Aqueous Two-Phase Systems of Mixture of Triblock Copolymer (EO

Article pubs.acs.org/jced

Aqueous Two-Phase Systems of Mixture of Triblock Copolymer (EO)13(PO)30(EO)13 (L64) and Sulfate Salts at Different Temperatures Diego Nunes Faria,† Angélica Siqueira da Silva,† Luciano Sindra Virtuoso,§ Kelany S. Nascimento,‡ Celso Shiniti Nagano,‡ and Anderson Fuzer Mesquita*,† †

Grupo de Macromoléculas e Surfactantes, Departamento de Química, Universidade Federal do Espírito Santo (UFES), 29075-910 Vitória, Espírito Santo, Brazil § Grupo de Pesquisa em Química de Colóides, Instituto de Ciências Exatas, Universidade Federal de Alfenas (UNIFAL), Rua Gabriel Monteiro da Silva, 700, 37130-000 Alfenas, Minas Gerais, Brazil ‡ Laboratório de Espectrometria de Massa, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Universidade Federal do Ceará (UFC), 60455-970 Fortaleza, Ceará, Brazil ABSTRACT: Phase equilibrium of aqueous two-phase systems (ATPS) containing triblock copolymer, L64, (EO)13(PO)30(EO)13 and sulfate salts were investigated in this work. It was analyzed the effect of the temperature at 278.15 K to 298.15 K and type of salt. The rise of the temperature increased the slope of the tie line (STL). The sodium sulfate showed better capability to induce phase separation.

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INTRODUCTION The aqueous two-phase systems (ATPS), which can be formed by adding two different hydrophilic polymer solutions by polymer solution and salt solution or by two salt solutions under specific thermodynamics conditions, has been proved to be efficient for the separation and purification of biomaterials such as proteins, cell organelles, membranes, DNA, and other biological particles. Who first showed the potentiality of using aqueous two-phase systems as a separating medium of complex mixtures of biomolecules was Albertsson in the mid 1960s.1−4 Most ATPS phase diagrams reported in the literature are composed of poly(ethylene oxide) (PEO) + inorganic salt + water or PEO + dextran + water.5 The former has some strategic and advantageous features over the latter, such as low cost, low viscosity, and a short period of time for phase segregation. The very high cost, in an industrial scale, makes it unviable to use of dextran. Furthermore, ATPS formed by PEO + salt presented some limitations, particularly for biological compounds, namely denaturation of biomolecules when salt concentrations were very high.6 An alternative to overcome these limitations are the ATPS formed by thermally separable triblock copolymers and polymers. Aqueous solutions of such thermosensitive macromolecules undergo phase separation when submitted to moderate temperature variations. An example is nonionic copolymer (EO20PO80). This copolymer has a cloud point of 18 °C and is used in purification of glucose + phosphate © 2015 American Chemical Society

dehydrogenase, hexokinase, and 3-phosphoglycerate kinase from bakers’ yeast.7,8 Despite being thermally separable, the phase separation temperature of poly(ethylene oxide) (PEO), above 373.15 K, can lead to a loss of biological activity. ATPS formed by triblock copolymers are excellent options for PEOATPS.9 These copolymers have been employed mainly due to the extent of the aqueous biphasic application for the extraction from the hydrophobic solutes into the polymer-enriched phase. In addition, under critical temperatures and concentration conditions, aqueous solutions of triblock copolymers form micelles via a self-assembly process. The micelles consist of a crown of hydrophilic units of PEO blocks and a core of hydrophobic units of PPO, essentially water free, which is capable of interacting with hydrophobic compounds.10−12 In this paper, the experimental liquid−liquid equilibrium data for ATPS composed of (EO)13(PO)30(EO)13 (L64) and salt were prepared and the phase compositions measured. The salts used were Na2SO4, MnSO4, and (NH4)2SO4 to investigate the effect of the salt cations. Equilibrium data at (278.15, 288.15, and 298.15) K were determined to study the influence of temperature in the phase composition. Received: December 2, 2014 Accepted: May 5, 2015 Published: May 21, 2015 1722

