Carbohydrate Polymers 90 (2012) 73–77

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Products from microwave and ultrasonic wave assisted acid hydrolysis of chitin
Anawat Ajavakom a , Sulaleewan Supsvetson b , Aimjit Somboot b , Mongkol Sukwattanasinitt a,∗
a
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
b
Program of Petrochemical and Polymer Science, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Hydrolysis of ␣-chitin in concentrated hydrochloric acid under elevated temperature is a general prepa-
Received 30 March 2012 ration of a nutrapharmaceutical glucosamine hydrochloride (GlcN.HCl). In this study, the microwave and
Received in revised form 26 April 2012 ultrasonic wave assisted acid hydrolysis of shrimp shell ␣-chitin are investigated with an aim to improve
Accepted 27 April 2012
the reaction rate and selectivity. Microwave heating shortens the hydrolysis time from 120 min in the
Available online 8 May 2012
conventional heating process to 12 min to afford GlcN.HCl in 57% yield. Sonication can be used to assist
chitin dissolution in 38% HCl prior to the hydrolysis at 120 ◦ C for 120 min to produce GlcN.HCl in 62%
Keywords:
yield. The selective hydrolysis of glycosidic bond of chitin is achievable at 30 ◦ C for 4 h to give N-acetyl
Chitin
Chitosan
glucosamine (GlcNAc) in 37% yield.
Glucosamine © 2012 Elsevier Ltd. All rights reserved.
Hydrolysis
Microwave chemistry
Sonochemistry

1. Introduction usually prepared from the hydrochloride salt (GlcN.HCl) which

is industrially produced by the acid hydrolysis of chitin using
As the second most abundant and renewable natural polysac- concentrated hydrochloric acid. GlcN.HCl also has potential appli-
charide, chitin is considered as an unlimited source of nitrogen cations in cosmetics (Szego & Makk, 1982), antiviral drugs (Floc’h
containing organic compounds. It is the structural constituent & Werner, 1976; Rashad, Hegab, Abdel-Megeid, Micky, & Abdel-
of the exoskeletons of marine and terrestrial arthropods as well Megeid, 2008), anti-cancer (Chesnokov, Sun, & Itakura, 2009),
as cell walls of fungi and yeasts (Muzzarelli, 2012; Muzzarelli wound healing (Mackay & Miller, 2003), and as a substrate in the
et al., 2012). Frozen and processed seafood industries generate synthesis of glycoproteins (Menon, Mayor, Ferguson, Duszenko,
marine biomass byproduct containing billions of tons of chitin & Cross, 1988) and glycolipids (Mayor et al., 1990). The yield of
each year. There will be huge economic and environmental bene- GlcN.HCl obtained from the acid hydrolysis of chitin is depended
fits if these marine biomass wastes are efficiently utilized (Pillai, on reaction conditions including acid concentration, acid to solid
Paul, & Sharma 2009; Rinaudo, 2006; Sakai, 1995). One of the ratio, and reaction time (Ingle, Vaidya, & Pai, 1973; Kamasastri &
brightest applications of chitin is as a raw material for produc- Prabhu, 1961). In general, concentrated (38%, w/w) hydrochloric
tion of the amino sugar, glucosamine (GlcN). GlcN has been proven acid solution is required for solubilizing the solid chitin and high
effective in numerous scientific trials for easing osteoarthritis pain yield of glucosamine hydrochloride is obtained by refluxing this
(D Ambrosio, Casa, Bompani, Scali, & Scali, 1981; Gladman & acidic solution at a temperature about 100 ◦ C for at least 90 min
Farewell, 1999; Hauselman, 2001; Mankin, Brandt, & Shulman, (Gandhi and Laidler, 2002; Mojarrad, Nemati, Valizadeh, Ansarin, &
1986; Matheson & Perry, 2003), and has long been prescribed as Bourbour, 2007; Novikov, 2004). Under this condition, both amidic
a safe nutraceutical alternative to nonsteroidal anti-inflammatory and anhydroglucosidic bonds are hydrolyzed to simultaneously
drugs (NSAIDs) for osteoarthritis pain and inflammation treat- affect the deacetylation and depolymerization. An alternative to
ment (Da Camara & Dowless, 1998; Ishiguro, Kojima, & Poole, the acid hydrolysis of chitin is an enzymatic hydrolysis which is
2002; Todd, 2002; White & Stegemann, 2001). This has led to used for chitin degradation to chito-oligosaccharides and/or its
worldwide consumption of large amounts of a great variety of monomer, N-acetyl-d-glucosamine (GlcNAc) (Klaikherd, Jayanta,
over-the-counter GlcN. Most of GlcN products for arthritis are Boonjawat, Aiba, & Sukwattanasinitt, 2004; Pichyangkura, Kudan,
Kuttiyawong, Sukwattanasinitt, & Aiba, 2002; Sashiwa et al., 2003;
Sukwattanasinitt, Zhu, Sashiwa, & Aiba, 2002). Despite low selectiv-
ity between depolymerization and deacetylation, acid hydrolysis is
∗ Corresponding author. Tel.: +66 819010730; fax: +66 22187598. still attractive mainly due to its cost effectiveness compared to the
E-mail address: msukwatt@gmail.com (M. Sukwattanasinitt). enzymatic hydrolysis.

