Study On A Novel Separation Technology Combining Metal Chelating Affinity Partitioning And Temperature-induced Phase Separation With Aqueous Two-phase Partitioning

The recent progresses and key problems in aqueous two-phase partitioning (ATPP) were reviewed. Especially, the principle, characteristics and utilization of metal chelating affinity partitioning and temperature-induced phase separation were discussed. A novel separation technology combining metal chelating affinity partitioning and temperature-induced phase separation was proposed, which utilizes the metal chelating affinity partitioning for selective partitioning of target protein and the temperature-induced effect to achieve the recycling of phase-forming polymer and affinity ligand. This doctoral dissertation consists of there parts: preparation of metal chelating affinity ligand, liquid-liquid equilibria behavior of aqueous solutions containing metal chelating affinity ligand, and affinity partitioning behavior and purification process of proteins in ATPS combined with metal chelating affinity partitioning and temperature-induced phase separation.The synthesis route of polyethylene glycol - iminodiacetic acid - Cu (PEG-IDA-Cu2+) was investigated. The hydroxyl group activation methods and IDA coupling conditions was optimized. Using thionyl chloride as the chloridating agent, a high value of synthetic yield was reached at 1.54 mol Cl/mol PEG, but the coupling efficiency of IDA was only 55%. Under the optimized conditions of epichlorohydrin method, IDA content in the product of PEG-IDA was 0.93 mol/mol PEG and the Cu2+ content in the product of PEG-IDA-Cu(II) could reach 0.83 mol/mol PEG.A novel affinity ligand for metal chelating affinity separation with temperature-induced effect was prepared successfully using the appropriate synthesis route of PEG-IDA-Cu(II). The random copolymer of ethylene oxide and propylene oxide (EOPO) was chosen as the thermoseparating polymer, and the metal chelating function group (IDA) was coupled to the polymer after suitable activation. The optimal conditions for preparing EOPO-IDA-metal were explored. The conditions of the reaction of epichlorohydrin to EOPO were as following: BF3C2H5OC2H5, 0.05mL/gEOPO; 40% NaOH, 0.2mL/g EOPO; 20h for first sub-step and 5h for second sub-step with temperature controlling at ice water. The condition of the reaction of IDA to EOPO-epoxide were: pH 10-10.5, mol ratio of IDA to EOPO is 2, 24h, reaction at 48. The Cu2+ content in EOPO-IDA-Cu(II) could reach 0.70 mol/mol EOPO and Ni2+ content in EOPO-IDA-Ni(II) is 0.45 mol/mol EOPO.The liquid-liquid equilibrium of PEG/(NH4)2SO4 PEG/hydroxypropyl starch(PES), EOPO/Na2SO4 and EOPO/PES aqueous two-phase systems(ATPS), with some amount of the PEG or EOPO was substituted by metal chelating affinity ligand, were measured at 20.The formaldehyde titration method was used for the determine of 4, the optical rotation method for PES, the indirect chelting titration for and vacuum-drying method for water. The phase diagrams of these systems were similar to those without affinity ligands. The effects of the metal chelating affinity ligand on the tie lines were investigated. It was found that the slope of tie line decreases slightly with increasing concentration of affinity ligands, while the concentration of polymers in the top phase were increased a little. The changes of liquid-liquid equilibrium of PEG containing ATPS is more than EOPO containing ATPS and polymer/salt ATPS more than polymer/polymer ATPS as affinity ligand added.The cloud point of three polymer solutions [EOPO, EOPO-IDA, EOPO-IDA-Cu] were studied. The results indicated that the cloud point changed only a little with the coupling of IDA or IDA-Cu (II) to EOPO. EOPO solution showed the highest cloud point and the cloud point of EOPO-IDA was the lowest. The differences of cloud point mainly due to the change of hydrophilic characteristic of EOPO when different group was coupled. All three polymers tested have appropriate cloud point for biomaterial separation. The cloud point of all three polymer/H2O systems decreased with the addition of Na2SO4. The phase diagrams of EOPO/Na2SO4 at different temperatures were measured. The critical conce...