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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
b
School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
c
School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu, China

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

Article history: Acid hydrolysis and subsequent quantication of glucosamine (GlcN) are widely used for chitosan quan-
Received 6 August 2012 tication. Degradation of GlcN during chitosan hydrolysis was the main reason for the decrease of recov-
Received in revised form 6 November 2012 ery, which made the method improper for the quantication of chitosan. Ten milligram of chitosan
Accepted 12 November 2012
hydrolyzed with 10 mL mixed acid solution of HClH3PO4 (75:25 in molar ratio) showed the highest
Available online 21 November 2012
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
Keywords:
and derived with 9-uorenylmethoxycarbonyl chloride (FMOC-Cl) before HPLC quantication. The opti-
Chitosan
Hydrolysis
mum ratio of FMOC-Cl:GlcN was 53:1, with excess FMOC-Cl induced by the high ionic strength of the
Glucosamine solution. This quantication procedure was then validated and proved to be specic, with good linearity,
Browning accuracy, and precision, making it well-suited for the determination of chitosan.
9-Fluorenylmethoxycarbonyl chloride 2012 Elsevier Ltd. All rights reserved.
Derivation

1. Introduction ports, the acid concentration and hydrolysis time were carefully
optimized to completely hydrolyze chitosan and to minimize the
Chitosan, the N-deacetylated derivate of chitin, is a heteropoly- degradation of GlcN.
saccharide consisting of linear b-1,4-linked glucosamine (GlcN) In the present study, hydrolysis of chitosan with HClH3PO4
and N-acetyl-glucosamine (GlcNAc) units.1 Both chitin and chito- was studied and the optimum hydrolysis condition with minimum
san are widely used in food,2,3 biotechnology, material science,4 degradation of GlcN and highest recovery of chitosan was studied
pharmaceutics,57 and gene therapy.8 and compared with HCl hydrolysis. The derivation of the hydroly-
Several colorimetric assays, using ninhydrin,9 o-phthalalde- sate was also optimized. A modied acid hydrolysis and HPLC
hyde10 (OPA), or Cibacron Brilliant Red 3B-A,11,12 have been used method for the determination of chitosan was then established
to the quantify chitosan. However, the response during these reac- and validated.
tions depends strictly upon the degree of deacetylation (DD) of
chitosan13 which limited their application. 2. Results and discussion
Many other quantication methods entail a total hydrolysis of
chitosan into GlcN followed by the subsequent characterization 2.1. Inuence of acid on the recovery of chitosan
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 Chitosan (10 mg) was hydrolyzed with 10 mL mixed acid com-
hydrolysis of the glycosidic linkage (depolymerization) and the posed of HCl and H3PO4 containing 6 M H+. One mol of H3PO4 was
N-acetyl linkage (deacetylation) of chitosan.27 In most of these re- estimated to contain about 1 mol H+, because the pKa1 and pKa2
ports (except Ref. 17), GlcN was spiked to chitosan samples to eval- values of phosphoric acid were 2.15 and 7.09, respectively.30 After
uate the recovery. However, the recovery rate was generally about a 24 h hydrolysis at 110 C, GlcN in the hydrolysates were derived
90% (Table 1), which was improper for the determination of chito- and determined. The recovery of chitosan was evaluated by the
san. The degradation of GlcN in the acid hydrolysis was responsible amount of GlcN released after hydrolysis versus the amount of
for this decrease of recovery. Millard reaction,28,29 and deamina- chitosan.
tion16 were estimated as main degradation pathway. In most re- As shown in Figure 1, the recovery of chitosan varied with the
composition of acid. Chitosan hydrolyzed with HClH3PO4 (75:25
Corresponding author. Tel.: +86 510 85919121; fax: +86 510 85329057. in molar ratio) showed highest recovery, signicantly higher
E-mail address: xiaws@jiangnan.edu.cn (W. Xia). (p < 0.05) than that with HCl. Meanwhile, the recovery

0008-6215/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.11.005
B. Li et al. / Carbohydrate Research 366 (2013) 5054 51

