Abstract
The present study investigated liquid-liquid equilibria of polyvinylpyrrolidone (PVP) K30 + tri-sodium citrate + urea (5% and 10% mass) aqueous two-phase systems at 25°C and pH values of 6.2, 7.4, and 10.0. Results showed that the binodal was displaced toward higher concentrations as the urea concentration increased. The effect of pH, polymer/salt (w/w) ratio, urea concentration, tie line length, and slope of tie line on the partition behavior of urea was examined. The viscosity, density, electrical conductivity, and refractive index of PVP (K30) + tri-sodium citrate + urea + water two-phase systems were measured versus pH value. The relation between tie line length and the density and viscosity of the aqueous two-phase systems was also examined.
Keywords:
Aqueous two-phase systems; Phase diagram; Urea; Viscosity; Polyvinylpyrrolidone
INTRODUCTION
An aqueous two-phase system (ATPS) is a liquid-liquid extraction strategy that can efficiently separate biological materials such as recombinant protein and enzymes (Albertsson, 1986Albartson P.A. Partition of cell particles and macromolecules, 3nd ed., New York: Willy(1986).; Hatti-Kaul, 2000Hatti-Kaul, Rajni (Ed.), Aqueous two-phase systems. Methods and Protocols. New York: Humana Press (2000).). Polyethylene glycol (PEG) is an important component of two-phase partitioning. PVP is a water soluble polymer that can be applied with suitable salts to form ATPSs. This polymer is used in industrial production, medicine, and pharmacology (Bühler, 2005Bühler, V., Excipients for Pharmaceuticals-Povidone, Crospovidone and Copovidone, Springer, Berlin, Heidelberg, New York, 1-254(2005).; Wang et al.2000Wang, Z.H., Song, M. and Ma, Q., Two-Phase Aqueous Extraction of Chromium and its Application to Speciation Analysis of Chromium in Plasma, Mikrochim. Acta, 134, No 1, 95(2000).; Zafarani-Moattar et al. 2015Zafarani-Moattar ,M.T., Karimi,N and Asadzadeh, B. Investigations of the Interactions in Aqueous Poly (vinyl pyrrolidone) + Di-sodium Hydrogen Citrate Solutions Using Vapor-Liquid Equilibria Studies at Different Temperatures, J. Mol. Liq, 211, No 12,767(2015).). PVP is a biocompatible alternative that is an inexpensive and stable polymer with good potential for aqueous two-phase extraction (Zafarani-Moattar and Zaferanloo, 2009Zafarani-Moattar, M.T and Zaferanloo, A., Measurement and Correlation of Phase Equilibria in Aqueous Two-Phase Systems Containing Polyvinylpyrrolidone and Dipotassium Tartrate or Di-potassium Oxalate at Different Temperatures, J. Chem. Thermodynamics, 41, No7, 864 (2009).).
Citrates have applications in biochemical and chemical reactions. They are used as substitutes for inorganic salts to form ATPSs along with PVP for protein extraction. They are biodegradable, nontoxic, and can be directly discharged into biological wastewater treatment plants (Sadeghiet al.2006Sadeghi, R, Rafiei,H.R and Motamedi, M., Phase Equilibrium in Aqueous Two-Phase Systems Containing Poly(vinylpyrrolidone) and Sodium Citrate at Different Temperatures-Experimental and Modeling, Thermochimica Acta, 451, No 1, 163 (2006).). Table 1 lists some ATPS containing PVP.
