Collection Mémoires et thèses électroniques
AccueilÀ proposNous joindre

Chapitre 4: Electromigration of Chitosan D-Glucosamine and Oligomers in Dilute Aqueous Solutions

Table des matières

Les travaux présentés dans ce chapitre sont une contribution aux connaissances fondamentales des propriétés électrophorétiques de standards de la D-glucosamine et d’oligomères de chitosane avec un degré de polymérisation variant entre 2 et 6. L’étude a été réalisée dans un milieu dilué. Jusqu’à présent, aucune étude n’avait été réalisée sur ce sujet. En modifiant les conditions du milieu, en particulier le pH, la force ionique et le type de sel ajouté, les mobilités des molécules mentionnées ont été mesurées.

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. Il a été supervisé avec une grande implication par le Dr. Laurent Bazinet de l’Université Laval sans qui ce travail ne serait pas réussi. 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(17), pp 6352-6357.

Le but de ce travail était d'étudier le comportement électrophorétique de la D-glucosamine et des oligomères de chitosan avec un degré de polymérisation de 1 à 6 dans des systèmes aqueux dilués contenant du NaCl et du KCl à 0.01, 0.05 et 0.1 M aux valeurs de pH de 2 à 9. Les résultats ont montré que l'électromigration de la D-glucosamine et des oligomères de chitosan n'a pas été affectée par le type de sel et que le pH a eu un effet significatif sur la direction de la migration de ces molécules sous l’effet du champ électrique externe. En outre, l'augmentation de la force ionique du milieu a causé une diminution significative sur la valeur absolue de la mobilité électrophorétique et les valeurs les plus élevées de cette mesure ont été observées dans l'eau  qui a été considérée comme étant le milieu avec zéro force ionique ajoutée. Cependant, la force ionique n'a eu aucun effet significatif sur les mobilités électrophorétiques à pH 2 en comparaison avec les autres valeurs de pH. Le dimère a montré la mobilité électrophorétique la plus élevée. Cette différence a été plus accentuée dans la zone alcaline de pH. Aux valeurs du pH inférieur au pKa (6.2-6.5) de la D-glucosamine où les groupes amine sont protonés et portent une charge positive, la D-glucosamine et les oligomères de chitosane avaient émigré vers l'anode. À des valeurs du pH plus élevées que le pKa, la D-glucosamine et les oligomères de chitosane ont émigré vers l'anode quoiqu'elles n'aient aucune charge électrique. La contribution de la différence de constantes diélectriques du solvant et du soluté à ce phénomène a été démontrée. Il été également démontré que la partie glucose contribue à la direction de migration de la D-glucosamine et des oligomères chitosan dans des conditions alcalines de pH et que la différence entre la constante diélectrique du glucose et du solvant expliquerait ce comportement électrophorétique.

Electromigration behavior of chitosan D-glucosamine and oligomers with degree of polymerization from 1 to 6 in dilute aqueous systems containing either NaCl and KCl salts at 0.01, 0.05 and 0.1 M at pH values from 2 to 9 was evaluated. The results showed that the electromigration of the chitosan D-glucosamine and oligomers did not change by changing the type of salt in the running medium and that the pH had a significant effect on the direction of migration under external electric field. In addition, the increase in the ionic strength of the medium caused a significant decrease on the absolute value of the electrophoretic mobility and the highest values of the electromobility were observed in water. However, the ionic strength had no significant effect on the electrophoretic mobilities at pH 2 in comparison with the other pH values. The dimer showed the highest electrophoretic mobility in the alkaline zone of the pH. At pH values lower than the pKa of the D-glucosamine, the chitosan D-glucosamine and oligomers migrated towards the anode where the amine groups are protonated and carry positive charge. At higher pH values, chitosan D-glucosamine and oligomers migrated towards the anode even though they did not carry any electric charge. The contribution of the difference in the dielectric constants between the solvent and the solute to this phenomenon was highlighted. It was shown that the glucose moiety contributes to the direction of migration of the chitosan D-glucosamine and oligomers under alkaline conditions and that the difference in the dielectric constant of glucose and the solvent accounts for the direction and the extent of electromobility.

Keywords: Electrophoretic mobility, chitosan D-glucosamine, oligomers, dielectric constant.

