Salt Phase

Article pubs.acs.org/jced

Influence of the Molecular Weight of PEG on the Polymer/Salt Phase Diagrams of Aqueous Two-Phase Systems Kamila Wysoczanska and Eugénia A. Macedo* Associated Laboratory of Separation and Reaction EngineeringLaboratory of Catalysis and Materials (LSRE-LCM), Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal ABSTRACT: Binodal data for systems composed by polyethylene glycol (PEG) (4000, 6000, or 8000) and salt (potassium citrate, potassium sodium tartrate) were obtained using the cloud point method, at T = 298.15 K. Merchuk equation was applied for the fitting of binodal curves. Tie-lines for systems of PEG (4000, 6000)− potassium citrate and PEG (4000, 6000, 8000)−potassium sodium tartrate were determined by electrical conductivity and lyophilization, and correlated using Othmer−Tobias and Bancroft equations, to verify the reliability of the measured tie-lines. Data were compared with that of previous studies reported in the literature, giving an indication of which system is more efficient in terms of separation, taking into account possible combinations of salt’s cations and PEGs of different molecular weight chosen for ATPS. 1450,15 1500,17 2000,18 3350,13,15 4000,17 6000,19,20 and 800021 g/mol), potassium citrate and PEG (2000,22 6000,23 and 800021 g/mol), sodium tartrate and PEG (600, 1000, 2000, 4000, 6000, and 800024 g/mol), potassium tartrate and PEG (400025 g/mol), and potassium sodium tartrate and PEG (400025 g/mol). It was previously reported in the literature for other ATPSs, that the higher is the PEG’s molecular weight, the higher is the two-phase region.26,27 Taking into account that with an increase of the PEG’s molecular weight density also increases, and that at an industrial level this can cause some problems, the impact of this molecular weight, on the size of biphasic regions, is of importance. In this work, physicochemical properties of aqueous two-phase systems composed by PEG of different molecular weights and salts were studied at T = 298.15 K. Different phase diagrams for systems of PEG (4000, 6000, 8000) and salt (potassium citrate, potassium sodium tartrate) were obtained. Tie-lines for PEG (4000, 6000)−potassium citrate and PEG (4000, 6000, 8000)−potassium sodium tartrate ATPSs were measured. As the binodal data for PEG 8000− potassium citrate ATPS measured here, agrees with the values reported by Silvério et al.,21 in this work we did not measure the tie-line data for this system, and we used those given by Silvério et al.21 The fitting of binodal curves was done using the Merchuk equation and reliability of the tie-lines was determined by Othmer−Tobias and Bancroft equations.28,29 This is, to our best knowledge, the first study that reports LLE data for systems composed by PEG 4000−potassium citrate,

1. INTRODUCTION Extraction in aqueous two-phase systems (ATPS) was devised to replace conventional techniques that employ hazardous, volatile organic solvents.1 Because of the high content of water, aqueous biphasic systems are considered an environmentally friendly separation media. Different mixtures of polymer−polymer or salt−polymer based solutions that are able to form two immiscible phases have been studied, and the list of specific applications of ATPS is still expanding.2−5 Aqueous biphasic systems present nondenaturing and mild conditions for the extraction of biomolecules, and it has been found that among the best systems for this purpose are those formed by polymer and salt.6 So far, various combinations of those components were used to study the (liquid−liquid) phase equilibria of the two-phase mixtures. To qualify the system and its potential applications, phase diagrams need to be known, as they present the working area for any given ATPS. Equilibrium data are obtained under specific conditions: pressure, temperature, salt concentration, polymer molecular weight, pH, among others. Polyethylene glycol (PEG) is widely used, nontoxic, and a relatively low-cost polymer. There are some studies in the literature on physicochemical properties of systems formed by PEG of different molecular weights and different salts, for example, sulfates,7−9 phosphates.10−12 Even though some of these salts, commonly used in low concentrations to obtain the biphasic systems, are only marginally toxic, at an industrial scale, they may cause some environmental distress. However, these salts convey many advantages in terms of separation. For that reason, more recent studies point out citrate and tartrate salts as an effective alternative. Liquid−liquid equilibrium measured at different conditions, can be found in the literature for systems composed of sodium citrate and PEG of different molecular weights (400,13 600,14,15 1000,13,15,16 © XXXX American Chemical Society

Special Issue: Proceedings of PPEPPD 2016 Received: July 5, 2016 Accepted: August 24, 2016

A

DOI: 10.1021/acs.jced.6b00591 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

Article

The slopes of the tie-lines (STL) were obtained using linear regression. Binodal curve and tie-line determination were made using the methodology previously reported in the literature.21

PEG (6000, 8000)−potassium sodium tartrate and that shows a comparison with other systems studied and presented in this assay.

