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Chapitre 8: Effect of Solution Flow Velocity and Electric Field Strength on Chitosan Oligomer Electromigration Kinetics and their Separation in an Electrodialysis with Ultrafiltration Membrane (EDUF) system

Table des matières

Suite aux résultats présentés au chapitre 7, l’efficacité de l’électrodialyse combinée avec des membranes d’ultrafiltration à séparer un mélange d’oligomères de chitosane a été améliorée par la combinaison du pH du milieu et du temps de traitement.

Il nous a paru pertinent d’étudier l’effet du champ électrique qui est la force motrice du procédé et la vitesse de circulation (débit) des solutions sur le taux d’électromigration des oligomères de chitosane et la possibilité d’exploiter les résultats pour les séparer. Basée sur les résultats obtenus aux chapitres 6 et 7, une membrane d’ultrafiltration avec un seuil de coupure (MWCO) de 10000 Da a été utilisée. Dans cette partie de la thèse, le pH du milieu a été fixé à 4 et le temps de traitement à 4h.

La planification de ce travail et la rédaction de cet article ont été réalisées par l’étudiant Mohammed Aider qui est aussi auteur principal. Il a été supervisé avec beaucoup d’attention par le Dr. Laurent Bazinet qui a contribué au succès de ce travail par sa grande implication. Serge Brunet de ISM Biopolymer a contribué au succès de cet article par ses corrections et commentaires. Ils sont co-auteurs de ce travail. Les résultats présentés dans ce chapitre sont en attente de soumission dans Journal of Membrane Science.

Dans cette étude, l'effet de la vitesse de re-circulation des solutions dans chaque compartiment et du champ électrique appliqué à la cellule d'électrodialyse avec membrane d’ultrafiltration sur les taux d'électromigration des oligomères de chitosane et l'impact de ces facteurs sur la possibilité de séparer les oligomères ont été étudiés. Les résultats ont montré que la vitesse de re-circulation des solutions n'avait pas d’effet significatif sur le taux d’électromigration de chaque oligomère, alors que le champ électrique avait un effet significatif à la fois sur le taux d’électromigration de chaque molécules ainsi que sur la possibilité de les séparer. En appliquant un champ électrique de 2.5 V/cm avec une durée de traitement de 2h, il était possible d’obtenir une solution composée uniquement du dimère et du trimère. En augmentant la valeur du champ électrique jusqu'à 5 et 10 V/cm, il n'était pas possible de séparer les oligomères de chitosane. Dans ce travail, les nombres de transport des oligomères de chitosane ont été mesurés. Ces molécules contribuent à transporter 7 à 9 % du courant électrique total. Les membranes utilisées ont gardé leur intégrité et aucun colmatage significatif n'a été détecté.

The aim of the present work was to study the effect of solution flow velocity and electric field strength applied to an electrodialysis with ultrafiltration membrane cell on chitosan oligomer electromigration kinetics and on chitosan oligomer mixture fractionation. It was shown that the effect of solution flow velocity was not significant, while the electric field strength showed a significant effect on each chitosan oligomer electromigration rate. The effect of the electric field strength was also significant on the separation possibility of the studied oligomers. It was shown that by using electric field strength of 2.5 V/cm, it was possible to obtain a solution composed only of the dimer and trimer until an operating time of 2 h. By increasing the electric field up to 5 and 10 V/cm, it was no more possible to separate the chitosan oligomers. Chitosan oligomer transport numbers were measured. It was found that they contribute to about 7% of total electric current carrying, while about 93% of the total electric current could be carried by the electrolytes. The system performance was also evaluated through electric field intensity measurement and membrane integrity evaluation. It was found that the membranes kept their integrity and no significant fouling was detected.

Keywords: Chitosan oligomers; Electroseparation; Electromigration; Transport number.

Electrokinetic separation process is one of the most promising in-situ technologies for the removal, separation and purification of bioactive charged molecules from complex solution made of charged and non-charged components (Nagarale et al., 2006; Bazinet, 2004; Bazinet et al., 1998). A separation by an electrokinetic phenomenon takes place when an external electric field is applied to a system in which a porous barrier was introduced (Lee & Hong, 2000). In such system, electrophoresis becomes the principal electrokinetic phenomenon for charged molecules migration (Eyal & Bressler, 1993). Based on physicochemical and electrokinetic properties of solutes in the feed solution, some of these molecules could be removed from the feed solution in order to obtain pure or enriched fractions (Loh & Moody, 1990).