DOI: 10.1021/je501094z J. Chem. Eng. Data 2015, 60, 1722−1726

Journal of Chemical & Engineering Data

Article

Table 1. Liquid−Liquid Equilibrium Data for L64 + Sodium Sulfate + Water Systems from 278.15 K to 298.15 K and Tie Line Length Values total tie line

tie line length

wL64

wS

top phase wW

1 2 3 4

31.38 34.54 42.36 45.75

20.01 21.52 23.51 24.95

5.20 5.60 5.92 6.32

74.79 72.88 70.57 68.73

1 2 3 4

37.59 41.79 43.03 44.97

22.55 23.26 23.97 25.98

5.49 5.60 5.79 6.10

71.96 71.14 70.24 67.92

1 2 3 4

34.25 45.63 51.57 55.36

20.01 22.14 24.16 25.62

5.18 5.49 5.90 6.21

74.81 72.37 69.94 68.17

wL64 278.15 K 32.38 34.66 41.22 44.55 288.15 K 37.79 41.85 42.41 45.86 298.15 K 35.11 45.82 52.35 56.65

bottom phase

wS

wW

wL64

wS

wW

2.05 1.73 1.38 1.23

65.57 63.61 57.40 54.22

2.35 1.68 0.31 0.44

11.14 12.00 12.36 13.38

86.51 86.32 87.33 86.18

1.29 0.95 0.94 0.82

60.92 57.2 56.65 53.32

1.26 1.16 0.59 2.14

10.19 10.50 11.09 11.36

88.55 88.34 88.32 86.50

3.37 2.33 1.81 1.38

61.52 52.35 45.84 41.97

1.22 0.75 1.47 2.07

8.37 9.47 10.20 10.63

90.41 89.78 88.33 87.30

Table 2. Liquid−Liquid Equilibrium Data for L64 + Manganese Sulfate + Water Systems from 278.15 K to 298.15 K and Tie Line Length Values total tie line

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tie line length

wL64

wS

top phase wW

1 2 3 4

30.08 31.55 34.36 37.46

21.40 22.83 24.29 25.88

5.78 6.17 6.57 7.00

72.82 71.00 69.14 67.12

1 2 3 4

26.34 31.51 38.67 43.79

21.49 23.22 25.13 26.05

5.40 5.82 6.27 6.68

73.11 70.96 68.60 67.27

1 2 3

41.05 47.71 52.81

23.81 24.89 26.55

5.70 5.98 6.69

70.49 69.13 66.76

wL64 278.15 K 32.40 34.49 37.43 40.36 288.15 K 29.70 35.34 41.13 46.18 298.15 K 44.53 50.05 56.07

EXPERIMENTAL SECTION Materials. The copolymer L64, (EO)13(PO)30(EO)13, containing 40 % of the EO unit and average molar mass of 2900 g mol−1, was purchased from Aldrich (U.S.A.). Sodium sulfate, Na2SO4, manganese sulfate, MnSO4, and ammonium sulfate, (NH4)2SO4, were obtained from Vetec (Brazil). All chemicals employed in the present work were of analytical grade and used as received. Milli-Q II water (Millipore, U.S.A.) was used for the preparation of all aqueous solutions. Experimental Procedure. The biphasic systems were prepared by weighing appropriate amounts of L64, salt [Na2SO4, MnSO4, and (NH4)2SO4], and water on an analytical balance (Radwag AS220 with an uncertainty of ± 0.0001 g). Aqueous two phase systems were prepared by mixing appropriate amounts of the stock solutions of polymer, salt, and water, in glass vessels, according to the global compositions desired. Typically, 10 g of each system was prepared. The systems were gently shaken, in order to ensure complete mixing, and then left undisturbed for 24 to 48 h at the operational temperature (278.15, 288.15, and 298.15) K in a temperature-controlled bath (Nova ética, modelo: 521-2D, with a temperature uncertainty of ± 0.1 K). The equilibrium state