2. CHEMICALS Polyethylene glycol (PEG), with an average molecular weight (Mw) of 4000 g/mol (lot no. BCBP8299 V), Mw of 6000 g/mol (lot no. BCBQ5326 V), Mw of 8000 g/mol (lot no. SLBJ5906 V), potassium citrate tribasic monohydrate (K3C6H5O7·H2O, ACS reagent, ≥ 99%, Mw = 324.41 g/mol) and potassium sodium tartrate tetrahydrate (C4H4KNaO6·H2O, ACS reagent, ≥ 99%, Mw = 282.23 g/mol) were obtained from SigmaAldrich. Dilutions were performed using double distilled deionized water. All materials were used as received without further purification. For the weighing purposes, an Adam Equipment balance model AAA250L, with an uncertainty of ±0.2 mg was used.

4. RESULTS AND DISCUSSION The binodal data were measured for aqueous two-phase systems composed by PEG (4000, 6000, 8000) and a salt (potassium citrate, potassium sodium tartrate), at T = 298.15 K and p = 0.1 MPa, and are presented in Figures 1 and 2. For the

3. METHODS Binodal curves were determined using the cloud point technique. Systems of different polymer and salt compositions were prepared in assay tubes, thoroughly vortexed (VWR, model VV3) and placed in a thermostatic bath (Techne, Tempette TE-8D) at T = 298.15 ± 0.2 K. Water was added dropwise to the tubes until one homogeneous phase was achieved. Curve fitting to the experimental data points was made using the equation given by Merchuk et al.:28 y = a exp(bx 0.5 − cx 3)

(1)

where x and y are the salt and polymer compositions in mass fraction, respectively, and a, b, and c are adjustable parameters that are estimated by nonlinear least-squares regression. To determine the tie-lines, systems with different compositions of PEG and salt (potassium citrate, potassium sodium tartrate) solutions were prepared in 15 mL tubes. Tubes were vigorously mixed in a vortex mixer and left for 48 h in a thermostatic bath, at T = 298.15 ± 0.2 K, to establish phase separation. The concentration of PEG and salt was determined in both phases, and samples were withdrawn using a syringe and suitably diluted with water. Salt concentration was measured at T = 298.15 K, by electrical conductivity using a Crison GLP31 meter with a platinum conductivity cell Crison 52 93, with a precision of ±0.5%. The calibration curve was constructed by measuring the electrical conductivity of standard solutions, having known salt concentration, within the range where no polymer interference was observed (0.01−0.4%). A final salt concentration was determined using three measurements corrected with the dilution factor (standard deviations < 0.2%). The polymer concentration was measured using a gravimetric method, after lyophilization. Samples of the top and bottom phase were withdrawn, diluted with water, frozen at T = 255 K for a minimum of 24 h and placed into a freeze-dryer (standard deviation of the average dry weight < 0.2%). To calculate the total polymer concentration in the top and bottom phase, salt concentration was subtracted from the average dry weight. The tie-line lengths (TLL) were calculated using the following equation: TLL =

WTPEG,

T B T B 2 (WPEG − WPEG )2 + (Wsalt − Wsalt )

