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Chapitre 6: Electroseparation of Chitosan Oligomers by Electrodialysis with Ultrafiltration Membrane (EDUF) and Impact on Electrodialytic Parameters

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Les résultats présentés aux chapitres 4 et 5 ont démontré qu’il serait possible d’exploiter les propriétés électrophorétiques des oligomères de chitosane pour les séparer dans un système d’électrodialyse combiné avec des membranes d’ultrafiltration. Cette approche repose sur le succès des travaux rapportés par (Labbé & Bazinet, 2006; Bazinet et al., 2005a, b) qui ont étudié l’électromigration de polyphénols de tabac et de catéchines de thé vert. Différentes membranes d’ultrafiltration ont été utilisées afin d’étudier la cinétique d’électromigration d’oligomères de chitosane et la possibilité des les séparer pour obtenir des frcations pures ou enrichies.

La planification de ce travail et la rédaction de cet article ont été réalisées par l’étudiant Mohammed Aider qui est l’auteur proncipal. Il a été supervisé par le Dr. Laurent Bazinet et la contribution de ce dernier au succès de ce travail a été d’une grande importance. Serge Brunet de ISM Biopolymer a contribué au succès de cet article par ses corrections et commentaires. Ils sont co-auteurs. Les résultats présentés dans ce chapitre seront soumis pour publication dans Journal of Membrane Science.

L'électrodialyse avec des membranes d'ultrafiltration (EDUF); une nouvelle technique de séparation à grande échelle de molécules organiques chargées a été utilisée. Dans cette partie de la thèse, l'effet de la taille de pores (MWCO) de membranes d'ultrafiltration empilées dans une cellule d’électrodialyse (ED) sur les taux d’électromigration des oligomères de chitosane contenus dans un mélange typique et sur les paramètres électrodialytiques du système a été étudié. Une différence de potentiels de 5 V, un pH 4 et une vitesse de circulation des solutions de 300 mL/min ont été utilisés. Les taux d'électromigration des oligomères de chitosane variaient entre 1.05 et 14.45% dépendamment des conditions opératoires, à savoir la taille des pores de la membrane utilisée ainsi que de la durée de traitement. Contrairement à la membrane d’ultrafiltration de 20000 Da qui était perméable à tous les oligomères de chitosane, la membrane avec une porosité de 500 Da n'a montré aucune perméabilité. La membrane d’ultrafiltration de 1000 Da a montré une rétention du tétramère pendant 3h de traitement, tandis que les membranes avec des tailles de pores de 5000 et 10000 Da avaient retenu le tétramère pendant les deux premières heures du traitement d’électroséparation. Dans cette étude, l'efficacité du système d’électrodialyse avec membrane d’ultrafiltration (EDUF) pour l'électroséparation des oligomères de chitosane a été démontrée.

The physiological activities and functional properties of chitosan oligomers depend on their molecular weight and chain length. Therefore, it is important to produce these bio-ingredients with a high degree of purity and homogeneity. Electrodialysis with ultrafiltration membrane (EDUF), a new method for separation of organic charged molecules at large scale, was tested. The effect of the molecular weight cut-off (MWCO) of the ultrafiltration membrane stacked in the electrodialysis (ED) cell was studied as well as the performances of the process in terms of oligomer migration rate and electrodialytic parameters. Electro-migration rates of the chitosan oligomers varied between 1.05 and 14.45% expressed as a percentage of the initial content in the feed solution depending on the operating conditions (membrane MWCO and processing time). Contrary to the 20000 Da MWCO UF-membrane which was permeable to all chitosan oligomers, the 500 Da MWCO UF-membrane did not exhibit any permeability. The 1000 Da membrane was selective to the tetramer during 3 h of operation, whereas the 5000 and 10000 Da membranes were selective to the tetramer during the first 2 hours of the treatment. It was demonstrated in this study the effectiveness of EDUF for the electro-separation of chitosan oligomers.

Keywords: Chitosan oligomers, Electrodialysis with ultrafiltration membrane (EDUF), Electromigration rate, conductivity, membrane fouling.

