双水相萃取技术研究进展

双水相萃取技术作为一项新的分离技术日益受到重视,它与传统的萃取方法相比有独特的优点,作者综述了又水相萃取技术基本原理、特点、应用及热力学模型,并对双水相萃取技术存在的问题和发展趋势作了论述.     

Separation selectivity of aqueous polyethylene glycol-based separation systems: DSC, LC and aqueous two-phase extraction studies

The distribution behavior of n-alcohols, ketones and nitroalkanes in aqueous liquid chromatography with a column packed with polyethylene glycol (PEG) gel, TSKgel Ether-250, was compared with that in aqueous two-phase systems (ATPSs) formed from PEG and Na₂SO₄ or (NH₄)₂SO₄. The plots of the distribution data obtained for the PEG gel system against those for the ATPS reveal that the separation selectivities exerted by the PEG gel system and the PEG-based ATPS are approximately the same. Differential scanning calorimetry studies on aqueous PEG solutions suggest that PEG polymer forms a hydration structure of which the composition is 50% (w/w) PEG or the hydration number per ethylene oxide is 2.4 and the separation selectivity of the PEG-water systems can be attributed to partition of solute compounds into the hydrated PEG polymer structure.     

Single step recovery of lipase from Penicillium cyclopium by aqueous two-phase extraction

Recovery of lipase from Penicillium cyclopium by aqueous two-phase extraction was studied with maximal possible crude enzyme loads. In polyethylene glycol/dextran and polyethylene glycol/salt systems the influences of molecular weight and concentration of polyethylene glycol, phase-forming salt and phase volume ratio were evaluated. Lipase partition coefficient 9 followed by the top phase yield 95.7% and purification factor 3.4 were achieved in 15% (w w^?1) polyethylene glycol 4000/15% (w w^?1) KH₂PO₄/70% (w w^?1) crude enzyme. Efficient single-step recovery of lipase followed by partial enzyme purification indicated possible integration of production and primary bioseparation step by aqueous two-phase extraction. By varying phase volume ratio, the concentration of phosphate was reduced without decrease in lipase recovery.     

双水相中亮绿的萃取分离及测定

建立聚乙二醇-2000(PEG)-(NH4)2SO4双水相体系萃取光度法测定亮绿(BG)的新方法。测定了PEG相中亮绿吸收光谱和荧光光谱,研究了溶液酸度、盐用量、PEG用量及共存物质对体系测定的影响。结果表明,在溶液酸度pH=6,PEG用量5.0 mL,硫酸铵加入量2.0 g、测定波长630 nm时,PEG相中BG有最大吸光度。线性回归方程Y=0.005 83+0.054 53X,相关系数R=0.999 3,线性范围为0.01～0.17μg/mL。方法具有快速、灵敏、简便的优点,用于天然水中BG测定结果较好。用加入不同类型表面活性剂和光谱方法初步探讨了PEG与BG的相互作用。     

Role of water structure on phase separation in polyelectrolyte-polyethyleneglycol based aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems (ATPS) has emerged as one of the important downstream processing techniques in bioprocess technology. The phase separation behavior of polyelectrolyte-polyethyleneglycol (PEG) based ATPS have been studied to elucidate the mechanism controlling phase behavior. The effect of various inorganic salt additives revealed the importance of water structure as a major factor controlling phase separation in these systems. Nitrate and potassium (water structure breaking ions) elevated the binodial line while sulphate, phosphate and sodium (water structure making ions) depressed the binodial line in both polyacrylic acid-PEG as well as polyethylenimine-PEG based ATPS. The effect of increase in concentration of either of the constituent polymers in both systems (at constant salt concentration) always led to a greater propensity towards phase separation. These results point to a mechanism in which salt-assisted polymer-modified water structure interactions play a central role in phase separation in ATPS.     