The electrokinetic processes were successfully applied to a variety of molecules. It was reported that organic molecules such as benzene, toluene, ethylene, xylene and trichloroethylene, could be moved using electrokinetic processes (Thang et al., 2005; Yi et al., 2005). The electro-transport and removal of polar organic molecules such as phenols, acetic acid, butyric, valeric, adipic, caproic and oxalic acids have been also investigated (Wang et al., 2006; Nagarale et al., 2004; Novalic et al., 2000). In food and biotechnology fields, several authors successfully used hybrid electrokinetic processes for separation and purification of bioactive molecules. Hence, it wasreported that an electrophoretic membrane contactor was used for electroseparation of binary mixtures made of poly(L-glutamic) acid, α-lactalbumin and bovine haemoglobin (Galier & Roux-de Balmann, 2004). They showed that solute flux through the ultrafiltration membrane used increased by increasing the product of electric field strength and the residence time. In this study, the solution flow velocities used were fixed at 1.66 and 83.33 mL/min, respectively. Cellulose ester ultrafiltration membrane was stacked in an electrodialysis cell for simultaneous separation of bioactive peptides produced by an enzymatic hydrolysis of β-Lactoglobulin (Poulin, 2007; Poulin et al., 2006a,b). It was shown that with one ultrafiltration membrane, increasing the overall applied voltage by a factor 2 and 4 resulted in increasing of the final peptide concentration by the same factor. Concerning the solution flow velocity (rate) effect, the following values were used: 100, 150, 200 and 250 mL/min (Poulin, 2007). The author showed that the solution flow velocity had no effect on the total peptide migration in the permeate solution. However, at the highest value tested, the selectivity of the process was influenced and the migration of some peptides was lower at 250 mL/min.

Chitosan oligomers are bioactive molecules. They are widely used for different purposesin biotechnology and medicine (Vishu-Kumar et al., 2005). It was demonstrated amongst others that chitosan oligomer tetramer has a significant effect to inhibit the adhesion of three strains of enterophathogenic Escherichia coli up to 30% of the level of adhesion seen in the controls (samples without chitosan oligomer) (Rhaodes et al., 2006). Some electrokinetic behaviors of chitosan oligomers (electrophoretic mobility) under different conditions have been studied (Aider et al., 2006a). Because of their cationic behavior in an acidic medium (Aider et al., 2006a,b), electrodialysis with ultrafiltration membrane (EDUF) procedure was successfully used for chitosan oligomer mixture separation (Aider et al., 2007a,b).

In a context of EDUF optimization for separation of chitosan oligomers, the aim of the present work was to study the effect of feed solution flow velocity and applied voltage to the electrodialysis with ultrafiltration membrane (EDUF) cell on the electromigration kinetics of the chitosan oligomers and impact on their separation possibility in order to produce pure or enriched fractions.

Electroseparation by electrodialysis with ultrafiltration membrane (EDUF) treatments were carried-out on 200 mL of chitosan oligomer solution in a batch process at three applied voltages (5, 10 and 20 V) which corresponded to three average electric field strengths of 2.5, 5 and 10 V/cm. Solutions flow velocities of 2.77, 8.33 and 13.88 cm/s which corresponded to 100, 300 and 500 mL/min, respectively, were used. The chitosan oligomer mixture solution was obtained by dissolving chitosan oligomer mixture in HPLC grade water to obtain a final concentration of 3% (w/v). Its initial pH was adjusted to pH 4 by adding 1 N HCl. This pH value was selected following previous work on the effect of the pH value on chitosan oligomer electrophoretic mobility (Aider et al., 2006b). The pH of the 200 mL KCl solution in the adjacent compartment was also adjusted and was the same one as in the chitosan oligomer solution compartment. Samples of chitosan oligomer and KCl solutions were drawn at the beginning of the process before applying the external electric field and every 60 min. during the treatment. EDUF was stopped after 4 h of treatment. During treatment, pH and conductivity values were recorded in the KCl and chitosan oligomer solutions. Measurements of the current intensity were used for the calculation of the total system resistance. Thickness and electrical conductivity of the membranes were measured before and after each treatment to check for their integrity and verify their potential fouling. All samples were analysed by HPLC to determine the electromigration rate of the three chitosan oligomers.

