Equilibrium Phase Behavior of Aqueous Two-Phase Systems

Article pubs.acs.org/jced

Equilibrium Phase Behavior of Aqueous Two-Phase Systems Containing Ethylene Oxide−Propylene Oxide of Different Molecular Weight (2500, 12000) and Sodium Citrate Salt at Various Temperatures and pH Raziyeh Ghahremani and Farshad Rahimpour* Biotechnology Research Lab, Chemical Engineering Department, Faculty of Engineering, Razi University, Kermanshah 67149-67346, Iran ABSTRACT: The liquid−liquid equilibrium (LLE) experimental data, including the binodal curve and the tie line of the aqueous two phase systems (ATPS) for two different molecular weights of ethylene oxide− propylene oxide copolymer (2500, 12000) and sodium citrate salt, are presented in two categories: first at different pH values 5, 7, and 8.2 at a constant temperature 25 °C and second at a constant pH of 8.2 with different temperatures of 25 °C, 30 °C, and 40 °C. It is found out that increases in the molecular weight of the polymer, pH, and temperature cause an increase of the two-phase region. Moreover, the experimental binodal data of the considered systems are successfully fitted to the Merchuk equation with significant R2 and low standard deviation.

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INTRODUCTION The aqueous two-phase system (ATPS) is a valuable method for partition and purification of proteins, cell organelles, and other biomolecules. These systems could be attained by the mixing of two different structural polymers or a polymer and a salt in water more than a definite concentration.1,2 Partitioning of biomolecules in ATPS is a selective process in which the concentration and purification could be performed in one step.3 These systems are capable of being applied in a large scale and provide a gentle environment for biomolecules because of their high water content. Furthermore, their performance could be controlled and optimized by altering system conditions.4 Traditional systems are polyethylene glycol (PEG)/dextran and PEG/salt in which the former could be commonly used for large-scale enzyme extraction because of their inexpensive phase forming, lower viscosity, and rapid phase spilitting.5 The biomolecule purification efficiency in aqueous two-phase systems could be increased by applying thermoseparating polymers like ethylene oxide−propylene oxide (EOPO). EO50PO50 thermoseparating copolymer is not soluble in water after heating above the lower critical solution temperature (LCST) and can be separated out of the solution. Therefore temperature-induced phase separation will occur, and two phases including the polymer and water phases appear. PEG has a high LCST (more than 100 °C), so it cannot be used for the separation of biomolecules as a thermoseparating polymer. Some random copolymers of ethylene oxide and propylene oxide (EOPO) could be used for the separation of biomolecules due to their low enough LCST. As a result the desired protein could be partitioned to a new water phase, and © 2013 American Chemical Society

the recycling of the copolymer will be possible. These copolymers could be used instead of PEG in the common polymer−salt systems because of their thermoseparating properties.6 It should be mentioned that in these systems two phases could be formed with less amounts of phase-forming components because of the high hydrophobicity of EO50PO50 thermoseparating copolymer in comparison with the polyethylene glycol.6−8 By applying the nontoxic and biodegradable citrate salt instead of ordinary sulfate and phosphate salts, which might be released into a biological wastewater treatment plant, the environmental factors are improved. Hence these systems are economical and reduce environmental problems.9,10 It is noteworthy that finding the data of the compositions and properties of phase systems is vital for modeling and optimization of separation processes, comprehension of system factors, determination of partitioning of components in ATPS, as well as the development and assay of thermodynamic and transport phenomena models of aqueous two-phase systems. So far, there has been little literature study about experimental liquid−liquid equilibrium (LLE) data for aqueous thermoseparating EO50PO50−salt mixtures.10,11 In the present study the phase diagrams data are evaluated for EO50PO50−sodium citrate salt with a copolymer molecular weight of both 2500 and 12000. Furthermore, the influence of pH as well as temperature on the tie lines and binodal curves was investigated, since these results are not reported in the Received: March 3, 2013 Accepted: December 12, 2013 Published: December 19, 2013 218

dx.doi.org/10.1021/je400489c | J. Chem. Eng. Data 2014, 59, 218−224

Journal of Chemical & Engineering Data

Article

literature. The obtained results could be applied for selection of the proper purification system for biomolecules.

