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Abstract
A simple sensitive and rapid colorimetric method has been developed, and herein described, for the qualita-
tive and quantitative chemical assessment of the commercially available chitosan products. The described
method relies on the reactivity of the basic amino function of chitosan with the acid dye bromocresol purple.
The applied technique allows assessment of variability and selectivity changes in the quality of the marketed
chitosan products.
completed to volume with 1N sodium hydroxide solution. tercept and slope values of DEAE-cellulose in compari-
The developed blue color for each sample concentration son to those obtained with chitosan products tip off the
is used for absorbance measurement at 589 nm. analyst to the performance properties, efficiency and
capacity of such polymers as anionic exchangers relative
2.6. Calibration Graphs to a different match of known operational parameters.
The reported methods for the evaluation of chitosan
5 mL aliquots from the liberated sample solutions were products target measuring the deacetylation degree (DD)
transferred into 50 mL volumetric flasks. The reaction to reflect the quantity of free amino group on the chito-
flasks are completed to volume with 1N NaOH and the san matrix relative to the fully acylated chitosan (chitin).
solutions were measured spectrophotometrically at the However, those methods do not gauge the factual capac-
max of 589 nm and the recorded absorbances for dif- ity or working efficiency of the polymer. Accordingly, it
ferent samples of each sample were used to construct has been viewed as more sensible to compare chitosan
calibration graphs (Figure 2). products as functional rather than chemical operators.
Synthetic ion-exchangers of known chemistry, capacity
3. Results
The product from acetylating chitosan was treated with
the dye solution the same way as described for chitosan.
Although the acetylated chitosan matrix have acquired a
red color once wetted with bromocresol purple, yet, the
acidic solution used to drive out the adsorbed dye did not
release any noticeable color intensity. Moreover, when
the collected acidic wash solution was treated with so-
dium hydroxide and the measured at the same wave-
length, such solutions did not record any appreciable
absorbance readings. This finding illustrates that the
presence of the free amino groups are essential for the
working-efficiency of chitosan as an ion-exchanger. All
chitosan samples, however, have produced abstracted
appreciable quantities of the dye and released consider- Figure 2 Calibration curve for chitosan.
able color intensities upon acidification. The sensitivity
of color-measurement is further enhanced by shifting the
pH to the alkaline side with sodium hydroxide. The
quantities of the dye freed from the chitosan-dye com-
plex are then proportional to the quantities of chitosan
used and hence calibration graphs were constructed. The
good linearity and slope of the calibration graphs for the
different chitosan batches indicate the efficient and
strong sensitivity of the chitosan polymer in binding
acidic compounds. Alternatively, when DEAE cellulose
was treated with the dye solution, it showed a better
linearity and a superior capacity. This data suggests that
DEAE cellulose retains better functional characteristics
that observed with chitosan polymers (Figure 3).
4. Discussion
The high value of the correlation coefficient and the in-
tercept value were used to evaluate the linearity of the
calibration curves. Regression analysis of these plots
using the method of least squares had produced correla-
tion coefficients (r) equal to 0.9904-0.998 indicating a
good linearity (Figure 2). The sensitivity of the method
to different batches is evident from the slope and inter- Figure 3 Comparison of chitosan samples and DEAE cellu-
cepts values in the calibration curves. Moreover, the in- lose.
and performance, like DEAE cellulose, can pose as a [2] G. A. F. Roberts, Thirty Years of Progress in Chitin and
yardstick reference chemical to weigh against the effi- Chitosan, Progress on Chemistry and Application of
ciency of similar compounds. The applied method is also Chitin, Vol. 13, 2008.
valuable as a routine tool for the quantitative analysis of [3] S.-O. Fernandez-Kim, Physicochemical and Functional
very small amounts of chitosan products while depend- Properties of Crawfish Chitosan as Affected by Different
ing on the selective ion-exchange capacity of the poly- Processing Protocols, M. S. Dissertation, Seoul National
University, Seoul, 2004.
mer. The observed linearity and sensitivity of the meth-
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of such polymers. Pechyonkin and N.G. Balabushevich, Colorimetric As-
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lytes by Using O-Phthalaldehyde, Carbohydrate Poly-
5. Conclusions mers, Vol. 75, No. 4, 2009, pp. 724-727.
[5] J. J. Park, Development of Biomems Device and Pack-
Simple, quick and inexpensive colorimetric assays serve age for a Spatially Programmable Biomolecule Assem-
as convenient means to rapidly and straightforwardly bly, Ph.D. Dissertation, Faculty of the Graduate School
assess product quality in a cost-efficient transaction. The of the University of Maryland, College Park, 2006.
reaction of chitosan with anionic dyes has instantly pro- [6] K. van de Velde and P. Kiekens, Structure Analysis and
duced stable and colored adducts. This has triggered de- Degree of Substitution of Chitin, Chitosan and Dibutyryl
veloping anionic dyes like bromocresol purple as an Chitin by FT-IR spectroscopy and Solid State 13C NMR,
analytical reagent for assessing the quality of chitosan. Carbohydrate Polymers, Vol. 58, No. 4, 1998, p. 409.
Anionic dyes such as bromocreaol purple and bro- [7] R. A. A. Muzzarelli, Colorimetric Determination of
mocresol green form colored dye-matrix complexes with Chitosan, Analytical Biochemistry, No. 260, 1998, pp.
chitosan to produce a colored product. The dye-binding 255-257.
method produces a colored product which shed and [8] C. Wischke and H. Borchert, Increased Sensitivity of
equivalent amount of dye which is linearly proportional Chitosan Determination by a Dye Binding Method,
to chitosan quantity. Colorimetric tests are rapid and easy Carbohydrate Research, Vol. 341, No. 18, 2006, pp.
to perform. The reagents and equipment for colorimetric 2978-2979.
tests are inexpensive, environmentally safe, and are ideal [9] T. A. Khan, K. K.Peh and H. S. Chng, Reporting De-
for use in non-very well-equipped labs. gree of Deacetylation Values of Chitosan: The Influence
of Analytical Methods, Journal of Pharmacy & Phar-
maceutical Sciences, Vol. 5, No. 3, 2002, pp. 205-212.
6. References
[10] K. M. Picker-Freyer and D. Brink, Evaluation of Powder
[1] M. N. V. R. Kumar, A Review of Chitin and Chitosan and Tableting Properties of Chitosan, AAPS Pharma-
Applications, Reactive and Functional Polymers, Vol. ceutical Science and Technology, Vol. 7, No. 3, 2006.
46, No. 1, 2000, pp. 1-27.