Carbohydrate Research 366 (2013) 5054

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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres

Determination of chitosan with a modied acid hydrolysis and HPLC

method
Bo Li a,b, Jiali Zhang c, Fen Bu b, Wenshui Xia a,
a

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
c
School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu, China
b

a r t i c l e

i n f o

Article history:
Received 6 August 2012
Received in revised form 6 November 2012
Accepted 12 November 2012
Available online 21 November 2012
Keywords:
Chitosan
Hydrolysis
Glucosamine
Browning
9-Fluorenylmethoxycarbonyl chloride
Derivation

a b s t r a c t
Acid hydrolysis and subsequent quantication of glucosamine (GlcN) are widely used for chitosan quantication. Degradation of GlcN during chitosan hydrolysis was the main reason for the decrease of recovery, which made the method improper for the quantication of chitosan. Ten milligram of chitosan
hydrolyzed with 10 mL mixed acid solution of HClH3PO4 (75:25 in molar ratio) showed the highest
recovery, signicantly higher than HCl hydrolysis. Further study revealed that the optimum conditions
involved the hydrolysis with HClH3PO4 (4.5:1.5 M) for 24 h at 110 C. The hydrolysate was neutralized
and derived with 9-uorenylmethoxycarbonyl chloride (FMOC-Cl) before HPLC quantication. The optimum ratio of FMOC-Cl:GlcN was 53:1, with excess FMOC-Cl induced by the high ionic strength of the
solution. This quantication procedure was then validated and proved to be specic, with good linearity,
accuracy, and precision, making it well-suited for the determination of chitosan.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Chitosan, the N-deacetylated derivate of chitin, is a heteropolysaccharide consisting of linear b-1,4-linked glucosamine (GlcN)
and N-acetyl-glucosamine (GlcNAc) units.1 Both chitin and chitosan are widely used in food,2,3 biotechnology, material science,4
pharmaceutics,57 and gene therapy.8
Several colorimetric assays, using ninhydrin,9 o-phthalaldehyde10 (OPA), or Cibacron Brilliant Red 3B-A,11,12 have been used
to the quantify chitosan. However, the response during these reactions depends strictly upon the degree of deacetylation (DD) of
chitosan13 which limited their application.
Many other quantication methods entail a total hydrolysis of
chitosan into GlcN followed by the subsequent characterization
of the monomer.14 Acid hydrolysis with HCl was the most widely
used, as shown in Table 1, because of its effectiveness in both the
hydrolysis of the glycosidic linkage (depolymerization) and the
N-acetyl linkage (deacetylation) of chitosan.27 In most of these reports (except Ref. 17), GlcN was spiked to chitosan samples to evaluate the recovery. However, the recovery rate was generally about
90% (Table 1), which was improper for the determination of chitosan. The degradation of GlcN in the acid hydrolysis was responsible
for this decrease of recovery. Millard reaction,28,29 and deamination16 were estimated as main degradation pathway. In most re Corresponding author. Tel.: +86 510 85919121; fax: +86 510 85329057.
E-mail address: xiaws@jiangnan.edu.cn (W. Xia).
0008-6215/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.11.005

ports, the acid concentration and hydrolysis time were carefully

optimized to completely hydrolyze chitosan and to minimize the
degradation of GlcN.
In the present study, hydrolysis of chitosan with HClH3PO4
was studied and the optimum hydrolysis condition with minimum
degradation of GlcN and highest recovery of chitosan was studied
and compared with HCl hydrolysis. The derivation of the hydrolysate was also optimized. A modied acid hydrolysis and HPLC
method for the determination of chitosan was then established
and validated.
2. Results and discussion
2.1. Inuence of acid on the recovery of chitosan
Chitosan (10 mg) was hydrolyzed with 10 mL mixed acid composed of HCl and H3PO4 containing 6 M H+. One mol of H3PO4 was
estimated to contain about 1 mol H+, because the pKa1 and pKa2
values of phosphoric acid were 2.15 and 7.09, respectively.30 After
a 24 h hydrolysis at 110 C, GlcN in the hydrolysates were derived
and determined. The recovery of chitosan was evaluated by the
amount of GlcN released after hydrolysis versus the amount of
chitosan.
As shown in Figure 1, the recovery of chitosan varied with the
composition of acid. Chitosan hydrolyzed with HClH3PO4 (75:25
in molar ratio) showed highest recovery, signicantly higher
(p < 0.05) than that with HCl. Meanwhile, the recovery

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B. Li et al. / Carbohydrate Research 366 (2013) 5054

Table 1
Application of acid hydrolysis in the quantication of chitosan
Ref.