bottom phase

wS

wW

wL64

wS

wW

1.75 1.47 1.24 0.95

65.85 64.04 61.33 58.69

4.87 6.03 6.84 6.72

13.87 15.09 16.87 17.42

81.26 78.88 76.29 75.86

3.11 2.36 1.71 1.59

67.19 62.30 57.16 52.23

4.48 5.23 4.18 4.12

10.73 11.68 13.11 13.79

84.79 83.09 82.71 82.09

1.77 1.38 1.04

53.7 48.57 42.89

4.30 3.26 4.43

9.93 10.76 12.09

85.77 85.98 83.48

was characterized by the absence of turbidity in both top and bottom phases. Samples were carefully withdrawn from both phases using a micropipette for the top phase and a syringe with stainless steel needle for the bottom phase. Construction of Phase Diagrams. The salt concentration was determined by conductivity (Gehaka, CG 2000, Brazil) of the electrolyte in the range of (10−3 to 10−2) % (w/w). The salt solutions showed the same conductivity in water or in the diluted polymer solution [(0.1 to 0.01) %]. The standard deviation of the salt mass percentage from this method was ± 0.10 %. The concentration of triblock copolymer (L64) was determined by measurements at 298.15 K using a refractometer (Krüss optronic GmbH). Because the refractive index of the phase depends on the total concentration (copolymer and salt concentrations) and is an additive property, copolymer concentration was obtained by subtracting from the total solution composition (refraction index) the salt concentration measured by conductivity. The standard deviation in the block copolymer mass percent was ± 0.005 %. The water content was determined by difference of mass fraction of each component (ww = wtotal − ws − wL64). All analytical measurements were performed in triplicate. 1723



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Table 3. Liquid−Liquid Equilibrium Data for L64 + Ammonium Sulfate + Water Systems from 278.15 K to 298.15 K and Tie Line Length Values total tie line

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tie line length

wL64

wS

top phase wW

1 2 3 4

23.94 31.09 39.08 45.68

21.25 22.65 24.32 26.11

6.67 7.06 7.57 8.08

72.08 70.29 68.11 65.81

1 2 3

29.26 40.64 48.05

17.53 18.19 19.02

7.91 8.50 9.48

74.56 73.31 71.50

1 2 3

47.63 49.23 55.91

24.98 28.20 30.45

6.01 6.42 6.89

69.01 65.38 62.66

wL64 278.15 K 29.62 32.97 39.43 46.16 288.15 K 32.40 41.60 48.74 298.15 K 47.39 50.05 55.92

RESULTS AND DISCUSSION Tables 1 to 3 present the composition of the upper and lower phases and the tie-line lengths (TLL) for the L64 + Na2SO4 + H2O (1), L64 + MnSO4 + H2O (2), and L64 + (NH4)2SO4 + H2O (3) systems at 278.15 K, 288.15 K, and 298.15 K. All concentrations are expressed in mass percentages. According to the systems, three to four tie-line lengths (TLL) were determined. It was impossible to get the fourth tie-line for system (2) at 298.15 K and for system (3) at 288.15 to 298.15 K. Although phase separation was observed, phase compositions could not be analyzed, because the polymer-rich phase had gel-like character and could not be manipulated.13 The tielines were obtained by linear regression of the corresponding set of total, bottom phase, and top phase concentrations. An increase in copolymer and salt segregation with an increase in total composition and consequently in the TLL are observed. On the other hand, the concentration of copolymer increased in the top phase, and that of the salt increased in the bottom phase. This behavior is in agreement with other different aqueous two-phase systems.14−20 The temperature effect on the binodal position of the ATPS is presented in Figures 1, 2, and 3. For the L64 + (NH4)2SO4 system, Figure 1, a decrease in temperature causes a small

bottom phase

wS

wW

wL64

wS

wW

4.65 4.55 3.70 2.81

65.73 62.48 56.87 51.03

4.26 3.37 2.26 2.61

13.20 14.08 15.78 16.58

82.54 82.55 81.96 80.81

5.23 3.74 3.06

62.37 54.66 48.20

3.62 1.87 1.98

10.51 12.24 14.13

85.87 85.89 83.89

2.35 2.35 2.14

50.26 47.60 41.94

0.62 1.86 1.14

11.33 12.43 13.30

88.05 85.71 85.56

Figure 2. Temperature effects on the phase diagram for the L64 + Na2SO4 + H2O systems at 278.15 K (▲) and 298.15 K (○).

Figure 3. Temperature effects on the phase diagram for the L64 + MnSO4 + H2O systems at 278.15 K (▲) and 298.15 K (○).

reduction in the biphasic region. This behavior confirms that the phase-separation process is endothermic. However, L64 + Na2SO4 and L64 + MnSO4 systems showed a decrease in the

Figure 1. Temperature effects on the phase diagram for the L64 + (NH4)2SO4 + H2O systems at 278.15 K (▲) and 298.15 K (○). 1724



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biphasic area with an increase in temperature, indicating that the phase separation process is exothermic.16,17,19 Another considerable change was also observed in the tie line slope (STL). The temperature effect in the phase equilibrium composition can also be analyzed by applying the slope of tie-line concept. The STL can be calculated by the following equation STL =

