P-02
DEGRADATION OF CHITIN WITH HYDROGEN PEROXIDE
N A MICROWAVE FIELD
Anna Wojtasz-Pająk, Joanna Szumilewicz1
Sea Fisheries Institute in Gdynia,
ul. Kołłątaja 1, 81-332 Gdynia, Poland
e-mail: [email protected]
1 Technical University of Łódź, Department of Physical Chemistry of Polymers,
ul. Żeromskiego 116, 90-543 Łódź, Poland
1. Introduction
Chitin is a natural, non-toxic, biodegradable polymer (1,4-β-)-2-acetamido-2-deoxy-Dglucopyranose). This polymer is usually used to obtain the derivative chitosan. Due to its
biological activity and functional characters, chitosan has applications in many areas [1 - 5].
The biological and functional properties of chitosan are determined primarily by its chemical structure and the degree of polymerization [6 - 14].
The chemical and physical properties of target chitosan can be modified by altering the molecular weight of the source chitin material and the time and temperature of the deacetylation
reaction [15]. Chitin is degraded before the process of hydrolyzing the amide bonds. The
method applied should permit reducing the molecular weight without substantially altering
the chemical structure and should not pose an environmental threat. To date, investigations
have indicated that methods meeting these criteria include the thermal degradation of chitin
in a solid state. This method permits the waste-free modification of molecular weight within
a wide range [16]. However, reducing the degree of polymerization substantially does require that chitin is processed thermally at high temperatures for long periods of time. A more
effective method may be to reduce the molecular weight of chitin with hydrogen peroxide in
a microwave field. This is indicated by the results of chitosan degradation tests in which hydrogen peroxide and microwave radiation were applied simultaneously [17]. Hydrogen peroxide and water are relatively inexpensive, readily accessible, and environmentally friendly
compounds; thus a process based on them will meet the criteria of Green Chemistry.
There is no information in the literature regarding the degradation of chitin with hydrogen
peroxide in a microwave field. What is described is the possibility of using hydrogen peroxide during various stages of the technological process by which chitin and chitosan are
derived for bleaching raw materials, semi-processed products, and products [18].
Polish Chitin Society, Monograph XII, 2007
13
A. Wojtasz-Pająk, J. Szumilewicz
The aim of the investigations presented in the current paper was to determine the impact of
the simultaneous application of hydrogen peroxide and microwaves on the chitin degradation process.
2. Materials and methods
2.1. Chitin
The material investigated was chitin obtained from common shrimp shells (Crangon crangon [L.]) according to Wojtasz-Pająk and Szumilewicz [16] and that derived from krill
shells (Euphausia superba) according to technology developed at the Sea Fisheries Institute
in Gdynia [19]. The krill chitin was cleaned of mineral remains with 3% hydrochloric acid
(1:5 w/v) for 30 min. at a temperature of approximately 18°C. It was then rinsed several
times in tap water (1:10 w/v) and deproteinized in 5% NaOH (1:5 w/v) for 18 h at a temperature of approximately 21°C. After deproteinization, the chitin was rinsed several times
in tap and distilled water (1:10 w/v) and then dried for 15 h at a temperature of 80°C. It was
then ground in a Cyclotec 1093 Sample Mill manufactured by Tecator.
The polymers were held in a desiccator until they were tested.
2.2. Chitin degradation
The ground chitin was suspended in water, and 30% hydrogen peroxide was added in quantities to achieve H2O2 concentrations of 1%, 5%, 9% (1:25 w/v). These were subjected to
600 W microwave radiation for 10 to 30 min. This process was performed in an RM800
microwave reactor with a reflux condenser manufactured by Plazmatronika. After degradation, the chitin was rinsed several times with hot distilled water. The final concentration of
hydrogen peroxide in the samples did not exceed 10 mg/l. Indicator paper was used to check
this, and semi-quantitative determinations were performed with Quantofix® Peroxide 100
by Macherey-Nagel GmbH & Co. KG. Next, the chitin was dried for 3.5 h at a temperature
of 80 °C, ground in a Cyclotec 1093 Sample Mill manufactured by Tecator, and then dried
again at the same temperature for 4.5 h. The polymers were held in a desiccator until testing.
The degradation of krill chitin was also conducted with the addition to the water suspension
of FeSO4·7H2O in quantities that ensured there was 200 or 300 µg Fe2+ per 1 g of polymer.
The samples were then mixed for five minutes, and the appropriate amount of hydrogen
peroxide was added. The rest of the procedure was executed as described above.
