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Chapitre 5: Electromigration Behavior of a Mixture of Chitosan Oligomers at Different Concentrations

Table des matières

Les travaux présentés dans ce chapitre sont une continuité de ceux obtenus dans le chapitre précédent et constituent également une contribution aux connaissances fondamentales des propriétés électrophorétiques d’un mélange d’oligomères de chitosane composé de dimères, trimères et tétramères. Jusqu’à présent, aucune étude n’avait été réalisée sur ce sujet. L’étude a été réalisée à différentes concentrations du mélange d’oligomères. L’objectif de cette étude était de comparer les résultats avec ceux obtenus dans un milieu dilué. Une gamme de pH allant de 3 à 12, d’une force ionique ajoutée (NaCl) de 0.01, 0.05 et 0.1 M ont été étudiées. Des mesures dans l’eau ont été réalisées comme milieu de référence.

La planification de cette partie de la thèse et la rédaction de cet article ont été réalisées par l’étudiant Mohammed Aider qui est l’auteur principal et supervisé avec beaucoup d’attention par le Dr. Laurent Bazinet de l’Université Laval. Le Dr. Mircea-Alexandru Mateescu de l’UQAM et le Dr. Joseph Arul de l’Université Laval ont contribué au succès de ce travail par leurs commentaires et corrections. Serge Brunet de ISM Biopolymer a contribué à la rédaction de l’article et a apporté une bonne contribution par ses corrections. Tous ces collaborateurs sont co-auteurs de ce travail. Les résultats présentés dans ce chapitre ont été publiés dans Journal of Agricultural and Food Chemistry, (2006), Vol. 54(26) pp. 10170-10176.

Dans ce travail, l'effet de la concentration d'un mélange d'oligomère de chitosan sur son comportement électrophorétique a été étudié en fonction du pH et de la force ionique. Il a été démontré que la concentration a un effet significatif sur la mobilité électrophorétique moyenne du mélange d'oligomères de chitosan et sur son point isoélectrique. À une concentration de 3% d’oligomères de chitosan, la concentration ionique n'a eu aucun effet sur le comportement électrophorétique du mélange d'oligomères de chitosan. En diminuant la concentration du mélange d'oligomères de chitosan, la force ionique a montré un effet significatif sur la mobilité électrophorétique moyenne mais non pas sur le point isoélectrique. Le déplacement le plus important du point isoélectrique du mélange étudié a été enregistré dans l'eau à une concentration de 0.003% du mélange d'oligomères. Sous ces dernières conditions, le point isoélectrique était à pH 5 tandis qu'il était à pH 8 à la concentration de 3% du mélange. Des mesures de mobilités électrophorétiques ont été également réalisées dans un milieu aqueux composé d'eau et d’éthanol. En ajoutant l'éthanol au milieu, la mobilité électrophorétique moyenne a diminué. Ceci aurait été provoqué par l'augmentation de la viscosité du milieu. En augmentant la concentration de l'éthanol dans le milieu, le point isoélectrique mesuré dans l'eau s'est déplacé de pH 5 jusqu'aux pH 6, 7 et 8, dépendamment du ratio eau/éthanol et de la concentration du mélange d'oligomères de chitosan. Le premier et deuxième sous-objectif de cette thèse ont été d’un intérêt fondamental et ont fait la base du second volet à caractère pratique.

In this study, the effect of the concentration of a chitosan oligomer mixture on its electrophoretic behavior was studied as a function of pH and ionic strength. It was shown that the concentration has a significant effect on the average electrophoretic mobility of the chitosan oligomers mixture and on isoelectric point. At a concentration of 3%, the ionic strength did not show any effect on the electromigration behavior of the chitosan oligomers mixture. By decreasing the concentration of the chitosan oligomers mixture, ionic strength showed a significant effect on the average electrophoretic mobility but not on isoelectric point. The highest shift of isoelectric point was recorded in water at 0.003% concentration of the oligomers mixture. Under these conditions, the isoelectric point was at pH 5 whereas it was at pH 8 at 3% concentration of chitosan oligomers mixture. Electrophoretic measurements were also taken in water/ethanol aqueous medium. By adding ethanol to the medium, the average electrophoretic mobility decreased. This would have been caused by the increase in viscosity of the medium. Increasing ethanol ratio in the running medium, isoelectric point moved from pH 5 in water up to pH 6, 7 and 8 dependently on chitosan oligomers mixture concentration and ethanol content of the medium.

