Partitioning behavior of erythrocytes in aqueous two-phase systems containing hydroxypropyl starch and polyethylene glycol

The partitioning behavior of erythrocytes in Reppal PES 200 (a hydroxypropyl starch produced by Reppe Glykos AB, V?xj?, Sweden)-polyethylene glycol (PEG) and in dextran (Dx)-PEG aqueous phase systems made isotonic with phosphate is similar in a number of ways: (i) There is a correlation between the relative electrophoretic mobilities and partition ratios, P, of red blood cells from different species; (ii) The cell P is reduced when, at constant polymer concentrations, phosphate is systematically replaced by sodium chloride (with the total concentration isotonic); (iii) The cell P is increased with reduced polymer concentrations (decreased interfacial tensions); (iv) Treatment of erythrocytes with neuraminidase results in a reduced P value; (v) Rat red cells of different ages can be fractionated by counter-current distribution; and (vi) Differences between red blood cells from genetically distinct rats or between humans can be detected. Aquaphase (a hydroxypropyl starch marketed by Perstorp AB, Lund, Sweden) has been tested as in ii-iv above with analogous results. The partitioning behavior of erythrocytes in PES-PEG and Dx-PEG aqueous phase systems containing sodium chloride differs in a number of ways: (vii) The correlation, apparent in Dx-PEG systems, between the P value of red blood cells from different species and the ratio of their membrane poly- to monounsaturated fatty acids is absent in PES-PEG systems. It is replaced by a correlation as in i; (viii) The increase in P value in Dx-PEG observed from red blood cells after treatment with neuraminidase is replaced by a decrease in P value in PES-PEG or Aquaphase-PEG systems. We conclude that PES (and Aquaphase) can be substitutes for dextran in cell partitioning studies when charge-sensitive phases are used (e.g., those containing phosphate) while separations based on properties reflected by Dx-PEG systems containing sodium chloride are not duplicated by PES-PEG (and probably not by Aquaphase-PEG). The hydroxypropyl starch-PEG systems containing sodium chloride, unlike the analogous Dx-PEG systems, have a significant electrostatic potential difference between the phases.     

Partitioning of l-methionine in aqueous two-phase systems containing poly(propylene glycol) and sodium phosphate salts

The partitioning behavior of l-methionine has been studied in aqueous two-phase systems of (poly(propylene glycol) + sodium phosphate salts + H₂O) at different temperatures. The salts used were sodium di-hydrogen phosphate (NaH₂PO₄), di-sodium hydrogen phosphate (Na₂HPO₄) and tri-sodium phosphate (Na₃PO₄). The effects of tie line length, salt type, and temperature on the partition coefficient of this amino acid have been studied. In addition, thermodynamic parameters (ΔH°, ΔS° and ΔG°) as a function of temperature were calculated. The results showed that increasing tie line length led to decreasing of the partition coefficient. We also showed that the partition coefficients of the amino acid in the systems containing Na₃PO₄ are greater than the other two salts. Moreover, it is verified that increasing temperature led to decreasing the partition coefficient. The experimental partition coefficient data are correlated using a modified virial-type model.     

Liquid-liquid phase equilibria of aqueous two-phase systems containing salts and polyethylene glycol

Liquid-liquid phase equilibria of the ternary systems: (a) polyethylene glycol - ammonium sulfate- water and (b) polyethylene glycol - sodium carbonate -water have been determined experimentally at 15°, 25°, 35° and 45°C and for two different molecular weights of the polymer (Avg. M.W. 1000 and 2000). Details of the glass cell and of the equilibration and analytical procedures used are described. Equilibrium data along with phase diagrams are presented. Finally the effect of temperature and of the molecular weight of the polymer are also discussed.     

Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems

This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker’s yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 2⁵ factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (Pur _TOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (K _Prot ) was 0.47.     