Since the early 1990’s, glucosamine has been widely promoted as an active molecule for the treatment of osteoarthritis and subjected to placebo-controlled studies. Glucosamine is a bio-active amino sugar that is present in all human tissues and is thought to promote the formation and repair of cartilage and has been shown to reduce the progression of diseases like osteoarthritis, and significantly lessen pain from arthritis (Homandberg et al., 2006; Xing et al., 2006; Uitterlinden et al., 2006; Bazian Ltd., 2005; Phoon & Manolios, 2002; Kendra & Hadwiger, 1984). This substance is the principal compound of the glucosaminoglycans that form the matrix of the connective tissues. Glucosamine could be combined with other glucosaminoglycans since it helps to maintain the viscosity in the articulation and stimulates the cartilage recovery (Gomez-Diaz & Navaza, 2005). Glucosamine and chitosan oligomers of low molecular weight (with degree of polymerization up to 7), were shown to be absorbed easily into the human intestine (Jeon et al., 2002) because of their low molecular weight. Their use as dietary supplement in human food has become quite common, and for this reason, the study of the various characteristics of these bio-molecules is of great interest (Domard &Cartier, 1989).

There are two principal methods for the production of chitosan oligomers: acid and enzymatic hydrolysis (Zhang et al., 1999; Ohtakara &Izume, 1987). Acid hydrolysis is non-specific and leads to the formation of chitosan oligomers with a low degree of polymerization. They are generally monomers and oligomers with a low degree of polymerization, as well as polymers of high molecular weights. On other hand, the enzymatic hydrolysis of the chitosan by an enzyme such as a chitosanase makes it possible to produce oligomers of desirable range of polymerization, and the product of the hydrolysis is a mixture of oligomers of a narrow range of molecular weights (Chen et al., 2005; Shahidi &Abuzaytoun, 2005; Aiba, 1994a,b). Considering the interest for chitosan oligomers of specific molecular weights for food, nutraceutical and bio-pharmaceutical industries, effective separation technologies for their production are needed. For this purpose, chromatographic techniques are generally required but they are expensive. Alternative techniques such as preparative electrophoresis and membrane filtration are at the present time being studied for the production of bio-active molecules (Bargeman et al., 2002a; Nashabeh & El-Rasii, 1990). Hence, membrane techniques could be used at a large scale to selectively separate these bioactive ingredients from complex solutions by exploiting the interactions between the compounds and the membrane surface as demonstrated for peptides (Bargeman et al., 2002b) and for glycine solutions using electrodialysis (Shaposhnik et al., 2000). Also, the separation of the mixture of chitosan oligomers can be carried out by exploiting the difference between their electrophoretic mobility. This can be carried out by adjusting the pH and/or the ionic strength of the solutions and the operating conditions of the separation system which allows separation of small compounds with a high selectivity. An effective separation of chitosan oligomers by electrophoretic techniques requires a good understanding of their behavior under electric field (Shaposhnik et al., 2000; Nashabeh &El-Rasii, 1992; Nashabeh &El-Rasii, 1990). Thus, it is necessary to study the electrophoretic mobility of each oligomer in electrolytic solutions at different pH values and ionic strengths (Mechref et al., 1997; El-Rassi, 1996). The charged functional groups (NH3 +) in their structures (at some pH values below pKa) would theoretically enable their separation (Lagane et al., 2000) by exploiting their potential differential electrophoretic mobilities.

In this context, the goal of this study was to investigate the electrophoretic mobility of chitosan D-glucosamine and oligomers with degree of polymerization from 1 to 6 subjected to an electric field. We report here the effects of pH (2.0 to 9.0), type of salt (NaCl and KCl) and ionic strength (0.0 M to 0.1 M) on the electromigration of chitosan D-glucosamine and oligomers.

A charged molecule having an effective electric charge Z, placed in an electric field E, is subjected to an electrical force, F (Garbow et al., 2004; Lagane et al., 2000):

(4.1)

The molecule is subjected to acceleration but will not accelerate indefinitely because of retardation of the molecule experiences due to viscous forces that oppose the acceleration until a constant velocity (v) is reached. If the molecule is a sphere of radius, r, then the frictional force opposing its motion is given by Stokes law:

(4.2)

where η is the viscosity of the medium.

Balancing the electrical force acting on the charged molecule and the resistance force opposing the motion, we obtain (Cross & Cao, 1997):

(4.3)

From equations (4.1), (4.2) and (4.3), electrophoretic mobility μ can be written as:

(4.4)

where μ is the electrophoretic mobility of the charged molecule defined as the distance traveled by the charged molecule per unit time under unit electric field. From equation (4.4), it is evident that the electrophoretic mobility is proportional to its charge and inversely proportional to its size. The mobility is also affected by solvent medium characteristics such as viscosity and the presence of electrolytes due to ionic atmosphere surrounding the charged molecule (Marinova et al., 1996),and temperature of the medium (Li, 1993; Charlionet & Rivat, 1990; Babskii et al., 1989).

Figure 4.1 shows the electrophoretic mobilities of the chitosan D-glucosamine (monomer) and chitosan oligomers in water.