Statistical analysis of the data was carried-out to check the degree of significance of the studied variables; applied average electric field (E) and solution flow velocity (d), as well as their interaction on the chitosan oligomer electromigration rate. Analysis of the data revealed that the interaction E*d was not significant (P>0.381). At the same time, data analysis showed that the effect of the solution flow velocity was not significant (P>0.785). The effect of the applied average electric filed (E) was significant (P<0.001). Because of the non significant effect of the solution flow velocity, the presented data on the electromigration rate of each chitosan oligomer as function of the processing time and applied voltage will be the averaged values obtained at the studied three flow velocities. The electromigration rate of each oligomer was expressed as a percentage of the total amount contained in the feed chitosan oligomer mixture solution.

Dimer electromigration rate as function of the processing time and applied voltage is shown in Figure 8.2. Independently of the applied voltage, the dimer electromigration rate showed linear kinetics. By applying 2.5 V/cm to the cell, the dimer electromigration rate behavior was correlated to a linear equation by a regression coefficient of R2=0.9668 and a slope of 2.36 representing the speed of the dimer electromigration through the 10000 Da cellulose ester ultrafiltration membrane used. At the end of the electroseparation process using 2.5 V/cm, the dimer electromigration rate reached 9.92 ± 2.45%. The dimer electromigration rate increased by increasing the applied electric field strength (voltage). At 5 and 10 V/cm, the dimer electromigration behavior was correlated to linear equations by regression coefficients of R2=0.9842 and 0.9974, respectively. The slopes of the corresponding equations were 6.54 at 10 V and 11.80 at 20 V. Electromigration rates at the end of the process were 26.79 ± 0.51 and 47.86 ± 3.12% when 5 and 10 V/cm, respectively.

Trimer electromigration rate evolution as a function of the processing time and applied voltage to the electrodialysis with ultrafiltration membrane (EDUF) cell is shown on Figure 8.3. At 2.5 V/cm, the trimer electromigration rate kinetic was correlated to a curvilinear function with a regression coefficient R2=0.9909. The average electromigration rate at the end of the electroseparation in this electric field condition reached a mean value of 8.58 ± 1.03%. When a voltage of 5 or 10 V/cm was used, the electromigration rate kinetic of the trimer followed a linear behavior. At 5 V/cm, the electromigration kinetic of the trimer was correlated to a linear function with a coefficient R2=0.9919 and a slope of 5.13. Electromigration rate was 20.28 ± 1.56% at τ = 4h. In the case when a voltage of 10 V/cm was used, the trimer electromigration rate was correlated to a linear function with R2=0.9953. The slope of the corresponded equation was 9.14. Under this applied voltage, trimer electromigration rate was 36.19 ± 2.24% at τ = 4h of the electroseparation procedure.

The electromigration behavior of the tetramer is shown on Figure 8.4. At an applied voltage of 2.5 V/cm, the tetramer was found in the adjacent KCl compartment only after 3h of treatment with an average electromigration rate of 5.06 ± 0.61%, which increased up to 6.46 ± 0.67% at the end of the electroseparation process. By increasing the applied average electric field up to 5 and 10 V/cm, respectively, the tetramer was found in the adjacent KCl compartment since the first hour of treatment. In both cases, it migrated with a linear kinetic. At 5 and 10 V/cm, the kinetics were fitted to linear curve regression coefficients of R2=0.9669 and 0.9950, respectively. The equations slopes indicating the electromigration speed of the tetramer through the 10000 Da MWCO ultrafiltration membrane were 3.75 and 6.86, respectively. At the end of the process, electromigration rates of the tetramer under these conditions were 14.46 ± 1.52 and 26.39 ± 2.33%, respectively.

Effect of solution flow velocity (flow rate) is directly related to the residence time of a given molecule in the side of the separation chamber. The higher the solution flow velocity the lower the residence time. Solution flow velocity (flow rate) must ensure a minimum time to the molecule to reach the membrane surface to be able to penetrate it. Such explanation was previousely reported in the literature (Galier & Roux-de Balmann, 2004; Roux-de Balmann et al., 1998).