C PT − C PB CST − CSB

(1)

where CTP and CBP are the polymer concentrations in the top and the bottom phases, respectively, and CTS and CBS are salt concentrations in the top and bottom phases, respectively. As shown in Table 4, an increase in the temperature promotes an Table 4. Tie Line Slope Values for L64 + MnSO4, L64 + (NH4)2SO4 and L64 + Na2SO4 Systems at 278.15 K, 288.15 K, and 298.15 K tie line 1 2 3 4 1 2 3 4 1 2 3 4

278.15 K

288.15 K

L64 + MnSO4 −2.27 −3.31 −2.09 −3.23 −1.96 −3.24 −2.04 −3.45 L64 + (NH4)2SO4 −2.97 −5.45 −3.11 −4.67 −3.08 −4.22 −3.16 L64 + Na2SO4 −3.30 −4.10 −3.21 −4.26 −3.73 −4.12 −3.63 −4.15

Figure 4. Influence of cation on the phase diagram of the L64 + sulfate salt systems at 288.15 K; Na2SO4 (▲), MnSO4 (○), and (NH4)2SO4 (■).

298.15 K −4.93 −4.99 −4.67

were obtained at different temperatures from (278.15 to 298.15) K. For the L64 + (NH4)2SO4 system, a decrease in temperature causes a small reduction in the biphasic region. This behavior confirms that the phase-separation process is endothermic. The phase separation process for the systems L64 + Na2SO4 and L64 + MnSO4 is exothermic. An increase in the STL with an increase in the temperature was also observed. The efficacy of the salts in inducing phase segregation follows the order Na2SO4 > MnSO4 > (NH4)2SO4.

−5.21 −4.78 −4.91

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−6.78 −6.31 −6.06 −5.90

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +55-27-40097946. Funding

The authors thank Fundaçaõ de Amparo a Pesquisa do Estado ́ Santo (FAPES), Coordenaçaõ de Aperfeiçoamento do Espirito ́ Superior (CAPES) and Fundaçaõ Cearense de Pessoal de Nivel ́ de Apoio ao Desenvolvimento Cientifico e Tecnológico (FUNCAP).

increase, in absolute value, in the STL. A possible explanation for this STL change is the spontaneous diffusion of water molecules from the top to the bottom phase, increasing the copolymer concentration at the top phase and decreasing the salt concentration at the bottom phase.1,17,20 Figure 4 presents the effects of electrolyte nature to induce phase segregation at 288.15 K. The three salts induce the phase separation in ATPS in the order: Na2SO4 > MnSO4 > (NH4)2SO4. This order is the same at all temperatures. The phase separation process may be explained by the model studied by da Silva and Loh.19 According to this model, when the macromolecule and sulfate salts are mixed, cations interact with the EO groups of macromolecule (copolymer), releasing some water molecules that solvate EO groups in a process that is driven by increase in entropy. This cation binding continues as more electrolyte is added until a separation point, after which no more entropy gain may be attained and phase splitting becomes more favorable. In the other words, the L64-Na2SO4 interaction energy is higher than that of the L64-(NH4)2SO4 apparent enthalpic interaction.16,21,22 This model can be expanded to understand the phase separation in triblock copolymers and salts.

Notes

The authors declare no competing financial interest.

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REFERENCES

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CONCLUSIONS Equilibrium data for the systems L64 + Na2SO4 + H2O (1), L64 + MnSO4 + H2O (2), and L64 + (NH4)2SO4 + H2O (3) 1725