0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2012.04.064
74 A. Ajavakom et al. / Carbohydrate Polymers 90 (2012) 73–77

Ultrasonic wave and microwave irradiations have 2.4. Determination of degree of hydrolysis
recently become useful energy sources in various
chemical processes (Capelo-Martinez, 2009; Kidak & Ince, 2006; The hydrolysate was neutralized with NaOH solution (10%, w/w)
Srogi, 2006; Mats & Kristofer, 2006; Mutyala et al., 2010). The and was then centrifuged at 2000 rpm for 20 min. The supernatant
benefits of these irradiations in chemical processes include faster was removed and the solid residue was washed with DI water
reaction rate, higher product yield and reduced energy consump- (40 mL) and ethanol (95%, 40 mL). The solid was dried under vac-
tion. Due to the lack of research in hydrolysis of chitin under these uum and the degree of hydrolysis was calculated from 100 × (mass
irradiations, we therefore decided to investigate the products of starting chitin − mass of residue)/mass of starting chitin.
from ultrasonic wave and microwave assisted acid hydrolysis of
chitin and the condition optimization for greater product yields as
reported herein.
2.5. Monitoring of hydrolysis reaction by ESI-MS

The hydrolysate (2 mL) was pipetted into absolute ethanol

2. Materials and methods
(25 mL) at each time interval. The resulting cloudy mixture was kept
in a refrigerator at 4 ◦ C overnight and then centrifuged at 2000 rpm
Shrimp chitin flakes (∼0.4 mm2 ) were obtained from Taming
for 20 min. The precipitate was collected by decanting the liquid
Enterprises (Thailand). Concentrated HCl (38%, w/w) and activated
supernatant off. The precipitate was dissolved in DI-water (4 mL),
charcoal were purchased from Merck (Germany).
added with activated charcoal (4 mg) and stirred for 30 min. The
mixture was filtered through a filter paper (Whatman No. 1) and
then a 0.45 ␮m PTFE filter. The filtrate volume was adjusted to 5 mL
2.1. Hydrolysis of chitin by microwave heating
by DI-water in a 5 mL volumetric flask and analyzed by an electro-
spray ionization mass spectrometer (ESI-MS). The solution sample
38% HCl (50 mL) was pre-warmed by conventional microwave
(∼1 ppm, each 1.5 mL) was injected into the mass spectrometer
oven (Samsung, M183GN) at 850 watts (W) for 30 s. Shrimp chitin
using the following injection and ionization parameters; i.e. the
(30 g; chitin/acid ratio = 1:2, w/w) was added quickly into the pre-
voltage at capillary, extractor and RF lens were 40 kV, 3 V and 0 V,
warmed acid. After 1 min, when chitin was fully submerged, the
respectively. The cone voltage was 30 and 35 V for GlcN and GlcNAc,
microwave irradiation was continued for the designated period
respectively. Under MS scan mode, these parameters were adjusted
of time. After stopping the irradiation, the resulting slurry was
to give the highest signals corresponding to glucosamine (GlcN) and
allowed to cool to room temperature and filtered to obtain a brown
N-acetyl glucosamine (GlcNAc). GlcN was detected as the signal of
precipitate containing GlcN.HCl. All reactions were performed in
[GlcNH.H2 O]+ at m/z = 162 and GlcNAc was detected as the signal
triplicates.
of [GlcNAcH.H2 O]+ at m/z = 204. The relative abundance of these
signals was plotted vs the hydrolysis time.
2.2. Purification of GlcN.HCl