Inclusion body refolding is a vital step in the production of recombinant proteins. Solubilization of aggregated protein with denaturants such as guanidine hydrochloride and urea has been reported to be essential to recovery of active protein from the inclusion bodies (Clark, 2001Clark, ED., Protein Refolding for Industrial Processes, Curr. Opin.Biotechnol.12, 202 (2001).). Urea is a powerful protein denaturant as it disrupts the noncovalent bonds in the proteins (Parnica and Antalika, 2014Parnica, M. and Antalika, M., Urea and Guanidine Salts as Novel Components for Deep Eutectic Solvents, J. Mol. Liq. 197, No 1 , 23(2014).). Several articles have been written about the application of urea in ATPS for the initial recovery step (Rahimpour et al., 2010Rahimpour, F and Pirdashti, M. Effective Parameters on the Partition Coefficient of Guanidine Hydrochloride in the Poly(ethylene glycol) + Phosphate + Water system at 298.15 K, Iran. J. Chem. Eng. 7, No.1, 67(2010).; Rämsch et al., 1999Rämsch, Ch. Kleinelanghorst, L.B., Knieps. E.A., Hommes, J. and Kula, M.-R., Aqueous Two-Phase System Containing Urea; Influence on Phase Separation and Stabilization of Protein Conformation by Phase Components, Biotechnol. Prog. 15, No 3, 493(1999).), but little has been published about the complex problem of how the urea affects the phase diagram behavior or about determining its partition coefficient in ATPS.
Despite these favorable features, ATPSs have not been extensively adopted for either industrial or commercial applications. The main reason is lack of knowledge about the mechanisms involved in partitioning equilibria of macromolecules, the lack of a comprehensive theory that can predict experimental trends, and the empirical nature of the method. This lack of knowledge has motivated researchers to study ATPSs (Yan-Min et al., 2010Yan-Min, L., Yan-Zhao, Y., Xi-Dan, Z., Chuan-Bo, X., Bovine Serum Albumin Partitioning in Polyethylene glycol (PEG)/Potassium Citrate Aqueous Two-Phase Systems. Food Bioprod. Process, 88, No.1, 40 (2010).; Rocha and Nerli, 2013Rocha, M.V and Nerli, B.B. Molecular Features Determining Different Partitioning Patterns of Papain and Bromelain in Aqueous Two-Phase Systems. Int. J. Bio. Macromol., 61, No 2, 204(2013).).
The present study obtained phase equilibrium data for PVP (K30) + tri-sodium citrate (pH = 6.2, 7.4, and 10.0) + urea (5% and 10% (mass) + water at 25°C. The effect of pH, PVP/salt ratio, urea concentration, tie line length (TLL), and slope of tie line (STL) on the partition behaviors of urea were also studied. The viscosity, density, electrical conductivity, and refractive index of the PVP + tri-sodium citrate + urea aqueous two-phase system and the top and bottom phases of the two-phase system were determined. The relation between TLL and the density and viscosity of the ATPSs was also examined.
EXPERIMENTAL
Materials
PVP (K30; average MW = 60000; moisture content < 5.0% by mass fraction) was obtained from Aldrich. Tri-sodium citrate (anhydrous GR for analysis > 99%), sodium hydroxide (NaOH; mass purity > 0.99%) and sulfuric acid (95% to 97% H2SO4; GR for analysis > 95.0%) were obtained from Merck(Germany) and used without further purification. Urea was purchased from Sigma-Aldrich. Distilled, deionized water was used for the preparation of all solutions. All other materials were of analytical grade.
Apparatus and Procedure
The biphasic systems were prepared by mixing PVP (K30), tri-sodium citrate, and urea at the required pH. The composition of the mixture was determined by mass. Feed samples (10 g) were prepared by mixing appropriate amounts of polymer, salt, urea, and water in 15 ml graduated tubes using an analytical balance (A&D model GF300; Japan) with a precision of ±10−4 g at 25°C. To maintain a constant temperature (25°C) with an uncertainty of 0.05°C, the tubes were placed in a thermostatic bath (Memert model INE400; Germany). The pH values of the salt solutions were adjusted by mixing the appropriate ratio of tri-sodium citrate, sodium hydroxide, and sulfuric acid. The pH values of the solutions were measured precisely with a Metrohm 827pH lab meter (Switzerland). For each mentioned system, 5% and 10% (w/w) samples of urea were produced.