Acid and concn

15

6 M HCl
100
10 M HCl
105
6 M HCl
100
8 M HCl
110
6 M HCl
115
72% (v/v) H2SO4, 90 min at room temperature, then 121 C for 1 h with diluted H2SO4
6 M HCl
121
6 M HCl
100
H2SO4 72% (v/v) 3 h room temp., then H2SO4 2 mol L1, 100 C, 4 h
10 M HCl
140
6 M HCl
100
4 M HCl
100

16
17
18
19
20
21
22
23
24
25
26
a

Temp (C)

Time (h)

Recovery (%)

48
6
10
4
1

86
99103
100.52a
90.198.7
96.5
92.4
92.0
93
80
135.2
86
106

7
4
1
7
3

Chitosan was used as standard spiked to matrix.

hydrolyze both glycoside and the acetyl groups,27,32 HCl was essential to ensure the complete hydrolysis of chitosan to GlcN.

dramatically decreased with higher H3PO4 ratio. To further prove

the inuence of acid, GlcN, the hydrolyzed product of chitosan,
was also hydrolyzed with HClH3PO4. Its recovery showed similar
trends as chitosan (Fig. 1). As HClH3PO4 (75:25 in molar ratio)
showed highest recovery for chitosan and GlcN, it was used in
further hydrolysis of chitosan.
The degradation of chitosan in acid hydrolysis was studied to
further reveal the inuence of acid composition on the recovery
of chitosan. Chitosan hydrolyzed with HClH3PO4 (75:25 in molar
ratio) showed least absorbance at 280 nm, and the absorbance signicantly increased with higher H3PO4 ratio, as shown in Figure 1.
This proved that the degradation varied with the ratio of HCl
H3PO4 and thus affected the recovery of chitosan.
In the hydrolysis of chitin, N-acetyl-D-glucosamine-1-phosphate was formed in H3PO4 hydrolysis31 while glucofuranosyl
oxazolinium was formed in HCl hydrolysis.32 This variation of
intermediates with the acid used in hydrolysis was responsible
for the change of degradation and recovery of chitosan in different
HClH3PO4 ratios. As the degradation products were believed to be
too complex,28 the degradation products and pathway of degradation in HClH3PO4 were not further studied in the present work.
The decrease of recovery with high H3PO4 ratio was more severe
for chitosan than GlcN, because the deacetylation of GlcNAc in
H3PO4 was limited.31,33 Considering that HCl was proved able to

120

Previous reports proved that the acid concentration could inuence the hydrolysis of chitosan16,18 and the degradation of GlcN.19
In the present study, the concentration of HClH3PO4 was optimized with the evaluation of chitosan recovery. Chitosan was
hydrolyzed using a mixed acid solution of HClH3PO4 (75:25 in
molar ratio) with different [H+] at 110 C for 24 h. The relative yield
of GlcN was studied. As shown in Figure 2, hydrolysis of chitosan
with 6 M [H+] showed optimum recovery for 98.8%. Lower [H+]
showed 64.6% yields of GlcN because of the incomplete hydrolysis
of chitosan; and 90.3% for 9 M [H+] due to the degradation of GlcN.
Therefore, the mixed acid solution with 6 M [H+], HClH3PO4
(4.5:1.5 M) in other words, was used in further research.
In comparison, chitosan was hydrolyzed with different concentrations of HCl. Highest recovery of chitosan was also found to be at
6 M. Meanwhile, chitosan hydrolyzed with 6 M HCl and 9 M HCl
showed lower recovery than hydrolyzed with HClH3PO4 (75:25
in molar ratio) having same [H+]. This further proved that HCl
H3PO4 (75:25 in molar ratio) could inhibit the degradation of GlcN
and improve the recovery of chitosan.

Recovery of Chitosan
Recovery of GlcN
A280 of Chitosan

2.5

A280 of GlcN

2.0

80
1.5

A 280

Recovery (%)

100

2.2. Inuence of acid concentration on the recovery of chitosan

60
1.0
40
0.5

20

0
100:0

75:25

50:50

25:75

0:100

0.0

HCl-H3PO4
Figure 1. Recovery of chitosan and GlcN in the mixture of 6 M HCl and 6 M H3PO4 (n = 3).