The crude precipitate was dissolved in distilled water 2.6. Purification of GlcNAc
(30 mL/10 g initial chitin), stirred for 30 min with activated char-
coal (20 mg/10 g initial chitin). The decolorized solution was stirred The brown slurry obtained from the hydrolysis was diluted with
at room temperature for 30 min. After the removal of activated 95% ethanol (40 mL) to precipitate a part of impurities. The ethano-
charcoal and insoluble residue by filtration, the clear filtrate was lic slurry was centrifuged at 2000 rpm for 20 min to remove the
evaporated under reduced pressure to recover GlcN.HCl as light remaining chitin and impurities. The obtained filtrate was evapo-
yellow solid. The solid was then dispersed in absolute ethanol rated and the residue was dispersed in 10 mL of water and filtered
(10 mL/10 g initial chitin), stirred for 30 min at room tempera- to remove the water insoluble residue. The filtrate was then evap-
ture and the slurry was then filtered. The white solid obtained orated by rotary evaporator to dryness. The pH of supernatant
was dried under vacuum for 24 h to yield pure GlcN.HCl. For was adjusted from pH ≤ 1 to neutral by filtering through NaHCO3
purity determination by acid-base titration, GlcN.HCl solution pre- powder (40 g). The neutral supernatant was then concentrated to
pared by dissolving GlcN.HCl salt in Milli-Q water (∼0.01 M) was 1/3 of volume by rotating evaporator and dropped into absolute
titrated with a standardized NaOH solution (∼0.01 M) using phe- ethanol (50 mL) while stirring to form a cloudy suspension and left
nolphthalein as the indicator. NaOH solution was standardized in a refrigerator at 4 ◦ C overnight to complete the precipitation.
by potassium hydrogen phthalate (KHP) solution (∼0.01 M) using The precipitate was separated by centrifugation at 2000 rpm and
phenolphthalein as the indicator. All titrations were performed in dried in desiccators under vacuum to afford solid A. The super-
triplicates. natant was concentrated to 1/3 of volume by rotating evaporator
and dropped into cool absolute ethanol (50 mL) to form cloudy sus-
pension. The precipitate (solid B) was collected by centrifugation at
2.3. Hydrolysis with ultrasonic wave treatment 2000 rpm. The supernatant was decolorized by stirring with acti-
vated charcoal for 45 min, filtered off to yield a clear solution that
Chitin (10 g divided into 5 portions) was added portion wise into was concentrated in a rotating evaporator and then freeze-dried to
38% HCl (50 g) immersed in a controlled temperature ultrasonic provide a white solid (solid C).
bath (Elmasonic S30H, 50/60 Hz, 275 W, England). The addition of
chitin was performed while the acid was sonicated at designated
temperature. Each chitin portion was added after complete disso-
2.7. 1H NMR spectroscopy
lution of the previous portion was observed. Typically, all portions
could be completely dissolved within 30 min. The chitin solution
1 H NMR spectra of the product samples, GlcN.HCl and GlcNAc
was allowed to stir at controlled temperature for a designated
period of time and the product was monitored and isolated as standards were acquired from the deuterium oxide (D2 O) solutions
described below. on Varian Mercury 400 NMR spectrometer at 400 MHz.
 A. Ajavakom et al. / Carbohydrate Polymers 90 (2012) 73–77 75