2.3. Viscometric measurements
Viscosity measurements were done at a temperature of 25±0.01°C using an AVS-350
(Schott Geräte) with a type 53213/Ic Ubbelohde viscometer with a constant of K=0.02912.
The chitin concentration in N,N-dimetyloacetamide (DMAC) with 5% LiCl varied from
0.0001 to 0.0003 g/ml of solvent.
2.4. Infrared spectroscopy
The chitin was processed into pellets with KBr in a proportion of 1 mg chitin per 150 mg
KBr. The spectra were recorded with a Perkin-Elmer System 2000 at a resolution of 4 cm-1.
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Polish Chitin Society, Monograph XII, 2007
Degradation of chitin with hydrogen peroxide in a microwave field
Three spectra were recorded for each sample; these were averaged for each sample and
normalized so that the absorption band at 1070 cm-1 equaled 1. Normalization did not alter
the proportion of signals in the original spectra.
2.5. Determination of the degree of acetylation
The degree of acetylation (AD) of undegraded and degraded chitin was determined by the
method of adsorption of C.I. Acid Orange 7 (Sigma) dye in an acidic environment [20].
Adsorption was tested by measuring absorbance at a wavelength of 484 nm using a Unicam
UV2-100 spectrophotometer. The reference used was 0.1 N acetic acid.
3. Results and discussion
3.1. Impact of chitin degradation performed with hydrogen peroxide in a microwave
field on chemical structure of the polymer
The tests were performed by determining the degree of acetylation (AD) of chitin and its
degradation products. The results are presented in Table 1.
The differences in the limiting viscosity numbers of the initial chitin and the derived products are evidence (Table 1). The products of degradation were characterized by a higher degree of acetylation than that in the initial chitin. However, the differences were small and did
not exceed 3% (Table 1). These results indicate that the number of amine groups decreased
during the process. The changes in the number of NH2 groups determined were higher as
the degree of chitin degradation increased.
It seems however that the simultaneous decrease in the number of amine groups and the
increase of degradation degree was not caused by the oxidation of the NH2 group. This
probably resulted from changes in content of NH2 groups while rinsing the polymer after
degradation. The low-molecular fraction containing D-glucosamine could have been rinsed
Table 1. Properties of initial chitin and degraded with hydrogen peroxide and microwaves;
aCHA I – krill chitin (Euphausia superba) and CHA II, CHA III - common shrimp chitin (Crangon
crangon [L.]), bCHA I1-3 - product of degradation CHA I, cCHA II1-2 - product of degradation
CHA II, dCHA III1-3 – product of degradation CHA III.
Sample symbol
Applied degradation treatment
CHA Ia
AD, %
[η], ml/g
97.2
3020
CHA I1b
9% H2O2; 10 min.
98.4
1125
CHA I2b
5% H2O2; 30 min.
99.4
657
CHA I3b
5% H2O2 + 349 μg Fe2+/ g CHA; 10 min.
99.4
533
96.8
3064
809
CHA IIa
CHA II1d
9% H2O2; 10 min.
98.7
CHA II2d
5% H2O2; 30 min.
99.6
508
96.5
3065
CHA III a
CHA III1c
1% H2O2; 10 min.
97.3
1840
CHA III2c
5% H2O2; 10 min.
98.7
816
CHA III3c
9% H2O2; 10 min.
99.5
661
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A. Wojtasz-Pająk, J. Szumilewicz
away during this procedure. This type of change in the fundamental chitin structure appears
to be possible due to the role played by the amine group in the degradation of polysacharides
by hydrogen peroxide. The glycoside bonds are more susceptible to split since there is an
amine group adjacent to the C2 pyranoses ring [21 - 22]
The lack of significant differences in the chemical structure of the initial chitin and the degradation products was confirmed by infrared spectral analyses (Figures. 1, 2, 3).
Figure 1. Infrared spectra of initial chitin CHA I (
and CHA I3 ( ).
) and degradation products CHA I2 (
)
Figure 2. Infrared spectra of initial chitin CHA II(
) and degradation product CHA II2 (
).
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Polish Chitin Society, Monograph XII, 2007
Degradation of chitin with hydrogen peroxide in a microwave field
Figure 3. Infrared spectra of initial chitin CHA III (
and CHA III3 (
).
) and degratation products CHA III2 (
)
All of the spectral bands that were recorded in the corresponding initial chitin appeared in
the chitin that was degraded with hydrogen peroxide in a microwave field.