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B. Li et al. / Carbohydrate Research 366 (2013) 5054

1.2

HCl-H3PO4(75:25)

derived in water
derived in matrix

HCl

100

1.0
0.8

AGlcN/AIS

Recovery (%)

80

60

0.6

40

0.4

20

0.2
0.0

0
4

10

50

Figure 2. Inuence of acid concentration ([H]+) on the recovery of chitosan.

Recovery of GlcN
100

Recovery (%)

80

60

40

20

0
0

10

20

30

100

150

200

250

300

FMOC-Cl (l)

Acid Concentration (mol/L)

40

Time (h)
Figure 3. Inuence of hydrolysis time on the recovery of chitosan (n = 2).

2.3. Inuence of hydrolysis time on the recovery of chitosan

The hydrolysis time was also studied by testing the yield of
GlcN with different hydrolysis times on chitosan samples. The
hydrolysis reactions were performed at 110 C with HClH3PO4
(4.5:1.5 M).
The results for chitosan hydrolyzed at different time intervals
are presented in Figure 3. The relative yield of GlcN for chitosan
samples treated with HClH3PO4 increased with time up to 24 h
and slightly decreased thereafter. The browning of the solution increased steadily for hydrolysis time longer than 24 h.
These results showed that chitosan could be completely hydrolyzed to GlcN after 24 h of the reaction. Glucosamine slightly degraded with extended hydrolysis time, with the recovery of GlcN
decreasing from 97.7% at 24 h to 90.0% at 36 h. Therefore, the
hydrolysis time was set to 24 hours to quantify the chitosan
samples.
2.4. Optimization of FMOC-Cl derivation of GlcN
Glucosamine does not contain a chromophore absorbing in the
wavelength range useful for liquid chromatography with UV detection.17,18 As a derivatization reagent, FMOC-Cl reacts with the primary amine of GlcN, which signicantly improved the UV
absorbance and the sensitivity in quantication of GlcN. In the

Figure 4. Inuence of FMOC-Cl volume to the derivation of GlcN (n = 3).

present study, a-aminobutyric acid was used as internal standard

(IS), which was derived with FMOC-Cl along with the derivation
of GlcN.
As H3PO4 in the hydrolysate could not be vaccum dried like HCl
in previous reports,24 the hydrolysate was neutralized before derivation. This neutralization dramatically increased the ion strength
of the matrix. To study the derivation of GlcN in matrix, the GlcN
solution was diluted with matrix and water, respectively. The solutions were then derived with 25300 ll FMOC-Cl solution, with
acetonitrile added to the same volume. As shown in Figure 4, GlcN
solution diluted with water showed a similar area within a range of
FMOC-Cl from 25 to 300 ll. But for GlcN solution diluted with matrix, the area of GlcN enhanced with the increasing of FMOC-Cl volume up to 100 ll and then decreased slightly. At the optimum
FMOC-Cl level, the molar ratio of FMOC-Cl and GlcN was approximately 53:1, much higher than previous reports.18,25 The derivation was performed with 100 ll FMOC-Cl in further research.
To further estimate the inuence of ion strength to derivation,
3 M NaCl solution was mixed with GlcN solution before and after
derivation. Results showed that the area of GlcN lowered with NaCl
added before derivation, and no signicant decrease of GlcN area
with NaCl added after derivation (data not shown). Therefore, the
derivation behavior was changed by the ion strength of the matrix.
To remove the excess derivation reagent and terminate the derivation reaction, acetonitrile- acetate acid solution (8:2, v/v) was
added to convert FMOC-Cl into FMOC-OH.34,35
2.5. Method validation
The recovery of chitosan was checked by applying the standard
addition technique and glucosamine hydrochloride was used as
standard. Five milligrams of chitosan sample (Sigma, St. Louis, United States, MW 607 kDa, DD 80.0%) was spiked with different
amounts of GlcN solution. The mixed samples were then hydrolyzed and determined as described in the experimental part. The
recovery using 6 M HCl hydrolysis was also studied. As shown in
Table 2, mean recoveries of HClH3PO4 hydrolysis was 96.9
98.6% for each level (n = 3), signicantly higher (p < 0.05) than
the recovery using 6 M HCl hydrolysis.
The precision was evaluated by assaying chitosan samples (Sigma, St. Louis, United States, MW 607 kDa, DD 80.0%) at 80, 100, and
120% levels (8, 10 and 12 mg, respectively). Table 2 summarized
the within run and between run precision. In this assay, the standard division of within run and between run precision ranged from
2.9% to 3.8% and from 3.0% to 3.9% for each level, respectively.

