Liquid-liquid phase equilibria of aqueous two-phase systems containing salts and polyethylene glycol

Liquid-liquid phase equilibria of the ternary systems: (a) polyethylene glycol - ammonium sulfate- water and (b) polyethylene glycol - sodium carbonate -water have been determined experimentally at 15°, 25°, 35° and 45°C and for two different molecular weights of the polymer (Avg. M.W. 1000 and 2000). Details of the glass cell and of the equilibration and analytical procedures used are described. Equilibrium data along with phase diagrams are presented. Finally the effect of temperature and of the molecular weight of the polymer are also discussed.     

聚乙二醇双水相萃取光度法测定镍

用水溶性高聚物、表面活性剂及有机物与无机盐形成的双水相体系萃取分离色素、蛋白质及测定金属离子已有报道[1-5].     

聚乙二醇双水相萃取光度法测定锌

A novel method of extraction spectrophotometric determination of Zn~(2+) in aqueous two-phase system formed by polyethylene glycol-2000,sodium sulfate and zincon has been established.Amounts of PEG solution,zincon solution,sodium sulfate added and the acidity of the system were optimized.The interference effects of various coexistent ions and the method to remove the interference were investigated.The Zn-zincon complex that had been formed in the presence of H_2BO_3-Na_2B_4O_7 buffer solution at pH 7.4 was extracted into the upper PEG-rich phase,and the absorbance of the extracted complex in the upper PEG-rich phase was measured at 630 nm.The apparent molar absorptivity was 7.3 × 10~4 L / (mol·cm).Beer's law was obeyed over the range of 0～2.0 μg / mL Zn~(2+).The analytical results obtained for Zn~(2+) in water samples were in good agreement with values measured by atomic absorption spectrometry.     

Downstream processing of virus-like particles with aqueous two-phase systems: Applications and challenges

The advancement of recombinant virus-like particle-based vaccines has attracted global attention owing to substantially safety and high efficacy in provoking a protective immunity against various chronic and infectious diseases in humans and animals. A robust, low-cost, and scalability separation and purification technology is of utmost importance in the downstream processing of recombinant virus-like particles to produce affordable and safe vaccines. Being a relatively simple, environmentally friendly, and efficient biomolecules recovery approach, aqueous two-phase systems have received great attention from researchers worldwide. This review aims to highlight the challenges and outlook in addition to the current applications of aqueous two-phase systems in downstream processing of virus-like particles. The efforts will confidently reinforce scholars’ knowledge and fill in the valuable research gap in the aspect of concerning recombinant virus-like particle-based vaccines development, particularly related to the virus-like particles downstream production processes.     

Influence of magnetic field on aqueous two-phase extraction of horse ferritin in the polyethylene glycol/hydroxyethyl starch system

The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL⁻¹. The lowest concentration was 0.42 mg mL⁻¹ in the system composed of 5% PEG 6000 and 1% HES.     

Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein

A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by ¹H nuclear magnetic resonance (¹H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV–vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV–vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins.     

Effect of agitation and aeration on production of hexokinase by Saccharomyces cerevisiae

A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was in oculated with cell suspension (about 0.7 g/L), and left ferm entingat 35°C and pH 4.0. The aeration and agitation were adjusted to attain k _La values of 15, 60, 135, and 230 h⁻¹. The highest hexokinase productivity (754.6 U/[L h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k _La of 60 h⁻¹. Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character of this enzyme. Hexokinase formation for k _La>60 h⁻¹ was not enhanced probably owing to saturation of the respiratory pathway by oxygen.     

丙酮-硫酸铵双水相体系分离纯化红花红色素

天然药用植物红花具有活血化淤、抗炎止痛的药用功效,其中的两种色素,红花红色素(Carthamine)和黄色素(Safflower yellow),还是天然色素的重要原料[1,2].     

Partitioning behavior of erythrocytes in aqueous two-phase systems containing hydroxypropyl starch and polyethylene glycol

The partitioning behavior of erythrocytes in Reppal PES 200 (a hydroxypropyl starch produced by Reppe Glykos AB, V?xj?, Sweden)-polyethylene glycol (PEG) and in dextran (Dx)-PEG aqueous phase systems made isotonic with phosphate is similar in a number of ways: (i) There is a correlation between the relative electrophoretic mobilities and partition ratios, P, of red blood cells from different species; (ii) The cell P is reduced when, at constant polymer concentrations, phosphate is systematically replaced by sodium chloride (with the total concentration isotonic); (iii) The cell P is increased with reduced polymer concentrations (decreased interfacial tensions); (iv) Treatment of erythrocytes with neuraminidase results in a reduced P value; (v) Rat red cells of different ages can be fractionated by counter-current distribution; and (vi) Differences between red blood cells from genetically distinct rats or between humans can be detected. Aquaphase (a hydroxypropyl starch marketed by Perstorp AB, Lund, Sweden) has been tested as in ii-iv above with analogous results. The partitioning behavior of erythrocytes in PES-PEG and Dx-PEG aqueous phase systems containing sodium chloride differs in a number of ways: (vii) The correlation, apparent in Dx-PEG systems, between the P value of red blood cells from different species and the ratio of their membrane poly- to monounsaturated fatty acids is absent in PES-PEG systems. It is replaced by a correlation as in i; (viii) The increase in P value in Dx-PEG observed from red blood cells after treatment with neuraminidase is replaced by a decrease in P value in PES-PEG or Aquaphase-PEG systems. We conclude that PES (and Aquaphase) can be substitutes for dextran in cell partitioning studies when charge-sensitive phases are used (e.g., those containing phosphate) while separations based on properties reflected by Dx-PEG systems containing sodium chloride are not duplicated by PES-PEG (and probably not by Aquaphase-PEG). The hydroxypropyl starch-PEG systems containing sodium chloride, unlike the analogous Dx-PEG systems, have a significant electrostatic potential difference between the phases.     

