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American Journal of Analyt ical Chemistry, 2010, 2, 91-94
doi:10.4236/ajac.2010.12012 Published Online August 2010 (http://www.SciRP.org/journal/ajac)
Copyright © 2010 SciRes. AJAC
A Simple Colorimetric Method for the Evaluation
Department of Ph arm ac og n os y , Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
Received June 19, 2010; revised August 7, 2010; accepted August 12, 2010
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
Keywords: Chitosan, Dye, Colorimetric, Bromocresol Purple
Chitosan is a natural biocompatible polymer derived
from the naturally-occurring polysaccharide-based bio-
polymer, chitin, by deacetylation with an alkali leaving
behind a free amino group (-NH2) (Figure 1) . Chito-
san naturally exists only in a few species of fungi but it is
mainly extracted from the cuticular and exoskeletons of
invertebrates like crustaceans, mollusks, crabs and sh-
rimp. Crabs obtained from seafood processing waste are
an important commercial source. Chitin is the second,
after cellulose, most abundant naturally occurring poly-
saccharide. Chitin is composed of 2-acetamido-2-deoxy-
β-D-glucose units that are combined by 1-4 glycosidic
linkages, forming long un-branched linear polymeric
chains. Therefore, the biopolymer chitosan is composed
of β-2-amino-2-deoxy-D-glucopyranose(glucosamine units)
and β-2-acetamido-2-deoxy-D-glucopyranose . Chi-
tosan possesses unique properties like its ability to form
films, and possesses a positive ionic charge which de-
velops its ability to chemically bind with negatively
charged fats, lipids and bile acids. Chitosan has a wide
range of applications in diverse fields like in health
(ranging from medical sutures to beauty aids), water pu-
rification (coagulants for waste treatment), biomedical
applications, agriculture (seed coatings), biotechnology,
nutrition (dietary supplements), and in the finishing
process of textile fibers . Chitosan is commercially
available from many suppliers in various grades of purity,
molecular weight, and degree of deacetylation. The de-
gree of deacetylation is one of the most important che-
mical characteristics as it reported to influence the phys-
icochemical properties and the performance of chitosan
in many of its applications. Chitosan versatility depends
mainly on the chemically reactive amino groups.
The degree of deacetylation of chitosan ranges from
56% to 99%, depending on the crustacean species and
the preparation method. Various methods have been re-
ported for the determination of the degree of deacetyla-
tion of chitosan. These included ninhydrin test, linear
potentiometric titration, infrared spectroscopy, near-
infrared spectroscopy, nuclear magnetic resonance spec-
troscopy, hydrogen bromide titrimetry, infrared spec-
troscopy, elemental analysis, colloidal titration, circular
dichroism, ultraviolet spectroscopy, pyrolysis-gas chro-
matography, gel permeation chromatography and ther-
mal analysis, acid hydrolysis, and X-ray diffraction
methods and first derivative UV-spectrophotometry [4-
6]. Chitosan could be also assayed colorimetrically using
Cibacron brilliant red 3B-A [7,8].
All the above mentioned methods employed for the
evaluation of chitosan slightly vary when measuring the
number of free amino groups in the structure (deacetyla-
tion degree, DD). Obviously, the DD values are highly
dependent on the analytical methods employed [9, 10].
Hence, we propose that the analytical method used for
the evaluation of chitosan products would also consider
the functional capacity of the matrix. This approach is
easily perceived when a reference matrix is taken into
A. S. MOHAMED 92
Copyright © 2010 SciRes. AJAC
Figure 1. Structure of Chitosan unit.
Colorimetric assays are often developed as simple and
sensitive practical procedures to rapidly evaluate drug
quality in rapid and inexpensive protocol. Chitosan pos-
sesses a free amino group that acts as a reactive binding
site for anionic dyes such as bromocresol purple to pro-
duce a color-bound complex. Therefore, our objective is
to develop and evaluate a colorimetric technique to
measure the functional capacity, which relates to deace-
tylation degree, so as to quantitatively assess these prod-
ucts in pharmaceutical preparations.
2. Methods—Experimental Data
2.1. Reagents and Apparatus
All reagents, hydrochloric acid, pyridine, acetic anhy-
dride and sodium hydroxide, were of analytical-reagent
grade, distilled or deionized water (DI) was used to pre-
pare all aqueous solutions. Pharmaceutical-grade chito-
san was used, bromocresol purple Indicator is a product
of Reidel-DeHaen Ag, Seelze-Hannover, sodium bicar-
bonate, and sodium hydroxide from Sigma-Aldrich (St.
Louis, MO, USA); Microgranular pre-swollen DEAE-32
Cellulose, Whatman, Springfield Mil, Madison, Kent,
Shimadzu UV 1601PC, UV/VIS double beam spectro-
photometer (Kyoto, Japan) was used and equipped with 1
cm quartz cells and connected to IBM compatible com-
puter. The software was UVPC personal spectroscopy
software version 3.7 (Shimadzu). The spectral width ap-
plied was set at 2 nm.
2.3. Sample Preparation
The colorimetric methods were evaluated in terms of
linearity, precision, and accuracy by using different
pharmaceutical batches of raw material compounds. Ac-
curately weighed quantities of three commercially avail-
able (1-60 mgs) chitosan powder were introduced into
small sintered funnel or filtration tubes. The chitosan
sample were soaked with 0.2 ml of water allow swelling
of the polymer matrix.