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B. Li et al. / Carbohydrate Research 366 (2013) 5054

Table 2
Recovery and precision of the HClH3PO4 hydrolysis and FMOC-Cl derivation HPLC
method (n = 3)

80.0%). Water was prepared using Milli-Q system. Other chemical

reagents were analytical grade.

GlcN added
(mg)

Recovery (% SD)

Recovery (% SD) HCl

hydrolysis

4.2. Hydrolysis of chitosan

3.03
5.05
7.07
Average

97.5 3.5
98.6 4.2
96.9 3.5
97.7 3.3

86.2 3.8
85.3 4.3
83.9 5.2
85.1 4.2

Level of
chitosan

Within run precision

(% SD)

Between run precision

(% SD)

80%
100%
120%
Average

95.1 3.7
95.5 3.8
96.7 2.9
95.7 3.4

96.3 3.0
95.9 3.4
97.1 3.9
96.4 3.3

Chitosan powder (10 mg) was accurately weighed and transferred to a hydrolysis tube. Five milliliters of water and 5 mL of
mixed acid solution of HClH3PO4 (prepared with 75 mL HCl and
25 mL 12 M H3PO4) were added to the tube, such that the nal concentrations of HCl and H3PO4 were 4.5 and 1.5 M, respectively. The
tube was vacuum-treated, ushed with N2 and then fuse-sealed to
avoid the inuence of oxygen. Acid hydrolysis was performed at
110 C for 24 h to generate GlcN. The tube was then cooled and
kept at 4 C till derivation and determination.
4.3. Derivation of GlcN in hydrolysate

Table 3
Determination of chitosan using HClH3PO4 hydrolysis and HPLC method (n = 3)
Chitosan sample

Chitosan (Sigma)
Chitosan
(Nantong)
Chitosan
(Zhejiang)

MW
(kDa)

DD
(%)

607
440
573

Content (% SD)
HClH3PO4
hydrolysis

HCl
hydrolysis

80.0
82.4

96.2 2.7
95.2 3.0

88.7 3.8
86.8 3.7

76.5

87.8 2.8

79.5 3.8

Glucosamine reference solution at a known concentration (20

2 mg/mL) was assayed as described above. A typical least squares
linear regression evaluation of the peak area ratio of GlcN and IS
(y) versus concentration of GlcN (x, mg/mL) relationship gave
y = 0.8427x + 0.01490 (r = 0.9983).
2.6. Application of the method to chitosan samples
The proposed method was applied to chitosan samples from different sources. HCl hydrolysis was also performed for comparison.
The content of different samples was 87.896.2%, signicantly
higher (p < 0.05) than that with HCl hydrolysis (Table 3).
3. Conclusion
In conclusion, hydrolysis of chitosan with a mixed acid solution
composed of HClH3PO4 (75:25 in molar ratio) could avoid the
degradation of GlcN and improve the recovery of chitosan. Hydrolysis of chitosan with HClH3PO4 (4.5:1.5 M) for 24 h at 110 C
proved optimum recovery of chitosan for 96.998.6%, signicantly
higher than HCl hydrolysis. After neutralization of the hydrolyzation, the derivation should be performed with [FMOC-Cl]/[GlcN]
for 53:1, due to its high ion strength. The modied method was
accurate and chitosan samples can be quantitatively determined.
4. Experimental

