Cloudy behavior and equilibrium phase behavior of triblock copolymer L64 + salt + water two-phase systems

Fluid Phase Equilibria 409 (2016) 439e446

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Fluid Phase Equilibria j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / fl u i d

Cloudy behavior and equilibrium phase behavior of triblock copolymer L64 þ salt þ water two-phase systems Yun Wang a, **, Yuanyuan Li a, Juan Han b, *, Jinchen Xia a, Xu Tang a, Tong Chen c, Liang Ni a a b c

School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, 212013, PR China School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, PR China Zhenjiang EntryeExit Inspection Quarantine Bureau, State Key Laboratory of Food Additive and Condiment Testing, Zhenjiang, 212013, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 September 2015 Received in revised form 30 October 2015 Accepted 31 October 2015 Available online 10 November 2015

The cloud point (CP) values of aqueous solutions of the triblock copolymer L64 were determined in the absence and presence of five salting-out salts (K2SO4, Na2SO4, (NH4)2SO4, K2CO3, K2HPO4) at different concentrations. All the five salts could decrease the CP values and form aqueous two phase systems (ATPSs) with L64. A notable phase inversion phenomenon occurred with the increasing salt concentrations and the phase inversion points were found. The salt-rich phase transferred from the top phase to the bottom phase because of the change of density. Liquideliquid equilibrium (LLE) data of these ATPSs were measured at 45  C, respectively. The consistency of the tie-lines was verified by using the empirical equations from the Othmer-Tobias and Bancroft correlation. The effects on the cloudy behavior and phase separation depended on the salting-out ability. The results showed that the salting-out ability of the cations followed the order: Naþ > Kþ > NH4 þ ; the anions followed the order: HPO4 2 > SO4 2 > CO3 2 . It could be concluded that using the Gibbs free energy of hydration of the ions (DGHyd) to estimate salting-out ability was not suitable for all of the ATPSs. © 2015 Elsevier B.V. All rights reserved.

Keywords: Cloud point Liquideliquid equilibrium Triblock copolymer L64 Phase inversion

1. Introduction It has drawn much attention since the aqueous two-phase systems (ATPSs) were first observed by Albertsson [1] in the early 1950s. ATPSs can be formed by compounding two incompatible hydrophilic polymers or by mixing a hydrophilic polymer and a salt in aqueous solution above a certain concentration. Owing to the advantages of high water content, fine biological compatibility, simple production process and easy to scale-up, aqueous twophase extraction (ATPE) replaces traditional methods of liquideliquid extraction to be extensively utilized, especially applied in the separation and purification of proteins [2], cell organelles [3], antibodies [4], nanoparticles [5] and other biomolecules [6]. Polymer-salt ATPSs are superior to polymerepolymer ATPSs in that the salt can reduce the costs, increase the interphase density differences, and shorten the phase separation time because of the lower viscosity. In terms of polymer-salt ATPSs, phase behavior of

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (J. Han). http://dx.doi.org/10.1016/j.fluid.2015.10.046 0378-3812/© 2015 Elsevier B.V. All rights reserved.

(polyethylene glycol) (PEG)-salt ATPS [7,8] has been commonly studied by previous workers. With further research, however, some shortcomings appeared. On the one hand, PEG faces the fact that its high cloud point gives rise to the too high temperature for phase separation (higher than 100  C). The tough extraction environment may break the biological activity of the target products and thus make the application of PEG-salt ATPS limited. On the other hand, purification of the target products from polymer-rich phase is of great difficulty and recycling polymer is also hard after the separation process. Contrapose these limitations, a new kind of thermoseparating polymer (triblock copolymer) formed by arrays of ethylene oxide and propylene oxide units (symbolized as (EO)ne(PO)me(EO)n) has been brought into people's horizons. With the amphiphilic character, triblock copolymer molecules in aqueous solution will form micelles via a self-assembly process under specific temperature and concentration conditions [9]. These micelles have a core of hydrophobic PPO units and a hydrophilic crown formed by PEO units. The triblock copolymer with the amphipathic structure could be used for both hydrophilic and hydrophobic materials extraction. More importantly, the unique structure of the triblock copolymer determines its thermo-

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Y. Wang et al. / Fluid Phase Equilibria 409 (2016) 439e446

Chemical Reagent Co., Ltd. (Shanghai, China). All the reagents were used without further purification. The chemicals specifications are described in Table 1. Double-distilled deionized water was used for the preparation of solutions.