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EXPERIMENTAL SECTION Materials. Poly(ethylene glycol-ran-propyleneglycol) (EO50PO50, Mn 2500, 12000), a random copolymer consisting of 50% ethylene oxide and 50 % propylene oxide (by weight) was purchased from Sigma Aldrich Co. (St. Louis, MO, USA), trisodium citrate (anhydrous GR for analysis, > 99 %), citric acid (anhydrous powder extra pure, > 99.5 %) were obtained from Merk (Darmstadt, Germany). The polymer and salt were used as received. Double distilled deionized water was used. All of the other reagents were of analytical grade. Apparatus and Procedure. The turbidometric titration method is applied to determine the binodal curves of an aqueous two-phase systems.12 For this purpose stock aqueous solutions of EO50PO50 (Mn ≈ 2500, 12000) of known concentration and trisodium citrate 25 % (w/w) were prepared. Salt solutions were adjusted to various pH (5, 7, 8.2) by the addition the appropriate ratio of citric acid, and the pH was measured using a pH meter JENWAY 3345. A salt solution was added dropwise to the polymer solution in 15 mL graduated tubes, and the system was well mixed after each addition step. The tubes were then put in a water bath (with known temperature) to reach equilibrium. The temperature of the thermostatted water bath was maintained constant and controlled within ± 0.1 °C. The presence of a biphasic area in the samples was determined with the initial appearances of turbidity. The turbidity of the system disappeared with the additions of some water to each sample. The weight of added salt and water solution was measured using an A&D GF-300 analytical balance with a precision of ± 1·10−4 g, and the composition of the system is calculated for each point on the binodal. For tie line length (TLL) calculation, different aqueous twophase systems with final mass of 10 g were prepared in 15 mL graduated glass tubes and kept in a thermostatic air bath with a constant temperature overnight. When the phase equilibrium was achieved, the samples of the two phases were taken with a syringe. A layer of solution at least 0.2 cm thick above the interface was left. Using the composition of both phases of the systems, the tielines were determined. Before the analysis, the samples were diluted as needed. Also the concentration of salt was calculated using atomic absorption spectroscopy (AAS) by Shimatzu AA6300 equipment. The average relative deviation of citrate concentration by AAS is nearly 0.2 (weight percent concentration). EO50PO50 concentration was evaluated by an ATAGO-DTM1 refractometer with a precision of ± 0.002. Also standard curves of refractive index vs polymer concentration were provided for different concentrations of salt because of the dependence of the refractive index of phase samples on both polymer and salt concentration.13−15 Each experiment was done twice, and the presented data were the average of them.

Figure 1. Experimental and correlated binodal curves of ATPSs of EO50PO50 2500 (1) + sodium citrate (2) + H2O (3) for different pH and T = 25 °C. The solid curves represent correlated binodals.

of the higher hydrophobic character of EOPO, less amount of phase-forming components were needed to form two phases for the EO50PO50/citrate ATPS in comparison with the PEG/ citrate aqueous two-phase systems with the same molecular weight of PEG. More economical and less environmental problems are ascribed to this process.10 Binodal data of EO50PO50 (Mn = 2500 and 12000) and sodium citrate ATPSs at 25 °C and various pH of 5, 7, and 8.2 are shown in Figures 1 and 2. The composition of binodal points of these systems at pH of 8.2 with different temperatures of 25, 30, and 40 °C are presented in Figures 3 and 4, respectively. The binodal data of the considered systems were fitted to the following equation presented by Merchuk:16 w2 = a exp(bw10.5 − cw13)

