widely acknowledged that the mechanism of low frequency
ultrasonic degradation of CS has been attributed mainly to the
action of shear forces (mechanical effect) caused during the
collapse of cavitation bubbles.13,14 The mechanical effect
combined with the cavitation eld of sonolysis not only breaks
the b-1,4-glucoside linkage of the CS chain, resulting in a lower
Mw, but also destroys hydrogen bonds among native CS, loos-
ening its tight structure and providing access for easy further
degradation.
The mechanism of degradation by H2O2 and Vc appears to be
via OHc because Vc is easily oxidized by H2O2 and generates HOc
according to the following equation:23,42
Fig. 6 ESR spectrum of DMPO–OH adduct formed compounds.
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observed for sonolysis in combination with H2O2 and Vc. The
results of the FT-IR and 13C NMR spectra indicated that the
chemical structure of the resulting degraded CS was
unchanged, indicating that the degradation mainly involved the
cleavage of b-glycosidic linkages without –COOH formation.
Moreover, intermolecular hydrogen bonds were broken, and the
crystallinity of degraded chitosan decreased. According to the
above analysis, the synergistic degradation of CS by sonolysis
and the H2O2/Vc redox reaction was attributed to mechanical
effects and HOc attack on the b-1,4-glucoside linkages of
glucosamine units. These results suggest that a combined
sonolysis/H2O2/Vc technique is promisingly suitable for large-
scale manufacture of chitooligosaccharides. The current inves-
tigation will also provide encouragement for further exploration
into the degradation of natural polysaccharides.
Fig. 7 Possible mechanism of sonolysis assisted H2O2/Vc action on
the CS chain.
Acknowledgements
(GlcN)m ꢀ (GlcN)n + HOc / (GlcNc)m ꢀ (GlcN)n + H2O
The authors would like to acknowledge the nancial support of
National Key Research and Development Program499
(2016YFD0400100) and Key Scientic Research Project of Zhe-
jiang Province, China (2014C02017).
where m and n are the numbers of glucosamine residues, and
GlcN represents the glucosamine residue in CS chain. These Rc
then undergo further reactions and break down CS molecules to
form smaller molecules by the scission of the glycosidic bond:
(GlcNc)m ꢀ (GlcN)n + H2O / (GlcN)m + (GlcN)n
References
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