sensitivity. With temperature rising, hydration degree of PEO chains in micellar shell decreases sharply and micelles gather to form more compact spherical structure. The triblock copolymer with this feature can be segregated from the target product and recycled by heating to a certain temperature after the primary extraction. Due to these advantages, the triblock copolymer can be an alternative. Triblock copolymers with relatively low cloud points provide a non-denaturing extraction environment for labile bio-compounds. Only a temperature beyond the CP will make the polymer solution become turbid and separate into two phases, otherwise, there will just be a single phase in the clear solution. So, the lower the CP is, the lower the separation temperature is. Previous papers [10,11] showed that CP was influenced by many factors. For instance, the addition of some salting-out salts would decrease the CP while salting-in salts increase the CP [12] and the change of CP depended on the property of the salt. The liquideliquid equilibrium (LLE) data, which can reflect the phase behavior of ATPSs, is essential for design and optimization of extraction processes [13]. In the copolymer-salt ATPSs, the copolymer firstly gathers in the bottom phase when salt concentration is very low. Then as the concentration of salt increases to a certain degree, the copolymer transfers from the bottom phase to the top phase while salt exhibits an opposite shift, which is called phase inversion. What needs to be emphasized is that the phase equilibrium can occur in an ATPS both before the phase inversion and after the phase inversion. In the previous studies [14,15], however, all the researchers paid much attention to the phase equilibrium and collected the LLE data from the ATPSs after the phase inversion. In fact, the phase equilibrium before the phase inversion is nonnegligible. To our best knowledge, researches about phase equilibrium of triblock copolymers-salt ATPSs are scarce, especially thermotemperature polymer (EO)13(PO)30(EO)13 (L64) [16e18]. Besides, the phase inversion behavior of L64-salt ATPSs has never been reported. To find the phase inversion behavior, a large number of tests on the phase separation of the ATPSs (L64-K2SO4/Na2SO4/ (NH4)2SO4/K2CO3/K2HPO4) show that 45  C is an appropriate temperature in this experiment. In addition, 45  C is higher than room temperature. This temperature makes the preparation of ATPSs based on the relatively high cloud point of L64 and low solubility of K2SO4 easier. In this work, the CP of L64 aqueous solution changing with the L64 concentration was studied. Five kinds of salting-out salts (K2SO4, Na2SO4, (NH4)2SO4, K2CO3, K2HPO4) were added to the L64 solution to investigate the effect of salts on the CP. The phase inversion points of L64-salt ATPSs were found. The LLE data of L64K2SO4/Na2SO4/(NH4)2SO4/K2CO3/K2HPO4 ATPSs were determined at 45  C respectively. The tie-line data before and after the phase inversion were both employed in the OthmereTobias and Bancroft equations [19] to correlate the binodal curves for the investigated systems. Furthermore, the effect of different salts on the LLE data and salting-out ability were assessed.

A density bottle (10 mL) was used to determine the density of the salt-rich phase and the L64-rich phase. In order to reduce the error, the density bottle was calibrated by redistilled water, the density of which was 0.990216 g cm3 at 45  C [21]. All of the samples were weighted on an analytical balance (Model BS 124S, Beijing Sartorius Instrument Co., China) with a precision of ±1.0  107 kg.

2. Experimental

2.5. Phase diagram measurements

2.1. Materials

Conductivity analysis (DDS11A Conductivity Meter, Shanghai Dapu Instrument Co., Ltd. China) with a maximum uncertainty of 0.10% was used to determine the salt concentrations in the two phases. Standard curves of the conductivity were determined with the salt concentrations in mass fraction ranging from 0 to 1%. In this range, the conductivity of salt solutions was independent of the polymer solution composition. The concentrations of L64 in the top and bottom phases were measured by refractive index method [7] after considering the effects of salt concentrations. The measurements were carried out at 25  C by a refractometer (2W AB0003,

Triblock copolymer L64, an (EO)13(PO)30(EO)13 with an average molar mass of 2900 g mol1, containing 40% EO was purchased from Aldrich (USA); the electrolytes potassium sulfate (K2SO4), sodium sulfate (Na2SO4) and ammonium sulfate ((NH4)2SO4), potassium carbonate (K2CO3) and dipotassium phosphate (K2HPO4) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The five salts above were of analytical grade. The dye bromocresol green was also purchased from Sinopharm