(1)

where a, b, and c denote the fitting parameters and w1/w2 represents the concentration of salt/polymer (in weight percent). In recent times, eq 1 has been effectively applied for presention of the binodal curves of some PEG + salt,17−19 PPG + salt systems,20−22 and UCON + (sodium or potassium) phosphate salt aqueous systems.23 Least-square regressions were applied to fit the binodal data of the considered systems to eq 1. The parameters a, b, and c together with their corresponding standard deviations for aqueous two phase systems at various temperatures and pH values are listed in Tables 1−4, respectively. From the obtained standard deviations and coefficient of determination (R2), it is concluded that eq 1 could be adequately applied to calculate the binodal data of these systems. The experimental and correlated binodal curves with the mentioned sequence are presented in Figures 1−4, respectively. Effect of the pH on the Phase Equilibrium. The binodal data for the systems with the two molecular weights of EO50PO50 at various pH values (5, 7 and 8.2) are determined. The influence of pH on the binodal curve of the systems with molecular weights of 2500 and 12000 of EO50PO50 were also presented in Figures 1 and 2, respectively. The results indicate that the two-phase region is broadened by increasing the pH of the system. As a result, less amount of the polymer is needed to obtained ATPSs. The ability of salt to encourage the water structure may cause this process. By dissolving a kosmotropic (water structure-promoting) salt like sodium citrate in an aqueous solution, the ionic hydration

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RESULTS AND DISCUSSION The binodal curve separated the one-phase region from the two-phase region. ATPSs, in which their total initial concentrations are higher than the compositions of the binodal curve, will divide into two phases. An analogous behavior was also observed for the other polymer/salts ATPSs.1,4,12 Because 219



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Figure 2. Experimental and correlated binodal curves of ATPS’s of EO50PO5012000 (1) + sodium citrate (2) + H2O (3) for different pH and T = 25 °C. The solid curves represent correlated binodals.

molecules. It seems that by adding a kosmotropic salt to an aqueous solution containing EO50PO50 copolymer, there is competition for the water molecules.20 The stronger affinity of salt ions to the water molecules rather than EO50PO50 copolymer causes a decrease in the hydration and solubility of the EO50PO50 copolymer. It is concluded that EO50PO50 is salted-out and discarded from the solution as a separated phase at certain concentrations.20,24 This may be related to the fact that the salt anions protonation degree is varied by a change of the medium pH.25 Increasing the aqueous medium pH causes the anions to be less protonated and leads to a higher valency for them. It should be mentioned that anions with a higher valence are more suitable salting-out agents with respect to those with a lower valence. A higher-valence anion can hydrate more water molecules, and as a result the repulsion between the anions and polyether functionality of EO50PO50 is increased. Hence, the anions with a higher valency which are in the basic medium pH can effectively force the phases to separate relative to minor valency anions found in the acidic medium pH. Increasing of medium pH, leads to increase of waterstructure-encourage ability of the salt ions which is in agreement with the observation that the water arrangement comes along with a reduction of the water acidity in pure water.25 Therefore it seems that the H3O+ ion, exist in the acidic pH, hinder the water structure. Effect of the Temperature on the Phase Equilibrium. The compositions of components in the systems with two molecular weights of EO50PO50 at three temperatures [(25, 30, and 40) °C] were determined and the impact of temperature on the binodal curve was illustrated in Figures 3 and 4 for EO50PO50 with a molecular weight of 2500 and 12000, respectively. Increasing the temperature causes an expansion of the twophase region for all the ATPSs. That is because the polymer− solvent interaction decreases with an increase in temperature and then results in a decrease in the solubility of EOPO in water.24 The water transfers from the polymer phase to the salt phase which leads to the reduction of the salt concentration in the salt-rich phase.24 Therefore, the binodal curve comes close to the origin. This tendency was also exhibited by other aqueous two-phase polymer/salt systems such as PEG/citrate

Figure 3. Experimental and correlated binodal curves of ATPSs of EO50PO502500 (1) + sodium citrate (2) + H2O (3) at different temperatures and pH = 8.2. The solid curves represent correlated binodals.

