International Journal of Biological Macromolecules
Some studies of crosslinking chitosan–glutaraldehyde interaction in a homogeneous system
Introduction
The reaction of glutaraldehyde with primary amine groups to produce covalent glutaraldehyde crosslinking is explored in many circumstances in order to detect the presence of free amine organic functions in simple or complex inorganic and organic compounds [1], [2], [3], [4], [5]. A large and clear application of this procedure can be illustrated with many polymeric matrices [6], [7]. However, the precise mechanism of reaction and the structure of the chemical compounds formed have not been studied in detail. Normally, three distinct structures are suggested. In order to interpret this behaviour, three propositions are considered: (a) there is formation of only one Schiff base, with one aldehyde group of the glutaraldehyde, the other aldehyde group remains free, and is commonly used for a subsequent reaction [2], [8]; (b) the crosslinking is formed with only one glutaraldehyde molecule and two chitosan unities, resulting in formation of two Schiff bases involving both aldehyde groups of the glutaraldehyde molecule [9]; (c) the crosslinking is formed with not only one glutaraldehyde molecule, but polymerization of glutaraldehyde, consequently forming a greater crosslinking chain [10], [11].
In general, the mechanism of this reaction is not discussed in many works. Nevertheless, Monsan et al. [4] studied the mechanism of interaction of glutaraldehyde with protein and Navarro and Manson [3] studied the mechanism of interaction of glutaraldehyde with microorganisms; they concluded that in a normal occurrence of glutaraldehyde reaction with a primary amine group, an imine bond is immediately formed. This bond is stabilized by resonance with the adjacent ethylenic double bond.
Chitosan is the product obtained from the deacetylation of the natural biopolymer chitin. Both biopolymers are chemically similar to cellulose, differing only in the functional group situated at carbon-2 of the monomeric unit. The presence of free amine groups in chitosan enhances the greater solubility and reactivity of this polymer than that of chitin and cellulose. Some products obtained by chemical modification of the chitosan have found multiple applications in various fields [12], [13], [14].
The interest in modifying chitosan by use of the glutaraldehyde has recently increased. The polymers obtained have been employed for many applications, mainly for the immobilization of the protein [15], [16], [17], [18], [19], [20], [21], [22], [23]. However, the mechanism of the chitosan and glutaraldehyde reaction and, much less, the structure of the chemical compounds formed were little studied.
Few investigations reported that the concentration of chitosan, glutaraldehyde and acetic acid, pH and temperature of the reaction medium are relevant features to be considered in the chitosan–glutaraldehyde reaction, mainly when these determinations are related to physical and chemical properties of the polymers obtained. The properties considered include dynamic storage modulus [11], the efficacy of metallic cation adsorption [24], [25], resistance to crush, solubility [9], enzyme immobilization, internal area [26], rate of gelation and colour formation [10]. On the other hand, the different degree of deacetylation of the chitosan seems to affect the formation of the covalent glutaraldehyde crosslinking on chitosans, an increased dynamic storage modulus was found within gels high in minor degree of deacetylation, suggesting that hydrophobic interactions are involved [11]. Recently, Crescenzi et al [27] noticed a counterproposal to the chitosan–monofunctional aldehyde interaction; the stoichiometry cannot be easily controlled with other aldehydes. Therefore, since the beginning this was a well-known complexity of this reaction [23]. The aim of this publication is to report some results connected to homogeneous chitosan–glutaraldehyde system reactions, in which the biopolymer chitosan is dissolved in acetic acid and reacted with glutaraldehyde in variable concentrations. From the collected data, a contribution to improve the understanding of the physical–chemical properties, the mechanism of this global reaction and the polymeric structure formed is proposed.
Section snippets
Chemicals
Chitin from shrimps shells was acquired from Fine Chemical Kito (Palhoça-SC-Brasil). Glutaraldehyde 25% solution in water (Aldrich), glacial acetic acid (Ecibra), hydrochloric acid (Nuclear), sodium borohydrate (Aldrich), EDTA (Nuclear) and copper nitrate (Vetec) or bidistilled water were used in the experiments.
Preparation of chitosan
Chitosan was prepared by deacetylation of chitin in alkali solution, NaOH 50%, during 1 h at 110°C. The solid was filtered, washed intensively with bidistilled water until a nearly
Degree of deacetylation of the chitosan
The degree of deacetylation was verified by infrared spectroscopy. The absorbance values found for amide I at 1655 cm−1 and hydroxyl groups at 3450 cm−1, gave 0.343 and 0.481, respectively. Applying these values in the correspondent equation gave 63.8%, which is an accepted good degree of deacetylation for this procedure [11]. This transformation changes the physical and chemical properties related to solubility of this natural organic polymer chitin, mainly in dilute acetic acid solutions [31]
Conclusions
Based on the obtained results, the reaction of chitosan dissolved in acetic acid at pH 3–4 with glutaraldehyde performed in a short time, less than 1 h. In this connection, the expected protonation of the amine groups of the chitosan does not affect the reaction, as observed by a series of the compounds obtained in this chitosan–glutaraldehyde system. However, the concentration of glutaraldehyde strongly affects the physical and chemical properties of the general CGX compounds formed. In the
Acknowledgements
The authors thank CNPq for fellowships and gratefully acknowledge FAPESP for financial support.
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