2.2. Determination of CP The method of determinating CPs was described by Albertsson [1] and modified by Blankschtein et al. [20], which consisted in visually identifying the cloudy temperature. Aqueous L64 solutions with different concentrations were prepared by weighing appropriate amounts on an analytical balance (Model BS 124S, Beijing Sartorius Instrument Co., China) with a precision of ±1.0  107 kg. Aqueous L64 solution in a glass tube equipped with a digital thermometer was heated in a thermostatic bath (DF101S, Yu Hua Instrument Co., Ltd., China) until the solution turned turbid. Then the turbid solution was removed from the bath and cooled at the room temperature. The CP of the solution was obtained visually by catching the catastrophe point where the turbidity disappeared. Temperature could be controlled within 0.1  C. Each value was determined 3 times. The effect of salts on the CP of L64 was evaluated by adding different amounts of salts to the glass tube. The final concentration of L64 was controlled in a constant value of 0.1% (w/w). The procedure above was repeated to get the other CPs. 2.3. ATPSs preparation The biphasic systems were prepared by weighing 1 g L64, appropriate quantities of salts (K2SO4, Na2SO4, (NH4)2SO4, K2CO3, K2HPO4) in 10 mL centrifugal tubes and adding water until the total weight of each system was 10 g. The mixture was shaken vigorously for 2 min, centrifuged for 5 min at 2000 rpm to speed the phases settling. In order to achieve the phase equilibrium, the samples were placed for 24 h at a constant temperature of 45  C in a temperature-controlled bath. The equilibrium state was characterized by the appearance of a clear interfacial boundary between the top and bottom phases and the absence of turbidity in the two phases. When the equilibrium was achieved, aliquots of both liquid phases were collected with a plastic syringe for determining the concentrations of L64 and salts. All determinations were carried out in triplicate and the average value was used. 2.4. Determination of density

Y. Wang et al. / Fluid Phase Equilibria 409 (2016) 439e446

441

Table 1 Purities and suppliers of chemicals used in the experiment. Chemical

Mn/g mol1

Source

Purification method

Final purity

L64 K2SO4 Na2SO4 (NH4)2SO4 K2CO3 K2HPO4 Bromocresol green

2900 174.26 142.04 132.14 138.21 174.18 698.01

Aldrich (USA) Sinopharm Chemical Sinopharm Chemical Sinopharm Chemical Sinopharm Chemical Sinopharm Chemical Sinopharm Chemical

e None None None None None None

e 0.990 0.990 0.990 0.990 0.990 0.990

Reagent Reagent Reagent Reagent Reagent Reagent

Co., Co., Co., Co., Co., Co.,

Ltd. Ltd. Ltd. Ltd. Ltd. Ltd.

(Shanghai, (Shanghai, (Shanghai, (Shanghai, (Shanghai, (Shanghai,

China) China) China) China) China) China)

Guiyang Xintian Optoelectronics Technology Co., Ltd. China) with a precision of ±0.0003. For appropriate diluted aqueous solutions of the phases, the relation between the refractive index (n) and the mass fractions of L64 (w1) and salt (w2) is given by Eq. (1)

n ¼ a0 þ a1 w1 þ a2 w2

TLL ¼


 

STL ¼ 

wt1  wb1

wt1  wb1 wt2  wb2

wt1 ,wb1 ,wt2

2



þ wt2  wb2

2 0:5

(2)



e GC GC GC GC GC GC

56

L64 L64 + 0.2 mol L-1 (NH4)2SO4

54

CP (oC)

52

L64 + 0.2 mol L-1 K2SO4⋅

50 48 46 44 42 40 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Concentration of L64 (w/w) Fig. 1. Effect of the concentration of L64 on the CP in the absence and presence of salts.



(3) wb2

where and represent the equilibrium compositions of L64 (1) and salt (2), in the top (t) and bottom (b) phases, respectively. 3. Results and discussion 3.1. Cloud points 3.1.1. Effect of L64 concentration As shown in Fig. 1, the CP values are affected by the concentration of L64 in the absence and presence of salts ((NH4)2SO4, K2SO4). With the increasing L64 concentration, the CP decreases going through a minimum at 0.3% with 52.7  C. This phenomenon is attributed to the rise in micelle concentration, which makes the content of free water decrease relatively and the micelles easier to meet with each other and gather. However, the CP then increased with the copolymer concentration above 0.3% and after a certain concentration (1.2%) the CP value becomes almost constant. This is

Table 2 Fitting parameter values of Eq. (1) for the investigated ATPSs. System

fraction) fraction) fraction) fraction) fraction) fraction)

58

(1)

where a0, a1 and a2 are constant fitting parameters. The values of these coefficients are given in Table 2. Dilution before determination is necessary because this equation is only valid within the range of w1  5% andw2  3%. This equation has been used for phase analysis of the ATPSs in many studies [22,23]. The tie-line length (TLL) and slope (STL) of the investigated systems at different compositions were calculated by Eq. (2) and Eq. (3), respectively:

(mass (mass (mass (mass (mass (mass

Analysis method

a0

a1

a2

100sda

L64-K2SO4 ATPS 1.3325 0.1347 0.1207 0.008 L64-Na2SO4 ATPS 1.3325 0.1397 0.1637 0.020 L64-(NH4)2SO4 ATPS 1.3325 0.1350 0.1970 0.016 L64-K2CO3 ATPS 1.3325 0.1370 0.1884 0.006 L64-K2HPO4 ATPS 1.3325 0.1420 0.1724 0.015 P a cal  nexp Þ2 =NÞ0:5 , where n and N represent the refractive index sd ¼ ð N i¼1 ðn and the number of the diluted aqueous solutions, respectively.

because a structured water-surfactant system appears at high copolymer concentration [24]. This structure breaks with the increase in temperature and most of the water molecules are attached to the micelle system. This forms a kind of buffer between micelles, which impedes the clustering of the micelles. Therefore, higher temperature is required to remove these “floating” water molecules which are barriers for micellar interaction. Once they move out at a higher temperature the interaction between micelles becomes easier and CP appears at a higher temperature [25]. Finally, with the barriers being removed at the high temperature 56.5  C, the micellar interaction will reach its limit so that the CP remains almost unchanged even though the L64 concentration keeps increasing. 3.1.2. Effect of addition of salting-out salts It also can be found from Fig. 1 that every temperature point of the curve in the presence of salt is lower than the corresponding one of the curve in the absence of salt. Additionally, the effect of K2SO4 is greater than (NH4)2SO4. Further studies are showed in Fig. 2 and Fig. 3. Fig. 2 represents the CP measurements of 0.1% L64 aqueous solutions with three kinds of sulfates whose concentration ranges from 0.1 mol L1 to 0.7 mol L1 except for K2SO4 (0.1 mol L1e0.5 mol L1) due to its low solubility in aqueous solution. Fig. 3 shows the CP measurements of 0.1% L64 aqueous solutions in the presence of three salts (K2HPO4, K2CO3, K2SO4), the concentration range of K2HPO4 and K2CO3 is 0.1 mol L1e0.7 mol L1, K2SO4 ranges from 0.1 mol L1 to 0.5 mol L1. It can be found that all the five salts lower the CP, and the downtrend in CP with the increasing molar concentration of salt is approximately a straight line. Regression equations and regression coefficients (R2) were found from Figs. 2e3 and shown

442

Y. Wang et al. / Fluid Phase Equilibria 409 (2016) 439e446 Table 5 Density (r) of two phases in different total salt concentration of L64-salt ATPSs at t ¼ 45  C and pressure p ¼ 0.1 MPa.a

60

K2SO4 Na2SO4

50

No.

(NH4)2SO4

o

CP ( C)

40

L64-K2SO4 system 1 0.21 2 0.22 3 0.28 4 0.30 L64-Na2SO4 system 1 0.24 2 0.25 3 0.27 4 0.30 L64-(NH4)2SO4 system 1 0.40 2 0.45 3 0.50 4 0.55 L64-K2CO3 system 1 0.23 2 0.24 3 0.30 4 0.35 L64-K2HPO4 system 1 0.22 2 0.23 3 0.25 4 0.30

30

20

10 -0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Electrolyte concentration (mol•L-1) Fig. 2. CP value of L64 solution (0.1%) in the presence of K2SO4, Na2SO4, (NH4)2SO4.

60

K2HPO4 K2CO3

50

K2SO4

o

CP ( C)

40

Total salt concentration (mol L1)

r (g cm3) Salt-rich phase

L64-rich phase

1.0370 1.0444 1.0645 1.0707

1.0542 1.0557 1.0575 1.0637

1.0614 1.0653 1.0800 1.0893

1.0703 1.0716 1.0737 1.0753

1.0509 1.0558 1.0637 1.0761

1.0587 1.0602 1.0611 1.0630

1.0369 1.0395 1.0515 1.0683

1.0441 1.0449 1.0475 1.0573

1.0570 1.0603 1.0697 1.0762

1.0621 1.0635 1.0654 1.0661

a Standard uncertainties u are u(r) ¼ 0.0001 g cm3, u(t) ¼ 0.05 u(p) ¼ 10 kPa.



C, and

30

concentration of these salts has a stronger effect on decreasing the CP. The abilities of individual salts to change CP are different. And the orders followed by the five salts in terms of decreasing the CP are Na2SO4 > K2SO4 > (NH4)2SO4 (Fig. 2) and K2HPO4 > K2SO4 > K2CO3 (Fig. 3).

20

10 -0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Eletrolyte concentration (mol•L-1)

3.2. Phase inversion point

Fig. 3. CP value of L64 solution (0.1%) in the presence of K2HPO4, K2CO3, K2SO4.