Also, taking into account the effectiveness of the promoter of turbulence to offer a particularly effective agitation of the solutions at the both sides of the ultrafiltration membrane used, it would be expected that the contribution of the solution flow velocity; which was the same in both chitosan oligomer mixture solution and KCl compartments to the hydrodynamic conditions at the interfaces membrane/solution was not important. The promoter of turbulence is mainly used to diminish the concentration polarisation phenomenon. The range of the used solution flow velocities was sufficient to offer this condition. Consequently, the effect of solution flow velocity on chitosan oligomer electromigration rate at a fixed value of the applied voltage was not significant. Nevertheless, it was reported that it is necessary that the solution flow velocity must be sufficient to make the role of the promoter of turbulence effective in order to minimise the concentration polarisation phenomenon. In our study, it seems that the used solution flow velocities offer this condition. This explanation is in good agreement with the information reported in the literature (Lteif et al., 1999). They showed that the solution flow velocity did not have any significant effect on the electrolyte transport through a membrane if the solution was well mixed. In their study (Lteif et al., 1999), magnetic bars were used to insure an adequate agitation of the solutions in the cell compartments. Varying simultaneously the solutions flow velocities, a series of experiments were carried out at 16.66 and 25 mL/min. They reported that taking into account the presence of a particularly effective magnetic agitation, the contribution of the solution flow velocity to the solute transfer through the membrane was not significant. In our study, an adequate agitation of the solution was obtained by the promoter of turbulence and the flow rate. At the other hand, the effect of solution flow velocity on the volumetric permeate flux of dissolved organic molecules through 15 kDa ceramic ultrafiltration membrane was evaluated (De la Rubia et al., 2006). In this study, it was reported that the solution flow velocity had a significant effect on solute permeation through the membrane used. The effect of solution flow velocity was expressed by Reynolds number. They used Reynolds number values varied between 2500 and 20000. They showed that decreasing the Reynolds number, the volumetric permeate flux decreased. At the difference with our study where the driving force of the process is an applied external electric filed, in the study described by (De la Rubia et al., 2006), the driving force of the system was a pressure gradient. The impact of modifying the flow velocity of the feed solution on the yield and selectivity of an electrodialysis with ultrafiltration (EDUF) process for the fractionation of peptides was studied (Poulin, 2007; Poulin et al., 2006a). Solution flow rates of 100, 150, 200 and 250 mL/min were used. In this study, it was shown that the solution flow velocity had no affect on the total peptide migration but on the migration of particular peptides when a solution flow rate of 250 mL/min and up was used. These authors explained that this behavior could be attributed to the limited electrophoretic mobility of these peptides.

