Modeling continuous aqueous two-phase systems for control purposes

A mathematical model was proposed to allow the analysis of steady-state and transient behaviors of single-stage continuous aqueous two-phase systems. Since the complete system of simultaneous equations contains more equations than unknown variables, a program based on the method of least squares was developed to solve the problem. The methodology was tested using a system composed of thaumatin, sodium chloride, and a contaminant protein. A poly(ethylene-glycol)/phosphate salt/water system was selected to isolate the thaumatin. For the steady-state and transient operations, a constrained optimization procedure--from the Matlab Optimization Toolbox (MathWork, Inc.)--was implemented after recasting the system of equations as a minimization problem. Euler's method was used in the transient case to discretize the differential equations. The steady-state concentrations agreed with published data. An input-output model based on a 4% step change decrease in the inlet stream flow rate showed that output variables such as concentrations of sodium chloride and phosphate salt settled to their final values in different time periods. The proposed analysis may be helpful in the dynamic control of large-scale commercial extractor units using advanced control schemes.     

Drop formation in aqueous two-phase systems

Extraction using aqueous two-phase systems (ATPSs) is a versatile technique for the downstream processing of various proteins/enzymes. The study of drop formation deals with the fundamental understanding of the behavior of liquid drops under the influence of various external body as well as surface forces. These studies provide a basis for designing of the extractions in column contactors in which liquid drops play a major role. Most of the drop formation studies reported so far is restricted to aqueous-organic systems. ATPSs, differ from aqueous-organic systems in their physical properties. In view of this, an attempt was made to develop a model for drop formation in ATPSs adopting the information available on aqueous-organic systems. In order to validate the model, experiments were performed by using polyethylene glycol (PEG)/salt systems of different phase compositions at various flow rates. At low flow rates the single stage model and at high flow rates the two stage model are able to predict the drop volume during its formation from tip of capillary. The experimental results were found to agree reasonably well with those predicted by the model.     

Cosolutes effects on aqueous two-phase systems equilibrium formation studied by physical approaches

The effect of urea and sodium salts of monovalent halides on the aqueous polyethyleneglycol solution and binodal diagrams of polyethyleneglycol–potassium phosphate (polyethyleneglycol of molecular mass 1500, 4000, 6000 and 8000) were studied using different physical approaches. The effect of these solutes on the binodal diagram for polyethyleneglycol–potassium phosphate was also investigated. The cosolutes affected in a significant manner the water structured around the ethylene chain of polyethyleneglycol inducing a lost of this. The equilibrium curves for the aqueous two-phase systems were fitting very well by a sigmoidal function with two parameters, which are closely related with the cosolute structure making or breaking capacity on the water ordered.     

Solvatochromic relationship: Prediction of distribution of ionic solutes in aqueous two-phase systems

Partition ratios of several ionic compounds in 20 different polymer/polymer aqueous two-phase systems (ATPS) containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4, were determined. The differences between the electrostatic properties of the phases in all the ATPS were estimated from partitioning of the homologous series of dinitrophenylated-amino acids. Also the solvatochromic solvent parameters characterizing the solvent dipolarity/polarizability (π*), solvent hydrogen-bond donor acidity (α), and solvent hydrogen-bond acceptor basicity (β) of aqueous media were measured in the coexisting phases of the ATPS. The solute-specific coefficients for the compounds examined were determined by the multiple linear regression analysis using the modified linear solvation energy relationship equation. The minimal number of ATPS necessary for determination of the coefficients was established and 10 ATPS were selected as a reference ATPS set. The solute-specific coefficients values obtained with this reference set of ATPS were used to predict the partition ratios for the compounds in 10 ATPS not included in the reference set. The predicted partition ratios values were compared to those determined experimentally and found to be in good agreement. It is concluded that the presented model of solute–solvent interactions as the driving force for solute partitioning in polymer/polymer ATPS describes experimental observations with 90–95% accuracy.     