Partitioning behavior of amino acids in aqueous two-phase systems containing polyethylene glycol and phosphate buffer

The partitioning behavior of four amino acids, cysteine, phenylalanine, methionine, and lysine in 15 aqueous two-phase systems (ATPSs) with different polyethylene glycol (PEG) molecular weights and phosphate buffers has been studied in the present paper. The phase diagrams of the systems are investigated together with the effect of the PEG molecular weight and pH of the phosphate solutions. The composition of these systems and some parameters such as density and refractive index are determined. The influences of salts in ATPSs, side chain structure of the amino acids, pH of ATPSs, and the PEG molecular weight on the distribution ratios of the amino acids have been studied. This work is useful for the purification of amino acids and the separation of some proteins whose main surface exposed amino acid residues are these four amino acids, respectively.     

Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI–His₆–GFP, together with the conjugate PEG–IDA–Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600–DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG–IDA–Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG–dextran system as a second extraction system, with 80–90% of pDNA partitioning to the bottom phase. This represents about 7.4 μg of pDNA extracted per 1 mL of pUC19 desalted lysate.     

利用双水相分离回收离子液体的研究进展

研究离子液体的分离与回收对于减少离子液体对环境的影响、提高离子液体的利用效率、降低离子液体的应用成本、促进离子液体的工业应用具有重要的意义.本文重点综述了利用无机盐-离子液体双水相、糖-离子液体双水相、聚合物-离子液体双水相和CO2诱导的离子液体双水相技术分离回收离子液体的研究进展,分析了影响离子液体分离回收的关键因素,评价了不同离子液体双水相体系的优缺点,展望了该领域的发展方向及面临的挑战.     

Correlations between distribution coefficients of various biomolecules in different polymer/polymer aqueous two-phase systems

Distribution coefficients for a variety of proteins and certain other biomolecules (peptides, amino acids, and carbohydrates) (overall 27 different solutes) were measured in aqueous two-phase systems (ATPSs) dextran (Dex)–polyethylene glycol (PEG) and Dex–Ucon 50-HB-5100 (Ucon—a random copolymer of ethylene glycol and propylene glycol) both containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4, at 23 °C. Distribution coefficients of some selected solutes were also measured in the above two-phase systems at three different polymer concentrations for each system. It was established that the distribution coefficients for all the proteins examined in the ATPSs are correlated according to the so-called Collander linear equation.     

Bioconversion of vanillin to vanillyl alcohol in a two-phase reactor

One of the next challenges in the use of biocatalysts (enzyme or microbial cells) is the upgrading of biological reactions of oxidoreduction. The oxidoreductases need cofactors that must be regenerated. Practical experience shows that this is most readily achieved by using living cells of microorganisms. Living cells ofSaccharomyces cerevisiae are able to bioconvert vanillin to vanillyl alcohol (1). By working with a two-phase reactor (dodecanol—feeding medium) it has been possible to use higher vanillin concentrations without inhibiting the bioconversion (2).     

Effective extraction of tylosin and spiramycin from fermentation broth using thermo-responsive ethylene oxide/propylene oxide aqueous two-phase systems

Recyclable aqueous two-phase systems with thermo-responsive phase-forming materials have been employed to separate macromolecules; however, these systems have achieved very limited separation efficiency for small molecules, such as antibiotics. In this study, aqueous two-phase systems composed of the ethylene oxide/propylene oxide copolymer and water were developed to extract alkaline antibiotics from the fermentation broth. In the aqueous two-phase systems with an ethylene oxide ratio of 20 and propylene oxide ratio of 80, the partition coefficients of tylosin and spiramycin reached 16.87 and 20.39, respectively, while the extraction recoveries were 70.67 and 86.70%, respectively. Coupled with mechanism analysis, we demonstrated the feasibility of extracting alkaline antibiotics using this aqueous two-phase system, especially for 16-membered macrolide antibiotics. The molecular dynamic simulation was employed to visualize the process of dual-phase formation and the partition behavior of antibiotics in an aqueous two-phase system. The dynamic simulation revealed the binding energy between the antibiotic and ethylene oxide/propylene oxide copolymers, which provides a simple indicator for screening suitable antibiotics in aqueous two-phase systems. Our recyclable aqueous two-phase systems provide a robust approach for the extraction of 16-membered macrolide antibiotics with ease of operation and high recovery rates, which is appropriate for large-scale extraction in the fermentation industry.     