Table 1
Application of acid hydrolysis in the quantication of chitosan

Ref. Acid and concn Temp (C) Time (h) Recovery (%)
15
6 M HCl 100 48 86
16
10 M HCl 105 6 99103
17
6 M HCl 100 10 100.52a
18
8 M HCl 110 4 90.198.7
19
6 M HCl 115 1 96.5
20
72% (v/v) H2SO4, 90 min at room temperature, then 121 C for 1 h with diluted H2SO4 92.4
21
6 M HCl 121 7 92.0
22
6 M HCl 100 4 93
23
H2SO4 72% (v/v) 3 h room temp., then H2SO4 2 mol L1, 100 C, 4 h 80
24
10 M HCl 140 1 135.2
25
6 M HCl 100 7 86
26
4 M HCl 100 3 106
a
Chitosan was used as standard spiked to matrix.

dramatically decreased with higher H3PO4 ratio. To further prove hydrolyze both glycoside and the acetyl groups,27,32 HCl was essen-
the inuence of acid, GlcN, the hydrolyzed product of chitosan, tial to ensure the complete hydrolysis of chitosan to GlcN.
was also hydrolyzed with HClH3PO4. Its recovery showed similar
trends as chitosan (Fig. 1). As HClH3PO4 (75:25 in molar ratio) 2.2. Inuence of acid concentration on the recovery of chitosan
showed highest recovery for chitosan and GlcN, it was used in
further hydrolysis of chitosan. Previous reports proved that the acid concentration could inu-
The degradation of chitosan in acid hydrolysis was studied to ence the hydrolysis of chitosan16,18 and the degradation of GlcN.19
further reveal the inuence of acid composition on the recovery In the present study, the concentration of HClH3PO4 was opti-
of chitosan. Chitosan hydrolyzed with HClH3PO4 (75:25 in molar mized with the evaluation of chitosan recovery. Chitosan was
ratio) showed least absorbance at 280 nm, and the absorbance sig- hydrolyzed using a mixed acid solution of HClH3PO4 (75:25 in
nicantly increased with higher H3PO4 ratio, as shown in Figure 1. molar ratio) with different [H+] at 110 C for 24 h. The relative yield
This proved that the degradation varied with the ratio of HCl of GlcN was studied. As shown in Figure 2, hydrolysis of chitosan
H3PO4 and thus affected the recovery of chitosan. with 6 M [H+] showed optimum recovery for 98.8%. Lower [H+]
In the hydrolysis of chitin, N-acetyl-D-glucosamine-1-phos- showed 64.6% yields of GlcN because of the incomplete hydrolysis
phate was formed in H3PO4 hydrolysis31 while glucofuranosyl of chitosan; and 90.3% for 9 M [H+] due to the degradation of GlcN.
oxazolinium was formed in HCl hydrolysis.32 This variation of Therefore, the mixed acid solution with 6 M [H+], HClH3PO4
intermediates with the acid used in hydrolysis was responsible (4.5:1.5 M) in other words, was used in further research.
for the change of degradation and recovery of chitosan in different In comparison, chitosan was hydrolyzed with different concen-
HClH3PO4 ratios. As the degradation products were believed to be trations of HCl. Highest recovery of chitosan was also found to be at
too complex,28 the degradation products and pathway of degrada- 6 M. Meanwhile, chitosan hydrolyzed with 6 M HCl and 9 M HCl
tion in HClH3PO4 were not further studied in the present work. showed lower recovery than hydrolyzed with HClH3PO4 (75:25
The decrease of recovery with high H3PO4 ratio was more severe in molar ratio) having same [H+]. This further proved that HCl
for chitosan than GlcN, because the deacetylation of GlcNAc in H3PO4 (75:25 in molar ratio) could inhibit the degradation of GlcN
H3PO4 was limited.31,33 Considering that HCl was proved able to and improve the recovery of chitosan.

120 Recovery of Chitosan 2.5


Recovery of GlcN
A280 of Chitosan
100
A280 of GlcN 2.0

80
Recovery (%)

1.5
A 280

60

1.0
40

0.5
20

0 0.0
100:0 75:25 50:50 25:75 0:100
HCl-H3PO4

Figure 1. Recovery of chitosan and GlcN in the mixture of 6 M HCl and 6 M H3PO4 (n = 3).
52 B. Li et al. / Carbohydrate Research 366 (2013) 5054

HCl-H3PO4(75:25) 1.2
derived in water
100 HCl derived in matrix
1.0

80
0.8
Recovery (%)

AGlcN/AIS
60
0.6

40 0.4

20 0.2

0 0.0
4 6 8 10 0 50 100 150 200 250 300
Acid Concentration (mol/L) FMOC-Cl (l)

Figure 2. Inuence of acid concentration ([H]+) on the recovery of chitosan. Figure 4. Inuence of FMOC-Cl volume to the derivation of GlcN (n = 3).