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(6) Goja, A. M.; Yang, H.; Cui, M.; Li, C. Aqueous Two-Phase Extraction Advances for Bioseparation. J. Bioprocess. Biotechnol. 2013, 4 (1), 1−8. (7) Persson, J.; Johansson, H.-O.; Galaev, I.; Mattiasson, B.; Tjerneld, F. Aqueous polymer two-phase systems formed by new thermoseparating polymers. Bioseparation 2000, 9 (2), 105−116. (8) Alred, P. A.; Kozlowski, A.; Harris, J. M.; Tjerneld, F. Application of temperature-induced phase partitioning at ambient temperature for enzyme purification. J. Chromatogr. A 1994, 659 (2), 289−298. (9) da Silva, M. d. C. H.; da Silva, L. H. M.; Paggioli, F. J. A novel micellar medium using triblock copolymer for cobalt determination. Anal. Sci. 2005, 21 (8), 933−937. (10) Virtuoso, L. S.; Vello, K. A. S. F.; de Oliveira, A. A.; Junqueira, C. M.; Mesquita, A. F.; Lemes, N. H. T.; de Carvalho, R. M. M.; da Silva, M. C. H.; da Silva, L. H. M. Measurement and Modeling of Phase Equilibrium in Aqueous Two-Phase Systems: L35 + Sodium Citrate + Water, L35 Sodium Tartrate + Water, and L35 + Sodium Hydrogen Sulfite + Water at Different Temperatures. J. Chem. Eng. Data 2012, 57 (2), 462−468. (11) Chu, B. Structure and Dynamics of Block Copolymer Colloids. Langmuir 1995, 11 (2), 414−421. (12) Alexandridis, P.; Alan Hatton, T. Poly(ethylene oxide)− poly(propylene oxide)−poly(ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling. Colloids Surf., A 1995, 96 (1−2), 1−46. (13) Rodrigues, G. D.; Teixeira, L. d. S.; Ferreira, G. M. D.; da Silva, M. d. C. H.; da Silva, L. H. M.; de Carvalho, R. M. M. Phase Diagrams of Aqueous Two-Phase Systems with Organic Salts and F68 Triblock Copolymer at Different Temperatures. J. Chem. Eng. Data 2009, 55 (3), 1158−1165. (14) Oliveira, R. M.; Coimbra, J. S. d. R.; Francisco, K. R.; Minim, L. A.; da Silva, L. H. M.; Rojas, E. E. G. Equilibrium Data of the Biphasic System Poly(ethylene oxide) 4000 + Copper Sulfate + Water at (5, 10, 35, and 45) °C. J. Chem. Eng. Data 2008, 53 (7), 1571−1573. (15) Carvalho, C. P.; Coimbra, J. S. R.; Costa, I. A. F.; Minim, L. A.; Silva, L. H. M.; Maffia, M. C. Equilibrium Data for PEG 4000 + Salt + Water Systems from (278.15 to 318.15) K. J. Chem. Eng. Data 2007, 52 (2), 351−356. (16) Martins, J. P.; de Oliveira, F. C.; dos Reis Coimbra, J. S.; da Silva, L. H. M.; da Silva, M. d. C. H.; do Nascimento, I. S. B. Equilibrium Phase Behavior for Ternary Mixtures of Poly(ethylene) Glycol 6000 + Water + Sulfate Salts at Different Temperatures. J. Chem. Eng. Data 2008, 53 (10), 2441−2443. (17) da Silva, L. H. M.; da Silva, M. d. C. H.; Mesquita, A. F.; do Nascimento, K. S.; Coimbra, J. S. R.; Minim, L. A. Equilibrium phase behavior of triblock copolymer + salt + water two-phase systems at different temperatures and pH. J. Chem. Eng. Data 2005, 50, 1457− 1461. (18) da Silva, M. d. C. H.; da Silva, L. H. M.; Júnior, J. A.; Guimarães, R. O.; Martins, J. P. Liquid-Liquid Equilibrium of Aqueous Mixture of Triblock Copolymers L35 and F68 with Na2SO4, Li2SO4, or MgSO4. J. Chem. Eng. Data 2006, 51 (6), 2260−2264. (19) da Silva, L. H. M.; Loh, W. Calorimetric Investigation of the Formation of Aqueous Two-Phase Systems in Ternary Mixtures of Water, Poly(ethylene oxide) and Electrolytes (Or Dextran). J. Phys. Chem. B 2000, 104, 10069−10073. (20) Virtuoso, L. S.; de S. Silva, L. M.; Malaquias, B. S.; Vello, K. A. S. F.; Cindra, C. P. R.; da Silva, L. H. M.; Mesquita, A. F.; da Silva, M. C. H.; de Carvalho, R. M. M. Equilibrium Phase Behavior of Triblock Copolymer + Sodium or + Potassium Hydroxides + Water Two-Phase Systems at Different Temperatures. J. Chem. Eng. Data 2010, 55 (9), 3847−3852. (21) 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. LiquidLiquid Equilibria of an Aqueous Two-Phase System Containing Poly(ethylene) Glycol 1500 and Sulfate Salts at Different Temperatures. J. Chem. Eng. Data 2007, 53 (1), 238−241.

(22) Huddleston, J. G.; Willauer, H. D.; Rogers, R. D. Phase Diagram Data for Several PEG + Salt Aqueous Biphasic Systems at 25 °C. J. Chem. Eng. Data 2003, 48 (5), 1230−1236.

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Aqueous Two-Phase Systems of Mixture of Triblock Copolymer (EO

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