Phase Of Aqueous Two-phase System Research Articles

Hydrogen Bond Arrangement Is Shown to Differ in Coexisting Phases of Aqueous Two-Phase Systems.

Analysis by attenuated total reflection–Fourier transform infrared spectroscopy shows that each coexisting phase in aqueous two-phase systems has a different arrangement of hydrogen bonds. Specific arrangements vary for systems formed by different solutes. The hydrogen bond arrangement is shown to correlate with differences in hydrophobic and electrostatic properties of the different phases of five specific systems, four formed by two polymers and one by a single polymer and salt. The results presented here suggest that the arrangement of hydrogen bonds may be an important factor in phase separation.

Surfactant as selective modulator in the partitioning of dyes in aqueous two-phase systems: A strategy for separation

In this work, aqueous two-phase systems (ATPSs) were employed to separate two model dyes, remazol golden yellow (RY) and methylene blue (MB), using the cetyltrimethylammonium bromide (CTAB) surfactant as a selective modulator in the partitioning. Experiments involving individual and simultaneous partitioning of the dyes were performed in the absence and presence of CTAB in ATPSs formed by a polymer (polyethylene glycol (PEG), polypropylene glycol (PPG), or poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) copolymer (L35)), a salt ((NH4)2SO4, MgSO4, Na2SO4, or Li2SO4), and water. In experiments of individual partitioning, RY and MB were transferred to the top phase in ATPSs formed using PEG in the absence of CTAB. However, in the presence of certain surfactant concentrations, the MB dye remained in the top phase, while the RY dye was transferred to the bottom phase. The same result was obtained in experiments of simultaneous partitioning. This was attributed to a specific interaction between CTAB and RY, which promoted the formation of RY-CTAB aggregates that were stabilized in the bottom phase. The amount of RY transferred to the bottom phase in the presence of CTAB was highly dependent on the polymer molar mass and the natures of the salt and the polymer. Interestingly, the surfactant did not promote the transfer of RY to the bottom phase in ATPSs formed by PPG or L35, due to competition between the polymer and the RY dye for the CTAB monomers. The best separation factor between the dyes was of 271.2 in the ATPS formed by PEG with molar mass of 1500 g mol−1, MgSO4, and water, hence demonstrating the potential of ATPSs for separation among dyes.

Role of Hydrophobic Interaction in Driving the Partitioning of Metal Ions in a PEG-Based Aqueous Two-Phase System

PEG-based aqueous two-phase systems (ATPS) for extraction and removal of metal ions from industrial wastewaters is a promising strategy to replace traditional organic-aqueous two phase systems, due to its environmentally friendly advantages and excellent separation efficiency from controllable phase structure and physicochemical properties. However, controversy still remains about the partitioning mechanism of metal ions into the PEG-rich phase in ATPS. The present contribution revealed that a specific hydrophobic interaction was a main driving force for the transferring of Cr(VI) ions into the PEG-rich phase in ATPS. The properties of aqueous media around the PEG molecules and Cr(VI) ions also played a crucial role in the partitioning of Cr(VI) ions in the PEG-base ATPS. The PEG-rich phase in the ATPS exhibited a specific affinity toward poorly hydrated HCrO4– ions, compared to the strongly hydrated CrO42– ions. The difference in the properties of aqueous media in the coexisting liquid phases was respons...

Determination of Benzene Series in Fish Based on Aqueous Two-Phase System for Samples Pretreatment

A novel method was developed for analyzing benzene series (benzene, toluene, ethylbenzene, xylene, and styrene; abbreviate as BTEXS) in fish sample by gas chromatography–mass spectroscopy (GC-MS), which was simplified the sample pretreatment process using matrix removal method by aqueous two-phase system (ATPS) of acetonitrile-K2HPO4-H2O. The interaction of protein with BTEXS was investigated via the equilibrium dialysis coupled with GC-MS. The results of equilibrium dialysis indicated that the combination among BTEXS and protein was very strong. In suitable concentration of acetonitrile aqueous solution, a slow but full protein denaturation takes place, which causes the unfolding of protein and the release of BTEXS. Then, the BTEXS were extracted into upper phase of ATPS of acetonitrile-K2HPO4-H2O with high extraction efficiencies. This method has been applied to the determination of seven benzene series (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, and styrene) residues in the fish sample. The detection limits were in the range of 1.1 ∼ 4.3 ng/g, and the spiked recoveries were 81.4 ∼ 97.8 %, with RSD 1.3 ∼ 5.1 %.

