Solute partitioning in polymer–salt ATPS: The Collander equation

The partition coefficients for several solutes (five nitrophenylated-monosaccharides and four proteins) were experimentally determined, at 23 °C, in three different tie-lines of two polymer–salt aqueous two-phase systems (ATPS): UCON-K₂HPO₄ and UCON-NaH₂PO₄. These partition coefficients together with others obtained from the literature for five dinitrophenylated-amino acids were used to investigate the suitability of the Collander equation to correlate partition coefficients in polymer–salt ATPS. This equation was first proposed to describe the linear correlation between partition coefficients of solutes in different water–organic solvent systems. More recently, it was proved that partition coefficients for several biomolecules in polymer–polymer ATPS can also be correlated with this equation. In this work, several correlations were tested: partition coefficients obtained for different tie-lines within the same system and also partition coefficients obtained from different systems. In both cases, a linear relation was observed, despite a less satisfactory correlation was found when different ATPS were compared. Overall, it was demonstrated that the Collander equation can be used to satisfactorily correlate solute partitioning in the studied polymer–salt ATPS.     

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phosphate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na(2)SO(4), pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively.     

外加盐作用形成的正负离子表面活性剂双水相

癸基三乙基溴化铵-癸基磺酸钠（C10NE-C10SO3）等摩尔混合均相体系（即使在表面活性剂总浓度高达0.2 mol•L－1时仍然可形成稳定的均相溶液）在外加盐NaF、Na2SO4和Na3PO4的作用下可自发分离成两个水相（双水相）.研究了该类双水相体系的形成、相行为及其对牛血清蛋白（BSA）的分配，并与普通的正负离子表面活性剂混合双水相体系进行了比较.结果表明，该类双水相体系克服了普通的正负离子表面活性剂混合双水相体系的一些不足，具有一些独特的优点.该类双水相体系的相行为可以通过外加盐进行调控,通过外加盐的种类来调控和优化BSA的分配行为.图1表2参8     

PEO-[M(CN)5NO](x-) (M = Fe, Mn, or Cr) interaction as a driving force in the partitioning of the pentacyanonitrosylmetallate anion in ATPS: strong effect of the central atom

The partitioning behavior of pentacyanonitrosilmetallate complexes[Fe(CN) 5NO] (2-), [Mn(CN) 5NO] (3-), and [Cr(CN) 5NO] (3-)has been studied in aqueous two-phase systems (ATPS) formed by adding poly(ethylene oxide) (PEO; 4000 g mol (-1)) to an aqueous salt solution (Li 2SO 4, Na 2SO 4, CuSO 4, or ZnSO 4). The complexes partition coefficients ( K complex) in each of these ATPS have been determined as a function of increasing tie-line length (TLL) and temperature. Unlike the partition behavior of most ions, [Fe(CN) 5NO] (2-) and [Mn(CN) 5NO] (3-) anions are concentrated in the polymer-rich phase with K values depending on the nature of the central atom as follows: K [ F e ( C N ) 5 N O ] 2 - > K [ M n ( C N ) 5 N O ] 3 - > K [ C r ( C N ) 5 N O ] 3 - . The effect of ATPS salts in the complex partitioning behavior has also been verified following the order Li 2SO 4 > Na 2SO 4 > ZnSO 4. Thermodynamic analysis revealed that the presence of anions in the polymer-rich phase is caused by an EO-[M(CN) 5NO] ( x- ) (M = Fe, Mn, or Cr) enthalpic interaction. However, when this enthalpic interaction is weak, as in the case of the [Cr(CN) 5NO] (3-) anion ( K [ C r ( C N ) 5 N O ] 3 - < 1), entropic driving forces dominate the transfer process, then causing the anions to concentrate in the salt-rich phase.     