The dimer showed a linear electromigration kinetic whatever the applied average electric field. This behavior indicates that the dimer migrated quite freely through the 10000 Da MWCO ultrafiltration membrane used, under the effect of the applied external electric field as a driving force. This is in a good agreement with the result reported in our previous studies (Aider et al., 2007a,b) in which it was shown that the dimer electromigration kinetic followed a linear behavior. In these studies, the cell configuration used was the same as in the present work with 10000 Da MWCO ultrafiltration membrane and a voltage of 5 V corresponded to average electric field strength of 2.5 V/cm. The trimer electromigration behavior followed a curvilinear type equation and showed an inflexion point after 2h of treatment, when an electric field strength of 2.5 V/cm was applied. But in the cases when electric field strengths of 5 and 10 V/cm were used, its electromigration behavior followed a linear kinetic. This behavior could be explained by an enhancement effect of the electric field on this molecule migration through the 10000 Da MWCO ultrafiltration membrane used. Indeed, by increasing the applied voltage, this molecule was subjected to a higher driving force that makes it possible to significantly minimize the friction exerted by the membrane. As result, the trimer migrated quite freely at the applied electric fields of 5 and 10 V/cm. The given explanation about the trimer is also applicable to the tetramer. Results explaining the effect of the applied voltage on solute electromigration through cellulose ester ultrafiltration membrane was previously reported (Roux de-Balmann & Sanchez, 1992). In a membrane contactor system, they studied a separation of model proteins. The studied mixture was composed of Poly-L-glutamic acid, α- lactalbumin and bovine haemoglobin. They showed that the solute migration trough the membrane increased by increasing the electric field. The electroseparation system used by Roux de-Balmann & Sanchez (1992) is similar to our system considering that both systems are based on the same principle (Figure 8.1). The difference is that in their study, the membrane active area was 32 cm2 and the separation was carried out at a fixed current density varied from 3 to 22 A/m2 corresponded to an electric field varying from 1 to 8 V/cm. Solution flow velocities of 1.66 and 83.33 mL/min were used. In our case, the active membrane area was 10 cm2, fixed electric field ranging from 2.5 to 10 V/cm was studied. Our study was carried out at solution flow velocities of 100, 300 and 500 mL/min. On the other hand, the electrophoretic transfer of proteins across 106 Da MWCO polyacrylamide membrane in an electrophoresis-based separation technique (Gradiflow technique) was studied (Rylatt et al., 1999). They reported that bovine serum albumin (BSA) transfer across a polyacrylamide membrane was directly proportional to the applied electric field strength. It was shown that as the electric field increased; there was a proportional increase of the amount of the BSA that migrated through the used 106 Da MWCO polyacrylamide membrane. In the study described by Rylatt et al. (1999), the electric voltages used were 25, 50, 100 and 200 V, and the assembled cartridge was 15 cm long, 3 cm wide and 1 cm deep. These voltages corresponded to average electric field strengths of 25, 50, 100 and 200 V/cm. In this study, the heat generated at higher voltages imposed a practical upper limit to the voltage which can be applied to the Gradiflow electro-separation system. A comparison of our study to the work of Rylatt et al. (1999), revealed some particularities. Our upper electric field was 10 V/cm while their lower one was 25 V/cm. The BSA is a protein with a molecular weight of 67000 Da (Oussedik & Mameri, 2001), isoelectric point (PI) of 4.9, negatively charged at pH values above the PI, and electrophoretic mobility of -1.7x10-6 m2/V.s (Takeda et al., 1992). The ratio of the polyacrylamide membrane MWCO used in this study to the solute (BSA) molecular weight is approximately 15, while in our study the ratio of the 10000 Da MWCO cellulose ester ultrafiltration membrane used to the dimer, trimer and tetramer molecular weight was 12.5, 17 and 25, respectively. In the contrary of the work of Rylatt et al. (1999), in our case, there was no significant heat generation because the applied voltage was lower. The temperature increased by 8-9 °C along the electroseparation procedure.

Concerning the separation possibility of the chitosan oligomers, it was not possible to obtain any separation at 5 and 10 V/cm because the applied electric field strength was sufficient for the tetramer to migrate through the membrane together with the dimer and trimer since the first hour of the treatment.

At the same time, each chitosan oligomer is submitted to the effect of the applied electric field and solution flow velocity. As consequence, the resultant molecule velocity is the sum of two vectors resulted from the electric filed strength and solution flow velocity effects. The following equation can be written (Doneddu, 1984):

(8.3.2.1)

being the molecule linear velocity caused by the applied external electric field, in a direction perpendicular to the membrane, (m/s).

being the molecule linear velocity caused by the applied solution flow velocity, in a direction parallel to the membrane, (m/s).

These above mentioned velocities can be expressed through chitosan oligomer electrophoretic mobility (μ), electric field strength (E), solution flow velocity (d) and area (S) calculated from compartment thickness and membrane length (Sun, 2004; Young et al., 2004):

(8.3.2.2)

(8.3.2.3)

By replacing Vx and Vy in eq.3.2.1, we obtained the following resultant velocity:

(8.3.2.4)

If we consider X as being the maximal available distance for each molecule to be in contact with the active membrane area, and τ the corresponding time, then, the following equation could be written:

(8.3.2.5)

By combining equations (3.2.4) and (3.2.5), the result will be given by the following equation:

(8.3.2.6)

From equation (3.2.6), the maximal time needed for a given molecule to reach the membrane surface will be as follows:

(8.3.2.7)

According to the equation (8.3.2.7), the above calculated time for a given chitosan oligomer in the feed solution compartment depends on the compartment geometry (X), solute electrophoretic mobility (μ) which is an intrinsic molecule characteristic at given concentration, pH value and ionic strength of the medium (Vishu-Kumar et al., 2005), the applied external electric field strength (E), surface (S) and solution flow velocity (d).