WTsalt,

W BPEG

Figure 1. (a) Binodal experimental data for (a) PEG 4000 + potassium citrate (―), PEG 6000 + potassium citrate (― ―) and PEG 8000 + potassium citrate (···) systems at T = 298.15 K, this work; (b) PEG 1500 + potassium citrate (―···―) at T = 296.15 K.21 Experimental cloud points: PEG (4000 ●, 6000 ○, 8000 ▼) + potassium citrate. Compositions are given in mass fraction. (b) Binodal data for PEG 8000 + potassium citrate system; (···) at T = 298.15 K, this work; (―) at T = 296.15 K.21

curve fitting of the experimental points, the Merchuk equation was successfully applied, and adjustable parameters are given in Table 1. Coefficients of determination for both systems were >0.999 and sd < 0.006. F-test values were higher than 4000. PEG’s molecular weight effect was evaluated for ATPSs formed by polymer and the two different salts, potassium citrate and potassium sodium tartrate. For both system types, presented in Figures 1 and 2, the same curve tendency can be

(2)

W Bsalt

where and are the top (T) and bottom (B) mass fractions of PEG and citrate or tartrate salt at equilibrium. B



Article

Figure 2. Binodal experimental data for: PEG 4000 + potassium sodium tartrate (―), PEG 6000 + potassium sodium tartrate (---) and PEG 8000 + potassium sodium tartrate (···) systems studied in this work, at T = 298.15 K. Experimental cloud points: PEG (4000 ●, 6000 ○, 8000 ▼) + potassium sodium tartrate. Compositions are given in mass fraction.

observed with just small differences in the diagram. A slightly higher “working area” was obtained when PEG of higher molecular weight was used. This means that the system formed by PEG 8000 has better phase forming ability and can be considered as a more efficient ATPS for separation. This was to be expected, because when the polymer’s molecular weight increases, hydrophobicity is higher and water solubility is lower. However, as density also increases, and the differences in the binodals are not significant, systems with PEG 4000 and 6000 can be envisaged as interesting and useful for extraction purposes, mainly in the separation of target solutes. Density being lower can have a positive effect on the decision of which PEG should be used. Because there are still only a few studies reported in the literature21−23 on liquid−liquid equilibrium of biphasic systems composed by potassium citrate and PEG, for comparison purposes, data from ref 21 was used, although measured at a slightly different temperature (T = 296.15 K). It can be observed that there is no significant difference in binodals for the system composed by PEG 8000, measured in this work and the results reported by Silvério et al.21 However, the size distribution of PEG is statistically characterized by its average molecular

Figure 3. (a) Binodal curves and tie-lines for the PEG 4000 + sodium citrate (···)30 and PEG 4000 + potassium citrate (―) systems, at T = 298.15 K. Compositions are given in mass fraction. (b) Binodal curves and tie-lines for the PEG 6000 + sodium citrate (···)30 and PEG 6000 + potassium citrate (―) systems, at T = 298.15 K. Compositions are given in mass fraction.

weight, so the LLE compositions found in the literature, even for PEGs with the same molecular weight, can differ among each other. A small displacement of the curve into a direction of higher PEG and salt concentrations, may be caused by temperature difference. As it was previously reported, the lower is the temperature, the lower is the heterogeneous region.23 The data

Table 1. Number of Solubility Points Used to Determine Each Binodal Curve (N), together with the Adjustable Parameters (a, b, and c) Obtained from the Merchuk Equation (eq 1), Respective Coefficients of Determination (r2), Standard Deviation (sd), and F-Test N

system PEG PEG PEG PEG PEG PEG a

4000−potassium 6000−potassium 8000−potassium 4000−potassium 6000−potassium 8000−potassium

citrate citrate citrate sodium tartrate sodium tartrate sodium tartrate

14 16 14 14 18 18

a 1.41 1.51 1.38 1.33 1.10 1.24

± ± ± ± ± ±

b −5.86 −6.24 −6.17 −5.67 −4.92 −5.54

0.07 0.07 0.08 0.04 0.06 0.05

± ± ± ± ± ±

c 0.11 0.12 0.13 0.07 0.09 0.10

224 281 348 177 264 312

± ± ± ± ± ±

21 18 34 15 18 17

r2

sda

F

0.9990 0.9990 0.9990 0.9995 0.9990 0.9993

0.005 0.004 0.006 0.005 0.004 0.003

4567 6747 4012 5324 7121 10689

Standard deviation: ⎛ N (100W cal − 100W exp )2 ⎞0.5 PEG PEG ⎟ sd = ⎜⎜∑ ⎟ N ⎠ ⎝ i=1 C