The contents of the test tube were rigorously vortexed for 10 min before being placed in a 25°C thermostatic bath for 2 h. To separate the resulting phases, the tubes were centrifuged (Hermle Z206A; Germany) at 6000 rpm for 5 min. The phases showed no turbidity and the top and bottom samples were easily separated. The electrical conductivity and refractive index for each sample were measured at 298.15 K using a JENWAY 4510 model with a precision of 0.01µS-1mS and a refractometer (Ceti; Belgium) with a precision of 0.0001 nD, respectively. All data measurements were conducted in duplicate and the average values were reported. The salt concentrations (Na3C6H5O7) were determined using atomic absorption spectroscopy (Shimatsu model AA-6300; Japan). The calibration plots of the refractive index and conductivity were prepared for the known polymer and urea compositions at the individual salt concentrations at 25°C. The measured values were then interpolated. The average relative deviation of the urea and polymer concentrations using this method was about 0.1% (wt).
RESULTS AND DISCUSSION
The viscosity, density, electrical conductivity, and refractive index of the PVPK30 + tri-sodium citrate + urea + water systems are shown in Table 2. Because the electrical conductivity and the refractive index of the phase samples depend on PVP (K30), urea, and salt concentration, calibration plots of the refractive index versus polymer concentration were prepared for different concentrations of salt and urea. Sample calibration plots for aqueous solutions of PVP (K30) + sodium citrate + urea + water are shown in Figure 1.
To increase the knowledge about ATPSs containing urea, phase diagrams of a range of systems based on PVP (K30) + sodium citrate + water at 298.15 K and pH values of 6.2, 7.4, and 10.0 in the presence of urea (5% and 10% mass) were determined. The experimental data of the binodal, tie line data, and STL for the PVP (K30) + sodium citrate + urea + water system at 25°C and the physical properties of the top and bottom phases are reported in Table 3. All concentrations are expressed as mass percentages.
Density(ρ), viscosity, refractive index (nD), and electrical conductivity (k) for aqueous single-phase system (PVP K30 (p) + tri-sodium citrate (s) + urea (u) + water system) at 298.15 K.
Phase composition, tie line data, STL, and physical properties for PVP (K30) + sodium citrate + urea (5% w/w) + water system at 25°C
Refractive index and electrical conductivity calibration curves for PVP (K30) + sodium citrate + urea + water at 25°C (salt = 10% w/w)
The experimental data of the binodal for PVP (K30) + sodium citrate + denaturant + water systems at 25°C and pH 6.2 at urea concentrations of 5% and 10% (w/w) are plotted in Figure 2.
Binodal of PVP (K30) + sodium citrate systems at 25°C and pH 6.2 versus urea concentration.