1,2,4-丁三醇的双水相萃取

对低分子有机溶剂/无机盐双水相体系萃取分离发酵液中1,2,4-丁三醇（1,2,4-butanetriol,BT）进行了深入研究。通过对不同双水相体系的筛选,最终选定无水乙醇/K₂HPO₄双水相体系来萃取分离BT。使用浊点法对以BT为溶剂的无水乙醇/K₂HPO₄双水相体系进行相图的绘制,发现在K₂HPO₄质量分数为19.83%~46.87%范围内均能成相。通过单因素实验,考察双水相体系中无水乙醇/K₂HPO₄质量分数、pH对BT在两相之间分配系数和萃取效率的影响,得到最佳萃取条件为：系统总量10g、pH 9.5,无水乙醇/K₂HPO₄的质量分数为28%/28%,分配系数和萃取效率分别可以达到18.35和95.87%。在最佳萃取条件下,进一步探究了放大实验对体系萃取效率的影响,发现其对分配系数和萃取效率影响较小,体系稳定性高,为工业提取发酵液中BT提供新思路。     

双水相分离提取胃蛋白酶和琼脂糖的初步研究

以聚乙二醇/硫酸钠双水相体系分离提取琼脂糖和胃蛋白酶.结果表明:琼脂糖的回收率高于90%,胃蛋白酶的回收率约为50%,两者最适宜的分离条件为:pH值为3,Na2SO4浓度为0.4 g·mL-1,PEG10000的浓度为0.002 mol·L-1.     

双水相体系萃取分离金属离子研究进展

双水相体系是一种绿色环保的新型分离技术,应用领域相当广泛,是近年来的研究热点。论述了双水相体系用于萃取分离金属离子的研究现状。根据所使用萃取剂的不同分3种情况进行了论述：不添加任何萃取剂直接萃取金属离子;以无机阴离子为萃取剂,依靠金属阳离子与无机阴离子形成的阴离子配合物（螯合物）而转移到萃取相,萃取金属离子;采用有机试剂作为萃取剂,依靠金属离子与萃取剂反应形成的中性配合物（螯合物）而转移到萃取相,萃取金属离子。论述了双水相体系萃取分离金属离子的发展趋势：建立金属离子在双水相体系中分配的机理模型;双水相体系成相物质的回收及再利用;通过无机盐水化能力的差异,或者通过双水相体系的温度诱导相分离,实现无机盐的分离与常温制备;开展双水相体系萃取分离金属离子的工程研究。     

Polyethylene glycol acquires certain compensatory solute properties when used to form aqueous two-phase extraction systems

The polyethylene glycols are currently considered an essential class of polymers with several applications in industry, medicine, and biology. The aqueous two-phase (ATP) extraction based on polyethylene glycol (PEG) and certain salts have gained the attention for further usage in protein purification as a simple and cheap technique; however, it is still under development. In this paper, the formation of ATP systems by PEG as a model polymer and sodium sulfate was studied focusing on (1) some of the conditions under which the polymer may have definite compensatory solute properties, and (2) the analysis of the crystallization degree and the conditions for forming di and triphasic systems. The results demonstrate that the polymer in some systems may have inherent compensatory solute-like behavior which becomes evident under certain conditions, including the type of the other solute molecules, solvent type, concentration, and pH of the system.     

Enhancing the recovery and concentration of polyphenols from camu-camu (Myrciaria dubia H.B.K. McVaugh) by aqueous two-phase flotation and scale-up process

In this study, polyphenols obtained from camu-camu pulp residue were recovered and concentrated from aqueous two-phase flotation (ATPF) by evaluating different operating conditions, such as solvent type, pH, ammonium sulfate concentration, flotation time, air flow, and volumetric ratio. Under optimal conditions, the polyethylene glycol 400/ammonium sulfate ATPF system showed an efficiency of 81.02% and a concentration coefficient two times higher than traditional aqueous two-phase extraction. Experiments were also carried out on a semi-pilot scale, using an apparatus with a 2.5-L working volume. The results showed that the semi-pilot system was comparable to the smaller scale system.     

Continuous aqueous two-phase systems devices for the recovery of biological products

Aqueous two-phase systems (ATPS) have proved to be a suitable technique for the recovery of biological products. Although ATPS have been in the field of primary recovery and purification of products for several years, the majority of the studies exploiting ATPS are usually based on batch mode operation. Reports on the potential of using continuous ATPS are not common. This review attempts to present a practical analysis of selected devices employed for ATPS continuous processing, from the conventional column contactors to novel designed mixer-settler units. A critical analysis of operational and design parameters that impact the system performance is presented. Current trends on the implementation of continuous ATPS approaches are discussed, together with the major challenges faced for the generic adoption of the technique. Conclusions are drawn on the major contribution of previous studies in the field to provide a better understanding of the technique for the newcomers.     