Figure 4. Experimental and correlated binodal curves of ATPSs of EO50PO5012000 (1) + sodium citrate (2) + H2O (3) at different temperatures and pH = 8.2. The solid curves represent correlated binodals.

process occurs in which the salt ions are encircled by a layer of water molecules. These water molecules are prearranged and immobilized. This causes a decrease of their salvation to other 220



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Table 1. Values of Parameters of eq 1 for EO50PO50 2500 (1) + Sodium Citrate (2) + H2O (3) System at T = 25°C and Different pH Values pH

a

b

c

SDa

R2

5 7 8.2

1.809 ± 0.101 1.496 ± 0.075 1.396 ± 0.108

−9.081 ± 0.361 −8.730 ± 0.341 −8.790 ± 0.604

56.42 ± 3.46 148.40 ± 5.67 250.80 ± 4.23

0.0054 0.0036 0.0118

0.9959 0.9968 0.9924

2 0.5 SD = (∑iN= 1(wcalcd − wexptl 1 1 ) /N) , where w1 and N represent the concentration (in weight percent) of polymer and the number of binodal data, respectively.

a

Table 2. Values of Parameters of eq 1 EO50PO5012000 (1) + Sodium Citrate (2) + H2O (3) System at T = 25°C and Different pH Values pH

a

b

c

SDa

R2

5 7 8.2

2.292 ± 0.224 1.029 ± 0.059 1.320 ± 0.089

−12.560 ± 0.650 −10.510 ± 0.523 −13.510 ± 0.620

43.25 ± 3.62 13.80 ± 1.81 39.38 ± 2.37

0.0052 0.0048 0.0049

0.9930 0.9931 0.9932

Table 3. Values of Parameters of eq 1 EO50PO502500 (1) + Sodium Citrate (2) + H2O (3) System at pH = 8.2 and Different Temperatures T (°C)

a

b

c

SD

R2

25 30 40

1.396 ± 0.108 1.200 ± 0.072 1.321 ± 0.048

−8.790 ± 0.604 −8.370 ± 0.487 −10.200 ± 0.310

250.80 ± 4.23 220.10 ± 3.71 172.50 ± 3.22

0.0118 0.0074 0.0025

0.9924 0.9941 0.9979

Table 4. Values of Parameters of eq 1 EO50PO5012000 (1) + Sodium Citrate (2) + H2O (3) System at pH = 8.2 and Different Temperatures T (°C)

a

b

c

SD

R2

25 30 40

1.320 ± 0.089 1.486 ± 0.102 1.356 ± 0.034

−13.510 ± 0.620 −14.810 ± 0.660 −16.460 ± 0.301

39.38 ± 2.37 189.70 ± 4.29 134.50 ± 4.88

0.0049 0.0051 0.0009

0.9932 0.9929 0.9989

Figure 6. Effect of temperature on the equilibrium phase compositions and on the slope and length of tie lines for EO50PO50 (1) + sodium citrate (2) + H2O (3), (solid line, MW = 2500), (dash line, MW = 12000).

Figure 5. Effect of pH on the equilibrium phase compositions and on the slope and length of tie lines for EO50PO50 (1) + sodium citrate (2) + H2O (3), (solid line, MW = 2500), (dash line, MW = 12000).

The slope of the tie line (STL) is calculated by the following equation:

and Ucon50-HB5100/citrate.10,11,26,27 On the basis of the Kjellander and Florin model,28 as a result of energetically unfavorable and extremely directional interactions like hydrogen-bonding between dissimilar molecules, and by decreasing the temperature, the entropically unfavorable structuring of water formed by EO50PO50 becomes dominant because of the huge reduction in enthalpy. The same results were also obtained in literature for an aqueous two-phase polymer/salt systems.29,30

STL = (C Ptop − C Pbottom)/(CSbottom − CStop)

(2)

which is the ratio of the differences of polymer concentration (CP) in top and bottom phase to the differences on salt concentration (CS) in these phases. The tie-line length (TLL), an experiential measure of the differences between compositions and intensive thermody221



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Table 5. Phase Compositions for EO50PO50 2500 (1) + Sodium Citrate (2) + H2O (3) System at Different pH and T = 25 °C total compositions pH 5