Table 3 Regression equations and coefficients for all electrolytes. Electrolytes

Regression equations

K2SO4 Na2SO4 (NH4)2SO4 K2CO3 K2HPO4

Y Y Y Y Y

¼ ¼ ¼ ¼ ¼

53.2000e60.6000X 52.2750e60.9286X 52.8833e35.1667X 52.7583e55.8810X 51.8583e62.5952X

Regression coefficient (R2) 0.9996 0.9986 0.9982 0.9989 0.9967

for all used electrolytes in Table 3. The average CP value of L64 was calculated with standard deviation as 52.6 ± 0.5  C. As a result of salting-out effect [26], the salt ions of the five electrolytes would compete with polymer molecules to bound water molecules, which makes micelle dehydrated and promotes micelleemicelle interactions. So, the phase separation of surfactant takes place at lower temperature in the presence of these salts and the higher

A phase inversion phenomenon was observed in the five investigated ATPSs at 45  C. On the basis of a same total mass (10 g) and a constant mass fraction of L64 (10%), the L64-rich phase transferred from the bottom phase to the top phase when the total salt concentration reached a certain concentration. All the phase inversion points of the five ATPSs were obtained and shown in Table 4. This unique phenomenon was considered to be caused by density changes and further studies on density of the two phases of ATPSs proved this view. Density data are shown in Table 5 and Fig. 4. It can be found that the density of the salt-rich phase increased sharply with the rise in the total salt concentration, relative to the L64-rich phase. When the density of salt-rich phase was bigger than L64-rich phase, the salt-rich phase sinked to the bottom of the tube while L64-rich phase moved to the top phase. In addition, the intersection of the two lines in Fig. 4 is the phase inversion point. The L64-K2SO4 ATPS is taken as an example in order to show the phase inversion more clearly. As we can see in Fig. 5, three samples with different K2SO4 concentrations used for photography were

Table 4 Phase inversion point of L64-salt ATPSs at 45  C and pressure p ¼ 0.1 MPa.a Salt Inversion point (10 a

3

1

mol L

)

K2SO4

Na2SO4

(NH4)2SO4

K2CO3

K2HPO4

240e273

256e259

481e499

257e285

231e234

Standard uncertainties u are u(c) ¼ 0.001 mol L1, u(t) ¼ 0.05  C, and u(p) ¼ 10 kPa.

Y. Wang et al. / Fluid Phase Equilibria 409 (2016) 439e446

443

1.075

1.090

Salt-rich phase L64-rich phase

1.070 1.065 1.060

1.080

ρ (g cm )

ρ (g cm )

Salt-rich phase L64-rich phase

1.085

1.055 1.050

1.075 1.070

1.045

1.065

1.040

a

1.035 0.20

0.22

0.24

0.26

0.28

b

1.060

0.30

0.24

0.25

0.26

0.27

0.28

0.29

0.30

Total salt concentration (mol•L )

Total salt concentration (mol•L )

(a) K2SO4

(b) Na2SO4

1.080 1.070 1.075

Salt-rich phase L64-rich phase

1.060

ρ (g cm )

ρ (g cm )

1.070

1.065

1.060

1.055

c

1.050 0.38

0.40

0.42

0.44

0.46

0.48

Salt-rich phase L64-rich phase

1.065

0.50

0.52

0.54

0.56

1.055 1.050 1.045 1.040

d

1.035 0.22

0.24

0.26

0.28

0.30

0.32

Total salt concentration (mol•L )

Total salt concentration (mol•L )

(c) (NH4)2SO4

(d) K2CO3

0.34

0.36

1.080

Salt-rich phase L64-rich phase

1.075

ρ (g cm )

1.070

1.065

1.060

1.055

e 0.22

0.24

0.26

0.28

0.30

Total salt concentration (mol•L )

(e) K2HPO4 Fig. 4. Density (r) of two phases in different total salt concentration of L64-salt (K2SO4/Na2SO4/(NH4)2SO4/K2CO3/K2HPO4) ATPSs at t ¼ 45  C.

dyed by bromocresol green, which mostly gathered in the L64-rich phase. Fig. 5(a) and (c) represent the phase state before and after the phase inversion, respectively. Fig. 5(b) represents a transition state where the density of the top phase and bottom phase are very close and the total K2SO4 concentration is near phase inversion point. In addition, the inversion points of the five salts are different for their discrepant properties. The lower the amount of salt was used, the stronger the ability of phase inversion was produced by salt [27]. It could be found that the effect of different salts on the phase inversion point was as follows: K2HPO4 > Na2SO4 > K2SO4 > K2CO3 > (NH4)2SO4. It is worth mentioning that the behavior of phase inversion in ATPS is an essential guide to study phase diagrams, and thus provides necessary theoretical data for design and optimization of extraction processes.

Fig. 5. Extraction photographs of the dye bromocresol green in three ATPSs formed by 10% L64 and K2SO4 with different concentrations: (a) 0.2 mol L1, (b) 0.24 mol L1 and (c) 0.3 mol L1 at 45  C.