Effect of salt additives on protein partition in polyethylene glycol–sodium sulfate aqueous two-phase systems

Partitioning of 15 proteins in polyethylene glycol (PEG)–sodium sulfate aqueous two-phase systems (ATPS) formed by PEG of two different molecular weights, PEG-600 and PEG-8000 in the presence of different buffers at pH7.4 was studied. The effect of two salt additives (NaCl and NaSCN) on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the proteins partition coefficients in ATPS with and without salt additives. The results obtained show that the concentration of buffer as well as the presence and concentration of salt additives affects the protein partition behavior. Analysis of ATPS in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning in PEG-600–salt and PEG-8000–salt ATPS cannot be explained by the protein size or polymer excluded volume effect. It is suggested that the protein–ion and protein–solvent interactions in the phases of ATPS are primarily important for protein partitioning.

Selective extraction of Hg(II), Cd(II) and Zn(II) ions from aqueous media by a green chemistry procedure using aqueous two-phase systems

A simple aqueous two-phase systems of polyethylene glycol (PEG)/sulphate salt (Na2SO4 or (NH4)2SO4) was proposed for the selective extraction of Hg(II), Cd(II) and Zn(II) ions from aqueous media, in presence of iodide as extracting agents. The extraction efficiency of metal ions from salt-rich phase to the polymer-rich phase was investigated as a function of the amount of added iodide extractant, nature of inorganic sulphate used as phase-forming salt, the volume ration between phases of aqueous two-phase system and the pH of salt solution. Maximum extraction efficiency of considered metal ions was obtained in the aqueous polyethylene glycol/ammonia sulphate two-phase system and a volume ration of the phases equal with 1.0. On the other hand, in mentioned experimental conditions, at iodide concentration higher than 0.06molL−1, the Hg(II) ions are quantitatively extracted (>99%) into polymer-rich phase, at all studied values of salt solution pH, while a quantitative extraction of Cd(II) ions was obtained only in strong acid media (salt solution pH lower than 3.0). The extraction efficiency of Zn(II) ions is insignificant (<15%) for all values of salt solution pH and iodide concentration added as extractant, they preferring to remain in the salt-rich phase of extraction system. Therefore, by adequate selection of the experimental conditions, and efficient and selective extraction of these metal ions could be obtained, and a separation procedure in two steps is proposed. The separation factors calculated for each case (SHg/Cd=(1.48±0.13)×103; SHg/Zn=(2.99±0.21)×103 and SCd/Zn=(3.16±0.17)×103, respectively) indicate that the proposed procedure is efficient and has a large viability for the selective extraction of Hg(II) and Cd(II) ions in presence of Zn(II) ions. In addition, this extraction procedure has the main advantage that is environmental safe.

Superparamagnetic lysozyme surface-imprinted polymer prepared by atom transfer radical polymerization and its application for protein separation

Molecular imprinting as a promising and facile separation technique has received much attention because of their high selectivity for target molecules. In this study, the superparamagnetic lysozyme surface-imprinted polymer was prepared by a novel fabricating protocol, the grafting of the imprinted polymer on magnetic particles in aqueous media was done by atom transfer radical polymerization (ATRP), and the properties of the imprinted polymer were characterized in detail. Its high selective adsorption and recognition to lysozyme demonstrated the separation ability of the magnetic imprinted material to template molecule, and it has been used for quick and direct separation of lysozyme from the mixture of standard proteins and real egg white samples under an external magnetic field. Furthermore, the elution of lysozyme from the imprinted material was achieved by PEG/sulphate aqueous two-phase system, which caused lysozyme not only desorption from the imprinted materials but also redistribution in the top and bottom phase of aqueous two-phase system. The aqueous two-phase system exhibited some of the extraction and enrichment effect to desorbed lysozyme. Our results showed that ATRP is a promising method for the protein molecularly imprinted polymer preparation.

Nitroprusside−PEO Enthalpic Interaction as a Driving Force for Partitioning of the [Fe(CN)5NO]2- Anion in Aqueous Two-Phase Systems Formed by Poly(ethylene oxide) and Sulfate Salts

Ions are known to concentrate in the salt-enriched phase of aqueous two-phase systems, with the only known exception being the pertechnetate anion, TcO(4)(-). We have discovered a second ion, nitroprusside anion (NP), which is markedly transferred from the salt phase to the polymer phase. The partitioning behavior of [Fe(CN)(5)NO](2-) anion was investigated in ATPS formed by poly(ethylene oxide) of molar mass 3350 and 35000 g mol(-1), and different sulfate salts (Na(2)SO(4), Li(2)SO(4), and MgSO(4)). On the basis of a model, the nitroprusside high affinity for the macromolecular phase was attributed to an enthalpic specific interaction between the anion and ethylene oxide unit. Partition coefficients increased exponentially with tie-line length increase, reaching values as high as 1000 and showing a relationship very dependent on the salt nature, but independent of the polymer molar mass.