Article

measured by the authors in ref 21 for ATPSs formed by PEG 1500 was also used for comparison and agrees with the previously drawn conclusions. The results of the comparison are shown in Figure 1a and Figure 1b. Tie-lines were obtained for systems of PEG (4000, 6000) and a salt (potassium citrate, potassium sodium tartrate), at T = 298.15 K and p = 0.1 MPa, and are presented in Figures 3a,b and 4a,b,c. As the binodal data measured in this work for PEG 8000 was in agreement with the previous study,21 the tie-line data reported in this work may be used for the comparison with the measured systems composed by PEG 4000 and 6000. Feed and phase composition, TLL, and STL are given in Table 2. In all cases the absolute values of STL decreases, while TLL increases. Regarding the effect of the salt presented in Figures 3 and 4, it can be seen that the cation has a stronger influence in the heterogeneous region. Nevertheless, from these figures, it is possible to observe that the difference is less than 5%. The reliability of the tie-lines was checked by Othmer-Tobias and Bancroft eqs 3 and 4: ⎛ 1 − WT ⎞ ⎛1 − WB ⎞ salt PEG ⎟ = k1 + n log⎜ ⎟ log⎜ B T ⎝ Wsalt ⎠ ⎝ WPEG ⎠

(3)

⎛ WT ⎞ ⎛ WB ⎞ ⎟ ⎜ water ⎟ k r log⎜ water = + log 2 B T ⎝ Wsalt ⎠ ⎝ WPEG ⎠

(4)

WTwater

W Bwater

where and are the mass fractions of water in the top and bottom phases in equilibrium, respectively. The results of the correlation were graphically presented in Figures 5a,b and 6a,b. Application of both equations gives good linearity of the LLE data. The fitted parameters k, n, and r and the corresponding coefficients of determination (r2) are given in Table 3.

5. CONCLUSIONS The binodal curves for systems composed by PEGs of different molecular weight (4000, 6000, 8000 g/mol) and two salts (potassium citrate or potassium sodium tartrate), at T = 298.15 K, were obtained experimentally. For the fitting of the experimental data, the Merchuk equation was applied. Tie-lines were measured for systems of potassium citrate with PEG (4000, 6000) and of potassium sodium tartrate with PEG (4000, 6000, 8000). Tie-line data for the system of PEG 8000 and potassium citrate was taken from previously reported studies, as the binodal curves were in agreement with our measurements. To check the reliability of the measured tie-lines, Othmer−Tobias and Bancroft equations were used, resulting in a very good linearization. It was observed that systems formed by PEG 8000 presented just slightly higher biphasic regions. This dependence

Figure 4. Binodal curves and tie-lines for the ATPS formed by PEG: (a) 4000, (b) 6000, (c) 8000 and tartrates, at T = 298.15 K; potassium sodium tartrate (―) (this work), sodium tartrate (···).24 Compositions are given in mass fraction.

Table 2. Feed, Top and Bottom Phase Compositions, Tie-Line Lengths (TLL) and Slopes (STL) for the PEG (1) + Potassium Citrate (2) and PEG (1) + Potassium Sodium Tartrate (2) Systems, at T = 298.15 K and p = 0.1 MPaa feed TLL

(1)

top phase (2)

(1)

I II III

0.1642 0.1688 0.1787

0.1393 0.1431 0.1520

0.296 0.317 0.348

I II

0.1754 0.1806

0.1412 0.1453

0.333 0.346

bottom phase (2)

(1)

PEG 4000 + Potassium Citrate 0.063 0.005 0.058 0.003 0.052 0.002 PEG 6000 + Potassium Citrate 0.053 0.003 0.051 0.002

D

(2)

STL

TLL

0.233 0.242 0.258

−1.71 −1.69 −1.68

0.337 0.363 0.403

0.244 0.254

−1.72 −1.70

0.381 0.399



Article

Table 2. continued feed TLL

(1)

top phase (2)

III

0.1860

0.1496

I II III

0.1295 0.1404 0.1502

0.1450 0.1645 0.1842

I II III

0.1618 0.1683 0.1747

0.1226 0.1268 0.1339

I II III

0.1636 0.1721 0.1781

0.1165 0.1231 0.1281

I II III

0.1505 0.1587 0.1700

0.1141 0.1204 0.1295

(1)

bottom phase (2)