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Y. Wang et al. / Fluid Phase Equilibria 409 (2016) 439e446

3.3. Phase diagram of ATPS 3.3.1. Liquideliquid equilibrium data (LLE) and correlation The knowledge of the equilibrium composition is of great importance for design and optimization of ATPE process, which can provide the basis for predicting phase behavior. The composition of each phase depends on the quantity and nature of the salting-out salts if the temperature and L64 concentration were kept constant. After being placed at 45  C for 24 h, the mixed solution separated into two phases. Table 6 shows the LLE data of L64K2SO4/Na2SO4/(NH4)2SO4/K2CO3/K2HPO4 ATPSs where all concentrations are expressed in mass fraction. Six tie-lines (TLs) of each system were obtained. The phase diagrams of the investigated systems can be seen in Fig. 6 and the phase inversion is highlighted as follows: black lines represent ordinary region tie lines and red lines represent inversion region tie lines. A four-parameter empirical equation (Eq. (4)) was used to correlate the binodal data of the systems which has been successfully put into use in the correlation of the binodal data for the polymeresalt ATPS [28].

  2 w1 ¼ exp a þ bw0:5 2 þ cw2 þ dw2

(4)

where w1 and w2 are the mass fractions of the L64 and salts; a, b, c and d are fitting parameters. The OthmereTobias (Eq. (5)) and Bancroft (Eq. (6)) equations widely applied for the assessment and correlation of LLE data in many similar block copolymer-salt systems [29,15] are used in this work. Table 6 LLE data of L64-salt ATPSs at t ¼ 45  C and pressure p ¼ 0.1 MPa.a No.

Total system w1

w2

L64-K2SO4 system 1 0.1000 0.0365 2 0.1000 0.0383 3 0.1000 0.0487 4 0.1000 0.0557 5 0.1000 0.0592 6 0.1000 0.0626 L64-Na2SO4 system 1 0.1000 0.0341 2 0.1000 0.0355 3 0.1000 0.0383 4 0.1000 0.0426 5 0.1000 0.0497 6 0.1000 0.0568 L64-(NH4)2SO4 system 1 0.1000 0.0528 2 0.1000 0.0594 3 0.1000 0.066 4 0.1000 0.0726 5 0.1000 0.0792 6 0.1000 0.0858 L64-K2CO3 system 1 0.1000 0.0317 2 0.1000 0.0331 3 0.1000 0.0414 4 0.1000 0.0483 5 0.1000 0.0552 6 0.1000 0.0621 L64-K2HPO4 system 1 0.1000 0.0383 2 0.1000 0.0400 3 0.1000 0.0435 4 0.1000 0.0487 5 0.1000 0.0522 6 0.1000 0.0574 a

Top phase

Bottom phase

TLL

STL

w1

w2

w1

w2

0.0388 0.0283 0.2740 0.3040 0.3542 0.3982

0.0412 0.0455 0.0307 0.0307 0.0301 0.0295

0.1486 0.1725 0.0143 0.0175 0.0163 0.0170

0.0330 0.0326 0.0567 0.0607 0.0653 0.0683

0.1102 0.1447 0.2610 0.2881 0.3398 0.3832

13.4303 11.1744 10.0004 9.5599 9.6009 9.8296

0.0198 0.0199 0.2683 0.3104 0.3450 0.3990

0.0411 0.0428 0.0231 0.0225 0.0219 0.0215

0.2190 0.2280 0.0175 0.0177 0.0160 0.0180

0.0240 0.0238 0.0468 0.0507 0.0548 0.0589

0.1999 0.2090 0.2519 0.2940 0.3305 0.3827

11.6355 10.9558 10.5649 10.3637 10.0247 10.2093

0.0192 0.0142 0.2586 0.2975 0.3349 0.3733

0.0595 0.0695 0.0405 0.0395 0.0388 0.0386

0.2300 0.2420 0.0156 0.0172 0.0182 0.0199

0.0411 0.0407 0.0799 0.0866 0.0939 0.0998

0.2117 0.2297 0.2462 0.2842 0.3215 0.3586

11.4035 7.8868 6.1683 5.9505 5.7547 5.7753

0.0327 0.0203 0.2050 0.2653 0.3215 0.3810

0.0346 0.0381 0.0283 0.0272 0.0273 0.0266

0.1396 0.1630 0.0114 0.0111 0.0115 0.0117

0.0295 0.0293 0.0518 0.0588 0.0653 0.0729

0.1070 0.1430 0.1950 0.2561 0.3123 0.3722

20.7476 16.2205 8.2278 8.0558 8.1598 7.9825

0.0073 0.0063 0.2018 0.2343 0.2614 0.2884

0.0455 0.0491 0.0309 0.0306 0.0302 0.0300

0.1780 0.1894 0.0052 0.0047 0.0039 0.0035

0.0316 0.0310 0.0548 0.0615 0.0657 0.0718

0.1713 0.1840 0.1981 0.2317 0.2599 0.2879

12.2835 10.1356 8.2367 7.4160 7.2594 6.8107

Standard uncertainties u are u(w) ¼ 0.0001, u(t) ¼ 0.05  C, and u(p) ¼ 10 kPa.