Chitosan oligomers are widely used in biotechnology, pharmaceutical and health food industry because of their bioactivity (Yamada et al., 2005; Zheng & Zhu, 2003; Shahidi et al., 1999; Yamada et al., 1993). Glucosamine, the monomer of chitosan, is widely used as an active molecule to promote the formation and repairing of cartilage, to reduce the progression of diseases like osteoarthritis, and significantly lessen pain from arthritis (Phoon & Manolios, 2002). Furthermore, oligomers of higher molecular weight had antitumoral, immunostimulating, antifungic and antimicrobial activities, and protect against pathogenic infections (Shahidi et al., 1999). It has also been demonstrated that the main physiological activities and nutraceutical properties of chitosan oligomers depend clearly on their molecular weight and chain length (Aiba, 1994a, b). However, in the two main methods usually used in the industry for chitosan oligomer production, acid hydrolysis and enzymatic hydrolysis (Kim & Rajapakse, 2005; Aiba, 1994a, b; Pelletier & Sygusch, 1992; Rogozhin et al., 1988; Bosso et al., 1986; Rupley, 1964) the final product is a mixture of molecules of different molecular weights and contains minerals (Domard & Cartier, 1989; Rogozhin et al., 1988; Horowitz et al., 1957).

Recently, in fundamental studies on chitosan D-glucosamine and oligomers electrophoretic mobility, it was demonstrated that these molecules migrated under the effect of an external electric field, according to the solution conditions, mainly pH (Aider et al., 2006a, b). Under pH conditions of 4, these molecules are positively charged because of the protonation of the amine group. Furthermore, the electrophoretic mobility of the dimer was higher than the one of trimer and tetramer due to their differences in molecular weight and electric charge. The exploitation of the electric properties of these molecules could offer a solution to separate mixtures of chitosan oligomer into pure or enriched fractions.

A new alternative method to separate bio-molecules into more homogeneous fractions and to demineralise them was recently developed. This new method is named electrodialysis with ultrafiltration membrane (EDUF). It is a hybrid process which combines conventional electrodialysis and ultrafiltration membranes. The ultrafiltration membrane is stacked as a molecular barrier in an electrodialysis cell and the driving force of the process is an external electric field. This new separation method was successfully used for tobacco polyphenol separation (Bazinet et al., 2005a,b), green tea catechin separation (Labbé et al., 2005) and for bio-active peptide purification (Poulin et al., 2006a, b).

The aim of the present work was to evaluate the effectiveness of EDUF process for separation and purification of chitosan oligomers. The effect of the molecular weight cut-off of the ultrafiltration membrane stacked in the electrodialysis (ED) cell was studied as well as the performances of the process in terms of oligomer migration rate and electrodialytic process parameters.

In the KCl compartment (Figures 6. 2), the general trend was the same for all UF-membranes.

The pH decreased drastically during the first 30 min as the electroseparation process progressed and then decreased slowly in a constant way. However, the variations in pH were not the same according to the MWCO of the membrane stacked. With 500 Da MWCO membrane, a total pH variation of 2.83 pH units was observed. The decrease in pH was similar (P>0.05) in the EDUF systems stacked with 1000, 5000 and 10000 Da MWCO UF-membranes for a global pH variation of 2.46 pH units, but different from the EDUF system stacked with the 500 Da MWCO UF-membrane. The pH evolution of the KCl compartment, when a 20000 Da MWCO UF-membrane was stacked in the ED cell, was significantly different from all the other MWCO with a pH variation of 1.22 pH unit.

In the chitosan oligomer compartment, the tendency was also the same for all UF-membranes used (Figures 6. 3).

The pH increased as the electro-separation process progressed and the pH variations between the beginning and the end of the process increased with a decrease in MWCOs. From the initial pH value of 4.0 ± 0.21, variations of 1.01, 0.97, 0.85, 0.69 and 0.49 pH units were observed for 500, 1000, 5000, 10000 and 20000 Da MWCO, respectively.

The differences in pH evolution of the chitosan oligomer solution observed between the MWCO could be explained by the differential electro-migration rates of the oligomers through the UF-membranes. Hence, when a 500 Da MWCO UF-membrane was stacked in the ED cell, the significant decrease of pH in KCl compartment or pH increase in the chitosan oligomer solution was caused by the fact that most part of the electric current through UF-membrane was transported by the protons because the membrane would be not permeable to the chitosan oligomer; due to its low MWCO (500 Da), this membrane could be classified as nanofiltration one (Hilal et al., 2005). With 20000 Da MWCO UF-membrane, the contribution of the protons (H+) to the transport of electric field through this membrane was small, and consequently, the variations of pH were also the lowest. In the case of the other MWCO membranes, the variations were intermediate because their selectivities to the oligomers were higher than that of 500 Da but lower than that of the 20000 Da. A similar behavior with 1000 Da MWCO UF-membrane was reported on green tea brewing by Labbé & Bazinet (2006).