7

8.2

a

top phase

bottom phase

salt w2

polymer w1

salt w2

polymer w1

salt w2

polymer w1

STLa

TLLb

0. 1 0.1 0.14 0. 1 0.1 0.14 0. 1 0.1 0.14

0. 15 0.2 0.17 0.15 0.2 0.17 0.15 0.2 0.17

0.0337 0.0183 0.0214 0.0245 0.0125 0.0143 0.0213 0.0138 0.0119

0.3051 0.423 0.494 0.3614 0.4501 0.5468 0.3763 0.4587 0.5579

0.1643 0.1682 0.2008 0.1587 0.1619 0.1892 0.1511 0.1579 0.1914

0.005 0.021 0.0117 0.0076 0.017 0.0129 0.0062 0.0092 0.0133

−2.298 −2.682 −2.688 −2.636 −2.899 −3.053 −2.851 −3.119 −3.034

32.729 42.904 51.458 37.84 45.814 56.182 39.22 47.643 57.342

Slope of the tie line. bTie line length.

Table 6. Phase Compositions for EO50PO5012000 (1) + Sodium Citrate (2) + H2O (3) System at Different pH and T = 25 °C total compositions pH 5

7

8.2

a

top phase

bottom phase

salt w2

polymer w1

salt w2

polymer w1

salt w2

polymer w1

STLa

TLLb

0. 1 0.1 0.14 0. 1 0.1 0.14 0. 1 0.1 0.14

0. 15 0.2 0.17 0. 15 0.2 0.17 0. 15 0.2 0.17

0.0174 0.015 0.0182 0.0109 0.013 0.0152 0.0085 0.005 0.0097

0.3412 0.4388 0.5235 0.3759 0.4672 0.5613 0.4017 0.4919 0.5719

0.1655 0.1715 0.2011 0.1603 0.1637 0.1867 0.1581 0.1562 0.1864

0.0096 0.0149 0.0067 0.0067 0.0139 0.0085 0.0054 0.0071 0.0133

−2.239 −2.709 −2.826 −2.471 −3.008 −3.223 −2.649 −3.206 −3.161

37.196 45.187 54.821 39.828 47.769 57.879 42. 36 50.783 58.588


Table 7. Phase Compositions EO50PO502500 (1) + Sodium Citrate (2) + H2O (3) System at Different Temperature and pH = 8.2 total compositions

bottom phase

salt w2

polymer w1

salt w2

polymer w1

salt w2

polymer w1

STLa

TLLb

25

0. 1 0.1 0.14 0. 1 0.1 0.14 0. 1 0.1 0.14

0. 15 0.2 0.17 0. 15 0.2 0.17 0. 15 0.2 0.17

0.0213 0.0138 0.0119 0.0154 0.013 0.0074 0.0093 0.005 0.0054

0.3763 0.4587 0.5579 0.4176 0.4839 0.6231 0.4856 0.5728 0.6681

0.1511 0.1579 0.1914 0.1415 0.1614 0.1827 0.1317 0.1475 0.1795

0.0062 0.0092 0.0133 0.005 0.0045 0.0065 0.0034 0.0064 0.0119

−2.851 −3.119 −3.034 −3.272 −3.230 −3.517 −3.94 −3.975 −3.769

39.22 47.203 57.342 43.144 50.184 64.103 49.749 58.405 67.890

30

40

a

top phase

temp °C


Table 8. Phase Compositions for EO50PO5012000 (1) + Sodium Citrate (2) + H2O (3) System at Different Temperature and pH = 8.2 total compositions