Partitioning of lactate dehydrogenase from bovine heart crude extract by polyethylene glycol-citrate aqueous two-phase systems

Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG)-citrate have been used for enzyme partitioning studies. The behavior of lactate dehydrogenase (LDH) from bovine heart crude extract was analyzed using a two-level factorial design in which the PEG molar mass and concentration, the citrate concentration were selected as independent variables, while the purification factor, the partition coefficient (K) and the activity yield were selected as responses. The statistical analysis revealed the effect of PEG molar mass on K. LDH exhibited a better partitioning toward PEG-rich phase and the highest K value (1079.81) was obtained with 42% (w/w) PEG 400 and 7.5% (w/w) citrate concentration. PEG molar mass also influenced the purification factor of the enzyme in the top phase. Possibly these ATPS remove inhibitors present in the extract affording higher enzyme yield.     

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].     

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.     

Affinity partitioning of proteins in aqueous two-phase systems containing polymer-bound fatty acids. I. Effect of polyethylene glycol palmitate on the partition of human serum albumin and alpha-lactalbumin

The partition of serum albumin between the two aqueous phases of a polyethylene glycol-dextran-water two-phase system is strongly influenced by replacing a small fraction of the polyethylene glycol by its palmitate ester. The partition of albumin is compared with the partition of polymer-bound palmitate using polyethylene glycol [1-14C]palmitate. The data indicate that a maximum ("saturation") effect on the partition of albumin corresponds to a binding of fewer than two palmitate groups per albumin molecule. The effect on alpha-lactalbumin corresponds to the maximum binding of 0.5 palmitate group per protein molecule. These data do not fit the most favoured available model for affinity partitioning. The deviation can be attributed to the observation that the palmitate groups probably form micelle-type aggregates in the polyethylene glycol-rich phase at the concentrations used for albumin extraction.     

Partitioning of biomaterials in aqueous two-phase systems of mixed cationic-anionic surfactants

In a previous paper, we have showed that, when aqueous solutions of cationicand anionic surfactants at certain concentrations were mixed, the solution separatedspontaneously into two immiscible phases (aqueous two-phases), one phase was rich,and the other was poor in the mixed surfactants. A clear interfacial boundary existsbetween two phases.     

聚乙二醇双水相萃取光度法测定锌

A novel method of extraction spectrophotometric determination of Zn~(2+) in aqueous two-phase system formed by polyethylene glycol-2000,sodium sulfate and zincon has been established.Amounts of PEG solution,zincon solution,sodium sulfate added and the acidity of the system were optimized.The interference effects of various coexistent ions and the method to remove the interference were investigated.The Zn-zincon complex that had been formed in the presence of H_2BO_3-Na_2B_4O_7 buffer solution at pH 7.4 was extracted into the upper PEG-rich phase,and the absorbance of the extracted complex in the upper PEG-rich phase was measured at 630 nm.The apparent molar absorptivity was 7.3 × 10~4 L / (mol·cm).Beer's law was obeyed over the range of 0～2.0 μg / mL Zn~(2+).The analytical results obtained for Zn~(2+) in water samples were in good agreement with values measured by atomic absorption spectrometry.     

铟在聚乙二醇-硫酸铵双水相体系中的分配

研究了稀散元素铟在有配合剂PAR（4－（2－吡啶偶氮）－间苯二酚）存在和无PAR存在的聚乙二醇PEG－（NH4）2SO4双水相体系中的分配行为。讨论了酸度、PEG分子量、PEG浓度及温度等因素对铟分配比的影响，发现酸度对分配比的影响最大，随着PEG分子量的增加及温度的上升分配比逐渐增大；随着PEG浓度的增加分配比逐渐减小。     

Partitioning and assembly of metal particles and their bioconjugates in aqueous two-phase systems

The behavior of metal nanospheres and nanowires and their bioconjugates in aqueous two-phase systems (ATPS) is described. The ATPS used in this work comprised poly(ethylene glycol) (PEG), dextran, and water or aqueous buffer. Au and Ag nanospheres less than 100 nm in diameter partition between the PEG-rich and dextran-rich phases on the basis of their surface chemistry and can be separated on this basis. Larger Au nanospheres and wires accumulate at the interface between the two aqueous phases. The influence of polymer molecular weight and concentration on interfacial assembly of Au wires is described. DNA-derivatized nanowires at the aqueous/aqueous interface retain the ability to selectively bind to fluorescent complementary DNA. In addition, Au nanoparticles have been bound to Au wires via selective DNA hybridization at the ATPS interface. Transmission electron microscopy and thermal denaturation experiments confirm that DNA-driven assembly is responsible for the formation of the nanosphere/wire assemblies. These results demonstrate the biocompatibility of the two-phase interface and point to future use as scaffolding in biorecognition-driven assembly.     