Composition, microstructure and rheology of aqueous two-phase cationic/anionic surfactant systems

Aqueous systems containing 1,3-propanediyl bis(dodecyl dimethylammonium bromide) (12-3-12) and sodium dodecyl sulfonate (AS) can separate into a concentrated phase and a dilute phase at certain conditions. The composition analysis demonstrates that the 12-3-12/AS/water system must be treated as a quaternary with four independent components, the equality relations between 2 m 12 − 3 − 12 2 + and m B r − , m A S − and m N a + , are not held in general for the two separated phases. Tie lines are only possible to be described in a three-dimensional phase diagram. Microstructures of some concentrated phases in the phase-separated systems were determined by negative stained TEM method. For the concentrated bottom phase in aqueous two-phase system (ATPS) with water or 0.10 mol kg −1 NaF brine as solvent, vesicles are easy to form. Whereas in 0.10 mol kg −1 NaCl brine, irregular aggregates appear in the concentrated top phase of ATPS. The rheological behavior was examined indicating that the systems studied exhibit thixotropic, antithixotropic, or mixed thixotropic and antithixotropic depending on the compositions and microstructures. Effects of added sodium halide have also been studied.

Size-exclusion chromatography applied to the purification of whey proteins from the polymeric and saline phases of aqueous two-phase systems

Size-exclusion chromatography (SEC) was applied to purify cheese whey proteins α-lactalbumin and β-lactoglobulin which were present, respectively, in the polymeric and saline phases of aqueous two-phase systems. The optimal operating conditions for SEC using a HR 10/10 ® column packed with Sephadex G-25 ® medium led to a high degree of protein purification, of 99.7% for the saline phase and 99.6% for the polymeric phase. The values of resolution and process productivity were, respectively, of 1.53 and 0.43 mg/(mL h) for the saline phase and 1.29 and 0.31 mg/(mL h) for the polymeric phase. A general rate model predicted successfully the whey protein separation using a preparative SEC column.

Process Design for Purification of Muscle Lactate Dehydrogenase by Affinity Partitioning Using Free Reactive Dyes

ABSTRACT It was shown that by using free reactive dyes as affinity ligands, lactate dehydrogen-ase (LDH) can be purified with affinity partitioning directly from rabbit muscle homogenization. The free reactive dyes not bound to polyethylene glycol (PEG) showed a strong tendency toward the top PEG-rich phase in aqueous two-phase systems, and thus enhanced the affinity partitioning effect. Wide-ranged reactive dyes were screened in terms of their partitioning abilities for LDH. The effects of various parameters on affinity partitioning behavior of LDH, such as phase composition, impurities in raw material, concentration of dyes, pH of the systems, and addition of salts, were studied. The optimized affinity extraction process has been carried out for the large-scale purification of LDH from rabbit muscle homogenization. The enzyme was recovered with a yield of 81*middot;3% and a purification factor of 7. Both PEG and dyes were recovered and reused directly without lowering the quality of the product.

Increased production of ?-cyclodextrin using an aqueous two-phase system

β-Cyclodextrin(β-CD) was produced by cyclodextrin glycosyltransferase(CGTase) in aqueous two-phase system. β-CD production from soluble starch was catalyzed by CGTase in dextran-rich bottom phase, and the β-CD produced was transferred to PEG-rich top phase in aqueous two-phase system, composed of 7% (w/w) polyethylene glycol(Mr 20,000) and 10% (w/w) dextran(Mr 38,900). Partition coefficients of β-CD and CGTase were 1.5 and 0.25, respectively. The total productivity of β-CD in aqueous two-phase system was about 3 times of that in dextran phase.

Electroextraction

Since both phases of aqueous two-phase systems are electrically conductive, application of electric fields in these systems gives rise to electrokinetic mass transfer of charged species. We have shown that an aqueous two-phase system can be used as a medium for electrophoretic separation with the liquid-liquid interface providing stability against convection and facilitating product recovery. Proteins have been directed into either the top or bottom phases of polyethylene glycol/dextran aqueous two-phase systems using 20-50 Vlcm electric fields perpendicular to the phase interfaces. Binary protein mixtures have been separated in both batch and continuous modes by operating between isoelectric points and directing oppositely charged proteins into separate phases. Preliminary results on focusing E. coli cells into one phase while directing a proteinaceous product into the other phase indicate that two-phase electrophoresis may prove useful for commercial-scale recovery of proteins from fermentation broth. Recent studies in our laboratory indicate that the electrostatic potential profile near the interface of conducting liquid-liquid systems can influence electrophoretic migration of charged species across the interface. We have observed that phase systems with high Donnan potentials hinder electrophoretic mass transfer across liquid-liquid interfaces, whereas phase systems with low Donnan potentials do not.