The TLL for the different compositionswith two phases were calculated as:
The STL is the ratio of the difference between the polymer concentration (CP) and the salt concentration (CS) in the top and bottom phases as:
Figure 3 shows the effect of urea concentration on equilibrium phase composition and on STL and TLL for PVP (K30) + sodium citrate + H2O system at a pH value of 6.2. The influence of urea concentration on the binodal curve for the PVP (K30) + sodium citrate ATPS at a constant pH is shown in Tables 2 and 3 and Figures 2 and 3. It can be observed that the binodal was displaced toward higher concentrations as the urea concentration increased and that the composition of the polymer-rich phase (and TLL) behaved in a slightly shorter manner. The STL decreased as the urea concentration increased. This effect has also been reported for urea (Creighton, 1993Creighton T. Proteins, 2nd ed.New York: W.H. Freeman and Co (1993).; Rämsch et al.1999Rämsch, Ch. Kleinelanghorst, L.B., Knieps. E.A., Hommes, J. and Kula, M.-R., Aqueous Two-Phase System Containing Urea; Influence on Phase Separation and Stabilization of Protein Conformation by Phase Components, Biotechnol. Prog. 15, No 3, 493(1999).; Silva and Meirelles, 2001Silva, L.H. M. D and Meirelles, A.J.D.A. PEG + Potassium Phosphate + Urea Aqueous Two-Phase Systems: Phase Equilibrium and Protein Partitioning, J. Chem. Eng. Data, 46, No 2, 251(2001).) and guanidine hydrochloride (Pirdashti and Rahimpour, 2010Rahimpour, F and Pirdashti, M. Effective Parameters on the Partition Coefficient of Guanidine Hydrochloride in the Poly(ethylene glycol) + Phosphate + Water system at 298.15 K, Iran. J. Chem. Eng. 7, No.1, 67(2010).; Pirdashti et al.2015Pirdashti, M., Movagharnejad, K., Curteanu, S., Dragoi, E.N and Rahimpour, F., Prediction of Partition Coefficients of Guanidine Hydrochloride in PEG-Phosphate Systems Using Neural Networks Developed with Differential Evolution Algorithm, J. Ind. Eng. Chem, 27, No 1,268(2015).).
The opposing components found in these systems are structure-making salts; urea is sometimes described as a structure-breaking agent. The combination of the two competing components on phase separation is interesting and cannot be predicted (Rämsch et al. 1999Rämsch, Ch. Kleinelanghorst, L.B., Knieps. E.A., Hommes, J. and Kula, M.-R., Aqueous Two-Phase System Containing Urea; Influence on Phase Separation and Stabilization of Protein Conformation by Phase Components, Biotechnol. Prog. 15, No 3, 493(1999)., Bertoluzzo et al. 2007Bertoluzzo, M.G., Rigatuso, R., Farruggia, B., Nerli, B. and Picó, G., Cosolutes Effects on Aqueous Two-Phase Systems Equilibrium Formation Studied by Physical Approaches, Colloids Surf B Biointerfaces, 59, No.2, 134 (2007) .). Urea has a similar structure-breaking effect on water and the preferential interaction with an aqueous interface (Annuziata et al. 2002Annuziata, O., Asherie, N., Lomakin, A., Pande, J., Ogun, O and Benedek, G.B., Effect of Polyethylene Glycol on the Liquid-Liquid Phase Transition in Aqueous Protein Solutions, PNAS, 99, No.22, 4165 (2002).). PVP is a hydrophilic polymer. The effects of urea on water structure increased the depletion force between the PVP-urea-water causing the ratio of PVP in the two phases to change and the phase regions to shrink.
Effect of urea concentration on equilibrium phase composition, STL, and TLL for PVP (K30) + sodium citrate + H2O system at a pH of 6.2 (■: total composition; : bottom phase composition; ●: top phase composition).
Urea added to ATPS as a solute is divided between the phases. Its partition coefficient, K, is defined as a function of the equilibrium concentrations of the solute in the upper and lower phases as:
Figure 4 is a diagram of the urea partition coefficient versus TLL. It can be seen that, at a constant TLL, the partition coefficient of urea increased as the urea concentration increased. The partitioning coefficient also increased as the TLL increased at a constant urea concentration. The slope of the partition coefficient of urea versus TLL decreased as the urea concentration increased.
The results from Table 3 indicate that, at a constant value of pH, the partition coefficient had a direct relation with the PVP/salt ratio. At a constant pH, the partition coefficient of urea increased as the PVP/salt ratio increased. In this case, the effect of urea concentration on the urea partition coefficient was more significant than the effect of the PVP/salt ratio. At a constant concentration of urea, the partitioning coefficient of urea decreased as the pH increased. Table 2 shows that, as the pH increased, the slope and length of the equilibrium tie lines for the biphasic system increased.
Effect of TLL and urea concentration on partition coefficient of urea at different pH values for the ATPS.