Recovery of GlcN
present study, a-aminobutyric acid was used as internal standard
100
(IS), which was derived with FMOC-Cl along with the derivation
of GlcN.
80 As H3PO4 in the hydrolysate could not be vaccum dried like HCl
in previous reports,24 the hydrolysate was neutralized before der-
Recovery (%)

ivation. This neutralization dramatically increased the ion strength


60
of the matrix. To study the derivation of GlcN in matrix, the GlcN
solution was diluted with matrix and water, respectively. The solu-
40 tions 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
20
FMOC-Cl from 25 to 300 ll. But for GlcN solution diluted with ma-
trix, the area of GlcN enhanced with the increasing of FMOC-Cl vol-
0 ume up to 100 ll and then decreased slightly. At the optimum
0 10 20 30 40 FMOC-Cl level, the molar ratio of FMOC-Cl and GlcN was approxi-
Time (h) mately 53:1, much higher than previous reports.18,25 The deriva-
tion was performed with 100 ll FMOC-Cl in further research.
Figure 3. Inuence of hydrolysis time on the recovery of chitosan (n = 2).
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
2.3. Inuence of hydrolysis time on the recovery of chitosan added before derivation, and no signicant decrease of GlcN area
with NaCl added after derivation (data not shown). Therefore, the
The hydrolysis time was also studied by testing the yield of derivation behavior was changed by the ion strength of the matrix.
GlcN with different hydrolysis times on chitosan samples. The To remove the excess derivation reagent and terminate the der-
hydrolysis reactions were performed at 110 C with HClH3PO4 ivation reaction, acetonitrile- acetate acid solution (8:2, v/v) was
(4.5:1.5 M). added to convert FMOC-Cl into FMOC-OH.34,35
The results for chitosan hydrolyzed at different time intervals
are presented in Figure 3. The relative yield of GlcN for chitosan 2.5. Method validation
samples treated with HClH3PO4 increased with time up to 24 h
and slightly decreased thereafter. The browning of the solution in- The recovery of chitosan was checked by applying the standard
creased steadily for hydrolysis time longer than 24 h. addition technique and glucosamine hydrochloride was used as
These results showed that chitosan could be completely hydro- standard. Five milligrams of chitosan sample (Sigma, St. Louis, Uni-
lyzed to GlcN after 24 h of the reaction. Glucosamine slightly de- ted States, MW 607 kDa, DD 80.0%) was spiked with different
graded with extended hydrolysis time, with the recovery of GlcN amounts of GlcN solution. The mixed samples were then hydro-
decreasing from 97.7% at 24 h to 90.0% at 36 h. Therefore, the lyzed and determined as described in the experimental part. The
hydrolysis time was set to 24 hours to quantify the chitosan recovery using 6 M HCl hydrolysis was also studied. As shown in
samples. Table 2, mean recoveries of HClH3PO4 hydrolysis was 96.9
98.6% for each level (n = 3), signicantly higher (p < 0.05) than
2.4. Optimization of FMOC-Cl derivation of GlcN the recovery using 6 M HCl hydrolysis.
The precision was evaluated by assaying chitosan samples (Sig-
Glucosamine does not contain a chromophore absorbing in the ma, St. Louis, United States, MW 607 kDa, DD 80.0%) at 80, 100, and
wavelength range useful for liquid chromatography with UV detec- 120% levels (8, 10 and 12 mg, respectively). Table 2 summarized
tion.17,18 As a derivatization reagent, FMOC-Cl reacts with the pri- the within run and between run precision. In this assay, the stan-
mary amine of GlcN, which signicantly improved the UV dard division of within run and between run precision ranged from
absorbance and the sensitivity in quantication of GlcN. In the 2.9% to 3.8% and from 3.0% to 3.9% for each level, respectively.
B. Li et al. / Carbohydrate Research 366 (2013) 5054 53

Table 2 80.0%). Water was prepared using Milli-Q system. Other chemical
Recovery and precision of the HClH3PO4 hydrolysis and FMOC-Cl derivation HPLC reagents were analytical grade.
method (n = 3)

