American-Eurasian Journal of Scientific Research 8 (2): 63-67, 2013

ISSN 1818-6785
© IDOSI Publications, 2013
DOI: 10.5829/idosi.aejsr.2013.8.2.7381

Preparation of Glucosamine Hydrochloride from Crustacean Shell

Waste and It’s Quantitation by RP-HPLC
1
G. Sibi, 1K. Dhananjaya, 1K.R. Ravikumar, 1H. Mallesha, 2R.T. Venkatesha,
2
Dwijendra Trivedi, 2Khum Prasad Bhusal, 2Neeraj and 2Krishne Gowda

1
R and D Centre, Robust Materials Technology Pvt. Ltd. Bengaluru, Karnataka, India
2
Dayananda Sagar College of Biological Sciences, Bengaluru, Karnataka, India

Abstract: Various products derived from crustaceans have food and medicinal values. Glucosamine is an amino
monosaccharide acting as substrate for the production of aggrecan and proteoglycans and thus have
therapeutic activity in osteoarthritis. The present study has been aimed to prepare glucosamine hydrochloride
(Glu-HCl) from various crustacean shells namely Penaeus monodon (Indian shrimp), Portunus pelagicus (blue
crab) and Portunus sanguinolentus (three spot crab) by acid hydrolysis and its quantitation by reversed phase
high performance liquid chromatography (RP-HPLC). The yield of chitin after demineralization with 0.5M HCl
was 87.83%, 89.18% and 51.11% and deacetylation of chitin with 2N NaOH resulted in the yield of 68.91%,
75.67% and 30% for P.sanguinolentus, P.pelagicus and P.monodon respectively. HPLC analysis of obtained
glucosamine hydrochloride revealed species of Portunus (21.64 mg g 1 and 21.83 mg g 1) were better source
of Glu-HCl than P.monodon (3.32 mg g 1). Further, this study describes the recycling of crustacean wastes to
a value added product which is having potential applications in the field of food and medicine.

Key words: Glucosamine HCl HPLC Crustaceans Chitosan Chitin

INTRODUCTION invertebrates and cell wall of fungi and is composed of

The major economically important group of
crustaceans includes lobsters, shrimps and crabs. About
40-50% of total weight of crustaceans goes as waste while
processing for human food and the slower degradation of
crustacean shell waste has become the major concern in
sea food processing industries [1, 2]. Further, it has
resulted in waste collection, disposal and pollution
problems [3]. Proper use of crustacean wastes allows
recovery of value added by products which are having
potential applications in the field of food and medicine
[4-8]. Glucosamine has been prepared from various
crustaceans [9-10]. Glucosamine produced by hydrolysis
of chitosan has therapeutic activity in osteoarthritis [11].
Methods have been described for the quantitation of
glucosamine in chitin by HPLC [12-15].
Chitin is mainly produced from cuticles of various
crustaceans mainly by crabs and shrimps. Chitin is the
major structural component of exoskeleton of
2-acetamido-2-deoxy- -D-glucose (N-acetyl glucosamine).
Deacetylated form of chitin is known as chitosan which is
composed primarily of 2-amino-2-deoxy- -D-glucose
(glucosamine). Glucosamine is an amino monosaccharide
acting as a preferred substrate for the constitution
of glycosaminoglycan chains. It is also a substrate for
the production of aggrecan and proteoglycans which
gives hydrophilicity to the cartilage thus beneficial in
treatment of osteoarthritis [16]. Glucosamine has anti-
cancer [17, 18], anti-inflammatory [19] and antibacterial
[20] effects.
The present study was aimed to prepare glucosamine
hydrochloride using crustacean (Penaeus monodon,
Portunus pelagicus and Portunus sanguinolentus) shell
waste involving demineralization, deproteination and
deacetylation processes. Both quality and quantity of
obtained Glu-HCl were determined by Fourier Transform
Infrared Spectroscopy (FT-IR) and Reversed Phase High
Performance Liquid Chromatography (RP-HPLC).

Corresponding Author: G. Sibi, R and D centre, Robust Materials Technology Pvt. Ltd., Bengaluru, Karnataka, India.