Figure 5 shows the effect of pH on equilibrium phase composition and on the STL and TLL for the PVP (K30) + tri-sodium citrate + H2O system. The TLL and STL increased as the pH increased, which is in agreement with the results of Perumalsamy and Murugesan (2009Perumalsamy, M and Murugesan, T. Phase Compositions, Molar Mass, and Temperature Effect on Densities, Viscosities, and Liquid-Liquid Equilibrium of Polyethylene Glycol and Salt-Based Aqueous Two-Phase Systems, J. Chem. Eng. Data , 54, No 4 , 1359(2009). ) and Shahbazinasab and Rahimpour (2012Shahbazinasab, M-K and Rahimpour, F. Liquid−Liquid Equilibrium Data for Aqueous Two-Phase Systems Containing PPG725 and Salts at Various pH Values, J. Chem. Eng. Data , 57, No 7, 1867(2012).).This could be the result of the decrease in hydrodynamic volume of the polymers in solution.Waziri et al. (2003Waziri, S.M., Abu-Sharkh, B.F., Ali, S.A. The Effect of pH and Salt Concentration on the Coexistence Curves of Aqueous Two-Phase Systems Containing a pH Responsive Copolymer and Polyethylene Glycol, Fluid Phase Equilib , 205, No 2, 275 (2003).) and Shahbazinasab and Rahimpour (2012)Shahbazinasab, M-K and Rahimpour, F. Liquid−Liquid Equilibrium Data for Aqueous Two-Phase Systems Containing PPG725 and Salts at Various pH Values, J. Chem. Eng. Data , 57, No 7, 1867(2012). reported that decreasing the pH decreased the intrinsic viscosity of the polymer solution. It is known that the hydrodynamic volume of polymers in solution is proportional to their intrinsic viscosity and that a decrease in pH creates a more compact structure for the polymer chains. It has been reported that, as the pH of the aqueous polymer-salt two-phase system increases, the concentration of the polymer-rich phase increases and the concentration of the salt-rich phase decreases.
Effect of pH on equilibrium phase composition, STL, and TLL for PVP (K30) + tri-sodium citrate + H2O system (: total composition; ■: bottom phase composition; ●: top phase composition).
Table 3 and Figure 6 show the relation between TLL and the density and viscosity of the ATPS. The difference in density between phases (Δρ) and viscosity between phases (Δη) increased as TLL increased.
Difference in viscosity (Δη), density (Δρ) and TLL for PVP (K30) + tri-sodium citrate + water vs. urea concentration at a pH value of 6.2.
CONCLUSIONS
Urea was applied as a denaturant for the initial recovery steps to produce recombinant proteins in an ATPS. The liquid-liquid equilibrium, effect of denaturant on phase diagram behavior, and partition coefficient in these systems and the physical properties of ATPS are relatively unknown. The effect of urea on the phase diagram behavior of the PVP (K30) + tri-sodium citrate ATPS at 25°C was investigated at different pH values (6.2, 7.4, and 10.0). The calibration method was applied to measure the refractive index and conductivity of the phases.
The binodal was displaced toward higher concentrations as the urea concentration increased; the composition of the polymer-rich phase (and TLL) behaved in a slightly shorter manner. The STL decreased as the urea concentration increased. The density, viscosity, electrical conductivity, and refractive index of the aqueous single-phase system of PVP (K30) (p) + tri-sodium citrate (s) + urea (u) + water) at 298.15 K with the properties of ATPS were measured at 25°C. The partition coefficient of urea was shown to have a direct relation with urea concentration, pH, TLL, and PVP/salt ratio. The partition coefficient of urea increased as the pH decreased and the PVP/salt (%w/w) ratio and urea concentration increased. The relation between TLL, density, and viscosity of the ATPS was also considered.
ACKNOWLEDGMENT
The authors would like to thank the anonymous reviewers and the editor for their insightful comments and suggestions.