The manner in which the process was conducted probably influenced the lack of fundamental differences in the chemical structure of the polymer. The chitin was subjected to
a high concentration of hydrogen peroxide (9%) for a short period of time (10 min.). The
degradation of polymers for a period three times longer was conducted with H2O2 at a lower
concentration of 4%. According to Kabal´nowa et al. [23], changes in the chemical structure of chitosan degraded under heterogenic conditions (pH 7, 70°C, 30 min.) occur when
the concentration of hydrogen peroxide concentration is 10%. Under such conditions, the
amine group is oxidized, which is illustrated in the increased intensity of infrared spectral
bands 1566, 1580, and 1630 cm-1. Carboxyl groups might also occur at low concentrations
of H2O2 and long reaction times of one hour or more [24]. The investigations confirmed that
the degradation time had a significant impact on the chemical structure of the chitosan [17].
Degrading chitosan for a short period (4 min.) with concentrated H2O2 (15%) produced
oligoglucosamine in spectra in which neither carboxyl nor aldehyde groups were recorded.
3.2. Impact of the degradation of chitin in hydrogen peroxide and a microwave field on
its degree of polymerization
The chemical and physical properties of the chitin subjected to degradation are presented
in Table 2.
Although the viscosity-average degrees of polymerization (
) of the chitins were similar, the contents of inorganic substances were different (Table 2).
Changes in the degree of polymerization that occurred in the chitin during the hydrogen peroxide and microwave process was evaluated by determining the limiting viscosity numbers
Polish Chitin Society, Monograph XII, 2007
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A. Wojtasz-Pająk, J. Szumilewicz
Table 2. Properties of initial chitin; aCHA I – krill chitin (Euphausia superba), bCHA II, CHA III
– common shrimp chitin (Crangon crangon [L.]).
Sample symbol
[η], ml/g
DPv
Ash, % s.m.
CHA Ia
3020
3559
0.26
CHA IIb
3064
3661
1.76
CHA IIIb
3065
3665
3.47
[η] of the initial polymers and the products of their degradation according to Terbojevich
and Cosani [25]. The pathway of the degradation of CHA I and CHA II chitin as a function
of time is presented in Figure 4.
[η], ml/g
The chitin derived from common shrimp shells under the conditions applied in the investigation was more susceptible to degradation than the polymer derived from the krill shells.
During the first ten minutes of the process, the limiting viscosity number of CHA II was
approximately 600 ml/g lower than that of CHA I. Subsequently, the speed of the process
decreased and after 30 min [η] CHA II differed from [η] CHA I by about 150 ml/g. The
higher changes of polymerization degree of chitin CHA II than CHA I stemmed from the
varied contents of inorganic substances in the polymers. This is indicated by the results of
the determinations of limiting viscosity numbers of the CHA I, II, and III chitins degraded
for 10 min in a 5% solution of hydrogen peroxide (Figure 5).
Time, min
Figure 4. Kinetics of the degradation of chitin from krill shells (CHA I) and common shrimp
shells (CHA II) in 5% H2O2.
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[η], ml/g
Degradation of chitin with hydrogen peroxide in a microwave field
Ash, % d.w.
Figure 5. Dependence of the limiting viscosity numbers of the products of degraded chitin from
krill ( p CHA I) and common shrimp (¢ CHAII, ˜ CHA III) shells upon the ash content of the
initial chitin; (time - 10 min., H2O2 concentration - 5%).
Changes in the degree of polymerization of the investigated chitin were more pronounced
the higher the ash content was in the dry weight. The lowest limiting viscosity number was
noted in the degradation product of CHA III chitin (Figure 5, Table 2). It was approximately
816 ml/g and was about as twice as low as the limiting viscosity number of the krill chitin
(CHA I) that was degraded under the same conditions.
The impact of inorganic contamination on the course of the process was also apparent in the
investigations of changes in the degree of polymerization in the CHA I, II, and III chitins as
the hydrogen peroxide concentration in the reaction mixture increased. The largest changes
in the limiting viscosity numbers were observed in the case of CHA III chitin that had the
highest dry weight ash content of all the H2O2 concentrations investigated (Figure 6).
Krill chitin, however, was the least susceptible to degradation (CHA I). The degree of polymerization in the investigated chitin decreased as the concentration of hydrogen peroxide
increased. The limiting viscosity numbers of the degradation products of CHA I, II, and III
chitin treated in 9% H2O2 for 10 min. were 1125 ml/g, 809 ml/g, and 661 ml/g, respectively.