Mathematical modelling of lignin pyrolysis

Seven lignins from different sources were pyrolysed (i) isothermally in vacuum over the temperature range 300–1300 °C and (ii) at a constant heating rate of 30 °C min^?1 and a pressure of 0.1 MPa over the temperature range 150–900 °C. The mass fraction of each product—char, tar and gas species—and the elemental composition of the char and the tar were determined for the flash pyrolysis experiments. The evolution rates of the gas species and the tar versus the dynamic temperature of pyrolysis were determined for the constant heating rate pyrolysis experiments. Although the amount of each product species varied from lignin to lignin, the evolution rates were insensitive to the lignin source and the extraction process. To model the data, modifications were made to a recently developed model of coal pyrolysis. The model proved to be successful in simulating both the data from vacuum flash pyrolysis and constant heating rate pyrolysis of Iotech lignin.     

双水相萃取法分离纯化黄酮类化合物的研究进展

综述双水相体系的组成、特点和双水相萃取法分离纯化黄酮类化合物的研究进展。双水相萃取是一项利用不复杂的设备,并在温和条件下进行简单的操作就可获得较高收率和有效成分的新型分离技术,该技术在黄酮类化合物的分离分析中取得了较好的效果,有望成为一种新型的黄酮类化合物的分离纯化方法。     

双水相萃取黄连生物碱的研究

利用聚乙二醇(PEG)和硫酸铵（(NH4)2SO4）形成的双水相体系萃取黄连生物碱。通过双水相体系的改变,分别对黄连中4种主要生物碱：小檗碱、黄连碱、巴马丁、药根碱在双水相中的分配行为进行研究,并结合紫外分光光度法检测筛选出黄连生物碱最佳萃取工艺。最佳萃取工艺参数：pH = 6,T = 20 ℃,PEG1000质量分数20%,(NH4)2SO4质量分数16%,小檗碱、黄连碱、巴马丁、药根碱的最高萃取率分别为99.79%,,98.04%,99.96%,99.39%。双水相萃取黄连生物碱高效环保可行。     

Recovery of B19 virus-like particles by aqueous two-phase systems

The development of process routes for the recovery of intracellular B19 virus-like particles by aqueous two-phase systems (ATPS) was investigated in this study. Two processes have been developed which may prove advantageous when compared to the conventional recovery processes. One process was designed to recover B19 particles from the bottom phase of a polyethylene glycol (PEG) 1000-magnesium sulphate system while removing cell debris and 31% of total protein with the top phase, interface and sediment. With regard to the analysis of VP1 and VP2 capsid protein after extraction, the yield of B19 particles in the bottom phase was 92.8% or 85.7%, respectively. In an alternative process, B19 particles were recovered from a clarified cell disruptate by interfacial partition. With regard to VP1 and VP2 proteins, 95.3% or 33.2%, respectively was recovered in the interface of a PEG 1000-magnesium sulphate system.     

Deep eutectic solvents aqueous two-phase system based ultrasonically assisted extraction of ursolic acid (UA) from Cynomorium songaricum Rupr.

Deep eutectic solvents (DESs) are new green solvents that have attracted the attention of the scientific community mainly due to their unique properties and special characteristics, which are different from those of traditional solvents. A method based on ultrasonically assisted deep eutectic solvent aqueous two-phase systems (UAE-DES-ATPS) was developed for extracting ursolic acid (UA) from Cynomorium songaricum Rupr. Four different types of choline chloride-based DESs were prepared. Choline chloride-glucose (ChCl-Glu) exhibited good selective extraction ability. An optimum DES-ATPS of 36% (w/w) ChCl-Glu and 25% (w/w) K₂HPO₄ was considered to be a satisfactory system for extracting UA. Response surface methodology (RSM) method was utilized to optimize the extraction of UA using UAE-DES-ATPS. The quadratic models were extremely significant (p?<?0.01) for all the parameters studied with high correlation coefficients (R²) of 0.9593. The optimum ultrasound-assisted conditions were as follows: solvent to solid ratio of 15:1 (g/g), ultrasound power density of 4.7?W·mL^?1, and extraction time of 54?min. Compared with the conventional UAE method, the yields were basically the same, but the presented method had higher purity. The structure of UA did not change between pure UA and UA in the upper phase using UV–vis and FT-IR. This approach using ChCl-based DES-ATPS as a novel extraction system and ultrasound as a source of energy provided a better choice for the separation of active components from other natural products.     