2.4. Bromocresol Purple Dye Solution
Dye solution is prepared by dissolving 100 mg of bro-
mocresol purple in 5 mL of ethanol.
2.5. Diethyl Amino Cellulose (DEA-32)
Diethylamino cellulose ion-exchanger was used as a
functional reference to relate the chitosan performance.
DEAE cellulose was selected because of its structural
resemblance to chitosan. Accurately weighed quantities
(10-80 mgs) of DEAE cellulose ion exchanger were
treated the same procedure like chitosan samples.
2.4. Preparation of Acetylated Chitosan
200 mgs from each chitosan samples were suspended in
0.5 ml of dry and distilled pyridine. 2 ml of acetic anhy-
dride were added and stirred for 4 hours at room tem-
perature then kept in dark for overnight. The reaction
mixture is then quenched with cold water and the product,
acetylated chitosan, was filtered off, washed with DI
followed by ethanol and dried in an oven at 60˚C for 15
2.5. Colorimetric Assay
Bromocresol purple was used for the colorimetric assay
and prepared at a concentration of 20 mg/mL in water.
For all chitosan samples (weights from 1mg and up to 50
mgs of chitosan or their equivalent amounts) were accu-
rately weighed. The chitosan samples were weighed ei-
ther directly into small sintered glass funnels, or in Pas-
teur pipettes (disposable pipettes) packed at the bottom
with glass wool. Secure the porosity of the filter pad,
especially when glass wool, to carefully hold and do not
allow passage of any of the loaded chitosan powder. The
powdered sample in each tube was then wetted with 0.2
mL of DI water and allowed to soak for 15 minutes to
allow possible swelling of the matrix. Approximately 0.3
mL of the dye solution is slowly passed through the sin-
tered funnels. Each tube is then loaded with 0.2 ml of the
dye solution. Excess dye solution is drained out and ex-
cess dye was washed out with 0.5 mL of DI followed by
95% ethanol till complete removal of all color in the
wash solution. The tube packed with chitosan-dye com-
plex is then contained in clean 20 mL volumetric flasks.
The chitosan-bound dye was then stripped off the bed by
20 ml of 1N HCl solution and completed to volume. The
acid solution is filtered through a 0.45 µ membrane filter.
Five milliliter aliquots were withdrawn from each sample
concentration into a separate 50 m volumetric flask and
93 A. S. MOHAMED
Copyright © 2010 SciRes. AJAC
completed to volume with 1N sodium hydroxide solution.
The developed blue color for each sample concentration
is used for absorbance measurement at 589 nm.
2.6. Calibration Graphs
5 mL aliquots from the liberated sample solutions were
transferred into 50 mL volumetric flasks. The reaction
flasks are completed to volume with 1N NaOH and the
solutions were measured spectrophotometrically at the
λmax of 589 nm and the recorded absorbances for dif-
ferent samples of each sample were used to construct
calibration graphs (Figure 2).
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-
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).
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-
cepts values in the calibration curves. Moreover, the in-
tercept and slope values of DEAE-cellulose in compari-
son to those obtained with chitosan products tip off the
analyst to the performance properties, efficiency and
capacity of such polymers as anionic exchangers relative
to a different match of known operational parameters.
The reported methods for the evaluation of chitosan
products target measuring the deacetylation degree (DD)
to reflect the quantity of free amino group on the chito-
san matrix relative to the fully acylated chitosan (chitin).
However, those methods do not gauge the factual capac-
ity or working efficiency of the polymer. Accordingly, it
has been viewed as more sensible to compare chitosan
products as “functional” rather than chemical operators.
Synthetic ion-exchangers of known chemistry, capacity
Figure 2 Calibration curve for chitosan.
Figure 3 Comparison of chitosan samples and DEAE cellu-
A. S. MOHAMED 94
Copyright © 2010 SciRes. AJAC
and performance, like DEAE cellulose, can pose as a
“yardstick” reference chemical to weigh against the effi-
ciency of similar compounds. The applied method is also
valuable as a routine tool for the quantitative analysis of
very small amounts of chitosan products while depend-
ing on the selective ion-exchange capacity of the poly-
mer. The observed linearity and sensitivity of the meth-
ods promotes its friendly application in routine analysis
of such polymers.
Simple, quick and inexpensive colorimetric assays serve
as convenient means to rapidly and straightforwardly
assess product quality in a cost-efficient transaction. The
reaction of chitosan with anionic dyes has instantly pro-
duced stable and colored adducts. This has triggered de-
veloping anionic dyes like bromocresol purple as an
analytical reagent for assessing the quality of chitosan.
Anionic dyes such as bromocreaol purple and bro-
mocresol green form colored dye-matrix complexes with
chitosan to produce a colored product. The dye-binding
method produces a colored product which shed and
equivalent amount of dye which is linearly proportional
to chitosan quantity. Colorimetric tests are rapid and easy
to perform. The reagents and equipment for colorimetric
tests are inexpensive, environmentally safe, and are ideal
for use in non-very well-equipped labs.
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