Interfacial Tension of Polyethylene Glycol/Potassium Phosphate Aqueous Two-Phase Systems

Abstract

The interfacial tension of Polyethylene glycol (PEG)/potassium phosphate two-phase systems was measured by the rotating drop method. The interfacial tension was as low as 0.001 dyne/cm and increased with increases in the total concentrations of both PEG and potassium phosphate in two-phase systems. The increase in the interfacial tension was a function of the concentration differences of PEG and potassium phosphate between the top and the bottom phases which was confirmed by the tie line analysis. The interfacial tension was affected also by the molecular weigth of PEG. At low PEG molecular weights, the increase in the molecular weight greatly increased the interfacial tension, but at high molecular weights, the interfacial tension varied less with the molecular weight.     

Two-step recovery process for tryptophan tagged cutinase: interfacing aqueous two-phase extraction and hydrophobic interaction chromatography

In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)4 was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K-Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to >95% purity according to silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a total yield of 83% during the two-step recovery process.     

Effect of Coriolis force on counter-current chromatographic separation by centrifugal partition chromatography

The effect of Coriolis force on the counter-current chromatographic separation was studied using centrifugal partition chromatography (CPC) with four different two-phase solvent systems including n-hexane-acetonitrile (ACN); tert-butyl methyl ether (MtBE)-aqueous 0.1% trifluoroacetic acid (TFA) (1:1); MtBE-ACN-aqueous 0.1% TFA (2:2:3); and 12.5% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) dibasic potassium phosphate. Each separation was performed by eluting either the upper phase in the ascending mode or the lower phase in the descending mode, each in clockwise (CW) and counterclockwise column rotation. Better partition efficiencies were attained by the CW rotation in both mobile phases in all the two-phase solvent systems examined. The mathematical analysis also revealed the Coriolis force works favorably under the CW column rotation for both mobile phases. The overall results demonstrated that the Coriolis force produces substantial effects on CPC separation in both organic-aqueous and aqueous-aqueous two-phase systems.     

Partitioning of Porcine Pancreatic Lipase in a Two-Phase Systems of Polyethylene Glycol/Potassium Phosphate Aqueous

The hydrolysis of triglycerides at the oil–water interface, synthesis of esters and transesterification in microaqueous conditions are catalysed by lipase. For its application, a proper purification method was necessary. This study examined the application of an aqueous two-phase system to partition porcine pancreatic lipase. The influence of molecular weight and concentration of polyethylene glycol (PEG), tie line length (TLL), potassium phosphate concentration, sodium chloride (NaCl) addition and temperature in the partition was studied. The enzyme was more efficiently purified in PEG 8,000 at 14.5 °C (PF?=?3.89-fold), presenting more recoveries at the top phase with shorter TLL and lower concentrations of PEG and potassium phosphate. Moreover, the increase of these variables repressed the purification and the further addition of NaCl did not promote the purification of the enzyme. These results demonstrated the efficiency of the aqueous two-phase system on lipase purification.     

Separation of no-carrier-added 99mTcO4 − from 99Mo–99mTc equilibrium mixture by PEG based aqueous biphasic separation technique using sodium/potassium salts of citric and tartaric acid

Extraction and separation of no-carrier-added (nca) ^99mTcO₄ ^? from ⁹⁹Mo–^99mTc equilibrium mixture was carried out by environmentally benign polyethylene glycol based liquid–liquid aqueous biphasic systems (ABS) consisting various inorganic salts. Among the various inorganic salt trisodium citrate and potassium sodium tartrate showed the suitable salt rich phase for the best separation in this report. The concentration variation of salt rich phase, temperature and PEG phase also exhaustively studied in paper for the achievement of high separation factor. At 40 °C temperature in 50 % (w/v) PEG-4000-2M Na₃citrate showed the highest separation factor (S _Tc/Mo) 1.2 × 10⁷.     

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.     

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.     

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.     