Simultaneous recovery of glycyrrhizic acid and liquiritin from Chinese licorice root (Glycyrrhiza uralensis Fisch) by aqueous two-phase system and evaluation biological activities of extracts

Aqueous two-phase system (ATPS) was used for simultaneous purification of glycyrrhizic acid (GA) and liquiritin (LQ) from crude extract of Chinese licorice root. It was revealed that 87% GA and 94% LQ were retrieved in the ATPS top phase, under the optimum conditions of 25% (w/w) ethanol, 30% (w/w) K₂HPO₄ and 4% (w/w) loading sample at 10–40°C. Compared with crude extract, the ATPS top-phase extract exhibited the highest antioxidative activity, but no tyrosinase inhibitory effect. Whereas, the ATPS bottom-phase extract was proved to be effective ABTS radical scavenger and tyrosinase inhibitor, suggesting the potency of the alcohol-salt ATPS purification for the different medicinal purposes.     

双水相萃取藻蓝蛋白的机理

采用荧光光谱的方法对双水相萃取藻蓝蛋白(PC)的机理进行了研究。不同分子量的聚乙二醇(PEG)对PC均有荧光猝灭作用。PEG与PC相互作用的热力学研究结果表明,PEG1000与PC的结合常数大于PEG2000和PEG4000,两者是熵驱动下的疏水作用力结合。不同的无机盐对PEG1000与PC结合的影响不同,其中Na₂SO₄使PEG1000-PC体系熵增效果最显著。同步荧光光谱的研究表明,PEG1000使PC色氨酸残基微环境的疏水性增强;Na₂SO₄通过促进PEG1000与PC的结合而使色氨酸残基微环境的疏水性进一步增强。单因素扫描实验的结果表明,PEG1000/Na₂SO₄体系双水相萃取纯化藻蓝蛋白(PC)的能力最强,符合PEG1000与PC分子相互作用的热力学规律。     

High throughput screening techniques in downstream processing: Preparation, characterization and optimization of aqueous two-phase systems

Merging aqueous two-phase systems (ATPS) with high throughput screening techniques is a promising approach which will certainly lead to a more frequent use of ATPS both in industry and in research. Despite the diverse possible applications of polymer-polymer or polymer-salt based extraction systems for biotechnological downstream processes the use of these systems has so far been limited. This is mostly due to the poor predictability and the large number of parameters influencing the partitioning result. Especially the large number of possible parameter combinations makes robotic high throughput screening an interesting alternative to manual experiments. Methods using the robotic pipetting station Tecan Freedom Evo 200 for the development of ATPS processes are described in this paper. Characteristic values such as tie lines, binodal curve and volumetric measurement are essential for the characterization of this kind of systems and can successfully be determined by the robotic system. Most important, the measurement of protein concentrations, partition coefficients and yields could also be automated and parallelized offering the possibility to design ATPS processes while screening a broad range of parameters with a high sample throughput.     

Supercritical carbon dioxide extraction of astaxanthin from Paracoccus NBRC 101723: Mathematical modelling study

Astaxanthin (AX) is a secondary metabolite that accumulates inside the cell during Paracoccus fermentation. The fermentation biomass was extracted using supercritical carbon dioxide (SC-CO₂). The solubility parameter of AX, CO₂ and entrainer solvents was calculated and validated with experimental results. The pressure and particle size of the biomass had a significant effect on the extraction of AX. A maximum recovery of 963.33 μg/g of AX was obtained after SC-CO₂ extraction at 40ºC, 350 bar and a run time of 60 min with ethanol (20% v/w) as an entrainer. Further, the experimental data has been modelled using non-linear regression analysis method.