(1)

PEG 6000 + Potassium Citrate 0.359 0.048 0.001 PEG 8000 + Potassium Citrateb 0.280 0.064 0.006 0.334 0.054 0.001 0.384 0.046 0.00023 PEG 4000 + Potassium Sodium Tartrate 0.226 0.085 0.015 0.254 0.075 0.010 0.286 0.066 0.004 PEG 6000 + Potassium Sodium Tartrate 0.226 0.082 0.012 0.264 0.071 0.006 0.287 0.064 0.002 PEG 8000 + Potassium Sodium Tartrate 0.216 0.080 0.005 0.253 0.069 0.002 0.290 0.061 0.001

(2)

STL

TLL

0.262

−1.67

0.417

0.210 0.239 0.267

−1.88 −1.79 −1.73

0.310 0.381 0.443

0.211 0.227 0.243

−1.67 −1.60 −1.59

0.246 0.288 0.333

0.206 0.225 0.236

−1.73 −1.68 −1.65

0.248 0.301 0.332

0.194 0.213 0.230

−1.84 −1.75 −1.71

0.240 0.290 0.335

a

Standard uncertainties u are u(T) = 0.2 K, u(p) = 0.005 MPa, u(WTPEG) = 0.0006, u(WTsalt) = 0.0002, u(WBPEG) = 0.0019, u(WBsalt) = 0.0015, for data obtained in this work. bTie-line data from ref 21.

Figure 5. Linearization of the liquid−liquid equilibrium data obtained for the PEG−potassium citrate ATPS using (a) Othmer−Tobias equation and (b) Bancroft equation: PEG 4000 (●); PEG 6000 (○).

Figure 6. Linearization of the liquid−liquid equilibrium data obtained for the PEG-potassium sodium titrate ATPS using (a) Othmer-Tobias equation and (b) Bancroft equation: PEG 4000 (●); PEG 6000 (○); PEG 8000 (▼).

area for the ATPS formed by PEG and potassium citrate was marginally higher than that for PEG with potassium sodium tartrate, which means that the first system may be somewhat better for separation. Taking into account the effect of the

is related to the higher hydrophobicity and lower solubility of the polymer of higher molecular weight. Through the comparison of the ability to form two-phases with the two types of salt studied within this work, it was observed that the biphasic E



Article

Table 3. Parameters Obtained Using Othmer−Tobias Equation (eq 3) and Bancroft Equation (eq 4) with the Corresponding Coefficients of Determination (r2) Othmer−Tobias ATPS PEG PEG PEG PEG PEG