Glucosamine in the hydrolysate was determined with FMOC-Cl

derivation and HPLC determination as Zhu18 and Zhou36 reported
with some modication.
For chitosan samples, the hydrolysate (2.0 mL) was transferred
to a volumetric ask and mixed with 1.0 mL internal standard (IS,
0.8 mg/mL a-aminobutyric acid solution). The mixture was neutralized with 2 M NaOH in an ice bath, mixed with 25 mL acetonitrile, and then diluted to 50 mL with water. The solution was
ltered with 0.45 lm membrane. Then 200 ll of the ltrate was
mixed with 200 ll 0.25 M borate buffer (pH 8.0) to adjust pH of
the solution. Glucosamine and IS was then derived with the addition of 100 ll FMOC-Cl solution (20 mM, in acetonitrile). The mixture was allowed to react at room temperature for 5 min. After the
derivation was complete, the reaction was stopped with the addition of 200 ll acetonitrile-acetate acid solution (8:2, v/v) to remove
excess FMOC-Cl.
For GlcN reference, 2.0 mL GlcN solution (1 mg/mL) was mixed
with 1.0 mL IS solution and 25 mL acetonitrile. The solution was
then diluted to 50 mL with water and treated in the same manner
as hydrolyzed chitosan solutions.
4.4. HPLC determination
Separation and detection of the FMOC-Cl derivatives were performed on a Waters liquid chromatography system consisting of a
1525 binary pump, a 717 plus autosampler, and a 2487 dual absorbance detector. Data were collected and processed using the
Waters Breeze software. Reversed-phase separation was carried
out on a Symmetry C18 column (4.6  150 mm, 5 lm, Waters
Co.). A solution of NH4Ac buffer (10 mM, pH 5.0) and acetonitrile
(80:20) was used as mobile phase A and acetonitrile was used as
mobile phase B, with gradient program shown in Table 4. The temperature of the column was maintained at 35 C and detection was
performed at 262 nm.
The IS was eluted at 9.4 min, and GlcN showed two peaks at 5.5
and 6.1 min after derivation, presumably represent the anomers of
FMOC-GlcN.37 GlcN concentration was quantied with the area ratio of GlcN at 6.1 min to IS. Chitosan content was calculated using
Eq. 1.15,18

4.1. Chemicals and materials

Glucosamine hydrochloride (>99.0%) and a-aminobutyric acid
(>98.0%) were purchased from Sinopharm Chemical Reagent
(Shanghai, China). 9-Fluorenylmethoxycarbonyl chloride (FMOCCl, >99.0%) was purchased from Sigma (St. Louis, United States).
Chitosan samples were from Jiangsu Shuanglin Marin Biological
Pharmaceutical Ltd. (Nantong, China, MW 440 kDa, DD 82.4%),
Golden-Shell Biochemical Co., Ltd (Yuhuan, China, MW 573 kDa,
DD 76.5%), and Sigma (St. Louis, United States, MW 607 kDa, DD

Table 4
HPLC gradient program for the determination of GlcN
Time (min)

A (%)

B (%)

Flow rate (mL/min)

0
10
11
15
16
20

80
50
20
20
80
80

20
50
80
80
20
20

1.2
1.2
1.2
1.2
1.2
1.2

54

Content %

B. Li et al. / Carbohydrate Research 366 (2013) 5054

Gsam  F  0:899
 100%
w  1  cwater

In Eq. 1, w represents the amount of chitosan weighed, and cwater

represents the water content of chitosan. Gsam represents the
amount of GlcN in hydrolysate, calculated based on the area ratio
of chitosan sample and GlcN reference. 0.899 represents the factor
of GlcN to chitosan, which was the MW of GlcN unit in chitosan
to that of GlcN. The deviation of the factor was lower than 0.2%
for chitosan with MW higher than 9.0 kDa (DP> 56) and 0.1% for
chitosan with MW higher than 18.1 kDa (DP> 112). F represents
the factor of GlcNAc and was calculated using the formula
F = (203.1  DD  42)/161.1, where DD is the degree of deacetylation of chitosan. In the present study, MW and DD of chitosan were
440607 kDa and 76.582.4%, respectively.
The water content and DD of chitosan were determined using
Karl Fischer method and rst derivative UV-spectrophotometry,38,39 respectively.
4.5. The degradation of chitosan in hydrolysis
Similar to previous report,29 brown products were formed in
the hydrolysis of chitosan. The UVvis spectrum of the hydrolysates was determined at 200800 nm using Shimadzu UV 2450.
The maximum absorbance was shown at about 280 nm and the
absorbance at 400800 nm was fairly low (<0.05) for most samples. Therefore, A280 was used to evaluate the degradation of
chitosan.
4.6. Statistical analysis
All experiments were carried out in duplicate or triplicate, and
average values or means standard deviations are reported. Means
of the main effects were separated by Duncans test with the
SPSS11.0 software package.
Acknowledgments
This research was supported by the National High Technology
Research and Development Program (863Program) of China
(2007AA091603), National Natural Science Foundation of China
(81202491), Fundamental Research Funds for the Central Universities (JKQ2011024), State Key Laboratory of food science and Technology Program (SKLF-MB-200805), National Twelfth Five-Year
Plan for Science & Technology Support (2011BAD23B04), and the
Fund Project for Transformation of Scientic and Technological
Achievements of Jiangsu Province (BA2009082).

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