Extractant Effect on the Retention of the Stationary Phase in a Rotating Coiled Column

The effects of physicochemical properties of two-phase liquid systems (interfacial tension and differences in density and viscosity) on the retention of the stationary phase in the column were examined. These effects mainly determine the separation parameters of compounds. Extractant/decane–aqueous phase systems were used; their physicochemical properties changed both as a result of adding an extractant (di-(2-ethylhexyl) phosphoric acid, trioctylamine, or tributyl phosphate) to the organic solvent and because of a change in the composition of the aqueous phase. Aqueous ammonium sulfate of varying concentration was used as a mobile phase. It was shown that interfacial tension substantially affects the behavior of the systems under consideration. An increase in the ammonium sulfate concentration only slightly affects the retention factor of the stationary phase in the column. With a proper choice of the stationary phase, countercurrent chromatography can be used for the extraction of components from salt solutions of various concentrations.     

One-step purification of alpha-amylase from the cultivation supernatant of recombinant bacillus subtilis by high-speed counter-current chromatography with aqueous polymer two-phase systems

Purification of alpha-amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography (HSCCC) in polyethylene glycol (PEG) 4000-inorganic salt aqueous polymer two-phase systems was studied. The effects of sodium chloride concentration on the partition coefficients of alpha-amylase and total protein were respectively tested in PEG4000-phosphate and PEG4000-citrate aqueous polymer two-phase systems to find the proper range of sodium chloride concentration for the HSCCC purification of alpha-amylase. Alpha-amylase was purified from the cultivation supernatant by HSCCC in PEG4000-phosphate system containing 2% (w/w) sodium chloride, yet with considerable loss of activity. PEG4000-citrate aqueous polymer two-phase system containing 2% (w/w) sodium chloride and supplemented with 0.56% (w/w) CaCl2 as protective agent was then successfully applied to purify alpha-amylase from cultivation supernatant by HSCCC to homogeneity and significantly increased the recovery of alpha-amylase activity from around 30 to 73.1%.     

The influence of carbon dioxide cosolvent on solubility in poly(ethylene glycol)

Supercritical carbon dioxide (CO₂) and poly(ethylene glycol) (PEG) can be utilized as an environmentally friendly biphasic solvent system for catalysis reactions and subsequent product separation. To efficiently implement this technology, it is important to understand how solutes partition between these phases as well as how dissolved CO₂ in PEG affects the solvent properties. The work presented here explores the influence of CO₂ on the solubility of four different solutes in PEG. The transferable potentials for phase equilibria-united atom force field and configurational-bias Monte Carlo molecular simulation were employed to determine the solubilities of ethylbenzene, 1-octene, 1-pentanol, and 2-pentanone at 323.15 K and 15?MPa in PEG-600 using an ideal vapor phase with a Poynting-corrected vapor pressure. The effect of CO₂ concentration within the PEG phase was determined by varying the amount from no CO₂ to the saturation limit. The results indicate that while there is preferential solvation of CO₂ around the solutes, solubility of non-polar solutes is unchanged whereas there is a modest increase for polar solutes as the concentration of CO₂ increases. Increased solubility is analyzed in terms of both modified solvent structure and direct solute–CO₂ interactions.     

Separation of Alkaloids from Datura Metel. and Sophora Flavescens Ait by High-Speed Countercurrent Chromatography

Abstract

High-speed countercurrent chromatography was successfully applied to the separation of alkaloids from two medicinal herbs. Matrine and oxymatrine were isolated from the root of Sophora flavescens Ait with a two-phase solvent system composed of chloroform-0.07 M sodium phosphate (pH 6.4), while atropine and scopolamine were purified from the flowers of Datura mete L. with a similar solvent system by modifying the phosphate buffer pH at 6.5. Identification of each alkaloid was made by either TLC or paper partition chromatography using authentic pure compounds.     

Horseradish peroxidase extraction and purification by aqueous two-phase partition

The effect of poly(ethyleneglycol) (PEG) molecular weight, system pH, and sodium chloride concentration on the partitioning behavior of horseradish peroxidase fromArmomcia rusticana root extract was investigated in poly(ethyleneglycol)/sodium phosphate systems. PEG molecular weight strongly affects the enzyme partition coefficient, whereas pH variation from 5.5 to 8.0 has little effect. The addition of sodium chloride (8% w/w) to a PEG 1540/phosphate system, pH 7.0, raises the peroxidase partition coefficient 13.5-fold without important changes in that of total horseradish root proteins. Moreover, these conditions allow direct homogenization of theA. rusticana roots with the selected aqueous two-phase system with the clear top phase containing over 90% of the enzyme and the purification factor being 4.8.     