4000 6000 4000 6000 8000

+ + + + +

potassium potassium potassium potassium potassium

citrate citrate sodium tartrate sodium tartrate sodium tartrate

k1

r2

r

k2

r2

1.7721 1.2578 1.7183 1.8025 1.8450

−0.5426 −0.3143 −0.4483 −0.5231 −0.579

0.9992 0.9999 0.9986 0.9997 0.9993

0.5706 0.8084 0.5701 0.5462 0.5520

0.3242 0.2735 0.2874 0.3147 0.3312

0.9991 0.9999 0.9980 0.9994 0.9996

(8) Martins, J. P.; Carvalho, C. D. P.; Silva, L. H. M. D.; Coimbra, J. S. D. R.; Silva, M. D. C. H. D.; Rodrigues, G. D.; Minim, L. A. Liquid− liquid equilibria of an aqueous two-phase system containing poly (ethylene) glycol 1500 and sulfate salts at different temperatures. J. Chem. Eng. Data 2008, 53 (1), 238−241. (9) Ferreira, L. A.; Teixeira, J. A. Salt effect on the aqueous two-phase system PEG 8000− Sodium sulfate. J. Chem. Eng. Data 2011, 56 (1), 133−137. (10) Cunha, E. V.; Aznar, M. Liquid− Liquid Equilibrium in Aqueous Two-Phase (Water+ PEG 8000+ Salt): Experimental Determination and Thermodynamic Modeling†. J. Chem. Eng. Data 2009, 54 (12), 3242−3246. (11) Amaresh, S. P.; Murugesan, S.; Regupathi, I.; Murugesan, T. Liquid− Liquid Equilibrium of Poly (ethylene glycol) 4000+ Diammonium Hydrogen Phosphate+ Water at Different Temperatures. J. Chem. Eng. Data 2008, 53 (7), 1574−1578. (12) Diederich, P.; Amrhein, S.; Hämmerling, F.; Hubbuch, J. Evaluation of PEG/phosphate aqueous two-phase systems for the purification of the chicken egg white protein avidin by using highthroughput techniques. Chem. Eng. Sci. 2013, 104, 945−956. (13) Marcos, J. C.; Fonseca, L. P.; Ramalho, M. T.; Cabral, J. M. S. Partial purification of penicillin acylase from Escherichia coli in poly (ethylene glycol)−sodium citrate aqueous two-phase systems. J. Chromatogr., Biomed. Appl. 1999, 734 (1), 15−22. (14) Gomes, G. A.; Azevedo, A. M.; Aires-Barros, M. R.; Prazeres, D. M. F. Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate. Sep. Purif. Technol. 2009, 65 (1), 22−30. (15) Tubío, G.; Pellegrini, L.; Nerli, B. B.; Picó, G. A. Liquid-liquid equilibria of aqueous two-phase systems containing poly (ethylene glycols) of different molecular weight and sodium citrate. J. Chem. Eng. Data 2006, 51 (1), 209−212. (16) Marcos, J. C.; Fonseca, L. P.; Ramalho, M. T.; Cabral, J. M. S. Variation of penicillin acylase partition coefficient with phase volume ratio in poly (ethylene glycol)−sodium citrate aqueous two-phase systems. J. Chromatogr., Biomed. Appl. 1998, 711 (1), 295−299. (17) de Oliveira, R. M.; Coimbra, J. S. D. R.; Minim, L. A.; da Silva, L. H. M.; Ferreira Fontes, M. P. Liquid−liquid equilibria of biphasic systems composed of sodium citrate+ polyethylene (glycol) 1500 or 4000 at different temperatures. J. Chem. Eng. Data 2008, 53 (4), 895− 899. (18) Murugesan, T.; Perumalsamy, M. Liquid-liquid equilibria of poly (ethylene glycol) 2000+ sodium citrate+ water at (25, 30, 35, 40, and 45) C. J. Chem. Eng. Data 2005, 50 (4), 1392−1395. (19) Zafarani-Moattar, M. T.; Sadeghi, R.; Hamidi, A. A. Liquid− liquid equilibria of an aqueous two-phase system containing polyethylene glycol and sodium citrate: experiment and correlation. Fluid Phase Equilib. 2004, 219 (2), 149−155. (20) Perumalsamy, M.; Bathmalakshmi, A.; Murugesan, T. Experiment and correlation of liquid-liquid equilibria of an aqueous salt polymer system containing PEG6000+ sodium citrate. J. Chem. Eng. Data 2007, 52 (4), 1186−1188. (21) Silvério, S. C.; Wegrzyn, A.; Lladosa, E.; Rodríguez, O.; Macedo, E. A. Effect of aqueous two-phase system constituents in different poly (ethylene glycol)−salt phase diagrams. J. Chem. Eng. Data 2012, 57 (4), 1203−1208.

cation in the salt, it has been noticed that this dependence has a higher impact for the LLE than that of the PEG’s molecular weight, in the range of Mw used in this study. However, at an industrial level, other features may have to be taken into consideration, and PEGs with a lower density and a smaller molecular weight can be preferred. Despite the difference between the diagrams for ATPSs formed by salts of different cations, all the salts compared in this work are very promising in terms of separation, as the “working area” is located in the region of small salt concentrations.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was financially supported by Project POCI-01-0145FEDER-006984, Associate Laboratory LSRE/LCM funded by FEDER funds through COMPETE2020, Programa Operacional Competitividade e Internacionalizaçaõ (POCI), and by national funds through FCT, Fundaçaõ para a Ciência e a Tecnologia. Kamila Wysoczanska acknowledges her Ph.D. grant of FCT (PD/BD/114315/2016). Notes

The authors declare no competing financial interest.

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Bancroft

n

REFERENCES

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Salt Phase

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