Extraction of humic acids in two-phase aqueous polymeric systems

Extraction of humic acids isolated from various soils was studied in polyethylene glycol(NH₄)₂SO₄, polyethylene glycol-dextran, and polyethylene glycol-dextran sodium salt two-phase aqueous polymeric systems. It was shown that, in all systems, humic acids are extracted into the polyethylene glycolrich phase. It was studied how the composition of the extraction system, molecular weight of the polymer, and the sample nature affect the distribution ratios of humic acids.     

Phase separation in aqueous two-phase systems containing poly(ethylene glycol) and magnesium sulphate at different temperatures

New experimental (liquid + liquid) equilibrium data have been determined for aqueous systems containing poly(ethylene glycol) of nominal molar mass 10,000 and magnesium sulphate at T = (295.15, 301.15, 305.15, and 311.15) K. The effect of temperature on the liquid compositions of coexisting phases is discussed. The experimental (liquid + liquid) equilibrium data of the systems were correlated by non-random two-liquid (NRTL) activity coefficient model. The interaction parameters of the NRTL activity coefficient model are obtained and reported. The calculated root mean square deviations (RMSD) showed that NRTL activity coefficient model can be used satisfactorily to correlate the (liquid + liquid) equilibrium data in aqueous solution of the {poly(ethylene glycol) + magnesium sulphate} system.     

Effect of temperature on the phase equilibrium of aqueous two-phase systems containing polyvinylpyrrolidone and disodium hydrogen phosphate or trisodium phosphate

Phase diagrams and liquid–liquid equilibrium (LLE) data of the aqueous polyvinylpyrrolidone–disodium hydrogen phosphate and aqueous polyvinylpyrrolidone–trisodium phosphate systems have been determined experimentally at 298.15, 308.15, 318.15 and 328.15 K. The effect of temperature and the type of salt on the binodal and tie-lines has been studied. It was found that, for the studied systems in the polyvinylpyrrolidone (PVP)-rich region, an increase in temperature caused the expansion of one-phase area; while, for the salt-rich region, expansion of the two-phase area was observed with increase in the temperature. It was also observed that the slope of all equilibrium tie-lines increased with increasing temperature. Furthermore, it was found that, as temperature is increased, these aqueous two-phase systems exhibit a phase inversion. Specifically, the PVP-rich phase – which at low temperatures is the less dense top phase – becomes the lower phase at elevated temperatures. Finally, an extended Flory–Huggins theory proposed in our previous paper has been used for correlation of LLE data.     

Equilibrium phase behavior of aqueous two-phase systems containing 17R4/L64 and citrates

The cloud points (CPs) of the copolymers 17R4 and L64 were first measured, and then the effects of salts ((NH₄)₃C₆H₅O₇, K₃C₆H₅O₇) on 17R4 and L64 were researched. After finishing the work described above, the temperature (278.15, 283.15, and 288.15) K of aqueous two-phase systems was determined, which consist of 17R4-(NH₄)₃C₆H₅O₇, 17R4-K₃C₆H₅O₇, L64-(NH₄)₃C₆H₅O₇, and L64-K₃C₆H₅O₇. Finally, the liquid–liquid equilibrium (LLE) data of binodal curve and the tie line for 17R4-(NH₄)₃C₆H₅O₇ aqueous two- phase systems (ATPSs) 17R4-K₃C₆H₅O₇ ATPSs, L64-(NH₄)₃C₆H₅O₇ ATPSs, and L64-K₃C₆H₅O₇ ATPSs were obtained. Nonlinear fitting of the empirical equation was used for making the diagram. The results showed that the change in the size of the two-phase areas increases with the increase of temperature. The capacity of the salts to induce phase segregation follows the Hofmeister series, that is, K₃C₆H₅O₇?>?(NH₄)₃C₆H₅O₇. In addition, the findings also showed that the phase separation ability of 17R4 is better than that of L64.     

