The Preparation Of Chitosan-based Amphoteric Membranes And Their Application In Protein Adsorption And Separation

Nowadays more attention has been paid in membrane chromatography systems that function as short, wide chromatography columns, as some of the major problems associated with packed bed chromatography can be solved by using macroporous membranes as chromatography media. The main advantage of macroporous membrane chromatography is the high flow rate through the membranes at low pressure drops and relatively short operation time.Different types of interactions have been utilized in membrane chromatography for bioseparation. One of them is ion-exchange, in which the separation can be achieved based on the electrostatic interaction between the surface charges of biomacromolecules and the charged groups on membranes. Amphoteric ion-exchange membranes contain both weak acidic (negative charge) groups and weak basic (positive charge) groups that are randomly distributed within the membrane matrix, and the sign of the net charge of the membrane can be controlled by environmental pH, resulting in some characteristic properties that can not be shown in a single-charged material.In view of growing public health and environmental awareness accompanied by an increasing number of ever stricter environmental regulations on discharged wastes, attention has been focused on the use of natural polymers from renewable resources as alternative to synthetic polymers. One kind of the most widespread natural polymers is polysaccharides, such as cellulose, starch, chitin and lignin. Among the commercially available polysaccharides that are prevailingly neutral or acidic, but chitin and its primary derivative chitosan (CS) are special in that they are basic. CS has both reactive amino and hydroxyl groups with common characteristics of polycation nature and good biocompatibility. These two functional groups offer several possibilities for derivatization and immobilization of biologically active species.In the present work, an amphoteric membrane matrix was prepared by a simple solution blending method of natural polyelectrolyte CS and carboxymethyl cellulose (CMC) or carboxymethyl chitosan (CMCS). These natural amphoteric membranes were studied in static adsorption and dynamic adsorption of proteins with different pIs. Both lysozyme and ovalbumin could be effectively separated from their binary mixture according to the difference between their pI.The CS/CMC blend membrane was prepared through mixing the CS and sodium CMC solution. Silica particles were used to generate the pores in the membranes. Glutaraldehyde and epichlorohydrin were used as crosslinking agents. The time for lysozyme to reach adsorption equilibrium was about 5 h and 6 h for ovalbumin. Both the protein and the CS/CMC blend membrane are amphoteric, so the charges on their surface vary according to the environmental pH, and as a result the adsorption capacity of the protein on membrane is different at different pH. The maximum adsorption of lysozyme and ovalbumin was found at pH 9.2 and 4.8, respectively. According to the different adsorption capacity between lysozyme and ovalbumin in the solution with same pH value, lysozyme and ovalbumin could be effectively separated from their binary mixture. Four cycles of adsorption-desorption were performed. The result shows the high desorption ratio and good reusability of the membrane.O-carboxymethyl chitosan was prepared in order to keep more amino groups of chitosan so the amino groups of chitosan and O-carboxymethyl chitosan could be cross-linked to increase the content of CMCS in CS/CMCS blend membrane. The preparation of CS/CMCS blend membrane was similar to that of CS/CMC blend membrane. CS/CMCS blend membranes could adsorb lysozyme and ovalbumin effectively under certain circumstance and Lagergren second-order kinetics model described the data better. The adsorption capacity varied with the change of environmental pH, and the maximum adsorption of ovalbumin and lysozyme was found at pH 5.2 and 8.0, respectively. Both Langmuir model and Freundlich model were suitable for describing the biosorption equilibrium of both proteins in the concentration ranges we studied. The stability and repeatability of the CS/CMCS blend membrane were found quite well after several adsorption-desorption cycles, which indicated the good reusability of the membrane. These membranes also could be effectively used to separate lysozyme-ovalbumin binary mixture only by changing the pH of the feed and the desorption solution. In addition, lysozyme could be separated from fresh chicken egg white by CS/CMCS blend membrane.The dynamic adsorption behavior of lysozyme and ovalbumin on CS/CMCS blend membrane was also investigated. The dynamic adsorption of individual protein on blend membranes was carried out by loading 0.5 mg/mL protein solution at a flow rate of 2 mL/min. The dynamic adsorption capacity for lysozyme was 9.19 mg/mL and for ovalbumin was 1.24 mg/mL. CS/CMCS blend membrane could be used repeatedly in the dynamic adsorption-desorption process of lysozyme and the desorption ratios were all about 90%. The dynamic adsorption behavior of ovalbumin on CS/CMCS blend membrane was affected by the swelling of membrane when it was used in the favorable pH range for ovalbumin adsorption. Thus the results for ovalbumin were not so satisfied compared with the results for lysozyme, but these data were still instructive for the further study. The desorption ratios for ovalbumin varied from 45% to 75% according to the desorption condition. Under proper condition the CS/CMCS blend membrane could separate each protein from lysozyme-ovalbumin binary mixture.