Академический Документы
Профессиональный Документы
Культура Документы
1
1/3I
CH
3
1/6I
H
2
H
6
n
100,
S.C. Tan et al. / Talanta 45 (1998) 713719 715
Table 1
Degree of deacetylation of commercial chitosan measured by rst derivative spectrophotometry, nuclear magnetic resonance, linear
potentiometric titration and ninhydrin test
Fluka Kasei Chitosan sample Puried Kasei
82.6690.74 85.8290.51 First derivative uv-spectrophotometry 98.0890.03 Degree of Deacetylation (%)
82.20* N/A Nuclear magnetic resonance N/A
89.2590.38 73.2990.62 75.0090.41 Linear potentiometric titration
44.9291.75 55.4192.91 Ninhydrin test 51.0191.88
* No replicate was done.
where I
CH
3
is the integral intensity of CH
3
and
I
H
2
H
6
is the summation integral intensities of H
2
,
H
3
, H
4
, H
5
, H
6
and H
6%
.
2.4. Linear potentiometric titration [15]
The determination of DD by LPT was a mod-
ication of the method of Ke and Chen. Chitosan
(0.200.25 g) was dissolved in 20 ml of standard-
ised 0.10 N HCl (to give a non-viscous solution)
and diluted with 10 ml of distilled water. The pH
of the solution was adjusted to :2 with standard
0.01 M NaOH and taken as the start point.
Under continuous stirring, 1 ml of standard
NaOH (0.5 ml for puried chitosan) was added,
allowed to equilibrate and the pH recorded. This
sequence was repeated until the pH reached a
value of 3. A value of f(x) of the corresponding
volume of NaOH added was calculated using the
following formula:
f(x) =
V
0
+V
N
B
([H
+
] [OH
]),
where V
0
is the volume of chitosan solution (ml);
V is the volume of NaOH added (ml); N
B
is the
concentration of NaOH (N); [H
+
] is the concen-
tration of H
+
(M); [OH
] is the concentration of
OH
] concentrations in the
analyte as the pH measured directly affects the
DD determined. The amine and thiol groups in
amino acids will affect the [H
+
] concentration of
the analyte, and therefore, the presence of protein
contaminant can also be a major interference.
1DUVS is very sensitive for GlcNAc detection,
the detectable concentration of GlcNAc in 0.01 M
acetic acid solution was found to be as low as
0.0005 mg ml
1
. The concentration of the chi-
tosan sample solution to be used for analysis was
standardised as 0.1000 mg chitosan per ml of
acetic acid solution. This permitted a wider range
of DD (050%) to be ascertained based on the
calibration plot ranging from 0.0050 to 0.0500 mg
GlcNAc per ml acetic acid solution. In this range,
the plot was linear and obeyed Beers law. This is
contrary to the concentration of 1 mg chitosan
per ml of acetic acid solution used by Muzzarelli
and Rocchetti [13]. Using this concentration, only
DD in the range 05% can be determined, which
is quite narrow. The dip of the 1DUVS spectrum
of chitosan solution is shifted to the right with
increasing chitosan concentration (Fig. 1). If the
concentration of chitosan solution is too high, the
dip of its rst derivative spectrum may be shifted
beyond the valid range. Therefore, high concen-
trations of chitosan solution for analyses are to be
avoided as it would require more sample which
may add more experimental errors whenever dilu-
tions are made. Chitosan solutions of too low
concentrations are also not advisable as this may
also incur larger experimental errors.
The reference curve for the correction of the
effect of GlcN on the H value is shown in Fig. 2.
Muzzarelli and Rocchetti showed that the pres-
ence of GlcN contributes to the H value, when the
GlcNAc to GlcN ratio is below 0.11 (GlcNAc is
less than 10%). However, our results showed that
even when the GlcNAc is 20%, contributions of
the GlcN to the H value is also present. The
discrepancy between our data and that of Muz-
zarelli and Rocchetti may lie in the sensitivity of
the UV-spectrophotometer used. Therefore, our
current data suggests that corrections up to 20%
are necessary.
Based on the results we obtained, the DD of
chitosan determined, varied with the methods
used. Each of these methods has its own advan-
tages and disadvantages. Therefore, it is difcult
to tell which method has given us the most accu-
rate results. However, all of them did show the
relative DD between different chitosan samples.