Keywords: Chitosan oligomerss mixture; Concentration; Isoelectric point; Ethanol; Dielectric constant, Electrophoretic mobility.

Chitosan is the N-deacetylated product of chitin found in the shells of crabs and shrimps, and as cell wall components of most fungi, yeasts and molds. Chitosan has applications in several fields such as biomedical, personnal care products, biotechnology, pharmaceutical, nutraceutical and food. Chitosan oligomerss with a degree of polymerization of about 6 are potentially useful as medicinal agents and as food ingredients because of their biological and therapeutic values (Chung et al., 2003; Lee et al., 2002; Wang &Hsieh, 2002; Suzuki et al., 1986). To produce these bioactive oligosaccharides, biocatalytical (enzymatic) and chemical procedures are used (Vishu-Kumar et al., 2004; Somashekar & Joseph, 1995; Ohtakara & Izume, 1987). The yield of the biocatalytical reaction is not high (Kurita et al., 2002). By combining physical methods (shear-force treatment) and acid-hydrolysis, the molecular weight of the chitosan oligomers can be decreased (Chung et al., 2003; Domard &Cartier, 1989; Austin, 1981). Although execution of these physical methods is not difficult, fast degradation rates and random reactions result in product variability (Kurita et al., 2002). In acid hydrolysis, 10% acetic acid is generally used as a solvent, with 5% NaNO3added for the deacetylation reaction. This method can decompose chitosan into units of one to six N-acetylglucosamines, and such products are soluble at pH 7 (Hirano et al., 1985). All these methods can serve to produce a mixture of molecules with various molecular weights.

In order to separate these chitosan oligomers mixtures to obtain pure or enriched oligomers, it is essential to find a suitable technique. The exploitation of the electric properties of these molecules could offer a solution (Mechref & El-Rassi, 1997). The chitosan oligomers have one or more amine functional groups which depend on the degree of polymerization (Nashabeh & El-Rassi, 1992). Under specific pH conditions, these groups are charged because of the protonation of the amine group (NH3 +). Therefore, they will migrate under the effect of an external electric field (Nashabeh & El-Rassi, 1992). The migration speed of each molecule should be different from others because of differences of their molecular weight, electric charge and concentration. That would make possible the separation of various chitosan oligomers by exploiting the electrophoretic properties of each molecule. To carry out this objective, it is important to study and understand the electrophoretic behaviors of the chitosan oligomers under various conditions of the medium and different concentrations of the mixture. In a previous study (Aider et al., 2006a), it was shown that electromobility of chitosan D-glucosamine (monomer) and individual chitosan oligomers (dimer, trimer, tetramer, pentamer and hexamer) in dilute media at a concentration of 10 μg/mL originated not only from the electrical charge but also from the glucose moiety through the difference in the dielectric constant of glucose and that of the medium at alkaline pH values.

The aim of this research was to study the electrophoretic behavior of chitosan oligomers mixture at different concentrations under varying conditions of pH, ionic strength and dielectric constant of the medium.

Figure 5.1 shows the electromigration behaviour of the chitosan oligomers mixture at a concentration of 3% in water (HPLC grade) and NaCl solutions with different ionic strengths as a function of pH, which had significant effect (P < 0.0001).