At pH 2, the dimer showed the highest mobility whereas there was no significant difference between the electrophoretic mobilities of the monomer, trimer, tetramer, pentamer and hexamer (P>0.05). At pH 3, the monomer showed a slightly lower mobility compared to the other molecules (P<0.04). At pH 4, the electrophoretic mobility of the dimer decreases considerably and its mobility highly increased at pH 5 compared with the others oligomers (P<0.001). The trimer, tetramer, pentamer and hexamer showed identical but lower mobilities. At pH 6, all oligomers migrated towards the anode. The monomer and the dimer showed the greatest electrophoretic mobilities. The mobilities of the trimer and the pentamer were identical but lower than those of the monomer and the dimer. At this pH, the tetramer and hexamer were quasi-motionless. At pH 7, the monomer showed a greater mobility. At pH 8, the monomer was always the most mobile compared to the other oligomers, and the hexamer was quasi-motionless. At pH 9, all molecules showed a migration towards the anode except the hexamer which was motionless. The monomer and the dimer were the most mobile.

The electrophoretic mobilities of chitosan D-glucosamine and oligomers were also measured in both NaCl and KCl at different ionic strength. Since there was no significant difference between the types of salt whatever the ionic strength, only results obtained with NaCl are reported here. Figure 4. 2 shows the electromigration behavior of the chitosan D-glucosamine and oligomers in NaCl 0.01 M.

At pH 2 and 3, all molecules showed identical electrophoretic mobilities (P>0.05). At pH 4, the monomer, trimer and pentamer migrated towards the cathode without significant difference between their mobilities, while the dimer and hexamer did not show any mobility. The tetramer migrated towards the anode with higher mobility than the other oligomers (P<0.001). At pH 5, only the dimer migrated towards the anode and at pH 6, the dimer showed a higher mobility than that of the hexamer (P<0.005). At pH 7, the dimer showed the highest electrophoretic mobility. By increasing the pH up to 9, the same phenomenon was observed. All the molecules migrated towards the anode and the dimer was the most mobile. The other chitosan oligomers migrated with identical electrophoretic mobilities (P>0.173). Figure 4. 3 shows the electromigration of the chitosan D-glucosamine and oligomers in NaCl 0.05 M.

At pH 2 and 3, D-glucosamine and all oligomers migrated with identical electrophoretic mobilities (P>0.778). At pH 4, the monomer, dimer and trimer migrated towards the anode without any difference between them. At this pH, electrophoretic mobilities of these molecules were significantly reduced. The tetramer, pentamer and hexamer migrated towards the cathode with identical but lower mobilities. At pH 5, the monomer, tetramer, pentamer were motionless while the dimer and trimer migrated towards the anode. At pH 6, the dimer showed the highest mobility. At pH 7, the pentamer showed a cationic behavior whereas the other molecules migrated towards the anode. At this pH, the dimer was the most mobile. At pH 8, chitosan D-glucosamine and all the chitosan oligomers did not show any mobility; and at pH 9, the dimer was more mobile and only the monomer showed mobility somewhat closer to that of the dimer. A significant decrease of the mobilities was recorded for all oligomers. In NaCl 0.1 M (Figure 4. 4), all the studied molecules showed identical mobilities at pH 2 and 3, whereas at pH 4, the dimer was motionless. At this same pH, the monomer, trimer, tetramer and pentamer showed a cationic behavior with higher mobilities compared with that of the dimer. With increasing pH (5, 6, 7, 8 and 9), chitosan D-glucosamine and all the chitosan oligomers showed identical electrophoretic mobilities.

The chitosan oligomers’ chain length had an effect on electromigration (P<0.013). In general, by increasing the degree of polymerization, the mobility decreased. Mobility of the monomer was different from that of the dimer; and their mobilities were significantly different from those of the other oligomers. Data analysis by the least squares means (Figure 4. 5) showed that there was no difference between the mobilities of the trimer, tetramer, pentamer and hexamer.

This means that the chain length had no impact on electromobility of the chitosan oligomers above the degree of polymerization of 3. Since the charge density is equal for all the oligomers, it is plausible that the mobility decreases with increase in the molecule size. However, the dimer showed the greatest mobility.

The data did not show any difference between the effect of NaCl and KCl presumably because the mean effective ionic diameter of Na+ and K+ are about the same (Cotton, 1999). In the contrary of the type of salt, ionic strength of the running medium affected the migration of the solutes. The electrophoretic mobility decreased as the ionic strength of the medium was increased. As the ionic strength of the medium is increased, the number of counter-ions around the migrating molecules increases. In the presence of an external electric field, migrating molecules move in one direction and the counter-ion atmosphere moves in the opposite direction, each carrying solvent molecules along with them. As result, the migration of the molecule is retarded by the screening effect of the counter-ions. This phenomenon was stronger when the chitosan oligomers migrated towards the anode with Na+ and K+ as counter-ions which are hydrated, but not with Cl- counter-ions which are not hydrated when the electromigration was towards the cathode. The highest values of the electrophoretic mobilities of the chitosan D-glucosamine and 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 solutions of salt with ionic strengths of 0.05 and 0.1 M, respectively, because the counter-ion screening effect is expected to be stronger at these conditions. This is in good agreement with the literature data (Mechref &El-Rassi, 1997; Mechref et al., 1997; El-Rassi, 1996).