双水相萃取结合液相色谱法分离蛋白质

建立了PEG/( NH4)2SO4双水相体系萃取富集,结合液相色谱分离分析多种蛋白质的方法.考察了无机盐种类和浓度、PEG分子量、pH值和温度等因素对双水相形成以及对细胞色素C、肌红蛋白、牛血清白蛋白、溶菌酶、胰蛋白酶分配行为的影响.结果表明,上述5种蛋白在室温、pH 3.5～9.0范围内,可在15％ PEG-4000/10％ (NH4)2SO4双水相体系中得到富集,且主要集中在下相.同样条件下,血清中的高丰度蛋白在上下相均有分配,下相分配量较大.通过双水相萃取分离蛋白质及对液相色谱一定时间段的色谱峰收集,可初步实现血清中高丰度蛋白质的分离去除.     

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.     

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].     

Xylanase recovery effect of extraction conditions on the aqueous two-phase system using experimental design

The partitioning of xylanase produced byPenicillium janthinellum in aqueous two-phase systems (ATPS) using poly(ethylene glycol) (PEG) and phosphate (K₂HPO₄/KH₂PO₄) was studied employing a statistical experimental design. The aim was to identify the key factors governing xylanase partitioning. The interactions of five factors (PEG concentration molecular weight, concentration of buffer K₂HPO₄/KH₂PO₄, pH, and NaCl concentration) and their main effects on the partition coefficient (K) were evaluated by means of a 2⁵ full-factorial experimental design with four center points. The %PEG, %NaCl, and pH were the most important factors affecting the response variable (K). Response surface methodology (RSM) was adopted and an empirical second-order polynomial model was constructed on the basis of the results. The optimum partition conditions were pH 7.0, PEG = 8.83% and NaCl = 6.02%. Adequacy of the model for predicting optimum response value was tested under these conditions. The experimental xylanase partition coefficient (K) was 2.21, whereas its value predicted by the model was 2.33. These results indicate that the predicted model was adequate for the process. PEG molecular weight and phosphate concentration did not affect the xylanase partition coefficient.     

Phase equilibrium and insulin partitioning in aqueous two-phase systems containing block copolymers and potassium phosphate

In this work, phase diagrams of aqueous two-phase systems (ATPS) containing PEO–PPO–PEO block copolymers and potassium phosphate as well as the partitioning behavior of insulin in these systems are presented. Experiments aimed at the identification of the effects of copolymer structure (by varying the number of EO units per polymer molecule), temperature (283.15 and 298.15 K) and pH (5.0 and 7.0) on the phase behavior of these systems were carried out. The results indicated the enlargement of the two-phase region upon increasing the temperature, pH and copolymer hydrophobicity (expressed as the PO/EO ratio in the copolymer molecule). Experimental measurements of the partitioning of human insulin in these ATPS also indicated the dependency of the partition coefficients on temperature, pH, and copolymer hydrophobicity, with the latter being the most influential factor. Finally, experimental data on the phase behavior and insulin partitioning were correlated using an excess Gibbs energy virial-type model modified in order to account for coulombic interactions and ionization equilibrium between the various forms of the phosphate ion.     

Extraction of the antimicrobial peptide cerein 8A by aqueous two-phase systems and aqueous two-phase micellar systems

Cerein 8A is an antimicrobial peptide with potential application against food spoilage and pathogenic bacteria. The partitioning of cerein 8A was investigated in two liquid-liquid extraction systems that are considered promising for bioseparation and purification purposes. Aqueous two-phase systems (ATPSs) were prepared with polyethylene glycol (PEG) and inorganic salts, and the addition of NaCl was investigated in this system. The best results concerning partition coefficients (K (b)) were obtained with PEG?+?ammonium sulphate, and K (b) value significantly increases when NaCl was added. Cerein 8A was effectively extracted into the micelle-rich phase in a 4% Triton X-114 medium. Recovery yield was higher for ATPS compared to micellar systems. Cerein 8A can be isolated from a crude suspension containing the bioactive molecule by ATPSs. Successful implementation of peptide partitioning represents an important step towards developing a low-cost effective separation method for cerein 8A.     