At this concentration, the chitosan oligomers showed the highest electrophoretic mobility at pH 2 and 3 with an average value of 2.009 ± 0.105 x 10-6 m2/V.s. At pH 4, 5 and 6, the oligomers showed a stable electrophoretic mobility but lower than that recorded at pH 2 with an average value of 1.225 ± 0.051 x 10-6 m2/V.s. At all the previous mentioned pH values, the chitosan oligomerss migrated towards the cathode. At pH 7, the electromigration was slower than at the preceding pH values (0.836 ± 0.080 x 10-6 m2/V.s). At higher pH values (8, 9 and 10), the oligomers mixture was motionless. At pH 11 and 12, we noticed a slight negative electromigration (towards the anode) with an average electrophoretic mobility of -0.331 ± 0.110 x 10-6 m2/V.s. In NaCl solutions (0.01-0.1 M), electromigration behaviour of the chitosan oligomers mixture was quite similar to that in water and the effect of the ionic strength compared to the data obtained in water was not significant (P > 0.971) .

In water mixed with ethanol at different ratios, the behavior of the chitosan oligomers mixture was different from what was observed in NaCl with various ionic strengths. Indeed, at a 3% concentration of chitosan oligomers mixture (Figure. 5. 2), with ethanol addition we observed a gradual fall of the absolute value of electrophoretic mobility was observed.

This fall was gradual. Highest mobilities were recorded in water. In water mixed with ethanol (W/Et-OH) at ratio of 75/25 (v/v) electrophoretic mobility had decreased and occupied intermediate values between what was observed in water and what was recorded in water with ethanol at ratios of 50/50 and 25/75 (v/v), respectively. Electrophoretic mobilities recorded in these two media (W/Et-OH at ratio 50/50 and 25/75 (v/v)) were not significantly different. The isoelectric point was the same under all conditions (PI=8). It is the same value which was recorded in the NaCl solutions at various ionic strengths. At pH values above the PI, there was no significant electrophoretic mobility of the chitosan oligomerss mixture.

Figure 5.3 shows the electromigration behaviour at a concentration of 0.3% of the chitosan oligomerss mixture in water and NaCl solution at different ionic strengths as function of pH.

Effect of pH was significant (P<0.001). In water, in the pH interval from 2 to 5, the electromigration of the chito-oligomers was stable and was towards the cathode with a mean value of 1.651±0.209x10-6 m2.V-1.s-1. A decrease of the electromobility was recorded at pH 6. At pH 7, the chitosan oligomers mixture was at the isoelectric point. By increasing the pH, the electromigration was towards the anode. In the interval of pH from 8 to 12, the electrophoretic mobility of the mixture was relatively stable and showed an average electrophoretic mobility of -1.029 ± 0.091 x 10-6 m2.V-1.s-1. By adding salt to the medium (Figure 5.3), the electromigration behaviour of the chitosan oligomers was affected (P<0.027). At ionic strength up to 0.01 M, a significant decrease of the electrophoretic mobility was recorded between pH 2 and 5, and increasing the ionic strength up to 0.05 or 0.1 M did not change the electromigration behaviour of the chitosan oligomers mixture.

At a concentration of 0.3% of chitosan oligomers mixture (Figure 5.4), highest mobilities were recorded in water.

In this case, the isoelectric point was moved compared to that recorded at a concentration of 3%, and at pH values above the isoelectric point (IP), negative electrophoretic mobilities were observed. By adding ethanol, at pH values below the isoelectric point, the same phenomenon was observed as in the case with a concentration of 3%. Electrophoretic mobility decreased gradually. At pH values above the isoelectric point, no significant difference was observed. The values recorded in water mixed with ethanol at ratio of 75/25 (v/v) were intermediate between those observed in water (the highest) and those recorded in water with ethanol at ratios of 50/50 and 75/25 (c/v), respectively. Mobilities in these two cases were the lowest. No significant difference was observed between electrophoretic mobilities in these two media. As regards the isoelectric point (IP), it moved up to pH 7.5 in water/ethanol at ratio of 75/25 (v/v) and up to pH 8 in water/ethanol at ratios of 50/50 and 25/75 (v/v), respectively.

Figure 5.5 illustrates the electromigration behaviour of the chitosan oligomers mixture at concentration of 0.03% under different conditions (water and NaCl).