Considering that the electrodialysis with ultrafiltration membrane (EDUF) cell is a multi-compartment separation chamber and that the electrical conductivity of the solutions in the different compartments are not the same, the electric field strength is consequently not the same in these compartments. Using the nominal averaged electric field strength in equation 8.3.2.7 is less precise. According to (Galier & Roux-de Balmann, 2004; O’Connor et al., 1996), the local electric field (E l ) can be calculated through the current intensity (I), active membrane surface (S) and solution electrical conductivity (κ) as follows:

(8.3.2.8)

By replacing the electric field in equation (3.2.7) by equation (3.2.8), we obtain the following equation:

(8.3.2.9)

For each chitosan oligomer, the equation (8.3.2.9) was plotted in 3D surface as function of the two studied variables (electric field strength and solution flow velocity). Only data of the dimer were shown because the tendencies were the same for all oligomers even if the calculated times were different. Figure 8.5 shows the time needed for the dimer to reach the membrane surface as a function of the electric field and solution flow velocity. We can see that the parameter influencing this time is the electric filed, and that the higher the electric field the lower the time needed independently on the solution flow velocity (rate). Using the first derivative of the equation (8.3.2.9) as a function of the solution flow velocity (d), it was possible to calculate the minimal solution flow velocity from which this last (flow velocity) is not significant. It was found that at a solution flow velocity of 0.83 cm/s corresponding to a flow rate of 30 mL/min and above, this parameter does not exert any effect on the time needed for the solute to reach the membrane surface. The same values were calculated for the trimer and tetramer and were found to be equal to 1.16 and 1.49 cm/s (42 and 54 mL/min), respectively.

Ions or charged organic molecules may carry drastically different portions of the total current. This depends on their respective electrophoretic mobilities, valences and concentrations (Usobiaga et al., 2000). Transport numbers of the studied chitosan oligomer mixture are shown in Table 8.2. The solution flow velocity and electric field strength did not affect the transport numbers of each given chitosan oligomer (P>0.171). At the same time, the chitosan degree of polymerisation had a significant effect on the transport number. Multiple comparisons of the chitosan oligomers transport numbers did not reveal any difference between the dimer and trimer transport numbers (P>0.299), while the tetramer transport number was significantly different from those of the dimer and trimer (P<0.006).

Results presented in Table 8.2 showed that approximately 7-8% of the total electric current passing through the electrodialysis with ultrafiltration membrane (EDUF) cell was transported by the chitosan oligomers. Considering that the total transport number can not exceed 1, we can deduce that approximately 92-93% of the total electric current was carried by the electrolytes present in the solutions (Na+, K+, Cl-, H+, OH-). The tetramer showed the lowest transport number because of its lower concentration in the feed solution compared to that of the dimer and trimer, its lower electrophoretic mobility as shown by (Aider et al., 2006a) and membrane restriction to its migration as shown in our previous study on the effect of membrane molecular weight cut-off on the chitosan oligomer electromigration kinetics (Aider et al., 2007a).

The EDUF system performance was evaluated according to the measurement of the limiting current density, global electrical resistance of the EDUF cell, ultrafiltration and anion-exchange membranes integrity by measuring their main characteristics (electrical conductivity and thickness) before and after each electroseparation process.

In the present study, it was found that the solution flow velocity did not exert any significant effect on the electromigration rate of chitosan oligomer, as well as on system performances. At the same time, it was found that the applied electric field strength had a significant effect on all chitosan oligomer electromigration rates. The dimer average electromigration rate after 4h of treatment varied from 9.92 ± 2.45 to 47.86 ± 3.12% for an electric field strength range of 2.5 to 10 V/cm (voltage from 5 to 20 V). The trimer migrated together with the dimer with electromigration rate of 8.58 ± 1.03 up to 36.19 ± 2.24% for the above mentioned values of the electric field. The tetramer showed the lowest electromigration rates. When electric field strength of 2.5 V/cm was applied, a solution composed only by the dimer and trimer was obtained until 2h of electroseparation treatment. At the pH used in the present study, no separation was possible at any time when electric field strengths of 5 and 10 V/cm were applied to the electroseparation system with the 10000 Da MWCO cellulose ester ultrafiltration membrane stacked in the EDUF cell.

The results obtained can be used to optimise the performance of the electroseparation process of the studied chitosan oligomers by increasing the effective ultrafiltration membrane area under applied electric field strength of 2.5 V/cm. This technique can be successfully used in the basic research laboratory as well as for commercially important scales for chitosan oligomers separation.

Other studies are needed to verify if the separated chitosan oligomers by EDUF technology keep their biological functions considering that soft conditions were used for their separation.

© Mohammed Aider, 2007