GlcN added Recovery (% SD) Recovery (% SD) HCl 4.2. Hydrolysis of chitosan
(mg) hydrolysis
3.03 97.5 3.5 86.2 3.8 Chitosan powder (10 mg) was accurately weighed and trans-
5.05 98.6 4.2 85.3 4.3 ferred to a hydrolysis tube. Five milliliters of water and 5 mL of
7.07 96.9 3.5 83.9 5.2
Average 97.7 3.3 85.1 4.2
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 con-
Level of Within run precision Between run precision
centrations of HCl and H3PO4 were 4.5 and 1.5 M, respectively. The
chitosan (% SD) (% SD)
tube was vacuum-treated, ushed with N2 and then fuse-sealed to
80% 95.1 3.7 96.3 3.0
avoid the inuence of oxygen. Acid hydrolysis was performed at
100% 95.5 3.8 95.9 3.4
120% 96.7 2.9 97.1 3.9 110 C for 24 h to generate GlcN. The tube was then cooled and
Average 95.7 3.4 96.4 3.3 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) Glucosamine in the hydrolysate was determined with FMOC-Cl
Chitosan sample MW DD Content (% SD) derivation and HPLC determination as Zhu18 and Zhou36 reported
(kDa) (%)
HClH3PO4 HCl
with some modication.
hydrolysis hydrolysis For chitosan samples, the hydrolysate (2.0 mL) was transferred
to a volumetric ask and mixed with 1.0 mL internal standard (IS,
Chitosan (Sigma) 607 80.0 96.2 2.7 88.7 3.8
Chitosan 440 82.4 95.2 3.0 86.8 3.7 0.8 mg/mL a-aminobutyric acid solution). The mixture was neu-
(Nantong) tralized with 2 M NaOH in an ice bath, mixed with 25 mL acetoni-
Chitosan 573 76.5 87.8 2.8 79.5 3.8 trile, and then diluted to 50 mL with water. The solution was
(Zhejiang)
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 addi-
tion of 100 ll FMOC-Cl solution (20 mM, in acetonitrile). The mix-
Glucosamine reference solution at a known concentration (20 ture was allowed to react at room temperature for 5 min. After the
2 mg/mL) was assayed as described above. A typical least squares derivation was complete, the reaction was stopped with the addi-
linear regression evaluation of the peak area ratio of GlcN and IS tion of 200 ll acetonitrile-acetate acid solution (8:2, v/v) to remove
(y) versus concentration of GlcN (x, mg/mL) relationship gave excess FMOC-Cl.
y = 0.8427x + 0.01490 (r = 0.9983). 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
2.6. Application of the method to chitosan samples then diluted to 50 mL with water and treated in the same manner
as hydrolyzed chitosan solutions.
The proposed method was applied to chitosan samples from dif-
ferent sources. HCl hydrolysis was also performed for comparison. 4.4. HPLC determination
The content of different samples was 87.896.2%, signicantly
higher (p < 0.05) than that with HCl hydrolysis (Table 3). Separation and detection of the FMOC-Cl derivatives were per-
formed on a Waters liquid chromatography system consisting of a
3. Conclusion 1525 binary pump, a 717 plus autosampler, and a 2487 dual absor-
bance detector. Data were collected and processed using the
In conclusion, hydrolysis of chitosan with a mixed acid solution Waters Breeze software. Reversed-phase separation was carried
composed of HClH3PO4 (75:25 in molar ratio) could avoid the out on a Symmetry C18 column (4.6  150 mm, 5 lm, Waters
degradation of GlcN and improve the recovery of chitosan. Hydro- Co.). A solution of NH4Ac buffer (10 mM, pH 5.0) and acetonitrile
lysis of chitosan with HClH3PO4 (4.5:1.5 M) for 24 h at 110 C (80:20) was used as mobile phase A and acetonitrile was used as
proved optimum recovery of chitosan for 96.998.6%, signicantly mobile phase B, with gradient program shown in Table 4. The tem-
higher than HCl hydrolysis. After neutralization of the hydrolyza- perature of the column was maintained at 35 C and detection was
tion, the derivation should be performed with [FMOC-Cl]/[GlcN] performed at 262 nm.
for 53:1, due to its high ion strength. The modied method was The IS was eluted at 9.4 min, and GlcN showed two peaks at 5.5
accurate and chitosan samples can be quantitatively determined. and 6.1 min after derivation, presumably represent the anomers of
FMOC-GlcN.37 GlcN concentration was quantied with the area ra-
4. Experimental tio of GlcN at 6.1 min to IS. Chitosan content was calculated using
Eq. 1.15,18
4.1. Chemicals and materials
Table 4
Glucosamine hydrochloride (>99.0%) and a-aminobutyric acid HPLC gradient program for the determination of GlcN

