| Chromatography is one of the most effective methods for bioseparation and has many advantages. Chromatography is widely used for the separation of biological targets, such as protein, genetic engineering drugs and so on. The matrix is one of the key factors on the chromatographic separation. In this thesis, ionic liquids would be used to directly dissolve cellulose for prepareing cellulose beads, then cellulose beads would be functionalied as anion-exchanger and new adsorbent for hydrophobic charge-induction chromatography (HCIC) and used for bioseparation. Main results include three aspects as following.Firstly, preparation of cellulose beads and pore enlarging. Three kinds of cellulose resources (refined cotton, cotton and microcrystalline cellulose) were used, and ionic liquid—BmimCl was chosen to dissolve cellulose to prepare cellulose viscose. Cyclohexane and starch were used as porogenic agent and the method of water-in-oil suspension regeneration and cooling solidification were developed. The preparation conditions were optimized, including cellulose resources, the ratio of oil to water, stirring speed and the method of process cooling and ethanol addition. The cellulose beads prepared showed good sphericity and had a logarithmic symmetrical particle size distribution of 80-300μm The wet density was about 1.01 g/ml, porosity was around 95%, average pore diameter was about 40 nm and the specific surface area was 90 m2/ml. It was also found that pore enlarging with cyclohexane showed a better pore structure, which was more suitable for the separation of biological macromolecules..Secondly, preparation of cellulose matrices and protein adsorption. With microcrystalline cellulose as the resource and different pore-enlarging methods, cellulose beads were coupled with anion-exchange ligand, DEAE. Three kinds of weak anion-exchange adsorbents were obtained and named as Cell-M-DEAE, Cell-S-DEAE and Cell-C-DEAE, respectively. The ion exchange capacity, static adsorption, dynamic adsorption and breakthrough behavior were studied. The results showed that ion-exchange capacities were about 250μmol/ml. The adsorption capacities of Cell-M-DEAE and Cell-S-DEAE were higher than that of Cell-C-DEAE. However, Cell-C-DEAE had the highest effective diffusion coefficient, the fastest adsorption rate and the highest dynamic adsorption capacity. Furthermore, HCIC adsorbents were prepared by coupling MMI ligand to the microcrystalline cellulose-cyclohexane pore enlarging cellulose beads, and named as Cell-C-MMI. It was found that at neutral pH condition, Cell-C-MMI had high adsorption capacity for IgY, up to 93.1 mg/ml. The adsorption capacity decreased significantly at pH 4. Adding appropriate amount of ammonium sulfate was helpful to the adsorption of IgY.Thirdly, separation of IgY from egg yolk powder with Cell-C-MMI. Using HCIC adsorbent Cell-C-MMI prepared, the pH of loading and elution for the separation of IgY were optimized. The results indicated that the appropriate loading pH was 8.0 and the elution pH was 4.0. The yield of IgY was 75.6% and the purity was 70.1%. It was demonstrated that the HCIC adsorbent prepared has a good performance on the separation of IgY, but the purity of IgY need to be improved.This thesis focused on the cellulose matrices, including cellulose beads preparation, functionaliztion and applications. With ionic liquid as the dissolution solvent for cellulose, the viscose preparation could be simplified and new processis more environmentally friendly. The new cellulose adsorbents prepared in the present work showed high adsorption capacity and good separation performance, which provides new material for the separation of biological macromolecules. |
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