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.     

Aqueous two-phase systems for protein separation: a perspective

Aqueous two-phase systems (ATPS) that are formed by mixing a polymer (usually polyethylene glycol, PEG) and a salt (e.g. phosphate, sulphate or citrate) or two polymers and water can be effectively used for the separation and purification of proteins. The partitioning between both phases is dependent on the surface properties of the proteins and on the properties of the two phase system. The mechanism of partitioning is complex and not very easy to predict but, as this review paper shows, some very clear trends can be established. Hydrophobicity is the main determinant in the partitioning of proteins and can be measured in many different ways. The two methods that are more attractive, depending on the ATPS used (PEG/salt, PEG/polymer), are those that consider the 3-D structure and the hydrophobicity of AA on the surface and the one based on precipitation with ammonium sulphate (parameter 1/m*). The effect of charge has a relatively small effect on the partitioning of proteins in PEG/salt systems but is more important in PEG/dextran systems. Protein concentration has an important effect on the partitioning of proteins in ATPS. This depends on the higher levels of solubility of the protein in each of the phases and hence the partitioning observed at low protein concentrations can be very different to that observed at high concentrations. In virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. Furthermore, true partitioning behavior, which is independent of the protein concentration, only occurs at relatively low protein concentration. As the concentration of a protein exceeds relatively low values, precipitation at the interface and in suspension can be observed. This protein precipitate is in equilibrium with the protein solubilized in each of the phases. Regarding the effect of protein molecular weight, no clear trend of the effect on partitioning has been found, apart from PEG/dextran systems where proteins with higher molecular weights partitioned more readily to the bottom phase. Bioaffinity has been shown in many cases to have an important effect on the partitioning of proteins. The practical application of ATPS has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. This separation and purification has also been successfully used for the separation of virus and virus-like particles.     

Construction of ternary phase diagrams in nonsolvent/solvent/PMMA systems

In this work, the ternary phase diagrams in three nonsolvent/solvent/PMMA systems (n-hexane/n-butyl acetate/PMMA, water/acetone/PMMA, and n-hexane/acetone/PMMA) were constructed by theoretical calculation and experimental measurement. Binodal curves were calculated by using the Flory–Huggins theory for three-component systems and measured by titrating the PMMA solution with nonsolvent until the onset of turbidity. By using concentration-dependent nonsolvent/solvent and solvent/PMMA interaction parameters and constant nonsolvent/PMMA interaction parameters, good agreement has been obtained between the calculation and the measurement. The values of nonsolvent/solvent interaction parameters were taken from the literature sources, and the values of solvent/PMMA and nonsolvent/PMMA interaction parameters were measured by vapor sorption and swelling equilibrium, respectively. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 607–615, 1998     

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.     

Interfacial tension of the aqueous two-phase systems of cationic-anionic surfactant mixtures

Interfacial tensions of the aqueous two-phase systems formed by cationic-anionic surfactant mixtures were measured using spinning drop method. The effects of surfactant structure, molar ratio of cationic to anionic surfactants, surfactant concentration, salt, and temperature on the interfacial tensions were investigated. It was shown that the values of the interfacial tensions of the aqueous two-phase were in the scale of ultra-low interfacial tensions at certain molar ratios of cationic to anionic surfactants. Three types of interfacial tension curves were observed. The first curve comprised two curves that were located on either side of 1:1 molar ratio, and the interfacial tension decreased with the increase of excessive surfactant components. The second one was a saddle-shaped curve that strode over the 1:1 molar ratio. The third type was a saddle-shaped curve that was located beside the 1:1 molar ratio. The types of interfacial tensions depended on the molecular structure of the surfactants such as the hydrophilic groups and the lengths and symmetry of hydrophobic chains.     