The acidification in the KCl compartment was caused by the H+ ions migrated from the chitosan oligomer solution compartment. However, the pH decrease in the KCl compartment was not equal to the pH increase in the chitosan oligomer solution compartment. The explanation will be as follows: the chitosan oligomer solution compartment was separated from the anodic electrolyte (NaCl solution) compartment by an anion-exchange membrane (Figure 6. 1). By applying an external electric field to the EDUF system, a drastic decrease of the pH in this compartment was observed because of electrochemical reactions at the anode. Within 10 min from the beginning of the EDUF process, the pH decreased from 5.33 ± 0.11 to 2.86 ± 0.07 in the anodic compartment. This phenomenon generates a lot of H+ ions. Even if theoretically the anionic exchange membrane is not permeable to the cations (H+ ions), proton escape is extremely probable because of the H+ amount generated in a few minutes. Cation, and especially H+ ions leakage through anionic exchange membrane is possible and was reported in the litterature (Labbé et al., 2005)in the study of catechin electro-migration process. The hydrogen ions could migrate easily towards the KCl compartment through UF-membrane (Figure 6. 1). Their migration could be stopped by the second anion-exchange membrane in contact with the KCl compartment. Consequently, the KCl compartment was enriched by H+ ions that came at the same time from the chitosan oligomer solution and from the anodic compartment. Combination of these H+ ions contributed to this pH decrease in the KCl compartment.

In the KCl compartment, the electrical conductivity increased with time (Figure 6. 4).

The average initial value of the electrical conductivity in KCl compartment was 3.33 ± 0.20 mS.cm-1. With UF-membranes of 500, 1000, 5000 Da MWCO, the increase in conductivity was statistically the same (P>0.05). At the end of the process, the average value reached a mean value of 6.05 ± 0.71 mS.cm-1. In the case of 10000 and 20000 Da MWCO UF-membranes, the increase in electrical conductivity was similar but different from lower MWCOs, and at the end of the EDUF process, an average value of 4.81 ± 0.38 mS.cm-1 was reached.

At the same time, electrical conductivity decreased linearly as a function of time in the chitosan oligomer solution (Figure 6. 5). However, the tendency was the same for all MWCO UF-membranes (P>0.063). Electrical conductivity decreased linearly with time and may be described by the following equation with R2= 0.8887, where: κ is the electrical conductivity (mS.cm-1), and τ is the process time, (min).

For the 1000, 5000, 10000 and 20000 Da MWCO UF- membranes stacked in an EDUF system, the increase in electrical conductivity in the KCl compartment was caused by the migration of different ionic species towards this compartment; chitosan oligomers and H+ ions migrated from the chitosan oligomer solution, and anions from NaCl compartment migrated through the anion-exchange membrane to maintain electro-neutrality in the KCl compartment. As a consequence, the KCl compartment was enriched by different ionic species and chitosan oligomers throughout the process. This contributed to an increase in the overall electrical conductivity of this compartment while the chitosan oligomer solution was being demineralised. In the case of EDUF system combined with 500 Da MWCO UF-membrane, the electrical conductivity increase was caused by the migration of H+ ions from chitosan oligomer solution compartment through UF-membrane and ions from NaCl compartment through anionic membrane to keep the electro-neutrality in KCl compartment. These results are in accordance with the cell configuration and the observations reported by Labbé et al. (2005) during electromigration of catechins from green tea brewand Poulin et al. (2006a)during EDUF of bioactive peptides from β-lactoglobulin hydrolysate.

The increase of the electrical conductivity in the KCl compartment was higher than the decrease of the conductivity in the chitosan oligomer solution. This could be explained as follows: during the electro-separation treatment, we noted that in the KCl compartment, the pH decreased with time, whereas it increased in the chitosan oligomer compartment. In order to maintain the electro-neutrality in the two compartments, anions must migrate to the KCl compartment, and others must leave the compartment of the chitosan oligomer solution. Consequently, the KCl compartment was enriched by H+ ions, which migrated from the chitosan oligomer compartment for the maintenance of the electroneutrality. Conductivity in the KCl compartment was highly enhanced through major contribution of the H+ ions (Koneshan et al., 1998), the most mobile and electrical conductive ionic species, and anions, which contributed to maintain the solution electroneutrality.

