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ABSTRACT
 The
N-succinil-chitosan is a chemically modified derivative of the
biopolymer chitosan. The succinic anhydride attached to the free
amino groups presented along the chitosan's polymer chain imparts
to the molecule different physicochemical properties not exhibited
before the modification. These chemical modifications enhance
chitosan's solubility in slightly acid, neutral and alkaline
media. These properties are related to the long alkyl chains
attached to hydrophilic parts. In this case the hydrophilic part
of D-glucosamine promotes stronger interactions with the water
molecules, and consequently, enhances the solubility of the
chitosan polymer. Non-modified free chitosan is soluble only in
acidic medium (pH < 5.5). These modifications made possible
new applications of chitosan in biotechnological area since the
solubility in neutral or slightly alkaline solutions is very
important in a biological field.
Key words: N-succinil-chitosan, succinic anhydride,
biopolymer, solubility
| Chitosan is the name given to the
polymers that are obtained by the deacetylation of chitin
(poly-b-1,4-D-N-acetylglucosamine).
In industrial-scale procedures, chitin is produced by
treating seafood waste, especially shells from crustaceans (shrimps,
crabs, lobsters, krills, etc). The most popular technique
for obtaining chitosan is alkaline deacetylation of chitin:
this involves boiling chitin in concentrated alkali (50%
w/v) for several hours. This will yield chitosan with a
degree of acetylation between 20-30%: the most popular
commercial available form of chitosan. In such a chitosan,
the acetyl groups are uniformly distributed along the
polymer chain. This is in contrast with the chitosan of
similar degree of acetylation, but isolated from fungal cell
walls in which the acetyl residues are grouped into
clusters. |
INTRODUCTION
Chitin is a biopolymer similar to cellulose and quite abundant in
nature. It is extracted mainly from crustaceous skin or shells. (Singla,
2001; Khan, 2002; Tangpasuthadol, 2003). Due to the natural origin
of chitin, different variants are found in environment. In
addition, when chitin is submitted to different chemical
processes, a series of polymers varying in the degree of
deacetylation (DD), molecular weight (MW), viscosity, pKa, etc,
may be generated (Chatelet, 2000; Canella, 2001; Singla, 2001).
Chitosan is a natural polysaccharide composed of
b(1-4)-D-glucosamine units originated
from the total or partial deacetilation of chitin in alkaline
solutions (Singla, 2001; Dee, 2004). It is soluble in acid medium
(pH=5.5) due to the presence of free amino groups along the
polymer chain. The presence of these amino groups allows the
synthesis of different chitosan derivatives (Khan,2002; Franco,
2004). The purpose of this study was to synthesize a
N-succinil-chitosan derivative based on a methodology proposed by
Yamagushi et al, 1981 with some modifications (Yamagushi,1981).
MATERIAL AND METHODS
Chitosan samples were obtained from shrimp's shells and purchased
from Farma Service Bioextract (São Paulo, Brasil). All chemicals
were of analytical grade.
Sample preparation
Two samples of N-succinil-chitosan were prepared by reaction of
chitosan (Chit) with succinic anhydride at 1:1 w/w (0.06:0.01,
mol/mol) and 1:3 w/w (0.06:0.03 mol/mol) proportions named as CS1:1
and CS1:3, respectively. These molar quantities of chitosan/anhydride
were smaller than what it was suggested in the original work of
Yamagushi et al. (1981), who used an excess of anhydride
(16,4moles/amino group). The sample was precipitated with ethanol,
not methanol as the authors proposed, without yield losses.
Moreover, the previous solubilization of succinic anhydride in
acetone was substituted by direct addition of the succinic
anhydride to the reactional medium.
Sample characterization
The
solubility of chitosan and its synthesized derivative was
evaluated at different pH: acid (pH=4.0; 3% CH3COOH
solution), neutral (pH=7.0, water) and alkaline medium (pH=10.0;
0.1 mol L-1 NaOH solution) at room temperature (25ºC).