Partitioning of Amino Acids in Surfactant Based Aqueous Two-Phase Systems Containing the Nonionic Surfactant (Triton X-100) and Salts

The partitioning of three amino acids, L-phenylalanine, L-tryptophan and L-tyrosine, has been studied in surfactant-based aqueous two-phase systems (ATPS) of Triton X-100 + salts + H₂O at 298.15 K. Sodium citrate as an organic salt and magnesium sulfate as an inorganic salt were used to prepare the ATP systems. The effects of tie-line length and salt type on the partitioning of amino acids have been discussed. The results show that the partitioning behavior of the amino acids in the systems containing MgSO₄ and Na₃C₆H₅O₇ are significantly different, because increasing the tie-line length in MgSO₄ solutions leads to a decrease of the partition coefficient, whereas the opposite trend is found in Na₃C₆H₅O₇ solutions.     

Partitioning in Aqueous Two-Phase Systems: Fundamentals,Applications and Trends

Over the last years, aqueous two-phase systems (ATPS) regained an increasing interest due to their potential in the downstream processing of biomolecules. After many years with only a few articles published, a lot of effort and work has been put into studying these systems for the partitioning of a range of compounds including proteins, organic low-molecular weight molecules or metal ions. Although several research and review articles appeared, a background review on ATPS partitioning fundamentals is needed. In this article, partitioning theories and main effects of several important factors for partitioning, such as molecular weight of the polymer, effect of added salts, pH, electrical charges, and temperature on phase diagrams, tie-line lengths, interfacial tension and settling time of the two aqueous phases are extensively reviewed. The trend in ATPS research is given compiling the recent 2008–2013 research articles published in the field.     

Nitroprusside-PEO enthalpic interaction as a driving force for partitioning of the [Fe(CN)(5)NO](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.     

System Establishment of ATPS for One-Step Purification of Glutamate Decarboxylase from <Emphasis Type="Italic">E. coli</Emphasis> After Cell Disruption

The partition of glutamate decarboxylase (GAD) from Escherichia coli in polyethylene glycol (PEG) and sodium sulfate aqueous two-phase systems (ATPS) has been explored with the purpose of establishing a phase system for the purification of GAD after cell disruption. The results showed that the partitioning of GAD was slightly influenced by PEG molecular weight (MW) but depended on the tie line length (TLL) and NaCl and loading sample concentrations. The optimum system obtained for GAD purification was composed of a PEG MW of 4,000, TLL of 63.5%, a volume ratio of 2.31, a loading sample concentration of 0.4 g/mL, which produced a GAD recovery of 90% with the purification fold of 73. Furthermore, the feasibility of directly purifying GAD from the cell disrupts using ATPS was evaluated. The established ATPS for GAD purification exhibited an efficient integrated purification process compared to the reported purification process in terms of purification efficiency and recovery.     

Specific ion effects on interfacial water structure near macromolecules

We investigated specific ion effects on interfacial water structure next to macromolecules with vibrational sum frequency spectroscopy (VSFS). Poly-(N-isopropylacrylamide) was adsorbed at the air/water interface for this purpose. It was found that the presence of salt in the subphase could induce the reorganization of water adjacent to the macromolecule and that the changes depended greatly on the specific identity and concentration of the salt employed. Ranked by their propensity to orient interfacial water molecules, sodium salts could be placed in the following order: NaSCN > NaClO4 > NaI > NaNO3 approximately NaBr > NaCl > pure water approximately NaF approximately Na2SO4. This ordering is a Hofmeister series. On the other hand, varying the identity of the cation exhibited virtually no effect. We also showed that the oscillator strength in the OH stretch region was linearly related to changes in the surface potential caused by anion adsorption. This fact allowed binding isotherms to be abstracted from the VSFS data. Such results offer direct evidence that interfacial water structure can be predominantly the consequence of macromolecule-ion interactions.     