The evolution of system global electrical resistance for a 500 Da MWCO UF-membrane stacked in an ED cell was different from the other MWCO membranes (P<0.001). With a 500 Da MWCO UF-membrane, the initial global system electrical resistance was 158.01 ± 24.57 Ω, decreased to 140.50 ± 18.42 Ω after 1 h of treatment, and remained quite constant at a mean value of 135 ± 2 Ω until the end of the EDUF process (Figure 6.6). For 1000, 5000, 10000 and 20000 Da MWCO UF-membranes, the general tendency was similar, but the variations were different. Data analysis showed that for 1000 and 20000 Da MWCO stacked in the ED cell, the global system resistance evolution were not significantly different (P>0.05), but were different from 5000 and 10000 Da MWCO UF-membranes. The total resistance of each system decreased as the process time progressed. In the case of the 1000 and 20000 Da MWCO UF-membranes, the system electrical resistances decreased linearly from 91.81 ± 12.22 and 89.38 ± 12.87 Ω at the beginning, respectively to 69.47 ± 6.08 Ω and 64.61 ± 6.60 Ω at the end of the electro-separation treatment. EDUF cells stacked with 5000 and 10000 Da MWCO UF-membranes showed higher system electrical resistances at the start with mean values of 128.73 ± 48.11 Ω and 113.66 ± 4.45 Ω, respectively which decreased to mean values of 80.56 ± 23.38 Ω and 78.75 ± 5.07 Ω at the end of process. Global system electrical resistances with these latter two MWCO UF-membranes were somewhat different from each other (P<0.025).

The system stacked with 500 Da MWCO UF-membrane showed a greater and stable resistance during the process compared with the other EDUF systems due to its lowest electrical conductivity (Table 6. 1). With other membranes (1000, 5000, 10000 and 20000 Da MWCO), the lower initial global system resistance can be explained by their higher intrinsic electrical conductivity (Table 6.1). While during the process the decrease in the total electric resistance of the system can be related to an increase in the conductivity of the KCl compartment, following the migration of the H+ ions; this phenomenon caused a decrease in the pH of this same compartment as well as to the migration of chitosan oligomers through UF membranes. These oligomers contributed to increase the electrical conductivity in the KCl compartment and consequently to the decrease of the total system resistance. A similar observation was reported in the litterature (Labbé et al., 2005)on the electromigration of green tea catechin where a 1000 Da MWCO membrane from the same company as that used in the present work was used.

Membrane fouling evaluation was based on the analysis of the data on the changes of the electric conductivity and thickness of each ultrafiltration membrane before and after electrodialysis with ultrafiltration membrane treatment (Tables 6. 1 and 6. 2).

Comparisons between the results of repetitions for each treatment were also used to evaluate the integrity of the membranes. In the case of the 500 and 1000 Da MWCO UF-membranes, significant difference of their electrical conductivity before and after EDUF treatment was found (P < 0.001). This could be caused by an accumulation of some chitosan oligomers inside or on the membrane. In the case of the other UF-membranes, there was no significant difference between electrical conductivity and thickness before and after treatments (P > 0.05). It means that membrane fouling was weak and could be removed by rinsing or by storing the membranes in distilled water between repetitions. The same observation was done by Poulin et al. (2006a)on 20 kDa UF-membranes after electroseparation of peptides where membranes of the same type as in our work were used. For the anion-exchange membranes used during the EDUF treatments, comparison of their characteristics (thickness and electrical conductivity) before and after 4 h of electroseparation treatment showed that no significant difference in fouling was detected (P>0.129). These results indicated that there was no fouling of the anion exchange membranes in both compartments (KCl and chitosan oligomers solutions) (Tables 6. 1 and 6. 2).

The ultrafiltration membrane MWCO (P<0.001), processing time (τ) (P<0.001), as well as the interaction of the two parameters MWCO*time (P<0.001), had significant effect on the electromigration rates of the chitosan oligomers. At the same time, chitosan oligomers chain length had also a significant effect (P<0.001) on the electro-migration rates.

