ANALYTICAL SCIENCES JUNE 2016, VOL.

32 701
2016 © The Japan Society for Analytical Chemistry

Notes

Determination of Chitin Based on the Colorimetric Assay of Glucosamine in

Acidic Hydrolysate

Hajime KATANO,† Masahiro TAKAKUWA, Hajime HAYAKAWA, and Hisashi KIMOTO

Department of Bioscience, Fukui Prefectural University, Eiheiji, Fukui 910–1195, Japan

A colorimetric method for the glucosamine (GlcN) assay was applied for the determination of chitin, which can be
hydrolyzed to produce GlcN. A  10-mg sample was mixed with 10 mL of a 5 mol/L HCl aqueous solution, and the
mixture was kept at 100° C for 12 h. Under these conditions, chitin was completely depolymerized and deacetylated to
produce GlcN, even when the sample was a crab shell. A 20-μL aliquot of the hydrolysate was mixed with 20 μL of a
5 mol/L NaOH aqueous solution and 200 μL of a 50 mmol/L Na2SiO3, 600 mmol/L Na2MoO4, 1.5 mol/L CH3COOH and
30% (v/v) dimethyl sulfoxide solution. The mixture was kept at 70° C for 30 min. In the mixture, GlcN reduced the
Mo(VI) species to form a blue molybdosilicate anion, which gave an absorbance maximum at around 750 nm. Since
N-acetylglucosamine and chitin oligosaccharides could not render the reaction mixture blue, GlcN in the hydrolysate
could be assayed colorimetrically with high selectivity. When a standard chitin sample was examined, the GlcN
concentration in the hydrolysate was determined to be 0.97 t 0.02 g/L (as hydrochloride salt), indicating that the sample
contained 10.0 t 0.2 mg chitin (as an N-acetylglucosamine homopolymer). Calcium cation, amino acids, and proteins did not
interfere with the GlcN assay. Thus, the proposed method was successfully applied to determine chitin in a crab shell sample.

Keywords Chitin, glucosamine, hydrolysate, molybdosilicate, colorimetry, crab shell

(Received January 13, 2016; Accepted February 23, 2016; Published June 10, 2016)