Exploitation of the coil-globule plasmid DNA transition induced by small changes in temperature, pH salt, and poly(ethylene glycol) compositions for directed partitioning in aqueous two-phase systems

In this study, the interplay of two linked equilibria is examined, one concerning an aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and salt employed to partition plasmid DNA (pDNA), and the other a potential structural transition of pDNA depending on PEG and salt concentration and other system parameters. The boundary conditions for pDNA partitioning are set by PEG and salt concentrations, PEG molecular weight, pH, and temperature. While investigating these parameters, it was found that a small increase/decrease of the respective values led to a drastic and significant change in pDNA behavior. This behavior could be attributed to a coil-globule transition of the pDNA triggered by the respective phase conditions. The combination of this structural change, aggregation effects linked to the transition process, and the electrostatic potential difference found in PEG-salt systems thus offers a sensitive way to separate nucleic acid forms on the basis of their unique property to undergo coil-globule transitions under distinct system properties.     

Prediction of the partitioning behaviour of proteins in aqueous two-phase systems using only their amino acid composition

The prediction of the partition behaviour of proteins in aqueous two-phase systems (ATPS) using mathematical models based on their amino acid composition was investigated. The predictive models are based on the average surface hydrophobicity (ASH). The ASH was estimated by means of models that use the three-dimensional structure of proteins and by models that use only the amino acid composition of proteins. These models were evaluated for a set of 11 proteins with known experimental partition coefficient in four-phase systems: polyethylene glycol (PEG) 4000/phosphate, sulfate, citrate and dextran and considering three levels of NaCl concentration (0.0% w/w, 0.6% w/w and 8.8% w/w). The results indicate that such prediction is feasible even though the quality of the prediction depends strongly on the ATPS and its operational conditions such as the NaCl concentration. The ATPS 0 model which use the three-dimensional structure obtains similar results to those given by previous models based on variables measured in the laboratory. In addition it maintains the main characteristics of the hydrophobic resolution and intrinsic hydrophobicity reported before. Three mathematical models, ATPS I-III, based only on the amino acid composition were evaluated. The best results were obtained by the ATPS I model which assumes that all of the amino acids are completely exposed. The performance of the ATPS I model follows the behaviour reported previously, i.e. its correlation coefficients improve as the NaCl concentration increases in the system and, therefore, the effect of the protein hydrophobicity prevails over other effects such as charge or size. Its best predictive performance was obtained for the PEG/dextran system at high NaCl concentration. An increase in the predictive capacity of at least 54.4% with respect to the models which use the three-dimensional structure of the protein was obtained for that system. In addition, the ATPS I model exhibits high correlation coefficients in that system being higher than 0.88 on average. The ATPS I model exhibited correlation coefficients higher than 0.67 for the rest of the ATPS at high NaCl concentration. Finally, we tested our best model, the ATPS I model, on the prediction of the partition coefficient of the protein invertase. We found that the predictive capacities of the ATPS I model are better in PEG/dextran systems, where the relative error of the prediction with respect to the experimental value is 15.6%.     

Hofmeister anion effect on aqueous phase behavior of heptaethylene glycol dodecyl ether

The aqueous phase behavior of heptaethylene glycol dodecyl ether (C12E7) was investigated in the presence of sodium salts of Cl-, I-, and ClO4-. Pseudo binary T-X phase diagrams were constructed for these mixtures by means of differential scanning calorimetry. The salting-out electrolyte NaCl expanded the Lalpha region toward higher temperatures and shrank the H1 region toward lower temperatures compared with the salt-free system. On the contrary, the salting-in electrolytes NaI and NaClO4 induced shrinkage of the Lalpha region and an expansion of the H1 phase. The influence of these salts on the mesophase regions was more pronounced for the Lalpha phase than for the H1 phase, and area of the Lalpha phase region decreased in the sequence of NaCl > none > NaI > NaClO4, consist with the Hofmeister series of the anions. This salt effect on the mesophase stability in aqueous nonionic surfactant mixture would be qualitatively interpreted in terms of the salt effect on the hydration of the polyoxyethylene chain in the surfactant molecules.     

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.