Condensation of self-assembled lyotropic chromonic liquid crystal sunset yellow in aqueous solutions crowded with polyethylene glycol and doped with salt

We use optical and fluorescence microscopy, densitometry, cryo-transmission electron microscopy (cryo-TEM), spectroscopy, and synchrotron X-ray scattering to study the phase behavior of the reversible self-assembled chromonic aggregates of an anionic dye Sunset Yellow (SSY) in aqueous solutions crowded with an electrically neutral polymer polyethylene glycol (PEG) and doped with the salt NaCl. PEG causes the isotropic SSY solutions to condense into a liquid-crystalline region with a high concentration of SSY aggregates, coexisting with a PEG-rich isotropic (I) region. PEG added to the homogeneous nematic (N) phase causes separation into the coexisting N and I domains; the SSY concentration in the N domains is higher than the original concentration of PEG-free N phase. Finally, addition of PEG to the highly concentrated homogeneous N phase causes separation into the coexisting columnar hexagonal (C) phase and I phase. This behavior can be qualitatively explained by the depletion (excluded volume) effects that act at two different levels: at the level of aggregate assembly from monomers and short aggregates and at the level of interaggregate packing. We also show a strong effect of a monovalent salt NaCl on phase diagrams that is different for high and low concentrations of SSY. Upon the addition of salt, dilute I solutions of SSY show appearance of the condensed N domains, but the highly concentrated C phase transforms into a coexisting I and N domains. We suggest that the salt-induced screening of electric charges at the surface of chromonic aggregates leads to two different effects: (a) increase of the scission energy and the contour length of aggregates and (b) decrease of the persistence length of SSY aggregates.     

On extension of UNIQUAC-NRF model to study the phase behavior of aqueous two phase polymer–salt systems

The UNIQUAC-NRF model for aqueous two-phase polymer–polymer systems is extended to correlate liquid–liquid equilibria of aqueous polymer–salt two-phase systems. The systems investigated are polyethylene glycol+ammonium sulfate+water and polyethylene glycol+potassium monohydrogen phosphate (K₂HPO₄)+water for five molecular weights of PEG. In this model, the nonrandom state is selected as a reference state. The model has six binary adjustable parameters that were determined by an optimization program. The Debye–Huckel equation based on Fowller–Guggenheim equation is used to calculate the long-range electrostatic interaction of the ions. The results obtained by this model are in good agreement with experimental data.     

Quaternary (liquid + liquid) equilibria of aqueous two-phase poly (ethylene glycol), poly (DMAM–TBAM), and KH2PO4: Experimental and generalized Flory–Huggins theory

Quaternary (liquid + liquid) equilibrium (LLE) data of the aqueous two-phase poly (ethylene glycol), poly (N,N-dimethylacrylamide-t-butylacrylamide) with abbreviation name poly (DMAM–TBAM) as a hydrophobic association water-soluble copolymer and KH₂PO₄ has been determined experimentally at T = 338.15 K. Furthermore, the generalized Flory–Huggins theory with two electrostatic terms (the Debye–Hückel and Pitzer–Debye–Hückel) was used for correlation of the phase behavior of the quaternary system and the interaction parameters between all species were calculated.It was found that addition of poly (DMAM–TBAM) copolymer as well as changing the temperature can shift the binodal curves of aqueous two-phase systems containing polyethylene glycol (PEG) and salt. Also, the phase behavior of the DMAM–TBAM copolymer with some salts containing sodium chloride, ammonium hydrogen phosphate, potassium hydrogen phosphate, and sodium carbonate were studied experimentally at T = 338.15 K and the effect of the salt type on the their binodal curves was determined.     