bottom phase

salt w2

polymer w1

salt w2

polymer w1

salt w2

polymer w1

STLa

TLLb

25

0. 1 0.1 0.14 0. 1 0.1 0.14 0. 1 0.1 0.14

0. 15 0.2 0.17 0. 15 0.2 0.17 0. 15 0.2 0.17

0.0085 0.005 0.0097 0.009 0.0053 0.0092 0.0047 0.0042 0.005

0.4017 0.4919 0.5719 0.4616 0.5279 0.6518 0.5173 0.618 0.6813

0.1581 0.1562 0.1864 0.141 0.1492 0.1856 0.1365 0.1476 0.1862

0.0054 0.0071 0.0133 0.0078 0.0077 0.005 0.002 0.0031 0.0048

−2.649 −3.206 −3.161 −3.438 −3.615 −3.667 −3.91 −4.288 −3.733

42.36 50.783 58.588 47.261 53.974 67.042 53.189 63.140 70.035

30

40

a

top phase

temp °C


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evaluated. The experimental binodal data for all of the considered systems were significantly modeled using the Merchuk equation. It was found out that by increasing the pH and temperature as well as polymer molecular weight, the two-phase region was expanded. In these considered systems fewer amounts of phase-forming components are needed for forming two phases in comparison with traditional systems, and this causes a reduction in environmental problems. Also, the biodegradability of the citrate anion and the thermo-separating properties of EO50PO50, make EO50PO50/citrate ATPSs a powerful and interesting alternative system for bioseparation.

namic properties of the two phases, could be evaluated according to eq 3: TLL =

(C Pbottom − C Ptop)2 + (CStop − CSbottom)2

(3)

where Cp and Cs represent polymer and salt concentration in the corresponding phases, respectively. Each corresponding set of total, bottom, and top-phase concentration points are connected to obtain the related tie line. All of the samples with feed compositions on a tie-line have the same top and bottom phase composition. According to the lever rule, the volume of each phase is related to the inverse proportion of the distances of the corresponding phase composition from the point of total composition.31 The tie lines of the considered systems with different pH values (8.2, 7 and 5) at constant temperature (25 °C) and with temperatures 25, 30, and 40 °C at pH 8.2 are depicted in Figures 5 and 6, respectively. The STL and TLL of the considered aqueous two-phase systems are calculated and summarized in Tables 5−8. According to Figure 5, increasing the pH leads to an increase in STL and TLL of aqueous twophase systems. From the obtained experimental data, it can be seen that an increase of pH causes an increase in the volume of the bottom phase and a decrease of the top phase volume. It could be related to the altering compositions of the equilibrium phases with changing pH. Increasing the pH of the system leads to an increase in the concentration of the top phase and a reduction in the concentration of the bottom phase. Note that with an increase in pH, the attractive forces between polymer and water molecules will reduce; hence, increasing pH of the EO50PO50−citrate ATPS causes migration of water molecules from the EO50PO50-rich phase to the bottom phase. As a result, the EO50PO50 concentration of the top phase increases, whereas the bottom phase concentration slightly decreased. This leads to the volume increase and decrease of the bottom and top phase, respectively. The influence of a temperature increase is approximately similar to that of the pH. With an increase of temperature of each considered system, the STL and TLL increases, which also has been observed for other polymer−salt aqueous two-phase systems.32 This might contribute to the increase of polymer hydrophobicity with increasing temperature, therefore the water molecules escape from the top to bottom phase and a decreasing salt concentration was observed.11 Effect of Polymer Molecular Weight on the Phase Equilibria. For most systems, especially those with higher molecular weight of the EOPO, the copolymer concentration in the bottom phase is negligible, and in some cases the copolymer is almost excluded from this phase while the opposite behavior is observed in the top phase. By increasing the ethylene oxide−propylene oxide molecular weight, binodal curves become more asymmetric and close to the origin, therefore a lower concentration of phase forming components are required for phase separation. An increase in the incompatibility between the copolymer and the salt, due to more hydrophobical character of ethylene oxide−propylene oxide of higher molecular weight, probably causes this behavior.26

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Tel.: +98-831-427 45 30. Fax: +98-831-427 45 42. Funding

This work was supported in part by the research vice department of Razi University. Notes

The authors declare no competing financial interest.

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REFERENCES

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CONCLUSION In the present study liquid−liquid equilibrium data for EO50PO50 (2500, 12000)−citrate at various temperatures of 25 °C, 30 °C, and 40 °C and pH values of 5.7 and 8.2) were 223



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Equilibrium Phase Behavior of Aqueous Two-Phase Systems

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