Fig. 3. GlcN.HCl obtained from chitin hydrolysis with microwave irradiation of

850 W for 12 min with and without mechanical agitation. The plots are the aver-
Fig. 1. GlcN.HCl yield obtained from chitin hydrolysis in 38% HCl with microwave
age values from three replicates with the error bars representing the maximum and
irradiation power of 850 W for 12 min at various chitin/acid ratios. The plots are the
minimum values.
average values from three replicates with the error bars representing the maximum
and minimum values.
stirring. Use of a mechanical stirrer improved the product yield
3. Results and discussion by 5–10% (Fig. 3). Under the optimum 1:3 chitin/38% HCl ratio,
GlcN.HCl was obtained in 57% yield with mechanical stirring. This
3.1. Chitin hydrolysis under microwave heating yield is comparable to conventional heating reported in literatures
but within much shorter time (Gandhi & Laidler, 2002; Ingle et al.,
First, we have investigated the effect of chitin/38% HCl ratio on 1973; Kamasastri & Prabhu, 1961; Mayor et al., 1990). Since agi-
the yield of GlcN.HCl. As illustrated in Fig. 1, the optimum chitin/38% tation of the reaction mixture can increase the product yield, the
HCl ratio is 1:3, w/w where GlcN.HCl can be obtained ∼50% yield. faster reaction is likely to be a result of the faster heating rate rather
The amount of HCl used affected the yield of GlcN.HCl in two man- than the superheating or selective heating. Microwave can thus
ners. With lower amount of 38% HCl (1:2 ratio), significant amount speed up and reduce the energy consumption in the acid hydrol-
of insoluble chitin was observed after the hydrolysis. Though the ysis of chitin to produce GlcN.HCl but it cannot improve product
reaction was done in reflux conditions, some HCl escaped from the yield.
reaction mixture leading to inadequate acidity to dissolve chitin in White crystalline powder of GlcN.HCl was obtained by using the
the initial state of the hydrolysis. When higher amount of 38% HCl decolorization-precipitation method (Gandhi & Laidler, 2002). The
(1:4 ratio) was used, less GlcN.HCl precipitate was obtained after isolated GlcN.HCl product obtained from the hydrolysis shows the
allowing the reaction mixture to cool to room temperature due to same 1 H-NMR spectrum pattern (Fig. S1) as the standard GlcN.HCl
dilution effect. (Fluka Chemicals, Ltd., ≥99% HPLC). The acid-base titration also
Fig. 2 shows a time course of GlcN.HCl yield obtained from the confirmed the percent purity of GlcN.HCl above 99%.
chitin hydrolysis with 38% HCl under microwave irradiation. The
isolated yield of GlcN.HCl increased with the hydrolysis time up to 3.2. Chitin hydrolysis with pre-sonication
12 min. The GlcN.HCl yield dropped slightly as the irradiation time
was extended beyond 12 min, probably due to the decomposition As described in the previous section that there was no selectiv-
of GlcN.HCl product. The optimum irradiation time for hydrolysis ity between glycosidic and amido bonds in the hydrolysis of chitin
is thus 12 min. either by microwave or conventional heating and only GlcN.HCl
To study the effect of heat transfer in this microwave assisted was obtained as the final product. It is important to note that
chitin hydrolysis, the reaction was conducted with and without chitin is dissolved in 38% HCl very slowly at room temperature
that a solution with concentration higher than 5% (w/w) cannot
be obtained within acceptable time, viz. <1 h, without warming the
%yield acid (∼60 ◦ C). In this part of study, we used ultrasonic wave to accel-
60 erate the dissolution of chitin at low temperature to obtain acidic
chitin solution with appreciable concentration. The hydrolysis of
50
chitin is expected to proceed with selectivity that should allow a
40 preparation of GlcNAc.
With sonication, 30 g of chitin was completely dissolved in
30 50 mL 38% HCl within 30 min even at 20 ◦ C. After the dissolution
20
Table 1
10 Degree of hydrolysis of chitin (10 g) in HCl (50 g) without and with sonication.

0 Temperature Without sonicationa With sonicationb

4 6 8 10 12 14 16
Residue (g) % hydrolysis Residue (g) % hydrolysis
Hydrolysis time (min) 20 C◦
9.7 3 8.8 12
40 ◦ C 4.3 57 2.0 80
Fig. 2. GlcN.HCl yield obtained from chitin hydrolysis in 38% HCl (1:1, w/w) using 60 ◦ C 0.9 91 0.3 97
microwave irradiation of 850 W at various irradiation times. The plots are the aver-
a
age values from three replicates with the error bars representing the maximum and Time = 120 min.
b
minimum values. Total time = 120 min with 30 min of sonication.
76 A. Ajavakom et al. / Carbohydrate Polymers 90 (2012) 73–77

Table 2
Purity and recovery mass of solid A, solid B and solid C from fractional precipitation.

Hydrolysis Solid A Solid B Solid C GlcNAc

conditions yield (%)

% % % % % %
purity mass purity mass purity mass

30 ◦ C, 4 h 75 27 77 13 >95 37 65
40 ◦ C, 2 h 65 27 73 30 >95 16 55
40 ◦ C, 3 h 69 20 67 28 >95 33 64
50 ◦ C, 2 h 67 30 75 20 92 10 43
60 ◦ C, 1 h 71 55 81 21 92 5 60