Effect of basic amino acids on photoreaction of ketoprofen in phosphate buffer solution

Photoreaction of ketoprofen (KP), one of the widely used nonsteroidal anti-inflammatory drugs (NSAIDs), was studied with transient absorption spectroscopy in phosphate buffer solution (pH 7.4) in the presence of basic amino acids of histidine (His), lysine (Lys) and arginine (Arg). Deprotonated form of KP (KP(-)) excited with UV-light irradiation gave rise to carbanion through a decarboxylation reaction. It was found that carbanion abstracted a proton from the side chain of the protonated amino acids to yield 3-ethylbenzophenone ketyl biradical (EBPH); however, no reaction was observed with alanine. The relative yield of EBPH by the proton transfer reaction with His was ca. 40 times larger than that of the other two basic amino acids, suggesting that the proton-donating ability of His (protonated His) should be quite high. The information on the photoreaction mechanism of NSAIDs with basic amino acids was essential to understand primary reaction of excited NSAIDs in vivo causing photosensitization on human skin.     

Solubility of amino acids in aqueous poly (ethylene glycol) solutions

The solubilities of amino acids have been measured in water and aqueous poly(ethylene glycol) (PEG) solutions as a function of temperature and PEG concentration. The free energies of transfer from water to aqueous PEG solutions forl-alanine,l-valine,l-isoleucine andl-leucine were positive, while those forl-phenylalanine andl-tryptophan were negative. The corresponding enthalpies of transfer were almost zero for all amino acids. The equilibrium constants of the binding of amino acids to PEG chain were estimated from the solubility data. Amino acids with larger hydrophobicity are bound more strongly to the PEG chain due to the hydrophobic interaction between the methylene groups of PEG and the side chain of amino acid. The equilibrium constants showed a correlation with the dynamic hydration number (n _DHN) which expresses the hydration properties of amino acids in aqueous solution.     

Concentration dependence of the interfacial tension for aqueous two-phase polymer solutions of dextran and polyethylene glycol

We studied the interfacial tension between coexisting phases of aqueous solutions of dextran and polyethylene glycol. First, we characterized the phase diagram of the system and located the binodal. Second, the tie lines between the coexisting phases were determined using a method that only requires measuring the density of the coexisting phases. The interfacial tension was then measured by a spinning drop tensiometer over a broad range of polymer concentrations close to and above the critical point. In this range, the interfacial tension increases by 4 orders of magnitude with increasing polymer concentration. The scaling exponents of the interfacial tension, the correlation length, and order parameters were evaluated and showed a crossover behavior depending on the distance to the critical concentration. The scaling exponent of the interfacial tension attains the value 1.50 ± 0.01 further away from the critical point, in good agreement with mean field theory, but the increased value 1.67 ± 0.10 closer to this point, which disagrees with the Ising value 1.26. We discuss possible reasons for this discrepancy. The composition and density differences between the two coexisting phases, which may be taken as two possible order parameters, showed the expected crossover from mean field behavior to Ising model behavior as the critical point is approached. The crossover behavior of aqueous two-phase polymer solutions with increasing concentration is similar to that of polymer solutions undergoing phase separation induced by lowering the temperature.     

Partitioning of alkali halides in aqueous dextran-ficoll two-phase system

Partitioning of alkali halides MeX in aqueous dextran-Ficoll two-phase system of fixed polymer and ionic composition was studied. It is found that NaCl and LiX concentrate in the Ficoll-rich phase, while all KX, RbX, and CsX concentrate in the dextran-rich phase. Partitioning of KX, RbX, and CsX in the presence of a large excess of NaCl is found to depend on the type of the halide anion X^–.