| Aqueous two-phase extraction system, a new liquid-liquid extraction technology for separation and purification of biomolecule, is consider to be the method which can save time, cost and have no influence on the activity of biomolecules. It can be a large scale of application. Aqueous two-phase system(ATPS) formed by deep eutectic solvent(DESs) and salt is a highly tunable and efficient extraction system which develops from polymer aqueous two-phase system. Compared with ionic liquid, the synthetic process of DESs is relatively simple, and the DESs display more superiority as they are nontoxic and biodegradable.This thesis deeply researches into the behavior of liquid-liquid equilibrium(LLE) of DES-aqueous two-phase system(DES-ATPS) and its phase separation abilities, so this provides sufficient theoretical foundation for its application. An ATPS based choline chloride-polyethylene glycol DES is studied for the first time for the extraction of amino acids and proteins. Firstly, choline chloride and polyethylene glycol were used to synthesized DES, then it was characterized by micro melting point apparatus, elemental analyzer, thermogravimetry, FT-IR and 1HNMR. A aqueous two-phase system compsed by DES and salt was build and study the nature of the phase diagram: binodal curves and tie-lines. The binodal curves were determined by the cloud-point method,the tie-line were performed mass balance calculations by determined the mass fraction of different equilibrium compositions in the top and bottom phases. The results show that the phase-separation abilities of the investigated four ATPSs are in the order of Na2CO3 > Na3C6H5O7 > K2HPO4 ≈ Na H2PO4 and aq w1= exp(a+bw20.5+cw2+dw2) can be successfully used to correlate the binodal curves of the investigated systems. And the tie-line lengths indicated that the compositions of two phases in DES-K2HPO4-H2 O system distinct a lot.Compared with the other three salts, the extracton of tryptophan with DES-Na2CO3 system under the same experiment condition is much larger. UV-vis spectrophotometer was used for both qualitative and quantitative analysis. And the concentration range of 0- 0.1 g/L for analyzing tryptophan guarantee good correlation coefficients. Based on the single-factor experiment, an initial serial investigative test was used to identify the optimal conditions of the extraction including the initial concertration of tryptophan, the amount of DES and salt, and the extraction temperature. Under the option condition, the extraction efficiency of DES-Na2CO3 system, DES-Na3C6H5O7 system, DES-K2HPO4 system, DES-Na H2PO4 system could reach up to 84.54%, 76.14%, 91.97%, 87.03%, respectively. The conformation of tryptophan was not affected after extracting into the DES-rich phase in terms of the determination by UV-vis and fluorescence spectra.The DES-K2HPO4 system was chosen for the extraction of Bull Serum Albumin(BSA) and papain(PAP). The concentrations of proteins were determined by measuring the absorbance using an ultra violet visible(UV–vis) spectrophotometer. And the concentration range of 0- 0.1 g/L for analyzing BSA and PAP both guarantee good correlation coefficients. Single factor experiments were used to research the effects of the process, such as the initial concertration of proteins, the amount of DES and K2HPO4, and the extraction temperature. Under the option condition, the extraction efficiency of BSA, PAP could reach up to 90.13%, 83.06%, respectively. The secondary strcture of protein during the experimental process was observed upon investigation using UV-vis spectroscopy, fluorescence spectrum and circular dichroism spectrum(CD spectrum). The results indicated that the DES-K2HPO4 system provided a moderate environment for bioactive molecules without chemical bonding and change of chemical structure. |
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