Extraction mechanism of sulfamethoxazole in water samples using aqueous two-phase systems of poly(propylene glycol) and salt

Based on the poly(propylene glycol)₄₀₀ (PPG₄₀₀)–salt aqueous two-phase system (ATPS), a green, economical and effective sample pretreatment technique coupled with high performance liquid chromatography was proposed for the separation and determination of sulfamethoxazole (SMX). The extraction yield of SMX in PPG₄₀₀–salt ATPS is influenced by various factors, including the salt species, the amount of salt, pH, and the temperature. Under the optimum conditions, most of SMX was partitioning into the polymer-rich phase with the average extraction efficiency of 99.2%, which may be attributed to the hydrophobic interaction and salting-out effect. This extraction technique has been successfully applied to the analysis of SMX in real water samples with the recoveries of 96.0–100.6%, the detection limits of 0.1 μg L⁻¹, and the linear ranges of 2.5–250.0 μg L⁻¹.     

Partitioning of alkali halides in aqueous dextran-ficoll two-phase system

Partitioning of alkali halides MeX in aqueous dextran-Ficoll two-phase system of fixed polymer and ionic composition was studied. It is found that NaCl and LiX concentrate in the Ficoll-rich phase, while all KX, RbX, and CsX concentrate in the dextran-rich phase. Partitioning of KX, RbX, and CsX in the presence of a large excess of NaCl is found to depend on the type of the halide anion X^–.     

Downstream processing of antibodies: Single-stage versus multi-stage aqueous two-phase extraction

Single-stage and multi-stage strategies have been evaluated and compared for the purification of human antibodies using liquid–liquid extraction in aqueous two-phase systems (ATPSs) composed of polyethylene glycol 3350 (PEG 3350), dextran, and triethylene glycol diglutaric acid (TEG-COOH). The performance of single-stage extraction systems was firstly investigated by studying the effect of pH, TEG-COOH concentration and volume ratio on the partitioning of the different components of a Chinese hamster ovary (CHO) cells supernatant. It was observed that lower pH values and high TEG-COOH concentrations favoured the selective extraction of human immunoglobulin G (IgG) to the PEG-rich phase. Higher recovery yields, purities and percentage of contaminants removal were always achieved in the presence of the ligand, TEG-COOH. The extraction of IgG could be enhanced using higher volume ratios, however with a significant decrease in both purity and percentage of contaminants removal. The best single-stage extraction conditions were achieved for an ATPS containing 1.3% (w/w) TEG-COOH with a volume ratio of 2.2, which allowed the recovery of 96% of IgG in the PEG-rich phase with a final IgG concentration of 0.21 mg/mL, a protein purity of 87% and a total purity of 43%. In order to enhance simultaneously both recovery yield and purity, a four stage cross-current operation was simulated and the corresponding liquid–liquid equilibrium (LLE) data determined. A predicted optimised scheme of a counter-current multi-stage aqueous two-phase extraction was hence described. IgG can be purified in the PEG-rich top phase with a final recovery yield of 95%, a final concentration of 1.04 mg/mL and a protein purity of 93%, if a PEG/dextran ATPS containing 1.3% (w/w) TEG-COOH, 5 stages and volume ratio of 0.4 are used. Moreover, according to the LLE data of all CHO cells supernatant components, it was possible to observe that most of the cells supernatant contaminants can be removed during this extraction step leading to a final total purity of about 85%.     

离子液体双水相萃取山楂黄酮和多糖的相行为研究

用分光度法研究了[Bmim]BF4/(NH4)2SO4双水相体系萃取山楂黄酮和多糖的相行为,考察了同时提取这两种组分时[Bmim]BF4的浓度、(NH4)2SO4的浓度、山楂的用量、超声萃取时间等因素对双水相的上下相体积以及分配系数的影响。结果表明:(1)离子液体浓度的增加,双水相的上、下相体积分别明显增大和减小。但黄酮和多糖在双水相中的分配系数仅有波动,均小于5%;(2)硫酸铵浓度的增加,双水相的上下相体积分别明显减小和增大。同时黄酮和多糖的分配系数均有较明显先降低后升高的趋势;(3)山楂质量的增减,不影响双水相的形成,但存在一个实验条件下的溶出饱和值,该值为0.15g,此时黄酮和多糖的分配系数最大;(4)超声萃取时间的延长或缩短,同样不影响双水相的形成,但存在对山楂有效成分达到饱和溶出的阈值,对于黄酮为25min,多糖为20min。     

醇-盐双水相技术去除废水中的酚类物质

A novel method for treatment of phenolic compounds in cokery wastewater by propyl-alcohol ammonium sulfate aqueous two-phase system was investigted.The effects of the amount of ammonium sulfate used,the amount of propyl-alcohol used,pH value on extractield was studied.Extraction was optimized using a L9(34)orthogonal test.The results indicated optimum extraction conditions were the amount of ammonium sulfate used of 18.0 g,the amount of propyl-alcohol used of 16 mL,and pH value of 3.0.A extraction yield of 99.37% was achived under optimum extraction conditions and via secondary extraction.