deacetylated to produce GlcN.17,18 Thus, chitin can be

Introduction
In a previous study,1 we developed a method for reducing
monosaccharide assay based on the color reaction in a
Si(VI)–Mo(VI) system. The Si(IV)–Mo(VI) solution is initially
yellowish due to the formation of yellow molybdosilicate
anionic species,2 and the monosaccharide reduces directly the
Mo(VI) species to form a mixed-valence molybdosilicate anion,
which renders the solution blue. This method is simple and
easy to carry out in comparison with the conventional reducing
saccharide assay using a copper(II) reagent, such as the
Somogyi–Nelson3,4 and Tauber–Kleiner5,6 methods. Also, even
in a ketose assay,7 our method shows higher reproducibility than
the conventional method using a phenol–acetone–borate reagent.8
Since di-, oligo-, and polysaccharides would not interfere with a
determination of the monosaccharide at the same concentration
level, the method was successfully applied to a microtiter plate
assay of saccharifying enzymes.1,9 The sensitivity of the
colorimetric assay differs among monosaccharides.1,7,9
Especially, glucosamine (GlcN) can be detected with very high
sensitivity.
Chitin is found in the shells of crustaceans, the cuticles of
insects, and the cell walls of fungi. It is well known that chitin
and its derivatives have wide-ranging applications in medicine
and industry.10–16 Thus, it is important to develop a suitable
method for determining chitin in bioresources. Briefly, chitin is
a long-chain polymer of N-acetyl-D-glucosamine (GlcNAc). By
an acidic hydrolysis, chitin can be depolymerized and
† CH3COOH aqueous solution was added slowly with stirring.
To whom correspondence should be addressed.
E-mail: hajime@fpu.ac.jp
determined from the amount of GlcN residue in the hydrolysate.
Usually, HPLC techniques have been employed for the GlcN
assay,19–21 although this application presents some difficulties.
For example, components in the hydrolysate affect the retention
characteristics, and the saccharide cannot be detected sensitively.
On the other hand, our colorimetric method would be applied in
the GlcN assay with higher sensitivity and without interference
by the components. Therefore, we studied the hydrolysis of
chitin and crab shell and the colorimetric assay of GlcN in the
hydrolysate.
Experimental
A technical-grade chitin powder was obtained from Wako Pure
Chemical Industries, and then washed with water. The chitin
sample was used as a standard material. We used 5 mol/L HCl
and 5 mol/L NaOH solutions for volumetric analysis in the
hydrolysis and neutralization processes, respectively. A reagent-
grade D(+)-glucosamine hydrochloride was obtained from
Wako, which was used as a standard material in the GlcN assay.
Sodium metasilicate enneahydrate, disodium molybdate(VI)
dihydrate, and dimethyl sulfoxide (DMSO) were obtained from
Wako, and were used without further purification to prepare a
Si(IV)–Mo(VI) solution. Other chemicals were of reagent
grade, and were used as received.
The Si(IV)–Mo(VI) solution was prepared as follows: 5 mL of
a 0.1 mol/L Na2SiO3 and 1.2 mol/L Na2MoO4 aqueous solution
was mixed with 3 mL of DMSO. Then, 1.5 mL of a 10 mol/L
The mixture was filled up to 10 mL to obtain a 50 mmol/L
702
Fig. 1 Absorption spectra of the Si(IV)–Mo(VI) reaction mixture
prepared with a test solution containing 1 g/L glucosamine (GlcN, as
hydrochloride salt) (a) before and (b) 30 min after heating at 70°C.
Path length, 1 cm.
Na2SiO3, 600 mmol/L Na2MoO4, 1.5 mol/L CH3COOH, and
30% (v/v) DMSO solution. The Si(IV)–Mo(VI) solution was
used more than 30 min after preparation.
The absorption spectra of the reaction mixtures were recorded
by a spectrophotometer (JASCO V-630) with a temperature
controller. In spectrophotometric measurements, the pass length
was 1 cm, and distilled water was used as a blank solution. In
the GlcN assay, the reaction mixtures were heated by a plate
shaker-thermostat (BioSan PST-100HL), and the absorbance
was recorded with a microplate reader (Bio-Rad iMark). In the
hydrolysis process, the reaction mixtures were heated at 100° C
under reflux.
Results and Discussion
Colorimetric glucosamine assay
In this section, we examined the x g/L GlcN (as hydrochloride
salt) and 5 mol/L HCl solution. A  20-μL portion of the test
solution was mixed with 20 μL of a 5 mol/L NaOH solution to
neutralize the HCl. This solution was mixed with 200 μL of the
Si(IV)–Mo(VI) solution consisting of 50 mmol/L Na2SiO3,
600 mmol/L Na2MoO4, 1.5 mol/L CH3COOH, and 30% (v/v)
DMSO. The mixture was kept at 70° C for 30 min. The reaction
mixture was then cooled to room temperature.
The Si(IV)–Mo(VI) solution was yellowish, due to the
formation of a yellow [SiMoVI 12 O40]
4– anionic species.22 The
1 g/L GlcN standard solution gave a bluish reaction mixture,
indicating that GlcN reduced directly the Mo(VI) species to
form a mixed-valence molybdosilicate anionic species,
[SiMoVMoVI 5–
11 O40] . Figure 1 shows the absorption spectra of the
reaction mixture before and after heating. The blue
molybdosilicate anionic species gave an absorbance maximum
at around 750 nm.
As shown in Fig. 2, the absorbance at 750 nm of the reaction
mixture in a microplate, A750, increased linearly to the
concentration of GlcN in the test solution, cGlcN, up to 1 g/L.
The regression line is given by
A750 = (0.826 t 0.009)(cGlcN/g L–1) + (0.0420 t 0.006), (1)
with a mean square of errors23 of 8 w 10–6. The detection limit