Qualitative observations of the samples submitted to the
solubilization tests were registered after 1, 5 and 10 hours.
The deacetylation degree (DD) of chitosan and its derivative was
determined by infrared (IR) spectroscopy and ninhydrin titration
The substitution degree (SD) was determined using the same
techniques. (Curotto, 1999; Canella, 2001; Khan, 2002). The
spectra of chitosan sample (in the forms of KBr disks) were
obtained using an IR Instrument (Bomem MB-100 FTIR, Germany) with
a frequency range of 4000-400 cm-1. The degree of
deacetilation of the chitosan sample by IR spectroscopy was
calculated using the equation proposed by Domszy and Roberts that
is given below:
DD=100-[(A
1665/A3450) x 100/1.33
where A
1665 and A3450 were the absorbance at 1655cm-1
of the amide-I band as a measure of the N-acetyl group content and
3450cm-1 of the hydroxyl band as an internal standard
to correct for film thickness or for differences in chitosan
concentration powder form. The factor '1.33' denoted the value of
the ratio of A 1665/A3450 for fully
N-acetylated chitosan. It was assumed that the value of this ratio
was zero for fully deacetylated chitosan.
The hygroscopic degree was measured under vacuum in 90.70%
humidity dessicators containing BaCl2 salt. Samples
were previously freeze-dried during 24 h, accurately weighted and
placed in the dessicator. The measurements were done in 1h
intervals for 5h, until constant weight for chitosan and its
derivative was reached.
RESULTS AND DISCUSSION
The
solubility measurements are shown in
Table 1 for
three different pHs. Chit was perfectly soluble in acetic acid
solution but precipitated at neutral and alkaline solutions.
This was expected due to the presence of amino groups along the
polymer chain which were protonated under low pH. SC1:1 became
partially solubilized in the entire pH range due to the increasing
substitution of the amino groups by carboxylic groups, which
became negatively charged above pH 6.0. The highly substituted SC1:3
appeared insoluble in pH 4.0 due to the predominance of carboxylic
groups compared to amino groups but totally solubilized at high
pH, where the complete dissociation of carboxylic acid groups
occured.
Deacetylation degree (DD) of original
chitosan sample was determined to verify how many amino groups
were available to react with succinic anhydride. Substitution
degree (SD) was determined for the modified samples. In
Figure 1
both results of DD for Chit and SD for SC1:1 and SC1:3 are shown.
It was possible to verify that the initial Chit presented a DD of
45% while the substituted samples, SC1:1 and SC1:3, presented a SD
of 10 and 20%, respectively. The increased substitution of amino
groups by carboxylic groups was directly related to the increase
in solubility related previously. The relatively low substitution
degree obtained for both modified samples could be explained in
part due to the hydrolysis of the reactant..
In
Fig. 2
the capacity of the samples to absorb humidity at room temperature
are shown. It was observed that the modified samples absorbed more
humidity than Chit.
All samples absorbed more humidity in the first hour followed by
shorter increase in the next 5h when the weight becomes constant.
Among all samples, SC1:3, which was the most substituted sample,
presented a larger hygroscopic degree as a function of time and
temperature.
CONCLUSION
The different physicochemical properties determined in this study
indicated that N-succinic-chitosan could be applied in many
biotechnological fields. The molar quantities of chitosan/anhydride
used in these study were quite lower than what it was done in the
original Yamagushi's 1981 work and, the modifications introduced
in the sample preparation resulted in optimization of the process,
economy of reactants without losses of physicochemical properties.
The solubility of substituted chitosan samples in neutral and
alkaline media increase the possibility of use in cosmetics and
pharmaceutical. The hygroscopic capacity of the modified samples
could be useful to film formation, to membranes formulated for
wound healing and as additive in cosmetics and toiletries.
ACKNOWLEDGEMENTS
The authors thank CAPES for the financial support and Farma
Service Bioextract for providing chitosan samples.
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