MWCO of the UF-membrane was the primary factor affected on the electromigration rate of the chitosan oligomers. When the 500 Da UF-membrane was used, no electromigration was observed throughout the treatments and that whatever the oligomer, while for UF-membrane with higher MWCO, the electro-migration of the chitosan oligomers was effective but different according to the MWCO, the chain length and also the duration of the process. In the case of the 1000 Da MWCO UF-membrane, during the first three hours of treatment, only the dimer and trimer migrated through this membrane while the tetramer was detected in the KCl compartment only at the end of the electro-separation process (Figure 6. 7).

The dimer showed a linear electromigration kinetic throughout the process. The electromigration rate of this chitosan oligomer was 0.58 ± 0.05% at τ = 1 h, and increased linearly with time to reach a value of 5.71 ± 0.95% at the end of the treatment. At the same time, the trimer also showed a linear electromigration kinetic behavior. Its electro-migration rate was 0.54 ± 0.15% at τ = 1 h, then increased linearly up to 1.93 ± 0.73% and 2.29% ± 0.12% at τ = 2 and 3 h, respectively. At the end of the process, the electro-migration rate of the trimer reached a mean value of 3.37 ± 0.98%. The tetramer was detected in the KCl compartment only after 4h of treatment and presented an electro-migration rate of 1.62 ± 0.93%. When the EDUF cell was stacked with a 5000 Da MWCO UF-membrane ((Figure 6. 8), the dimer and trimer showed also a linear electro-migration behavior similar to the one observed with the 1000 Da MWCO UF-membrane. The dimer and the trimer migrated from the beginning of the process, but the tetramer was detected in the KCl compartment only after 2 hours of the EDUF treatment. When 5000 Da MWCO UF-membrane was used, the dimer electro-migration rate increased linearly from 0.58 ± 0.18% of its initial content in the feed solution at τ = 1 h to up to 5.66 ± 0.29% at τ = 4 h (Figure 6. 8). The trimer showed electro-migration kinetics with a linear behavior with an electro-migration rate which passed from 0.33 ± 0.03% at τ = 1 h up to 2.89 ± 1.17% at the end of the process. No tetramer was detected during the first two hours of the treatment. The tetramer was found in the KCl solution at τ = 3 and 4 h with electro-migration rates of 0.82 ± 1.41% and 3.37 ± 1.56%, respectively.

During EDUF process with 10000 Da MWCO UF-membrane, no retention of both the dimer and the trimer was observed throughout the treatment (Figure 6. 9). The electro-migration rate of the dimer was 2.40 ± 1.12% at τ = 1 h and increased linearly as the process progressed to reach an average final value of 9.91 ± 1.37% at the end of the EDUF treatment. The electromigration rate of the trimer was 0.94 ± 0.34% at τ = 1 h and increased with time to reach a mean value of 7.50 ± 0.99% at τ = 4 h. When 10000 Da MWCO UF-membrane was used, at the contrary of the dimer, the electromigration kinetic of the trimer as a function of time showed an exponential behavior. The inflexion point of the electromigration curve was recorded after τ = 2 h. For the tetramer, data showed that this chitosan oligomer did not migrate during the first two hours of electroseparation treatment. Thereafter, at τ = 3 and 4 h, analyses of KCl solution showed electro-migration rates for the tetramer of 6.01 ± 0.82 and 6.52 ± 1.19%, respectively.

When the 20000 Da MWCO UF-membrane was stacked in the EDUF cell, the electromigration rate for all the chitosan oligomers increased with time (Figure 6. 10). From the start of the EDUF treatment, this membrane showed the highest electromigration rates of chitosan oligomers and was significantly different from the other membranes (P<0.001). The dimer electromigration rate was 3.39 ± 1.23% at τ = 1 h and linearly increased up to 14.45 ± 1.43%, at the end of the electroseparation treatment. On the other hand, electro-migration kinetic evolution of the trimer showed an exponential behavior. Its electro-migration rates were smaller than those of the dimer. The mean value of the trimer electro-migration rate was 1.15 ± 0.48% at the first hour and increased exponentially up to 12.19 ± 0.97% at the end of the EDUF process. In this case, the time-dependency electro-migration curve inflexion point was recorded at τ = 3 h. At the same time, the tetramer electro-migration rate increased from 0.85 ± 0.76% at the first hour of the treatment up to 11.58 ± 0.98% at the end of the process. The time dependency of the electro-migration rate of this chitosan oligomer (tetramer) showed also an exponential behavior and the inflexion point was recorded at τ = 3 h, similar to that of the trimer.