was calculated to be 3 w(8 w 10–6)1/2/0.826 ~ 0.01 g/L.
ANALYTICAL SCIENCES JUNE 2016, VOL. 32
Fig. 2 Plot of the absorbance at 750 nm of the reaction mixture in a
microplate, A750, against the concentration of glucosamine in test
solution, cGlcN. Error bar, the 95% confidence interval.
Table 1 A750-values for 1 g/L glucosamine, N-acetylglucosamine,
chitosan oligosaccharides, and chitin oligosaccharides
Saccharide A750
Glucosamine 0.870 t 0.008
N-Acetylglucosamine 0.046 t 0.005
Chitosan oligosaccharides 0.125 t 0.007
Chitin oligosaccharides 0.043 t 0.005
Blank 0.043 t 0.005
The test solution contained a high concentration of Cl– ion,
which interferes with the retention in the chromatographic GlcN
assay.20 On the other hand, because the present colorimetric
GlcN assay was not affected by the Cl– ion, the test solution
could be examined without the evaporation of HCl. As listed in
Table 1, the 1 g/L GlcNAc and (GlcNAc)n (n = 2 – 6) test
solutions gave A750 = 0.046 t 0.002 and 0.043 t 0.001,
respectively. The values were as low as that of a blank signal.
A  solution of 1 g/L chitosan oligosaccharides, (GlcN)n with
n = 2 – 9,24 gave A750 = 0.125 t 0.07, indicating that (GlcN)n
may give a small positive error in the GlcN assay. However, as
mentioned below, chitin can be converted into GlcN completely
under certain conditions. Thus, the present colorimetric method
could be applied advantageously to the determination of GlcN
in the hydrolysate of chitin.
An isomeric amino saccharide, galactosamine, gave A750 =
0.277 t 0.007, which is much smaller than A750 = 0.870 t 0.008
of GlcN. The value is smaller than A750 = 0.566 t 0.013 of a
saccharic acid, glucuronic acid. The difference in the reaction
rate among the positively and negatively charged saccharides
cannot be explained by the electrostatic interaction with the
[SiMo12O40]4– anion. Thus, the relationship between the reaction
rate and the structure of saccharides is not clear at present.
Hydrolysis of chitin and glucosamine assay in the hydrolysate
A 10-mg quantity of chitin powder was mixed with 10 mL of
a 5 mol/L HCl aqueous solution. The mixture was kept at
100°C under reflux for a given reaction time, t. Then, the
ANALYTICAL SCIENCES JUNE 2016, VOL. 32
Fig. 3 Plots of the determined cGlcN-values against the reaction time,
t, for 10 mL of the hydrolysate of 10 mg (a) standard chitin and (b)
crab shell samples.
mixture was cooled to room temperature. A  20-μL aliquot of
the hydrolysate was assayed by the present colorimetric method.
When t > 2 h, most of the powder was dissolved in the
solution. Plot (a) in Fig. 3 shows the determined cGlcN-value
plotted against t. The cGlcN-value increases with time, and
reaches a constant value at around t = 6 h. Using the determined
value of cGlcN = 0.97 t 0.02 g/L at t = 12 h, the amount of
(GlcNAc)n in the chitin sample was calculated to be 10.0 t 0.2
(=0.97 w(221.21/215.63)w 10) mg. These results indicate that
the chitin sample was completely depolymerized and
deacetylated to produce GlcN at t v 6 h, and that the present
colorimetric method can be utilized in the GlcN assay in the
hydrolysate.
A 10-mg quantity of chitin was mixed with 1 mL of a 5 mol/L
HCl aqueous solution at 100° C for 12 h. However, the chitin
powder did not dissolve completely. Thus, although a very
small volume of hydrolysate is assayed, a much larger volume
of acidic medium is required for the complete hydrolysis of chitin.
Determination of chitin in crab shell
Crab shells contain a large amount of calcium salts and
proteins. In the present colorimetry, the 1 g/L CaCl2 solution
gave a very low A750-value, the same as a blank signal. The
interference by amino acids and proteins was estimated as
follows: 10 mg of crab shell was mixed with 10 mL of a 1 mol/L
NaOH aqueous solution at 100° C for 2 h; the mixture was
centrifuged; 20 μL of the supernatant was neutralized by mixing
with an equivolume of 1 mol/L HCl aqueous solution; the
resulting solution was assayed by the present colorimetric
method. The solution gave an A750-value as small as the blank
signal. Thus, other components in the hydrolysate of crab shell
would not interfere with the GlcN assay.
A commercially available crab shell was examined without
drying. The cGlcN–t plot is shown as plot (b) in Fig. 3. The cGlcN-
value increased with time, until reaching a constant value at
around t = 12 h, indicating that the chitin in the crab shell
sample takes a longer time to hydrolyze completely. At t = 12 h,
cGlcN was determined to be 0.297 t 0.018 g/L. The water
content in the crab shell sample was determined to be 12.4 wt%
by an infrared moisture meter. If the water content was
excluded, the chitin content in the crab shell sample was
estimated to be 34 t 2% (w/w), which is in good agreement
703
with a reported value.25
In conclusion, our colorimetric method can be applied
advantageously to the GlcN assay in chitin hydrolysate, and the
chitin content in a crab shell can be successfully determined.
The present method will be useful for estimating the chitin
content in various bioresources. In this estimation, a microwave
pretreatment may be applied conveniently to the hydrolysis
process. Also, the present colorimetric method for the GlcN
assay will be useful to study the enzymatic production of GlcN
from bioresources. The experimental results will be described
elsewhere.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Numbers
26410226 and 26450099.
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