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 Am-Euras. J. Sci. Res., 8 (2): 63-67, 2013
MATERIAL AND METHODS
Demineralization: Crustacean (shrimp and crab) shells
were collected from local market and washed in cold water
tap water by removing dirt and loose tissues. Legs and
heads were separated and the shells were dried in hot sun
for 24 hours. After drying, the shells were treated with
0.5M HCl (1:14 w/v) for 8 hours at room temperature and
the squashy shells were rinsed in water to remove calcium
chloride.
Deproteination: The demineralized shells were treated
with 1N NaOH (1:12 w/v) at 90°C for 2 hours and the
residue was neutralized in running tap water. The
deproteinized shells were dried in sun to obtain the chitin
material which was further used to produce chitosan by
deacetylation process.
Deacetylation: Chitin was treated with 2N NaOH
(1:14 w/v) solution to remove the acetyl groups at room
temperature for 8 hours in a shaker. The resulting chitosan
was washed in running tap water followed by distilled
water and dried in sun.
Preparation of Glucosamine HCL: The chitosan material
was coarsely grinded and hydrolyzed with conc. HCl at
90°C for 75 minutes. The resultant brownish black material
was dissolved in distilled water and decolourized with
activated charcoal. The solution was filtered and the
filtrate was evaporated at 45°C to recover glucosamine
hydrochloride (Glu-HCl). The crystals were washed in
ethanol and dried at 50°C in hot air oven and analyzed by
high performance liquid chromatography.
FT-IR Spectrum: Standard glucosamine hydrochloride
and obtained Glu-HCl were compared using FT-IR
spectra for identification and comparing purity.
The FT-IR spectrum was recorded on a Cary 640 FT-IR
(Agilent Technologies) in the form of Kbr discs.
The resolution was in 2 cm 1 and the scanning range was
4000-800 cm 1.
HPLC Method: HPLC analysis was performed
using 1260 infinity series LC system installed with
a G1311C pump, a G1329B autosampler and a G13166
column compartment and G4212B DAD Detector.
Chromatographic separation was carried out on a Zorbax
Eclipse XDB-C8 Analytical column (4.6 × 250mm, 5 µ;
64
Agilent Technologies). The mobile phase used was
orthophosphoric acid (pH 2.5): acetonitrile (70:30) with the
flow rate of 0.6 ml min 1 in column at ambient temperature
with infusion volume of 20 µl in each experiment. Injection
volume was 10 µl and detection was by UV absorbance at
195 nm.
Stock standard solutions were prepared by accurately
adding 100 mg of glucosamine hydrochloride to 50 ml
mobile phase (orthophosphoric acid: acetonitrile - 70:30)
in a 100 ml volumetric flask, sonicated and made up to
final volume. Working standards were prepared in mobile
phase, sonicated and filtered through 0.22µm filter.
RESULTS AND DISCUSSION
Glucosamine is part of the structure of chitosan and
chitin which compose the exoskeletons of crustaceans,
arthropods and fungi. The hydrolysis of chitosan results
in monomers of -(1-4)-linked-D-glucosamine (GlcN), an
amino monosaccharide with physiological importance to
the human body.
In the present study, crustacean wastes were used to
produce chitin and chitosan by acid hydrolysis method.
The yield of chitin after demineralization was 87.83%,
89.18% and 51.11% and deacetylation of chitin with 2N
NaOH resulted in the yield of 68.91%, 75.67% and 30% for
P.sanguinolentus, P.pelagicus and P.monodon
respectively (Table 1).
The finger prints of the FT-IR spectra of standard and
prepared Glu-HCl does not show any excess peaks (Fig 1).
The absorption bands at particular wavelength are due to
the formation of NH3+ in Glu-HCl which was in accordance
with previous data [21].
Peak identification of glucosamine was done by
comparing with the retention time of pure standard. Purity
of the peaks was confirmed with the characteristic spectra
obtained from the detector. Fig 2 represents the
chromatograms glucosamine hydrochloride prepared from
crustacean shells. In the figures, y axis is milli absorbance
unit (mA) and x axis is retention time (RT) in minutes.
There were two peaks occurred close which are related to
adsorption of glucosamine in the standard chromatogram
(Fig 2). From the HPLC analysis, the yield of Glu-HCl
prepared from crustacean wastes was 21.64 mg g 1, 21.83
mg g 1 and 3.32 mg g 1.
Previous studies have revealed that acid hydrolysis
is the preferred method to release glucosamine from chitin
material [22]. Several factors such as time, temperature,
 Am-Euras. J. Sci. Res., 8 (2): 63-67, 2013

Fig. 1: FT-IR spectra of Glu-HCl

Fig. 2: HPLC chromatograms of glucosamine hydrochloride from crustacean waste

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 Am-Euras. J. Sci. Res., 8 (2): 63-67, 2013
Table1: Yield of chitin, chitosan and Glu-HCl from crustacean waste
Sample Chitin Chitosan
Portunus sanguinolentus 87.83% 68.91%
Portunus pelagicus 89.18% 75.67%
Penaeus monodon 51.11% 30%
pH and acid concentration are attributed to the release of
glucosamine from chitin or chitosan. Acid hydrolysis of
chitin with concentrated HCl for longer time leads to
breakdown of glucosamine and decreased recovery [23].
Low concentrations of hydrochloric acid slow down the
chitin hydrolysis. Mojarrad et al. [15] described the
optimized conditions for the preparation of glucosamine
HCl as 30% and 37% HCL (9:1 v/w) for 4 hours from
Metapenaeus monoceros. In the present study, HCl
(0.5M) treatment for 8 hours produced a better yield of
chitin (89.83 %) from P.pelagicus. Purchase and Braun
[24] isolated glucosamine from crab shells. Stacey and
Webber [25] described the simple production of
glucosamine from crab shells of low protein content.
The present findings revealed that exoskeleton of
P.sanguinolentus and P. pelagicus were better sources
of Glu-HCl than P.monodon under the described
conditions. Ferrer et al. [26] has obtained 80% yield of
glucosamine during the acid hydrolysis of shrimp shell
wastes. The yield of chitin, chitosan and Glu-Hcl from
P.monodon was comparatively lower than that of crab
species used in this study.
CONCLUSION
The present study was found as simple, efficient and
suitable for the preparation of glucosamine from
crustacean shells thereby recycling crustacean wastes.
Further, HPLC method allows the detection and
quantitation of glucosamine prepared by acid hydrolysis
of chitosan from exoskeleton of crustaceans and could be
used as routine method for analysis of Glu-HCl in raw
materials and pharmaceutical formulations.
ACKNOWLEDGMENT
The authors are grateful to Vision Group on Science
and Technology (VGST), Government of Karnataka, India
for the financial support under SPiCE scheme.
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