REFERENCES
- Albartson P.A. Partition of cell particles and macromolecules, 3nd ed., New York: Willy(1986).
- Annuziata, O., Asherie, N., Lomakin, A., Pande, J., Ogun, O and Benedek, G.B., Effect of Polyethylene Glycol on the Liquid-Liquid Phase Transition in Aqueous Protein Solutions, PNAS, 99, No.22, 4165 (2002).
- Bertoluzzo, M.G., Rigatuso, R., Farruggia, B., Nerli, B. and Picó, G., Cosolutes Effects on Aqueous Two-Phase Systems Equilibrium Formation Studied by Physical Approaches, Colloids Surf B Biointerfaces, 59, No.2, 134 (2007) .
- Bühler, V., Excipients for Pharmaceuticals-Povidone, Crospovidone and Copovidone, Springer, Berlin, Heidelberg, New York, 1-254(2005).
- Clark, ED., Protein Refolding for Industrial Processes, Curr. Opin.Biotechnol.12, 202 (2001).
- Creighton T. Proteins, 2nd ed.New York: W.H. Freeman and Co (1993).
- Fedicheva, N., Ninni, L and Maurer, G., Aqueous Two-Phase Systems of Poly(vinyl pyrrolidone) and Sodium Sulfate: Experimental Results and Correlation/Prediction, J. Chem. Eng. Data, 52, No.2, 1858(2007).
- Foroutan, M and Zarrabi, M. Activities of Water, Polymer and Salt in Liquid-Liquid Equilibria of Polyvinylpyrrolidone and (NH4)2HPO4/(NH4)H2PO4 Buffer Using the Flory-Huggins Model with Debye-Huckel and Pitzer-Debye-Huckel Equations and the Osmotic Virial Model: Effects of pH and Temperature, Fluid Phase Equilib, 266, No1, 164 (2008).
- Hatti-Kaul, Rajni (Ed.), Aqueous two-phase systems. Methods and Protocols. New York: Humana Press (2000).
- Perumalsamy, M and Murugesan, T. Phase Compositions, Molar Mass, and Temperature Effect on Densities, Viscosities, and Liquid-Liquid Equilibrium of Polyethylene Glycol and Salt-Based Aqueous Two-Phase Systems, J. Chem. Eng. Data , 54, No 4 , 1359(2009).
- Rahimpour, F and Pirdashti, M. Effective Parameters on the Partition Coefficient of Guanidine Hydrochloride in the Poly(ethylene glycol) + Phosphate + Water system at 298.15 K, Iran. J. Chem. Eng. 7, No.1, 67(2010).
- Rocha, M.V and Nerli, B.B. Molecular Features Determining Different Partitioning Patterns of Papain and Bromelain in Aqueous Two-Phase Systems. Int. J. Bio. Macromol., 61, No 2, 204(2013).
- Rämsch, Ch. Kleinelanghorst, L.B., Knieps. E.A., Hommes, J. and Kula, M.-R., Aqueous Two-Phase System Containing Urea; Influence on Phase Separation and Stabilization of Protein Conformation by Phase Components, Biotechnol. Prog. 15, No 3, 493(1999).
- Sadeghi, R., Measurement and Correlation of Vapor-Liquid Equilibria of the Poly(vinylpyrrolidone)+ NaH2PO4 + H2O System at Different Temperatures, CALPHAD, 30, No 1, 53 (2006).
- Salabat, A., Moghadasi, M.A., Zalaghi, P and Sadeghi, R., (Liquid + liquid) Equilibria for Ternary Mixtures of (Polyvinylpyrrolidone + MgSO4 + Water) at Different Temperatures, J. Chem. Thermodynamics,38, No 2, 1479 (2006).
- Sadeghi, R, Rafiei,H.R and Motamedi, M., Phase Equilibrium in Aqueous Two-Phase Systems Containing Poly(vinylpyrrolidone) and Sodium Citrate at Different Temperatures-Experimental and Modeling, Thermochimica Acta, 451, No 1, 163 (2006).