1  wt1 1  wb2 ¼ k 1 wt1 wb2 wb3 wb2

¼ k2

wt3 wt1

!n (5)

!r (6)

ehere wt1 and wt3 are the mass fractions of L64 and water in the top phase, respectively;wb2 and wb3 are the mass fractions of salt and water in the bottom phase, respectively; k1, n, k2 and r are the fitting parameters. The values of the fitting parameters along with the corresponding correlation coefficient (R2) values are given in Table 7. The data and the two models showed a good agreement. In addition, a special phenomenon can be found from Table 7 that the R2 for L64-K2SO4 is lower than those obtained from other ATPSs. This may be caused from the following two aspects. On the one hand, the solubility of K2SO4 is lower than other four salts (Na2SO4, (NH4)2SO4, K2CO3, K2HPO4). This fact made the preparation of L64K2SO4 ATPS hard and caused some measurement errors. On the other hand, the R2 was obtained from the six tie lines before and after the phase inversion point. It can be found from Table 4 that the phase inversion point of L64-K2SO4 ATPS is 240e273 mmol L1, the concentration range of which is bigger than other ATPSs. Due to the large difference of salt concentration, the six tie lines (two obtained before phase inversion point and four obtained after phase inversion point) are not concentrated and may result in some errors. Thus, the R2 for L64-K2SO4 ATPS obtained from the six tie lines is lower than other ATPSs. 3.3.2. Effect of salting-out salts on phase diagrams Cation and anion are generally believed as the fundamental elements of salting-out salt to affect the phase diagram of ATPSs. Fig. 7 shows the influence of cation on phase diagrams. It was proposed by da Silva and Loh [30] that the effect of sulfate salt to induce ATPS formation is due to the cationepolymer interactions. That is to say, cations interact with the ethylene oxide copolymer groups, releasing some water molecules that solvate EO groups when the polymer and sulfate salts are mixed [16,17]. The concentrations of sulfate salts are shown in terms of molality in order to clearly compare the effect of anion and cation on the binodal curves. The size of the biphasic area follows the order: Na2SO4 > K2SO4 > (NH4)2SO4. Based on the same anions in the investigated salts, the salting out ability of cations for separation can be found as follows: Naþ > Kþ > NH4 þ . Many workers related the effect of the ions on the phase separation with the Gibbs free energy of hydration of the ions (DGHyd) [31,32]. Hence, the ions with the higher kosmotropicity had a more negative DGHyd. The more negative the DGHyd values were, the stronger the ability of salting out was [33]. According to some literature [34,35], the DGHyd value of cations Naþ, Kþ and NH4 þ were 365 kJ mol1, 295 kJ mol1 and 285 kJ mol1, respectively. This was in great agreement with the result of the experiment. In addition, according to the tendency of ion hydration, salting out ability of electrolyte was described in some works [36] using the Hofmeister series. However, the experimental result in this work (Naþz Kþ > NH4 þ ) was not completely consistent with the Hofmeister series [37]. Fig. 8 presents the influence of anions on the induction of phase separation. The Hofmeisister series of anions [38] was arranged as follows: citrate3 > SO4 2 ¼ tartarate2 > HPO4 2 > CrO4 2 >  acetate > HCO3  > Cl > NO 3 > ClO3 . And the DGHyd value of anions [29,39] reported in the literature followed the order: DGHyd ðSO4 2 Þ ¼ 1080 kJ mol1 > DGHyd ðCO3 2 Þ ¼ 1350 kJ mol1 > DGHyd ðHPO4 2 Þ ¼ 1789 kJ mol1. However, the determination results in this study showed a little different. The

Y. Wang et al. / Fluid Phase Equilibria 409 (2016) 439e446

445

Fig. 6. Equilibrium data for aqueous systems formed by triblock copolymer L64 and salt (K2SO4/Na2SO4/(NH4)2SO4/K2CO3/K2HPO4) at 45  C. All concentrations are in mass fraction.