Increasing the concentration of hydrogen peroxide from 5% to 9% had less of an impact on
the degradation degree of the investigated polymers than did duration of the process. When
the process time was extended from 10 to 30 min. at H2O2 concentration of 5%, the limiting
viscosity numbers of the products derived from CHA I and CHA II chitin were lower at 657
ml/g and 508 ml/g, respectively.
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[η], ml/g
A. Wojtasz-Pająk, J. Szumilewicz
cHO,%
2
2
Figure 6. Dependence of the limiting viscosity numbers of chitin from krill (CHA I) and common shrimp
(CHAII, CHA III) shells upon hydrogen peroxide concentration; (degradation time - 10 min.).
That CHA III was more susceptible to degradation than CHA I or CHA II is evidence that
the metal ions that are components of the inorganic contamination may have a significant
impact on the process. This theory was tested by adding Fe2+ ions (177 and 349 µg/g CHA)
to the CHA I suspension as described in section 2.3. The Fe2+ ions resulted in the faster
degradation of krill chitin (Figure 7).
The limiting viscosity number of products obtained after adding of Fe2+in amount 177 µg/g
CHA was approximately 2.4-fold lower than that of chitin that had been degraded under the
same conditions but without Fe2+. Increasing the amount of Fe2+ ions nearly twofold resulted in a further decrease in the limiting viscosity number, although the changes were less
pronounced. The difference between the limiting viscosity numbers of degradation products
obtained at Fe2+ ion concentrations of 349 and 177 µg/g CHA was about 160 ml/g.
The results obtained indicate that the metal ions present in the inorganic contamination of
the investigated chitin had a significant impact on the kinetics of their degradation.
Chitin can contain ions of iron, copper, zinc, aluminum, cadmium, lead, and other metals.
They are bound by the polymer through both physical and chemical adsorption [26].
The quantities of these ions depends on the type of raw material, how the technological
process is conducted, the quality of the water used in the process, and the equipment. Metal
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[η], ml/g
Degradation of chitin with hydrogen peroxide in a microwave field
c Fe , ppm
2+
Figure 7. Dependence of the limiting viscosity numbers of chitin from krill shells (CHA I) degraded
in 5% H2O2 for 10 min. upon the concentration of Fe2+.
ions catalyze the breakdown of hydrogen peroxide into radicals or oxygen and water. Environmental pH and the metal ion complexing capabilities of the reagents, among other
factors, have an impact on the reaction [27 - 31]. Other factors that cause the formation of
free radicals and the breakdown of hydrogen peroxide into water and oxygen might be the
homolysis of the O-O bonds, temperature, and microwave radiation [24, 32 - 33]. Radicals
and oxygen initiate the splitting of the glycoside polysaccharides [17 - 18, 21, 24, 34 - 36].
Although the splitting mechanism of the chitin glycoside bonds was not addressed in the
current study, it can be inferred from a review of the literature that the degradation of chitin
under the conditions of the current investigation (pH of approximately 7) could have occurred with both free radicals as well as oxygen.
Chitin degradation performed with hydrogen peroxide in a microwave field was a more effective method for modifying molecular weight than thermal processing. This process was
quick (not exceeding 30 min.) at a temperature that did not exceed the boiling point of the
reaction mixture, and it caused significant changes in the degree of polymerization of chitin.
The limiting viscosity numbers of the degradation products were lower to those determined
for initial chitin from 15 to 83%, depending on the conditions under which the process
had been conducted. In the case of thermal degradation, changes in the limiting viscosity
numbers of 80% were not obtained even when thermal processing was conducted at a high
temperature (160 °C) for 15 hours; they were about 70% [16].
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A. Wojtasz-Pająk, J. Szumilewicz
4. Conclusions
Chitin degradation with hydrogen peroxide in a microwave field caused significant changes
in the molecular weight of the polymer in a short period (up to 30 min). The limiting viscosity
numbers of the degradation products were from 15 to 83% lower than those of the initial chitin.
The conditions under which the process was conducted (time, H2O2 concentration) and the
content of inorganic substances in the polymer impacted the degree of degradation of the
chitin. The rapidity of the process could be modified by changing the concentration of the
Fe2+ ions in the reaction environment.
Chitin degradation with hydrogen peroxide in a microwave field under the conditions applied in the current investigation did not cause significant changes in the chemical structure
of the polymer.
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