The electrophoretic mobility experiments on the chitosan D-glucosamine and oligomers revealed that the pH had a significant effect on the behavior of the molecules when they are subjected to an external electric field. The pH determines the charge of the molecule and consequently, the direction of migration will be towards the cathode when the molecule carries positive charge and the migration will be towards the anode if the molecule carries negative charge. Generally, the absolute electrophoretic mobility of a charged particle submitted to an external electric field is directly proportional to the charge/mass ratio (Eq.4.4). With increasing medium pH, the electrophoretic mobility of the oligomers decreased and passed through zero value which corresponds to the isoelectric point of each molecule in that medium. Chitosan D-glucosamine and oligomers are positively charged in acid medium since the amine groups are protonated and their migration towards the cathode is expected. However, it was interesting to see that the chitosan D-glucosamine and oligomers migrated towards the anode at some pH values near the pKa of glucosamine (pH<7), where the amine groups were always protonated, and at pH values greater than the glucosamine pKa value, where the amine groups are unprotonated and the chitosan D-glucosamine and oligomers did not carry any electric charge.

It was hypothesized initially that anionic character acquired by the chitosan D-glucosamine and oligomers may originate from the glucose moiety. To confirm that glucose moity contribute to the electromigration of chitosan D-glucosamine and oligomers, electrophoretic measurements were carried out in various aqueous media (Figure4.6).

Measurements of the electrophoretic mobility of the glucose were carried-out in the same conditions as previously for chitosan D-glucosamine and oligomers. Glucose showed a cationic behavior at pH 2 and 3 in water (Figure 4.6), with higher mobility at pH 2. At pH 4, glucose did not show any mobility. Above pH 4, this molecule migrated towards the anode. By adding salt to the medium, glucose showed migration towards the cathode only at pH 2 and 3. As in water, with increasing pH, glucose migrated towards the anode. Also by increasing the ionic strength of the medium, the absolute value of the electrophoretic mobility of the glucose decreased considerably. This phenomenon was greater at the alkaline pH values.

While the results confirmed that glucose moiety may contribute to the electromigration of chitosan D-glucosamine and oligomers towards the cathode at lower pH conditions (since glucose can form oxonium ion at low pH conditions) and towards the anode at higher pH conditions, it was not clear regarding the origin of its migration towards the anode. It was further hypothesized that glucose migration towards the anode may arise from the difference between the dielectric constant of the medium and the migrating molecule. In order to verify this possibility, electromigration of glucose, glucosamine (monomer) and a mixture of chitosan oligomers composed of dimers, trimers and tetramer (1:1:1) were determined in water without salt added, NaCl (0.01 M) and NaCl (0.01 M)/Ethanol in the ratio of 50:50 (v/v) at pH 7.0. Table 4.1 shows the results obtained for these analyses. Without ethanol addition, the monomer, glucose and chitosan oligomers mixture migrated towards the anode at pH 7 (Table 4.1). But the addition of 50% (v/v) of ethanol to the medium eliminated its migration towards the anode. Under these conditions, monomer (glucosamine), glucose and oligomers were motionless.

The addition of ethanol to the medium, whose dielectric constant is lower than that of water (Arnaud, 2004; Barrow, 1988) caused disappearance of electromigration towards the anode at pH 7.0. Since the addition of ethanol decreases the dielectric constant of the medium, the difference between the dielectric constant of the solvent and the solute is lowered. Molecules with permanent dipole moments are oriented under the effect of external electric field and can have induced dipoles. The oriented high polar solvent molecules may exert an electrophoretic effect on less polar solute molecules, leading to their electromigration. Ethanol addition lowers the dielectric constant of the solvent medium and diminishes the electromigration of glucose and chitosan oligomers.

At low pH values, while the amine functions of the oligomers are protonated, electrophoretic mobilities of these molecules were not significantly different. This would be probably due to the charge/mass ratio which is the same one for all the oligomers (Eq.4.4). At higher pH values, the dimer showed the greatest electrophoretic mobility in water and in electrolytic solution with an ionic strength of 0.01 M. The higher oligomers showed comparable mobility. Under low potential field, convective diffusion can occur and contribute to overall mobility of the smaller molecules. This is much apparent in alkaline medium here the amine function was uncharged. This is probably due to the fact that the hydroxyl ions of the medium are able to reach the dimer more easily than the other oligomers and consequently, the ionic atmosphere around the dimer is greater in comparison with the others oligomers .

© Mohammed Aider, 2007