positions of the binodals for the three kinds of potassium salts indicated that the salting-out ability of anions followed the order: HPO4 2 > SO4 2 > CO3 2 , which was consistent with the ability to decrease the CP. In this work, CO3 2 has a weaker salting-out ability than SO4 2 because the pKa2 value of CO3 2 is 10.329 while pKa value of SO4 2 is only 1.9. That means a large number of HCO 3 can be dissociated in aqueous solutions, which will not promote the phase separation. In PEG-salt ATPSs [7], the salting-out strength of

Table 7 Values of parameters of Eqs (5) and (6) for the investigated ATPSs at t ¼ 45  C and pressure p ¼ 0.1 MPa. System

k1

n

R21

L64-K2SO4 system L64-Na2SO4 system L64-(NH4)2SO4 system L64-K2CO3 system L64-K2HPO4 system

1.35E-03 0.0022 1.50E-02 0.0033 0.0345

2.7029 2.3663 2.1507 2.4288 1.6658

0.9758 11.6503 0.3549 0.9714 0.9922 13.3462 0.4142 0.9924 0.9981 7.1063 0.4668 0.9980 0.9992 10.5325 0.4117 0.9990 0.9949 7.7849 0.5904 0.9943

k2

r

R22

The values of k1, n, R21 were obtained from Eq. (5); the values of k2, r, R22 were obtained from Eq. (6).

the investigated salts of phase-forming was in the order: (NH4)2HPO4 > (NH4)2SO4 > (NH4)2C4H4O6 > (NH4)2CO3. This result was proved by the binodal curves in phase diagrams where the biphasic region of PEG-(NH4)2HPO4 system was the biggest and the biphasic region of PEG-(NH4)2CO3 was the smallest. Analogously, in polyvinylpyrrolidone-ammonium salt ATPSs [40], it was found that phase-forming abilities of the ammonium salts were in the order: (NH4)2HPO4 > (NH4)2SO4 > (NH4)2C4H4O6 > (NH4)2HC6H5O7 > (NH4)2CO3. It can be concluded that the salting-out ability did not absolutely depend on the Gibbs free energy of hydration of the ions in our study. What's more, the theory of DGHyd and Hofmeister series to evaluate salting-out ability is not suitable for all ATPSs. 4. Conclusions In this work, the CP of L64 solution in different concentrations was obtained. The effects of five salting-out salts which are K2SO4, Na2SO4, (NH4)2SO4, K2CO3, K2HPO4 on the CP of L64 in aqueous solution were investigated. The results showed that all the five

446

Y. Wang et al. / Fluid Phase Equilibria 409 (2016) 439e446

Acknowledgments

0.45

K2SO4

0.40

Na2SO4

0.35

(NH4)2SO4

0.30

w1

0.25 0.20

This work was supported by the National Natural Science Foundation of China (Nos. 31470434, 21406090 and 21576124), the Special Financial Grant from the China Postdoctoral Science Foundation (2015T80510), and the grants from the project of General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China (No. 2015IK139 and 2015IK142).

0.15

References

0.10 0.05 0.00 0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

w2/M2 (mol g-1) Fig. 7. Cation effect on the phase diagram for the triblock copolymer L64 þ sulfate salt at 45  C: (-) K2SO4; (C) Na2SO4; (:) (NH4)2SO4. All concentrations are in molality.

0.45

K2SO4

0.40

K2CO3 K2HPO4

0.35 0.30

w1

0.25 0.20 0.15 0.10 0.05 0.00 0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

w2/M2 (mol g-1) Fig. 8. Anion effect on the phase diagram for the triblock copolymer L64 þ potassium salt at 45  C: (-) K2SO4; (C) K2CO3; (:) K2HPO4. All concentrations are in molality.

salting-out salts could decrease the CP values with the increasing in salt concentration. As the salt concentration rose, the increase of the density in the top phase induced a peculiar phase inversion that the salt-rich phase transferred from the top phase to the bottom phase. The phase inversion point of every ATPS was investigated. The LLE data for the L64-salt ATPSs at 45  C were acquired. The consistency of the tie-lines was verified by using the empirical equations from the OthmereTobias and Bancroft correlation. The influence of the cations and anions on the process of phase separation was assessed. In the investigated systems, the greater twophase area indicated a better phase separation ability and was attributed to a stronger salting-out ability. The results pointed out that the compact of cations on the CP values and phase separation process followed the order: Naþ > Kþ > NH4 þ . And effects of anions followed the order: HPO4 2 > SO4 2 > CO3 2 . Even more remarkable, it could be concluded that using the Gibbs free energy of hydration of the ions (DGHyd) to estimate salting-out ability was not suitable for all of ATPSs. The selection and optimization of L64-salt ATPSs depend on the compositions of phases which may change with the cloudy behavior and phase behavior. This work provided such a good guide to employ these ATPSs for separation and purification of biomolecules.

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