Influence of ionic and polymer composition on the properties of the phases of aqueous two-phase systems formed by non-ionic polymers

Partitioning of a homologous series of sodium salts of dinitrophenylated amino acids with aliphatic side-chains in aqueous dextran—poly(ethylene glycol), dextran–polyvinylpyrrolidone and dextran–Ficoll two-phase systems of different polymer concentrations and various ionic compositions was studied. The differences in the abilities of aqueous media in the coexisting phases of the systems used to participate in ionic and hydrophobic hydration interactions with a partitioned solute are estimated. The hydrophobic hydration ability appears to depend mostly on the polymer composition of the phases, while the ionic hydration ability is governed mostly by the ionic composition. In particular, it is found that the ionic hydration and hydrophobic hydration abilities of water are interrelated. Both these characteristics of water in the two phases are considered with regard to the distribution of hydrogen-bond strengths and the relative stability of the hydrogen-bond networks in the phases.

Affinity‐Specific protein separations using ligand‐coupled particles in aqueous two‐phase systems: II. Recovery and purification of pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae

The novel approach of using aqueous two-phase systems for the elution of protein from ligand-coupled particles is investigated using pyruvate kinase and alcohol dehydrogenase from recombinant Saccharomyces cerevisiae and Cibacron blue F3G-A-coupled Sepharose CL6B (Blue-Sepharose) particles as a model system. The ligand-coupled particles distribute quantitatively to the polyethylene glycol-(PEG-) rich top phase and the recovered enzymes partition selectively to the dextran-(DEX-) rich bottom phase. An effective recovery and partial purification of pyruvate kinase and alcohol dehydrogenase from Blue-Sepharose particles using PEG8000-DEXT500 aqueous two-phase systems are demonstrated through a modest increase of salt concentration. The bioselective eluting agent, MgADP, which is useful in chromatographic operations, is not required for the process using aqueous two-phase systems. Recovery of pyruvate kinase, which is bound to ligand-coupled particles, in the DEX-rich bottom phase of aqueous two-phase systems can be up to 95% in one-step operations. The mixing time of ligand-coupled particles with aqueous two-phase systems is a major controlling variable. The salt concentration, the molecular weight of polymer, and the total volume of aqueous two-phase systems also influence the recovery of pyruvate kinase from ligand-coupled particles. The recovered enzymes in the DEX-rich bottom phase remain biologically stable over a long period of storage time. The concentration of product protein in a reduced volume and the easy separation from ligand-coupled particles are added advantages of the process using aqueous two-phase systems. Preliminary studies with goat polyclonal anti-pyruvate kinase-coupled Sepharose particles indicate that the process also may be applicable when a high-affinity ligand such as antibody is used. The experimental results and a theoretical derivation based on equilibrium models for binding/dissociation of ligands and proteins show that the process results in better recovery as compared to that of conventional bulk elution techniques.

The synthesis of water-soluble polymers of β-cyclodextrin and their use in aqueous two-phase systems

A highly water-soluble β-cyclodextrin polymer was prepared from β-cyclodextrin by crosslinking with epichlorhydrin under basic conditions. This polymer could, unlike β-cyclodextrin itself, be partitioned to one phase in aqueous two-phase systems. The binding of some organic compounds to the polymer in such systems is demonstrated.

Effect of polymer structure on affinity partitioning of lactate dehydrogenase in polymer—water two-phase systems

Lactate dehydrogenase from rabbit muscle (EC 1.1.1.27) was partitioned between the liquid phases of aqueous two-phase systems containing two kinds of polymers. The partition was influenced by the use of a dye ligand, Procion Yellow HE3G, bound to a polymer. The ligand-carrying polymers were poly(ethyleneglycol), poly(vinyl alcohol), ethylhydroxyethylcellulose, hydroxypropyl-starch, Ficoll and dextran. Their influence on the partition of lactate dehydrogenase was studied in systems composed of the following pairs of polymers: dextran—poly(ethylene glycol), dextran—poly(vinyl alcohol), dextran—Ficoll and hydroxypropyl-starch—poly(ethylene glycol). The ratio between the change in the logarithmic partition coefficient of the enzyme, Δ log K, caused by the ligand and the logarithm of the partition coefficient of the ligand—polymer, log K L—polymer, was used as a measure of the number of ligand polymers bound per enzyme molecule under saturation conditions. Possible explanations for the observed effects are discussed.