Liquid–liquid equilibria of water/1-propanol/methyl ethyl ketone/potassium chloride

The liquid–liquid equilibrium of water/1-propanol/methyl ethyl ketone (MEK) at 25°C was significantly modified by the presence of dissolved potassium chloride. Water is salted out of the organic phase while MEK is more preferentially salted out of the aqueous phase than 1-propanol. These results in considerable enlargement of the two-phase region and enhancement of the extractive efficiency of MEK for the separation of 1-propanol from its aqueous mixture. Good correlation of the liquid–liquid equilibria (LLE) of the system in the presence of potassium chloride up to saturation was obtained with Tan’s modified NRTL phase model for multicomponent solute–solvent mixtures with the solute–solvent interaction parameters expressed as a third-order polynomial function in salt concentration. Similar to the observation reported for vapour–liquid equilibrium (VLE) of solvent–solute mixtures, salting-in and salting-out of the solvent components from the respective phases can be predicted according to the relative solute–solvent interaction parameters of the solvent components in the two phases.     

Effects of ammonium sulfate and sodium chloride concentration on PEG/protein liquid-liquid phase separation

When added to protein solutions, poly(ethylene glycol) (PEG) creates an effective attraction between protein molecules due to depletion forces. This effect has been widely used to crystallize proteins, and PEG is among the most successful crystallization agents in current use. However, PEG is almost always used in combination with a salt at either low or relatively high concentrations. Here the effects of sodium chloride and ammonium sulfate concentration on PEG 8000/ovalbumin liquid-liquid (L-L) phase separation are investigated. At low salt the L-L phase separation occurs at decreasing protein concentration with increasing salt concentration, presumably due to repulsive electrostatic interactions between proteins. At high salt concentration, the behavior depends on the nature of the salt. Sodium chloride has little effect on the L-L phase separation, but ammonium sulfate decreases the protein concentration at which the L-L phase separation occurs. This trend is attributed to the effects of critical fluctuations on depletion forces. The implications of these results for designing solution conditions optimal for protein crystallization are discussed.     

New solvents for cellulose: Hydrazine/thiocyanate salt system

The hydrazine/thiocyanate system was found to be an excellent solvent for cellulose. The solubility and solution properties were investigated. Even at room temperature, the combinations of hydrazine and lithium, sodium, and potassium thiocyanate had high dissolution power for cellulose, up to an 18% (w/w) maximum, unrelated to the polymorph, whereas a combination with ammonium thiocyanate exhibited a solubility difference among celluloses I, II, and III. The effect of the temperature cycling of the system for the rapid dissolution of cellulose was investigated thermodynamically. In these systems, a high concentration of salts was necessary to effect the cellulose dissolution; this suggested that an undissociated salt–solvent complex played an important role in the cellulose dissolution as implied by electroconductivity measurements of the hydrazine/salt system. Gel and liquid‐crystal formation was observed in all systems above 4 and 6% (w/w) cellulose concentrations, respectively. The values of both critical concentrations were quite similar to those observed in the ammonia/ammonium thiocyanate system studied earlier in our laboratories. The gelation temperature was between approximately 10 and 50 °C, depending on the salt and cellulose concentration. The dependence of the cellulose solubility on the degree of polymerization was also examined. It is suggested that these solvent systems have great potential for the fiber and film formation of cellulose. © 2002 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 40: 601–611, 2002; DOI 10.1002/pola.10135     

Salt Effects on the Aqueous Two-Phase System of Gemini(12-3-12, 2Br<Superscript>−</Superscript>)/SDS/PEG

The effect of added salts (NaCl, KCl and NaBr) on the aqueous two-phase system (ATPS) formed in mixtures of Gemini(12-3-12, 2Br⁻)/sodium dodecyl sulfate/polyethylene glycol has been investigated. Phase diagrams of the aqueous systems containing Gemini(12-3-12, 2Br⁻), sodium dodecyl sulfate (SDS), polyethylene glycol(PEG) and a salt have been determined experimentally at 313.15 K. The results indicate that the addition of salts not only induces the appearance of ATPS-A (in which anionic surfactant is in excess), shortens the phase separation time, enlarges the regions of ATPS-C (in which cationic surfactant is in excess), and decreases the minimum concentration required for forming an ATPS, but also alters the matching between anionic and cationic surfactants. Extractive experiments also showed that these salts notably enhance the extraction ability of ATPS; the Gemini-rich phase exhibits prominent cohesive action with xylenol orange, regardless of whether or not it is the upper phase or the lower phase.