In all cases, the effect of pH was significant (P<0.001). In water, the mixture showed the greater electrophoretic mobility at pH 2, 3, 4 with a mean value of 1.779 ± 0.227 x10-6 m2.V-1.s-1. By increasing the pH up to 5, the electrophoretic mobility decreased. At all these pH values, the migration was towards the cathode. The isoelectric point was recorded at pH 6. By increasing the pH, the oligomers mixture completely changed its behavior and migrated towards the anode. At pH 7, the recorded electrophoretic mobility was -0.866 ± 0.245x10-6 m2.V-1.s-1. In the pH interval between 8 and 12, the electro-mobility of the oligomers reached a plateau with an average value of -1.386 ± 0.112 x10-6 m2.V-1.s-1. Data analyses showed that ionic strength influenced the electromigration behaviour of the mixture depending on pH range. Between pH 2 and 4, ionic strength had a significant effect on the magnitude of the electrophoretic mobility (P<0.0001), where the electrophoretic mobility decreased by increasing ionic strength to the medium. In the range of pH between pH 5 and 7 the ionic strength had no effect on the electrophoretic mobility of the chitosan oligomers mixture (P>0.877), and the isoelectric point was situated in this pH range. In this pH interval, the mixture was closer to the isoelectric point. Between pH 8 and 10, the electrophoretic mobility decreased by the addition of salt to water. In the pH range between pH 11 and pH 12, ionic strength did not show any effect on the electrophoretic mobility (P > 0.160).

Electrophoretic mobilities of the chitosan oligomer mixture at a concentration of 0.03% in water and water/ethanol at different ratios are shown in (Figure 5.6). At a concentration of 0.03% of chitosan oligomer mixture, the highest electrophoretic mobilities were recorded in water. This was valid at pH values below and above the isolectric point (IP). By adding ethanol to the medium, electrophoretic mobility of the chitosan oligomers mixture decreased gradually. The PI was observed at pH close to 6 in water and then it passed up to pH close to 8 in water mixed with ethanol at different ratios.

Figure 5.8 shows the electrophoretic behavior of the chitosan oligomers in water at a concentration of 0.003% and NaCl solution at different ionic strengths as a function of pH. The effect of pH was significant at all conditions (P<0.001). In water, the isoelectric point was recorded at pH 5. The cationic character of the chitosan oligomers was measured at pH 2, 3 and 4 and the electromigration was towards the cathode. In the pH interval between 6 and 12, the migration was towards the anode. The highest electro-mobility was at pH 12 with an average value of -1.555 ± 0.224 x10-6 m2.V-1.s-1. In NaCl solutions, data analysis showed three intervals for the effect of ionic strength. At pH between 2 and 6, the ionic strength had no significant effect on the electrophoretic mobility of the chitosan oligomers mixture (P>0.465). At pH between 7 and 9, ionic strength had a significant effect on the absolute value of the electrophoretic mobility (P<0.003). As seen in (Figure 5.8), the chitosan oligomers mixture was more mobile in water and by increasing the ionic strength, the electrophoretic mobility decreased. The effect of the ionic strength of 0.01 M was different from that of 0.05 or 0.1 M. In the pH range between 10 and 12, there was no significant differences between electrophoretic mobilities (P>0.622).

At a concentration of 0.003% of chitosan oligomers (Figure 5.8), no significant difference was observed between electrophoretic mobilities below the isoelectric point (IP). The IP was at pH 5 in water. By adding ethanol to the medium, the isolectric shifted up to pH 6 in water/ethanol at ratio of 75/25 (v/v) and up to pH 7 in water/ethanol at ratios of 50/50 and 75/25 (v/v), respectively. At pH values above the isolectric point, the highest electrophoretic mobilities were observed in water. By adding ethanol to the medium, electrophoretic mobility decreased gradually.