(>98.0%) were purchased from Sinopharm Chemical Reagent Time (min) A (%) B (%) Flow rate (mL/min)
(Shanghai, China). 9-Fluorenylmethoxycarbonyl chloride (FMOC- 0 80 20 1.2
Cl, >99.0%) was purchased from Sigma (St. Louis, United States). 10 50 50 1.2
Chitosan samples were from Jiangsu Shuanglin Marin Biological 11 20 80 1.2
Pharmaceutical Ltd. (Nantong, China, MW 440 kDa, DD 82.4%), 15 20 80 1.2
16 80 20 1.2
Golden-Shell Biochemical Co., Ltd (Yuhuan, China, MW 573 kDa,
20 80 20 1.2
DD 76.5%), and Sigma (St. Louis, United States, MW 607 kDa, DD
54 B. Li et al. / Carbohydrate Research 366 (2013) 5054

Gsam  F  0:899 References


Content %  100% 1
w  1  cwater
1. Franca, E. F.; Lins, R. D.; Freitas, L. C. G.; Straatsm, T. P. J. Chem. Theory Comput.
In Eq. 1, w represents the amount of chitosan weighed, and cwater 2008, 4, 21412149.
represents the water content of chitosan. Gsam represents the 2. Prashanth, K. V. H.; Tharanathan, R. N. Trends Food Sci. Technol. 2007, 18, 117
131.
amount of GlcN in hydrolysate, calculated based on the area ratio 3. No, H. K.; Meyers, S. P.; Prinyawiwatkul, W.; Xu, Z. J. Food Sci. 2007, 72, 87100.
of chitosan sample and GlcN reference. 0.899 represents the factor 4. Muzzarelli, R. A. A. Carbohydr. Polym. 2009, 76, 167182.
of GlcN to chitosan, which was the MW of GlcN unit in chitosan 5. Prabaharan, M. J. Biomater. Appl. 2010, 25, 319335.
6. Jayakumar, R.; Menon, D.; Manzoor, K.; Nair, S. V.; Tamura, H. Carbohydr. Polym.
to that of GlcN. The deviation of the factor was lower than 0.2%
2010, 82, 227232.
for chitosan with MW higher than 9.0 kDa (DP> 56) and 0.1% for 7. Nair, R.; Reddy, B. H.; Kumar, C. K. A.; Kumar, K. J. J. Pharm. Sci. Res. 2009, 2, 1
chitosan with MW higher than 18.1 kDa (DP> 112). F represents 12.
8. Jayakumar, R.; Chennazhi, K. P.; Muzzarelli, R. A. A.; Tamura, H.; Nair, S. V.;
the factor of GlcNAc and was calculated using the formula
Selvamurugan, N. Carbohydr. Polym. 2010, 79, 18.
F = (203.1  DD  42)/161.1, where DD is the degree of deacetyla- 9. Leane, M. M.; Nankervis, R.; Smith, A.; Illum, L. Int. J. Pharm. 2004, 271, 241
tion of chitosan. In the present study, MW and DD of chitosan were 249.
440607 kDa and 76.582.4%, respectively. 10. Larionova, N. I.; Zubaerova, D. K.; Guranda, D. T.; Penchyonkin, M. A.;
Balabushevich, N. G. Carbohydr. Polym. 2009, 75, 724727.
The water content and DD of chitosan were determined using 11. Muzzarelli, R. A. A. Anal. Biochem. 1998, 260, 255257.
Karl Fischer method and rst derivative UV-spectrophotome- 12. Wischke, C.; Borchert, H. H. Carbohydr. Res. 2006, 341, 29782979.
try,38,39 respectively. 13. Miralles, B.; Mengibar, M.; Harris, R.; Heras, A. Food Chem. 2011, 126, 1836
1839.
14. Prochazkova, S.; Varum, K. M.; stgaard, K. Carbohydr. Polym. 1999, 38, 115
4.5. The degradation of chitosan in hydrolysis 122.
15. Eikenes, M.; Fongen, M.; Roed, L.; Stenstrm, Y. Carbohydr. Polym. 2005, 61, 29
38.
Similar to previous report,29 brown products were formed in 16. Yan, X.; Evenocheck, H. M. Carbohydr. Polym. 2012, 87, 17741778.