- Shahbazinasab, M-K and Rahimpour, F. Liquid−Liquid Equilibrium Data for Aqueous Two-Phase Systems Containing PPG725 and Salts at Various pH Values, J. Chem. Eng. Data , 57, No 7, 1867(2012).
- Silva, L.H. M. D and Meirelles, A.J.D.A. PEG + Potassium Phosphate + Urea Aqueous Two-Phase Systems: Phase Equilibrium and Protein Partitioning, J. Chem. Eng. Data, 46, No 2, 251(2001).
- Parnica, M. and Antalika, M., Urea and Guanidine Salts as Novel Components for Deep Eutectic Solvents, J. Mol. Liq. 197, No 1 , 23(2014).
- Pirdashti, M., Movagharnejad, K., Curteanu, S., Dragoi, E.N and Rahimpour, F., Prediction of Partition Coefficients of Guanidine Hydrochloride in PEG-Phosphate Systems Using Neural Networks Developed with Differential Evolution Algorithm, J. Ind. Eng. Chem, 27, No 1,268(2015).
- Wang, Z.H., Song, M. and Ma, Q., Two-Phase Aqueous Extraction of Chromium and its Application to Speciation Analysis of Chromium in Plasma, Mikrochim. Acta, 134, No 1, 95(2000).
- Waziri, S.M., Abu-Sharkh, B.F., Ali, S.A. The Effect of pH and Salt Concentration on the Coexistence Curves of Aqueous Two-Phase Systems Containing a pH Responsive Copolymer and Polyethylene Glycol, Fluid Phase Equilib , 205, No 2, 275 (2003).
- Yan-Min, L., Yan-Zhao, Y., Xi-Dan, Z., Chuan-Bo, X., Bovine Serum Albumin Partitioning in Polyethylene glycol (PEG)/Potassium Citrate Aqueous Two-Phase Systems. Food Bioprod. Process, 88, No.1, 40 (2010).
- Zafarani-Moattar, M.T and Sadeghi, R., Measurement and Correlation of Liquid-Liquid Equilibria of the Aqueous Two-Phase System Polyvinylpyrrolidone-Sodium Dihydrogen Phosphate, Fluid Phase Equilib, 203, No 1, 177 (2002).
- Zafarani-Moattar, M.T and Sadeghi, R., Effect of Temperature on the Phase Equilibrium of Aqueous Two-Phase Systems Containing Polyvinylpyrrolidone and Disodium Hydrogen Phosphate or Trisodium Phosphate, Fluid Phase Equilib, 238, No 1, 129 (2005).
- Zafarani-Moattar, M.T and Seifi-Aghjekohal, P., Liquid-Liquid Equilibria of Aqueous Two-Phase Systems Containing Polyvinylpyrrolidone and Tripotassium Phosphate or Dipotassium Hydrogen Phosphate: Experiment and Correlation, CALPHAD, 31, No 4, 253 (2007).
- Zafarani-Moattar, M.T and Zaferanloo, A., Measurement and Correlation of Phase Equilibria in Aqueous Two-Phase Systems Containing Polyvinylpyrrolidone and Dipotassium Tartrate or Di-potassium Oxalate at Different Temperatures, J. Chem. Thermodynamics, 41, No7, 864 (2009).
- Zafarani-Moattar ,M.T., Karimi,N and Asadzadeh, B. Investigations of the Interactions in Aqueous Poly (vinyl pyrrolidone) + Di-sodium Hydrogen Citrate Solutions Using Vapor-Liquid Equilibria Studies at Different Temperatures, J. Mol. Liq, 211, No 12,767(2015).
Publication Dates
-
Publication in this collection
July 2017
History
-
Received
04 Oct 2015 -
Reviewed
13 Feb 2016 -
Accepted
15 Apr 2016