It appeared from these results that the 500 Da MWCO membrane was not permeable to any chitosan oligomer throughout the electroseparation treatments because of its MWCO which seemed to be smaller than the chitosan oligomer molecular hydrated shield and its high intrinsic electrical resistance as shown in Table 6. 1. As expected, chitosan oligomers electromigration rates increased by increasing membrane MWCO, however, the selectivity of the membranes decreased. The 1000 Da membrane was permeable to the dimer and trimer because of its MWCO and low electric resistance which supported the electromigration of these two molecules. At the same time, this membrane (1000 Da) was not permeable to the tetramer because of its MWCO which was close to the molecular weight of the tetramer of approximately 800 Da. In the case of the 5000 and 10000 Da UF-membranes, which MWCOs were favourable to electromigration of all oligomers, retention of the tetramer was noticed during the first two hours of the electroseparation treatment. This was probably due to the lower electrophoretic mobility of the tetramer compared to that of the dimer as it was shown in our previous study (Aider et al., 2006a) and also because of the high competition exerted by the other oligomers which are more concentrated in the feed solution. A 20000 Da MWCO membrane did not show any retention of the three oligomers. This is in good agreement with the general law of the membrane processes where the flow rate is proportional to the membrane pore size (Labbé & Bazinet, 2006; Hilal et al., 2005; Khayetet al., 2005). In the same context, Bargeman et al. (2002b) reported that during electro-ultrafiltration of casein hydrolysate, the transport rate of αs2-casein hydrolysate (fraction 183-207) was dependent on the MWCO of the UF membrane used. It was reported that for a MWCO of 10 kDa which is approximately 3 times the molecular weight of the αs2-casein fraction (f-183-207), the transport rate was more than a factor 3 lower than for the membranes with higher MWCO. This phenomenon was attributed to the higher friction of the peptide in the membrane pores when low MWCO membranes were used (Bargeman et al., 2002b). This friction resulted in a retardation of the peptide transport through the ultrafiltration membrane. When the MWCO used exceeded 20 kDa (approximately 6 times the molecular weight of αs2-casein fraction ( f-183-207), it was reported that this friction plays a less important role and the peptide migration through the membranes was higher (Bargeman et al., 2002b).

According to the previous results and the two general tendencies observed for the chitosan oligomer electro-migration kinetics, a linear kinetic through all the duration of the EDUF process or an exponential kinetics, a model could be proposed according to the chain length and the MWCO of the UF membrane used.

In the case of the dimer, for MWCO UF-membrane >500 Da used, the time-dependent increase of its electro-migration rate results in linear kinetics. The linearity would be due to the fact that, the dimer is not submitted to any significant resistance of the medium and it could freely migrate through all the ultrafiltration membranes used without very limited friction. The interaction, especially the friction with the UF-membrane is minimal because of its low molecular weight and its spatial conformation (Figure 6. 11a). The electro-migration flux of the dimer expressed by its migration rate through the UF-membrane would be only dependent on its electrophoretic mobility. Also, as a general tendency, the dimer electro-migration rate increased with an increase of the UF-membrane MWCO. However, some similarity was observed for the electro-migration rates of the dimer when 1000 and 5000 Da MWCO UF-membranes were used. This was probably due to the high global electrical resistance of the EDUF cell stacked with the 5000 Da MWCO UF-membrane compared to the system with a 1000 Da one, even if the porosity of the first 5000 Da membrane is theoretically 5 times greater than that of the 1000 Da MWCO. This is in a good agreement with some conclusions hypothesized by Bazinet et al. (2007) on the role of membrane material on electrical properties of the UF-membranes stacked in EDUF.