the hydrolysis of chitosan. The UVvis spectrum of the hydroly- 17. El-Saharty, Y. S.; Bary, A. A. Anal. Chim. Acta 2002, 462, 125131.
sates was determined at 200800 nm using Shimadzu UV 2450. 18. Zhu, X. L.; Cai, J. B.; Yang, J. Carbohydr. Res. 2005, 340, 17321738.
19. Jang, J. H.; Hia, H. C.; Ike, M.; Inoue, C.; Fujita, M.; Yoshida, T. Biotechnol. Lett.
The maximum absorbance was shown at about 280 nm and the 2005, 27, 1318.
absorbance at 400800 nm was fairly low (<0.05) for most sam- 20. Zamani, A.; Jeihanipour, A.; Edebo, L.; Niklasson, C.; Taherzadeh, M. J. J. Agric.
ples. Therefore, A280 was used to evaluate the degradation of Food Chem. 2008, 56, 83148318.
21. Mills, P.; Rotter, R. G.; Marquardt, R. R. Can. J. Anim. Sci. 1989, 69, 11051106.
chitosan.
22. Chen, G. C.; Johnson, B. R. Appl. Environ. Microbiol. 1983, 46, 1316.
23. Dallies, N.; Francois, J. J.; Paquet, V. Yeast 1998, 14, 12971306.
4.6. Statistical analysis 24. Chen, W. L.; Chiou, R. Y. Y. J. Agric. Food Chem. 1999, 47, 19992004.
25. Ekblad, A.; Nasholm, T. Plant Soil 1996, 178, 2935.
26. Pearson, C. H. Biochemistry 1963, 88, 540544.
All experiments were carried out in duplicate or triplicate, and 27. Einbu, A.; Varum, K. M. Biomacromolecules 2008, 9, 18701875.
average values or means standard deviations are reported. Means 28. Einbu, A. Characterisation of Chitin and a Study of its Acid-Catalysed Hydrolysis;
of the main effects were separated by Duncans test with the Norwegian University of Science and Technology: Trondheim, Norway, 2007. p.
29.
SPSS11.0 software package. 29. Knill, C. J.; Kennedy, J. F.; Mistry, J.; Miraftab, M.; Smart, G.; Groocock, M. R.;
Williams, H. J. J. Chem. Technol. Biotechnol. 2005, 80, 12911296.
Acknowledgments 30. ONeil, M. J.; Heckelman, P. E.; Koch, C. B.; Roman, K. J.; Kenny, C. M.; DArecca,
M. A. The Merck Index, 14th ed.; Merck: Whitehouse Station, United States,
2006. p. 1266.
This research was supported by the National High Technology 31. Vincendon, M. Carbohydr. Polym. 1997, 32, 233237.
Research and Development Program (863Program) of China 32. Einbu, A.; Vrum, K. M. Biomacromolecules 2007, 8, 309314.
33. Wu, T.; Zivanovic, S. Carbohydr. Polym. 2008, 73, 248253.
(2007AA091603), National Natural Science Foundation of China
34. Haynes, P. A.; Sheumack, D.; Greig, L. G.; Kibby, J.; Redmond, J. W. J. Chromatogr.
(81202491), Fundamental Research Funds for the Central Universi- 1991, 588, 107114.
ties (JKQ2011024), State Key Laboratory of food science and Tech- 35. Haynes, P. A.; Sheumack, D.; Kibby, J.; Redmond, J. W. J. Chromatogr. 1991, 540,
177185.
nology Program (SKLF-MB-200805), National Twelfth Five-Year
36. Zhou, J. Z. Q.; Waszkuc, T.; Mohammed, F. J. AOAC Int. 2005, 88, 10481058.
Plan for Science & Technology Support (2011BAD23B04), and the 37. Ibrahim, A.; Jamali, F. J. Pharm. Pharm. Sci. 2010, 13, 128135.
Fund Project for Transformation of Scientic and Technological 38. Muzzarelli, R. A. A. Carbohydr. Polym. 1985, 5, 461472.
Achievements of Jiangsu Province (BA2009082). 39. Tan, S. C.; Khor, E.; Tan, T. K.; Wong, S. M. Talanta 1988, 45, 713719.