Up to date there is still no standard method for
Fig. 2. Correction curve for GlcNac determination.
S.C. Tan et al. / Talanta 45 (1998) 713719 719
the determination of DD of chitosan yet. We
advocate 1DUVS to be used as the standard
method for routine determination of the DD of
chitosan due to its high sensitivity, minimal inter-
ference of results from contaminants and easy to
be performed.
Acknowledgements
The authors acknowledge the National Univer-
sity of Singapore for nancial sponsorship of this
work (RP 920633) and the award of a research
scholarship for S.C. Tan.
References
[1] P.A. Sandford, Chitosan: commercial uses and potential
application, in G. Skjak, T. Anthonsen, P. Sandford
(Eds.), Chitin and Chitosan: Sources, Chemistry, Bio-
chemistry, Physical Properties and Applications, Ele-
sevier, New York, 1988, pp. 5169.
[2] Chitin and Chitosan: Specialty Polymers for Food,
Medicine and Industry, Technical Insights, Engelwood,
NJ.
[3] M.G. Peters, Applications and environmental aspects of
chitin and chitosan, J. Mat. Sci. Pure Appl. Chem. A32
(1995) 629640.
[4] S. Mima, M. Miya, R. Iwamoto, S. Yoshikawa, Highly
deacetylated chitosan and its properties, J. Appl. Polym.
Sci. 28 (1983) 19091917.
[5] H. Miyoshi, K. Shimahara, K. Watanabe, K. Onodera,
Characterisation of some fungal chitosans, Biosci. Bio-
tech. Biochem. 56 (1992) 19011905.
[6] K. Nishimura, S. Nishimura, N. Nishi, I. Saiki, S.
Tokura, I. Azuma, Immunological activity of chitin and
its derivatives, Vaccine 2 (1984) 9399.
[7] K. Nishimura, S. Nishimura, N. Nishi, F. Numata, Y.
Tone, S. Tokura, I. Azuma, Adjuvant activity of chitin
derivatives in mice and guinea-pigs, Vaccine 3 (1985)
379384.
[8] A. Baxter, M. Dillon, K.D.A. Taylor, G.A.F. Roberts,
Improved method for IR determination of the degree of
N-acetylation of chitosan, Int. J. Biol. Macromol. 14
(1992) 166169.
[9] A. Domard, U. Rinaudo, Preparation and characterisa-
tion of fully deacetylated chitosan, Int. J. Biol. Macro-
mol. 5 (1983) 4952.
[10] T. Sannan, K. Kurita, K. Ogura, Y. Iwakura, Studies on
chitin: 7. IR spectroscopy determination of degree of
deacetylation, Polym. 19 (1978) 458459.
[11] T.D. Rathke, S.M. Hudson, Determination of the degree
of N-deacetylation in chitin and chitosan as well as their
monomer sugar ratios by near infrared spectroscopy, J.
Polym. Sci. 31 (1993) 749753.
[12] S. Aiba, Studies on chitosan: 1. Determination of the
degree of N-acetylation of chitosan by ultraviolet spec-
trophotometry and gel permeation chromatography, Int.
J. Biol. Macromol. 8 (1986) 173176.
[13] R.A.A. Muzzarelli, R. Rochetti, Determination of the
degree of acetylation of chitosan by rst derivative ultra-
violet spectrophotometry, Carbohydr. Polym. 5 (1985)
461472.
[14] H. Terayama, Method of colloid titration (a new titration
between polymer ions), J. Polym. Sci. 8 (1952) 243253.
[15] H. Ke, Q. Chen, Potentiometric titration of chitosan by
linear method, Huaxue Tongbao 10 (1990) 4446.
[16] F. Nanjo, R. Katsumi, K. Sakai, Enzymatic method for
determination of the deacetylation of chitosan, Anal.
Biochem. 193 (1991) 164167.
[17] A. Hiral, H. Odani, A. Nakajima, Determination of
degree of deacetylation of chitosan by
1
H NMR spec-
troscopy, Polym. Bull. 26 (1991) 8794.
[18] E. Curotto, F. Aros, Quantitative determination of chi-
tosan and the percentage of free amino groups, Anal.
Biochem. 211 (1993) 240241.
[19] A. Domard, Determination of N-acetyl content in chi-
tosan samples by cd measurements, Int. J. Biol. Macro-
mol. 9 (1987) 333336.
.