As the ionic strength of the medium was increased, the ionic atmosphere composed of counter-ions around the migrating molecules increased. Under the effect of the applied external electric field, migrating chitosan oligomers move in one direction and the counter-ion atmosphere moves in the opposite direction, each carrying solvent molecules along with them. As a result of this phenomenon, the electromigration of the molecule is retarded by the atmosphere of counter-ions, i.e., the screening effect of these counter-ions increases. In dilute aqueous medium, this phenomenon was stronger when the chitosan oligomers migrated towards the anode with Na+ as counter-ions which are hydrated (solvated), while the Cl- counter-ions are not hydrated when the electromigration was towards the cathode. The highest values of the electrophoretic mobilities of the chitosan oligomers were obtained in water because the screening effect of the counter-ions was lowest, followed by those recorded in solutions with an ionic strength of 0.01 M. The lowest values of the electrophoretic mobility were recorded in the salt solutions with ionic strengths of 0.05 and 0.1 M respectively. All these observations are in good agreement with previous results in the literature (Aider et al., 2006a; Mechref, et al., 1997; Nashabeh & El-Rassi, 1992). Also, by decreasing concentration of the chitosan oligomers mixture, the oligomers molecules are separated and the screening effect is reduced. The network formed between these oligomerss was lower because the interactions between chitosan oligomers molecules decreased (interaction by mean of the hydrogen bonds).

The above given explanation is not valid in the case of chitosan oligomer solution of 3% concentration. Ionic strength did not have any effect on the electrophoretic mobility of the chitosan oligomers mixture. At this concentration (3%), the hydrogen bonds formed between the molecules contribute to the formation of a network between the oligomers and thus the screening effect of the ionic strength was negligible. By decreasing the concentration, the ionic strength affected the electrophoretic mobility of the oligomers.

As it can be seen (Table 5.1), the isoelectric point and consequently the electrophoretic behaviour of the chitosan oligomers depend on the concentration. It was at pH 8 for a concentration of 3%, and then decreased by decreasing the concentration. At a concentration of 0.03% and below (0.003%), the isoelectric point was lower than the pKa of D-glucosamine. This indicated that the isoelectric point and in general the electrophoretic behavior of chitosan oligomers mixture depend on the concentration.

The dependence of the electrophoretic behavior of the chitosan oligomers mixture on the concentration could be explained as follows: at a concentration of 3%, the interactions between the molecules by the intermediary of the hydrogen bonds are stronger and the network is not highly hydrated. The molecules form a more compact network and consequently, the effect of the medium or more exactly the friction between the medium and the solute was less significant. On the other hand, by decreasing the concentration of the chitosan oligomers mixture, the network formed by the molecules becomes weak and the dielectric friction with the medium becomes stronger. This friction has caused the attraction of the chitosan oligomers molecules towards the anode as if they were anions. This phenomenon caused the displacement of the isoelectric point (IP) to pH values lower than the isoelectric point recorded at a concentration of 3% (Figure 5.1). The deprotonation of amine group was stronger in dilute media and thus the shift of the chitosan oligomers mixture isoelectric point was more pronounced.

In this study, it was demonstrated that the concentration has a significant effect on the isoelectric point and the electrophoretic behaviour of the chitosan oligomers mixture. By decreasing the concentration of the chitosan oligomers mixture from 3% to 0.003%, the isoelectric point was reduced from pH 8 to pH 5. The isoelectric point of the chitosan oligomers mixture is thus linearly contingent on the free hydronium (H+) ion concentration present in the medium. The interactions with the medium become increasingly significant by decreasing the concentration. The contribution of the difference in dielectric constant between the solute and the solvent was also demonstrated. In dilute medium, this phenomenon led to the migration of the chitosan oligomers towards the anode. Moreover, this study also made it possible to observe the effect of the ionic strength on the electrophoretic mobility at various concentrations of the chitosan oligomers mixture. At a concentration of 3%, no effect on the electrophoretic mobility of the ionic strength was showed. But, by decreasing the concentration, the effect of the ionic strength became significant. The pH of the medium showed a significant effect on the electrophoretic behaviour of the chitosan oligomers mixture at all concentrations because it determines the charge of the molecules and thus the direction of the migration.

These results are actually the basis for electro-separation of chitosan oligomers mixture by an electrodialysis-ultrafiltation (EDUF) system.

© Mohammed Aider, 2007