Synthesis and characteristic of the thermo- and pH-sensitive hydrogel and microporous hydrogel induced by the NP-10 aqueous two-phase system

A series of novel thermo- and pH-sensitive (NP10-AA TPS) hydrogels and microporous (NP10-AA MP) hydrogels, inducing by polyoxyethylene (10) nonyl phenyl ether (NP-10) aqueous two-phase system, was designed and fabricated with acrylic acid (AA) as the monomer for the first time. The resultant NP10-AA TPS hydrogel, compared with the traditional TPS hydrogel, was more advanced in both of the high swelling ratio and the variable lower critical solution temperature (LCST). A simple synthesis technique of the NP10-AA MP hydrogel was developed. The thermo-sensitivity of the NP10-AA TPS hydrogel including the initial swelling ratio, LCST, dehydrated efficiency, was depended strongly on the crosslinker (MBA), initiator (APS), NP-10 and AA concentration. The swelling rate of the NP10-AA MP hydrogel was much higher than that of AA hydrogel dehydrated in the same lyophilization condition.     

Phase transitions and phase separations in aqueous polyether systems

Detailed investigations on the structural and macroscopic properties of a hydrogel based on a polyether/water/LiCl system as a function of composition and temperature are reported. The investigations include the determination of the types of water by differential scanning calorimetry (DSC) measurements, the characterization of the polymorphism by microscopic observations in combination with DSC measurements as well as the determination of the optical and viscoelastic properties by spectroscopic and rheological measurements. The influence of the addition of salts on the water binding properties of the polyether are reported and the results are discussed on a molecular level. For the optical properties it was found that the transmission–temperature curves reflect the polymorphism of the samples. Two-phase regions at a phase transition or regions of coexisting phases (phase separation or mixing gaps) lead to the appearance of light scattering states, whereas homogeneous phase regions are highly transparent. The rheological measurements indicate the formation of hydrogels by cross-linking with LiCl, whereby in different phases the gelation occurs at different LiCl concentration ranges. Received: 3 August 2000 Revised: 19 November 2000 Accepted: 29 November 2000     

Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt

Based on aqueous two-phase systems (ATPS) consisting of 1-butyl-3-methylimidazolium chloride, a hydrophilic ionic liquid (IL), and K2HPO4, a new and simple extraction technique, coupled with a reversed-phase high performance liquid chromatography (RP-HPLC), was developed for the simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine. Under the optimal conditions, the extraction efficiencies for both analytes were 80-90% in a one-step extraction. The method required only 3.0 mL of urine and a single hydrolysis/deproteinization/extraction step followed by direct injection of the IL-rich upper phase into HPLC system for analysis. The method has been satisfactorily applied to the analysis of T and ET in human urine with detection limits of 1 ng/mL and linear ranges of 10-500 ng/mL for both compounds. Compared with conventional liquid-liquid extraction or solid phase extraction, this new method is much "greener" due to no use of volatile organic solvent and low consumption of IL. The proposed extraction technique opens up new possibilities in the separation of other drugs.     

Aqueous two-phase systems containing N-vinylpyrrolidone: Experimental results and correlation/prediction

Experimental results are presented for the liquid–liquid equilibrium of a ternary system (N-vinylpyrrolidone + sodium sulfate + water) and three quaternary systems composed of (N-vinylpyrrolidone + poly(vinylpyrrolidone) + sodium sulfate + water) at 25 °C. The quaternary systems differ in the number averaged molecular mass of the poly(vinylpyrrolidones). That molecular mass was between about 4 000 and 140 000. The experimental results are described with a semi-empirical model (VERS model) for the excess Gibbs energy.     