In the case of the trimer the electromigration behaviour was dependent to the MWCO of the UF-membrane used for MWCO > 500 Da. When 1000 and 5000 Da MWCO UF-membranes were used, the time-dependent increase of the respective electro-migration rates result in linear kinetic while when 10000 and 20000 Da MWCO UF-membranes were used, the trimer showed an exponential electro-migration behavior (positive non linear kinetic). In the case of the 1000 and 5000 Da, the linear kinetics recorded would be due to the fact that the general flux or electromigration phenomena was low, in relation with the steric restriction (obstruction) exerted by these membranes and the lower electrophoretic mobility of the trimer in comparison with the dimer. Another factor for low and linear migration would be the accumulation of this oligomer at the interface of the membrane, due to its hydrated radius or conformation. The migration of the trimer through the pore of the membrane would be linked to its conformation when arriving at the interface of the membrane; perpendicular or parallel (Figure 6. 11b). When arriving perpendicular, no restriction to its migration was present (Flux = J 1 ), while when parallel, its migration would be blocked (Flux ≈ 0). However, under the influence of the electric field, the parallel form could be changed to intermediate distorted forms allowing its very slow migration into the pore due to the friction (Flux = J 2 << J 1 ) after a certain time. This accumulation of parallel oligomers or the slow down migration of stretched forms would explained the low migration rate and the decrease in the electrical conductivity of the 1000 Da MWCO UF-membrane after treatment by about 24.57% of its initial value (Tables 6. 1 and 6. 2). For membrane with MWCO > 5000 Da, the exponential kinetic could be the result of a decrease in friction for the distorted forms due to the higher pore size, facilitating its migration through the membrane after a certain time (Figure 6. 11c); the J 1 migration rate would be due to the perpendicular form and the J 2 migration rate would be due to the distorted forms. The distorted form migrated after the deformation of the parallel form. The following same explanations could be applied to the tetramer, with an increase in the phenomena due to its larger hydrated radius, and the more numerous possibility of conformations and consequently of distorted forms due to the presence of 4 monomers on the structure

To complete the model explanation, another important point has to be underlined. Indeed, the analysis of the data of the electro-migration rate of the trimer showed that while using a 10000 Da MWCO UF-membrane, the electromigration rate recorded after 2 h of treatment was equivalent to that recorded with a 1000 Da MWCO UF-membrane but after 4 h of treatment. The same observation was reported in the case when a 20000 Da MWCO UF-membrane was used after 2 h of treatment compared with the membrane of 5000 Da MWCO but after an EDUF treatment of 4 h. It would be probable that the linearity of the electro-migration kinetics of the trimer recorded with 1000 and 5000 Da MWCO UF-membranes is only temporary because it well corresponds to the linear section of exponential recorded with the two other membranes (10000 and 20000 Da) whose pores are theoretically 4 to 5 times larger.

These results showed that chitosan oligomers of low molecular weight can migrate through UF-membranes of different MWCO under the effect of an external electric field. MWCO of the UF-membranes used, time duration of the EDUF process, interaction of these two parameters as well as chitosan oligomers chain length have significant effect on electro-migration rates of each oligomer. 500 Da MWCO UF-membrane was not permeable to the chitosan oligomers during the whole treatment, whereas the 20000 Da MWCO UF-membrane was permeable to all chitosan oligomers at any time. 1000 Da MWCO UF-membrane was permeable to the dimer and trimer during the first 3 h of treatment while the tetramer was found in the adjacent migration compartment only at 4 hours of the electro-separation process. 5000 and 10000 Da MWCO UF-membranes were not permeable to the tetramer only during the first 2 h of the EDUF treatment. The dimer showed a linear electro-migration kinetic in all cases. The electro-migration behavior of the trimer was linear when 1000 and 5000 Da MWCO UF-membranes were used and showed an exponential electro-migration behavior when the EDUF cell was stacked with 10000 and 20000 Da MWCO UF-membranes.

Results suggest that it is possible to separate chitosan oligomers by a combination of UF-membranes intrinsic parameters and electro-separation duration as well as differences between electrophoretic mobilities of the chitosan oligomers. The 10000 Da MWCO UF-membrane appeared to be the best membrane to carried-out separation of the tetramer and an increase in the membrane surface would increase the total quantity of both oligomers (dimer and trimer) migrated in the same laps of time.

EDUF method could be used as a powerful process for the separation of bioactive chitosan oligomers of interest from complex feed solution under mild conditions, and other applications in food, bio-pharmaceutical, and nutraceutical industries. However, further investigations are necessary to understand the intrinsic properties of the UF-membranes affecting electro-migration of chitosan oligomers even if the MWCO of membranes are large enough to permit transport of these molecules.

© Mohammed Aider, 2007