Fig. 4. MS signal intensities of GlcNAc during chitin hydrolysis in 38% HCl (1:5, w/w)
was responsible for the decrease of GlcNAc yield upon prolonged
at various temperature and times with 30 min pre-sonication.
hydrolysis.
The crude GlcNAc product was purified by fractional precipita-
of chitin by sonication at designated temperatures, the solutions
tion in absolute ethanol followed by decolorization as described in
were allowed to be stirred at controlled temperature for addi-
Section 2.6. The purity was measured by ESI-MS against the Glc-
tional 90 min. For comparison, another similar set of chitin/38%
NAc standard. The purities and recovery masses of the precipitate
HCl mixtures were allowed to stirred without pre-sonication at the
fractions (solid A, B and C) are summarized in Table 2. The solid C
same designated temperature for 120 min. The degree of hydrol-
which was the last precipitate fraction showed the highest purity. It
ysis, determined from the ratio of water soluble portion to the
is important to note that the low temperature (30 or 40 ◦ C) hydrol-
amount of initial chitin, was significantly higher with the sonication
ysis of chitin afforded higher GlcNAc purity and recovery mass of
pretreatment (Table 1). At 20 ◦ C, about 12% of chitin was hydrolyzed
solid C. With the hydrolysis at 30 ◦ C for 4 h, the total GlcNAc yield of
with pre-sonication while only 3% of hydrolysis was observed with-
65% was determined in the crude product which could be isolated
out the sonication pretreatment. At 40 ◦ C, the sonication process
by fractional precipitation to give 37% yield of high purity product
improved the degree of hydrolysis from 57% to 80%. However, high
(>95% pure). The high purity of GlcNAc was also confirmed by com-
degree of hydrolysis (>90%) could be obtained at the temperature
paring the 1 H NMR spectrum of the product with standard GlcNAc
60 ◦ C with and without sonication. The results confirmed that after
(Fig. S3).
the dissolution of chitin by sonication process, the hydrolysis of
chitin readily proceeded even at the temperature below 40 ◦ C.
4. Conclusion
To study the products obtained from the chitin hydrolysis, the
hydrolysates at various temperature were monitored by an elec-
The acid hydrolysis of ␣-chitin with 38% HCl to GlcN.HCl can
trospray ionization mass spectrometer (ESI-MS). The MS spectra of
be accelerated by microwave irradiation. The reaction rate accel-
the hydrolysate samples typically showed two major peaks cor-
eration is likely to be a result of the faster heating rate rather than
responding to GlcN at m/z = 162 ([GlcNH.H2 O]+ ) and GlcNAc at
the superheating or selective heating. The hydrolysis gave GlcN.HCl
m/z = 204 ([GlcNAc.H2 O]+ ) (Fig. S2). First the GlcNAc peak was mon-
comparable to the conventional heating within 12 min which is
itored at various hydrolysis temperature and the signal abundance
much shorter than 90 min normally required. Sonication brought
was plotted vs the hydrolysis time as shown in Fig. 4. At low hydrol-
about the dissolution of chitin in HCl even at 20 ◦ C that provides a
ysis temperature of 20 ◦ C, GlcNAc started to appear after 8 h and
method for selective acid hydrolysis of chitin at low temperature
increased slowly thereafter. Using hydrolysis temperature of 30 ◦ C,
to produce GlcNAc. This is the first report of microwave and ultra-
GlcNAc yield reached the maximum at 4 h and became constant
sonic wave assisted acid hydrolysis of chitin in the preparation of
afterward. At 40 ◦ C, the production of GlcNAc also reached the max-
amino monosaccharide, GlcN.HCl and GlcNAc.
imum at 4 h but gradually dropped afterward. For the hydrolysis
temperature of 50 ◦ C, GlcNAc yield reached the maximum within
Acknowledgements
2 h which then dropped quickly afterward. Similarly, the hydroly-
sis at 60 ◦ C gave the maximum GlcNAc yield within the first hour
The authors would like to acknowledge the financial support
which then dropped quickly. We hypothesize that the yield drop
from the National Research Council of Thailand (NRCT) in the
is caused by the deacetylation of GlcNAc. We monitored both GlcN
project of “Production of Amino Sugar Food Supplement from
and GlcNAc MS peaks obtained from the hydrolysis at 40 ◦ C and
Squid Pen”, the National Research University of CHE and the
found that the GlcN peak increased sharply after 4 h at the expense
Ratchadaphiseksomphot Endowment Fund (AM1006A), and the
of GlcNAc peak (Fig. 5). The results confirmed that the deacetylation
90th Anniversary of Chulalongkorn University Fund for the finan-
cial support. This work is part of the Project for Establishment of
Comprehensive Center for Innovative Food, Health Products and
Agriculture supported by the Thai government stimulus package 2
(TKK2555, SP2).

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at http://dx.doi.org/10.1016/
j.carbpol.2012.04.064.

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