Solvent properties governing protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of various proteins were measured in aqueous Dextran-Ficoll, Dextran-PES, and Ficoll-PES two-phase systems, containing 0.15M NaCl in 0.01 M phosphate buffer, pH 7.4. The acquired data were combined with data for the same proteins in different systems reported previously and known solvatochromic solvent properties of the systems to characterize the protein-solvent interactions. The relative susceptibilities of proteins to solvent dipolarity/polarizability, solvent hydrogen bond acidity, solvent hydrogen bond basicity, and solvent ability to participate in ion-ion and ion-dipole interactions were characterized. These parameters, which are representative of solute-solvent interactions, adequately described the partitioning of the proteins in each system. It was found that the relative susceptibilities of proteins to solvent dipolarity/polarizability are interrelated with their relative susceptibilities to solvent hydrogen bond acidity and solvent hydrogen bond basicity similarly to those established previously for small nonionic organic compounds.     

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.     

Membrane protein isolation by in situ solubilization, partitioning and affinity adsorption in aqueous two-phase systems. Purification of the human type 1 11beta-hydroxysteroid dehydrogenase

Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11beta-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg(-1) min(-1) of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg(-1) min(-1) was achieved.     

Thermodynamic analysis of polymerization-induced phase separation of a polystyrene in epoxy/monoamine–diamine systems. Effect of monoamine–diamine proportion on the phase diagram

Polymerization-induced phase separation of a polystyrene in various epoxy-amine systems where the amino groups were provided by a monoamine and a diamine mixed in different proportions was thermodynamically studied. A model based on the Flory–Huggins theory extended by Koningsveld and Staverman approach where the interaction parameter was dependent on temperature, composition and conversion, and polydispersity of the components was considered, was used. A general equation for the evolution during polymerization of the epoxy-amine species distributions according to the monoamine–diamine ratio was derived from the Stockmayer distribution. The interaction parameters continuously decreased with conversion. Phase diagrams of the blends were obtained and the critical composition was between 5 and 6 vol.% PS in all blends.     

Electrokinetic demixing of aqueous two-phase polymer/salt systems

Electrokinetic demixing of aqueous two-phase polymer/salt systems is demonstrated, resulting in significant enhancement in demixing rates by about 1-4-fold. The effect of field polarity, field strength, volume ratio, and phase composition on phase demixing has been studied. Further the influence of these parameters on phase demixing could be explained based on the hydrodynamic flow-electroosmotic flow (HEF) model.     

Effect of anionic structure on the phase formation and hydrophobicity of amino acid ionic liquids aqueous two-phase systems

Compared with the conventional ionic liquids, amino acid ionic liquids are more biodegradable and biocompatible, and can enhance stability of biomaterials. In this work, amino acid ionic liquids 1-butyl-3-methylimidazolium L-serine ([C(4)mim][Ser]), 1-butyl-3-methylimidazolium glycine ([C(4)mim][Gly]), 1-butyl-3-methylimidazolium L-alanine ([C(4)mim][Ala]) and 1-butyl-3-methylimidazolium L-leucine ([C(4)mim][Leu]) have been synthesized. These ionic liquids are found to form aqueous two-phase systems (ATPSs) by the salted-out of K(3)PO(4) in aqueous solutions. Phase diagram of the ATPSs and the Gibbs energies of transfer of methylene group from the bottom salt-rich phase to the top ionic liquid-rich phase have been determined at 298.15K and pH 14, and the effect of anionic structure of the ionic liquids on phase formation of the ATPSs and the relative hydrophobicity between the top and the bottom phases are then examined. In order to understand the effect of relative hydrophobicity of the phases in equilibrium in the ATPSs on the extraction/separation capability of biomolecules, the partition coefficients of cytochrome-c (as a model biomolecule) in the ATPSs are measured by spectrophotometry. It is suggested that hydrophobic interactions are mainly responsible for the higher partition coefficients of cytochrome-c in aqueous two-phase systems at pH 14, and the extraction and separation capacity of biomolecules can be improved by the modulation of the relative hydrophobicity of the phases and/or the pH of the system.     

A green deep eutectic solvent-based aqueous two-phase system for protein extracting

As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n = 3), 1.6057% (n = 3) and 1.6132% (n = 3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV–vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES–protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins.