Phase Equilibrium of PEG 2000 + Triammonium Citrate + Water

Article pubs.acs.org/jced

Phase Equilibrium of PEG 2000 + Triammonium Citrate + Water System Relating PEG Molecular Weight, Cation, Anion with Effective Excluded Volume, Gibbs Free Energy of Hydration, Size of Cation, and Type of Anion at (298.15, 308.15, and 318.15) K. Rajendran Govindarajan and Muthiah Perumalsamy* Department of Chemical Engineering, National Institute of Technology, Tiruchirappalli, Tamil Nadu, 620 015, India ABSTRACT: Phase equilibria of an aqueous two-phase system consisting of polyethylene glycol (PEG 2000) + triammonium citrate + water were determined at different temperatures (298.15, 308.15, and 318.15) K. The binodal curve moves toward the origin with an increase in temperature, resulting in an increase in the two-phase region with an increase in temperature. The effective excluded volume (EEV) of the above aqueous two-phase system was determined from the binodal data. The salting-out capacity of the system increases with the temperature and is explained by the increase of EEV with increase in temperature. The effect of PEG molecular weight, cation, and anion on the binodal curve was analyzed using EEV, Gibbs free energy of hydration (ΔGhyd), size of cation, and type of anions. The phase equilibrium compositions, tie-line length (TLL), and slope of tie-line (STL) were determined at (298.15, 308.15, and 318.15) K. The consistencies of the experimental equilibrium data were explained with the Othmer−Tobias and Bancroft equations. The salting-out coefficients were determined from tie-line compositions using Setschenow equation. The salting-out coefficient increases with the increase in temperature.

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INTRODUCTION The separation and purification step of biological products is considered to be of paramount importance, as it costs about (50 to 80) % of the total production cost.1 In addition, the biological activity of the product of interest must be retained during the purification process. These constraints necessitate the process industries to develop a cost-effective, efficient, and environmental friendly method for the extraction of biomaterials.2 The aqueous two-phase system (ATPS) has emerged as a promising downstream processing technique in recent years in the field of industrial biotechnology. ATPS is capable of separating potential products of interest such as proteins,3 pharmaceuticals,4 bionanoparticles,5 recovery of metal ions,6 phytochemicals7−9 etc. An aqueous two-phase system can be obtained by mixing two incompatible polymers, polymer and an inorganic salt, surfactant, and polyelectrolyte. The aqueous mixtures of these phase-forming components remain in a turbid state above certain compositions and settle down to two different phases when left uninterrupted. The top and the bottom phase are principally rich in one of the phase-forming components being utilized for the phase formation. Water forms the greatest part of these two phase systems (around 90 %) and it provides a gentle environment for the separation of biomolecules. The industrial application of ATPS containing polymer−polymer systems has been limited because of the high cost of the polymers. To overcome the limitations of the polymer− polymer system, the polymer−salt system has been extensively used in the ATPS extraction of biomolecules. In the latter © XXXX American Chemical Society

system, PEG is commonly used as a polymer, and phosphate or sulfate has been used as salts for developing aqueous two-phase systems. The continuous application of these salts in process industries leads to environmental pollution. Hence, in order to abate the toxicity of phosphate and sulfate salts, environmentally benign citrate salts were employed for the formation of ATPS.10−12 Liquid−liquid equilibrium of ATPS with various citrate salts has been previously established.13 Our research group has previously discussed the liquid−liquid equilibrium (LLE) of several citrate salts.14,15 However, further research on ATPS continues with the search for several new systems for ATPS formation.16 Liquid−liquid equilibrium of PEG 6000 + triammonium citrate + water was previously available in the literature.17 Recently, the phase behavior and density for binary and ternary solutions of the PEG 4000 + triammonium citrate + water ATPS system were discussed.18 In the present work, the phase equilibrium for aqueous PEG 2000 + triammonium citrate systems were determined at different temperatures (298.15, 308.15, and 318.15) K in order to show the effect of molecular weight. Various empirical correlations have been successfully fitted with the phase equilibrium data of the above-mentioned system. The effective excluded volume (EEV) was obtained by using the theoretical equation proposed by Guan et al.19 The effects of PEG molecular weight, cations, and anions on the binodal curve Received: April 15, 2013 Accepted: September 30, 2013

A

dx.doi.org/10.1021/je400355g | J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

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The uncertainty of the salt determination was ± 0.002 % (w/ w). This is followed by the determination of PEG concentration (for known salt concentration) using the refractive index. Cheluget et al.21 successfully correlated the refractive index, nD using the mass fraction of polymer, wp, and salt, ws, and is expressed by the following equation (eq 2).

were discussed. The tie-line compositions, tie-line length (TLL), and slope of tie-line (STL), were determined at (298.15, 308.15, and 318.15) K. Tie-lines were correlated using Othmer-Tobias and Bancroft equations. The Setschenow equation was applied to tie-line compositions, and the saltingout coefficients were determined.

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MATERIALS AND METHODS Materials. Polyethylene glycol 2000 [HO(C2H4O)nH], (PEG 2000) with average molar mass of 1800 g·mol−1 (Merck Chemicals, Germany), and triammonium citrate ((NH 4 ) 3 C 6 H 5 O 7 ) with molar mass of 243.22 g·mol −1 (Qualigens Chemicals, India) with a minimum purity of 0.99 were procured and used without further purification. Double distilled and deionized water was used throughout the experiments. Apparatus and Procedures. The titration method (cloud point method) was followed for the determination of binodal curve. The experiments were conducted in a 100 cm3 glass vessel with an external glass casing for thermoregulation using a thermostatic water bath (Schott-Gerate CT 52, Germany). Temperatures were maintained with an uncertainty of ± 0.05 K. The top portion of the binodal curve was obtained by titration from a known quantity of aqueous PEG 2000 (60%, wt/wt) against the aqueous triammonium citrate solution (40%, wt/wt). The titration was paused once the solution became turbid, and the amount of salt solution added was noted. The consecutive points were determined by adding water to the turbid solutions to become clear and retitrating. The resulting composition of the mixture was determined using an analytical balance (Sartorius, Germany) with a precision of ± 0.1 mg. Similarly the bottom portion of the binodal curve was obtained by titratation of the aqueous triammonium citrate solution against aqueous PEG 2000. The experimental phase equilibrium studies were carried out in graduated centrifuge tubes with a conical bottom (50 cm3). The systems were prepared on a 40 g basis by mixing a predetermined amount of PEG 2000, triammonium citrate, and water. The samples were mixed in a cyclomixer for about 10 min, and then maintained at constant temperature in a thermostatic water bath. The samples were once again mixed for about 2 min after a time interval of 30 min to ascertain the separation at the desired constant temperature. Finally the mixtures were left undisturbed to settle for 24 h at the desired temperature. The clear top phase was removed using a micropipet. A layer of salt solution in the top of the bottom phase along with the interface was discarded to prevent the cross contamination. The bottom phase left behind was collected separately using a pipet. The PEG and triammonium citrate concentrations in both phases were analyzed at 303.15 K using the refractive index14 (Digital Refractometer, Atago, Japan with an accuracy of ± 0.00005) and conductivity method17,20 (Eutech Instruments, Singapore, with an accuracy of ± 0.01) respectively. The conductivity for the triammonium citrate concentration ranges from [(2 to 14) % (w/w)] is measured and related by the following equation (eq1) K = b0 + b1ws

nD = a0 + a1wp + a 2ws

(2)

where a0, a1, and a2 denote the correlation coefficients. This equation has been used continuously for determining the PEG concentrations of several aqueous two-phase systems. A calibration curve linking the wide range of concentrations of aqueous solutions of PEG 2000 [(10 to 50) % (w/w)] and triammonium citrate [(0 to 10) % (w/w)] were plotted. The coefficients (a0, a1, and a2) of eq 2 are obtained through linear regression and are 1.3298, 0.1526, and 0.2076, respectively. The uncertainty of the PEG determination was ± 0.001 % (w/w).

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RESULTS AND DISCUSSION The Binodal Data and Its Empirical Correlation. The experimental binodal data of the PEG 2000 + triammonium citrate + water at different temperatures (298.15, 308.15, and 318.15) K are reported in Table 1 and are correlated to the following literature correlations. wp = a + bws0.5 + cws + dws2

(3)

wp = a + bws0.5 + cws

(4)

ln(wp) = a + bws0.5 + cws3

(5)

1 = a + bwp0.5 + cwp ws

(6)

The fitting parameters a, b, c, and d, and the square of correlation coefficient (R2) were determined by means of regression analysis of the experimental binodal data of the present investigated system and are reported in Table 2. Average arithmetic relative deviation (AARD) was also reported in Table 2. On the basis of the average arithmetic relative deviation (AARD) and square of correlation coefficient (R2) values obtained for the correlation of binodal data, it is concluded that the empirical correlation proposed by Hu et al.22 (eq 3) was the best fit. The average arithmetic relative deviation (AARD) for eq 3 was less than 1%. In addition Figure 1 confirms that predicted values (using eq 3) and experimental values of PEG 2000 mass fraction have a good line of agreement. It is followed by Jayapal et al.13 (eq 4) and Taboada et al.23 (eq 5) with satisfactory results. The least fit was obtained for the equation proposed by Graber et al.24 (eq 6). Similar best fit for binodal data was obtained for eq 3 in our previous study.18 Influence of Temperature on the Binodal Curve. The effect of temperature on the binodal curve for PEG 2000 + triammonium citrate + water system is depicted in Figure 2. The two-phase region increases with an increase in temperature. As the temperature increases, the water molecules move from the top phase to the bottom phase, increasing the hydrophobicity of the PEG. Therefore, at high temperature, the combined effect of hydrophobicity of PEG and the salting-out effect of salt is responsible for the two-phase formation, whereas at low temperature, the salting-out effect of salt alone

(1)

where, K represents the conductivity (μS·cm −1 ). The parameters of eq 1, bo and b1 for aqueous solutions of triammonium citrate are 46.14 and 455.3, respectively. After determining the conductivity of the phases, the salt concentration of the phases can be determined from eq 1. B



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in the salting-out effect of salt by way of the increase in EEV at higher temperatures. Influence of PEG Molecular Weight on the Binodal Curve. The effect of PEG molecular weight on the binodal curve for PEG + triammonium citrate + water at 298.15 K is shown in Figure 3 as an example. The two-phase region increases with the increase in the PEG molecular weight from 2000 to 6000. The increase in the PEG chain length increases its hydrophobicity and decreases its water solubility. Thus the mutual effect of hydrophobicity of high molecular weight PEG and the salting-out effect of salt reduces the amount of phase forming components for two-phase formation. The EEV of PEG (2000, 4000, 6000) + triammonium citrate + water system at (298.15, 308.15, and 318.15) K were reported in Table 3. The EEV increases with increase in the PEG molecular weight and the temperature. The PEG 6000 at 318.15 K has the highest EEV (58.5834 g·mol−1). This confirms the increased salting-out strength of salt at higher temperature, when combined with high molecular weight PEG. The effect of PEG molecular weight on the binodal curve has been previously confirmed by other authors.2,25 Influence of Cation and Anion of Salt on the Binodal Curve. The area of the two-phase region is affected by the cation and the anion of the salt being used for the ATPS formation. The citrate anion is kept constant and the cation (sodium,14 potassium,13 and ammonium) is changed to illustrate the cation effect and is shown in Figure 4. The salting-out capacity of salt decreases with the cation in the following order as is the Hofmeister series (Na+ > K+ > NH4+).20 In other words, with the citrate anion, the salting-out effect of anion (Na+) is greater than that of the NH4+. This phenomenon is explained with the Gibbs free energy of hydration (ΔGhyd) and the size of the cation. Marcus26 has previously reported the experimental values of Gibbs free energies of hydration for different ions. The ΔGhyd for Na+, K+, and NH4+ are −365, −295, −285 KJ·mol−1, respectively. This explains that the ion with more negative ΔGhyd requires less amount of its salt for two-phase formation. The size of the cation also contributes to the salting-out effect of salt. The monovalent cations such as Na+, K+, and NH4+ have ionic radii of 0.098, 0.133, and 0.137 nm, respectively.27 It is evident from Figure 4 that the area of the two-phase region decreases with an increase in the size of the cation. The binodal curves of tripotassium and triammonium citrate overlap at high concentrations of salt due to their close ionic radii. The anion has a major effect on the position of the binodal curve. The charge of the anion also affects the area of the two-phase region. The higher the valency is, larger is the area of the twophase region. The binodal curves of PEG 2000 + triammonium citrate + water and PEG 2000 + Diammonium Hydrogen citrate + water system20 is considered for this purpose. The salting-out effect of citrate anion is more than that of hydrogen citrate. This is for the reason that the triply charged citrate anion (C6H5O73‑) and the doubly charged hydrogen citrate anion (HC6H5O72‑) have ΔGhyd of −2793 and −968 KJ.mol−1 respectively.28,29 The more negative ΔGhyd of the citrate anion enclose more structured water lattice around it than the hydrogen citrate with less negative ΔGhyd and hence with less structured water lattice.29 In general, with ammonium as cation, the anion (Citrate, Hydrogen citrate,20 and Sulfate30) effect is considered and the salting-out effect follows the following order (SO42‑ >C6H5O73‑ > HC6H5O72‑) and is illustrated in Figure 4. In contrary, the sulfate anion30 (SO42‑) with ΔGhyd (−1080 KJ.

Table 1. Binodal Data for PEG 2000 + Triammonium Citrate + Water System at Different Temperaturesa,b 298.15 K

308.15 K

318.15 K

100ws

100wp

100ws

100wp

100ws

100wp

3.16 4.06 4.93 5.89 6.90 7.74 8.70 9.43 10.19 10.97 11.76 12.44 13.06 13.63 14.40 14.93 15.40 15.85 16.26 16.66 16.96 17.27 17.54 17.82 18.08 18.43 18.60 18.80 12.71 14.16 15.84 17.74 19.79 22.15

55.26 49.43 44.54 40.23 36.45 33.32 30.43 28.07 25.93 23.99 22.24 20.72 19.37 18.18 16.94 15.95 15.07 14.27 13.54 12.88 12.30 11.76 11.26 10.79 10.37 9.92 9.57 9.23 20.01 17.24 14.18 10.92 7.75 4.34

2.62 3.43 4.23 5.03 5.80 6.56 7.29 8.08 8.76 9.41 10.11 10.72 11.35 11.91 12.41 12.90 13.33 13.76 14.14 14.49 14.84 15.16 15.43 15.67 15.92 16.15 16.39 16.61 16.84 17.03 14.75 16.35 17.93 19.83 21.96

56.08 50.28 45.39 41.21 37.61 34.48 31.74 29.25 27.12 25.23 23.48 21.96 20.55 19.30 18.20 17.20 16.29 15.46 14.71 14.02 13.39 12.80 12.28 11.80 11.35 10.93 10.52 10.15 9.80 9.47 13.57 10.60 8.12 5.11 2.02

2.05 2.70 3.35 3.97 4.62 5.28 5.84 6.42 7.01 7.59 8.11 8.69 9.15 9.61 10.08 10.49 10.87 11.26 11.61 11.96 12.29 12.59 12.90 13.15 13.41 13.63 13.88 14.10 14.32 14.53 13.50 14.60 15.69 16.90 18.19 19.65 21.20

56.92 51.27 46.48 42.41 38.87 35.75 33.09 30.72 28.59 26.68 25.00 23.42 22.08 20.84 19.69 18.67 17.75 16.89 16.11 15.37 14.70 14.09 13.51 12.99 12.49 12.05 11.61 11.21 10.83 10.47 12.36 10.33 8.60 6.80 5.05 3.14 1.32

a ws, mass fraction of triammonium citrate; wp, mass fraction of PEG 2000. bStandard uncertainities u are u(T) = 0.05 K and u(100w) = 0.01.

contributes to the two-phase formation. On the basis of the statistical geometry, Guan et al19 developed a model to predict the coexistence curves of the polymer aqueous two-phase systems. The model (eq 7) predicts the coexistence curves using a single parameter, the effective excluded volume (EEV) and is extended to polymer-salt ATPS. ⎛ w ⎞ w * p ⎟⎟ + V123 * s =0 ln⎜⎜V123 Mp ⎠ Ms ⎝

(7)

where, the parameter, V123 * represents the effective excluded volume (EEV), Mp and Ms correspond to the molar mass of polymer (1800 g·mol−1) and the salt (243.22 g·mol−1), respectively. Using the experimental binodal data, the EEV were determined at different temperatures (298.15, 308.15, and 318.15) K and are reported in Table 3. It signifies the increase C



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Table 2. Binodal Curve Correlations and Their Parameter Values for PEG 2000 + Triammonium Citrate + Water System at Different Temperaturesa literature correlations Hu et al.

22

(eq 3)

Jayapal et al.13 (eq 4)

Taboada et al.23 (eq 5)

Graber et al.24 (eq 6)

a

T/K

a

b

c

d

AARD/%*

R2

298.15 308.15 318.15 298.15 308.15 318.15 298.15 308.15 318.15 298.15 308.15 318.15

1.1375 1.1040 1.0592 1.0233 0.9946 0.9884 0.1001 −0.1065 −0.0309 17.7103 15.7404 16.1650

−4.0816 −4.0953 −4.0648 −3.0540 −3.0705 −3.3629 −4.0729 −3.4231 −4.1743 −79.1545 −73.9289 −81.5256

4.5643 4.6949 4.5533 2.0971 2.1548 2.7319 −110.8985 −183.5184 −220.2886 126.3817 129.8821 152.1792

−3.7591 −4.0746 −3.1264

0.4405 0.4810 0.4785 1.3536 2.3651 2.3489 1.3608 3.0576 2.6129 6.2432 9.7185 12.6643

1.000 1.000 1.000 0.999 0.999 0.999 0.997 0.991 0.992 0.980 0.966 0.957

Standard uncertainty u is u(T) = 0.05 K. *Average arithmetic relative deviation (AARD) = (∑ |(expt − cal)/(expt)|)/N·100.

Table 3. Effective Excluded Volumes (EEV) for PEG + Triammonium Citrate + Water System at Different Temperaturesa (eq 7) EEV/g·mol−1

a

T/K

PEG 2000

PEG 400018

PEG 600017

298.15 308.15 318.15

24.8138 26.3867 28.6335

36.3581 38.9550 42.4475

45.8113 48.3751 58.5834

Standard uncertainty u is u(T) = 0.05 K.

Figure 1. Experimental versus predicted (eq 3) data of polymer mass fraction for PEG 2000 + triammonium citrate + water system: Δ, 298.15 K; ○, 308.15 K; □, 318.15 K; ―, line of agreement.

Figure 3. Effect of PEG molecular weight on the binodal curve for PEG + triammonium citrate + water system at 298.15 K: Δ, PEG 2000; ○, PEG 4000;18 □, PEG 6000.17

length (TLL) and the slope of the tie-line (STL) and are determined using eqs 8 and 9, respectively. Figure 2. Effect of temperature on the binodal curve for PEG 2000 + triammonium citrate + water system: Δ, 298.15 K; ○, 308.15 K; □, 318.15 K; ―, calculated from eq 3

TLL =

ΔX2 + ΔY 2

(8)

STL = ΔY /ΔX

(9)

where, ΔX = − and ΔY = − Here, ws and wp denote the mass fraction of polymer and salt. Superscript t and b denotes the top and bottom phase, respectively. As the temperature increases, water molecules are driven from PEG-rich top phase to salt-rich bottom phase.20,31 Thus, concentrating the PEG phase and diluting the salt phase. Accordingly, the concentration difference of salt between the top phase and bottom phase decreases and as a result, STL is increased. This is also evident from the fact that the average wts

mol−1)26 have a greater salting-out effect than citrate and hydrogen citrate anion. Liquid−Liquid Equilibrium Data and Its Empirical Correlation. The experimental liquid−liquid equilibrium data of PEG 2000 + triammonium citrate + water, along with the tieline length (TLL) and slope of tie-line (STL) at different temperatures (298.15, 308.15, and 318.15) K were reported in Table 4. The tie-lines are usually characterized using tie-line D

wbs

wtp

wbp.



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Figure 4. Effect of different salt on the binodal curve for PEG 2000 + salt + water system at 298.15 K. × , diammonium hydrogen citrate;20 Δ, triammonium citrate; ○, tripotassium citrate;13 □, trisodium citrate;14 +, ammonium sulfate.30

Figure 5. Ternary phase diagram of PEG 2000 + triammonium citrate + water system at 298.15 K. ▲, experimental binodal data; △−△, experimental tie-lines.

Table 4. Phase Compositions, Tie-Line Length, and Slope of Tie-Line of PEG 2000 + Triammonium Citrate + Water System at Different Temperaturesa,b feed

top

100 wp

100 ws

100 wp

16.50 18.00 19.50 21.00 22.50

16.00 16.50 17.00 17.50 18.00

30.49 34.69 38.45 41.28 44.85

15.00 16.50 18.00 19.50 21.00

15.50 16.00 16.50 17.00 17.50

29.51 34.33 37.03 40.57 43.43

15.00 16.50 18.00 19.50 21.00

15.50 16.00 16.50 17.00 17.50

34.68 38.36 41.63 44.48 47.24

bottom

100 ws

100 wp

298.15 K 8.75 1.61 7.40 1.46 6.37 1.43 5.64 1.33 4.94 1.24 308.15 K 7.98 1.35 6.63 1.23 5.98 1.10 5.19 1.02 4.61 0.97 318.15 K 5.38 0.84 4.75 0.68 4.13 0.61 3.67 0.52 3.23 0.44

100 ws

TLL

STL

23.84 25.52 27.11 29.03 30.40

32.58 37.85 42.44 46.29 50.50

−1.914 −1.834 −1.785 −1.708 −1.713

22.54 24.06 25.85 27.38 29.07

31.70 37.41 41.06 45.35 49.00

−1.933 −1.899 −1.808 −1.782 −1.736

22.58 23.97 25.46 27.08 28.68

37.96 42.30 46.23 49.80 53.27

−1.968 −1.961 −1.923 −1.878 −1.839

Figure 6. Ternary phase diagram of PEG 2000 + triammonium citrate + water system at 308.15 K. ●, experimental binodal data; ○−○, experimental tie-lines.

a

ws, mass fraction of triammonium citrate; wp, mass fraction of PEG 2000. bStandard uncertainty u are u(T) = 0.05 K, and u(100 w) = 0.01

STL changes from −1.791 to −1.914 as temperature increases from 298.15 K to 318.15 K. The ternary phase diagrams of PEG 2000 + triammonium citrate + water system at (298.15, 308.15, and 318.15) K were illustrated in Figures 5 to 7. The liquid−liquid equilibrium data has been successfully correlated using the empirical equations such as Othmer−Tobias (eq 10) and Bancroft equation (eq 11).32 The parameters of eqs 10 and 11 along with the correlation coefficient (R2) are reported in Table 5. The exactness of the tie-lines was assured with greater R2 (≈ 0.99) values. ⎛ 1 − wpt ⎞ ⎛ 1 − w b ⎞n s ⎜⎜ ⎟ ⎜⎜ ⎟⎟ K = ⎟ t b w w ⎝ ⎠ ⎝ ⎠ p s

Figure 7. Ternary phase diagram of PEG 2000 + triammonium citrate + water system at 318.15 K. ■, experimental binodal data; □−□, experimental tie-lines.

⎛ w t ⎞r ⎛ wb ⎞ w ⎜⎜ b ⎟⎟ = K1⎜⎜ wt ⎟⎟ ⎝ ws ⎠ ⎝ wp ⎠

(11)

where wtp and wbs denote the mass fraction of polymer and salt in the top and bottom phase respectively. wtp and wbw denote the mass fraction of water in the top and bottom phase respectively. K, n, K1 and r are the constants.

(10) E



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salting-out strength of the salt increases with increase in temperature.

Table 5. The Estimated Parameter Values of eqs 10 and 11 for the PEG 2000 + Triammonium Citrate + Water System at Different Temperaturesa temp

a

Othmer−Tobias equation (eq 10)

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CONCLUSIONS The experimental binodal curves were determined at different temperatures (298.15, 308.15, and 318.15) K and were successfully correlated using empirical correlations. The effective excluded volume (EEV) was determined for different PEG molecular weight and at different temperatures. The effect of PEG molecular weight was studied, and the ability for twophase formation using EEV was discussed. The type of the cation and its size affects the position of the binodal curve. The concentration of salt required for two-phase formation increases with the type of cation. The area of the two-phase region decreases with an increase in the size of the cation. Gibbs free energy of hydration (ΔGhyd) was also used to describe the effect of cations. The more negative ΔGhyd indicates the greater salting-out capacity of salt. The saltingout capacity of salt, with citrate as anion follows the order (Na+ > K+ > NH4+) as in the Hofmeister series. The type of anion and its charge greatly affects the two-phase area. The two-phase region increases for the anion with higher valency. The triply charged citrate anion holds more negative ΔGhyd than the doubly charged hydrogen citrate. The salting-out effect of salt follows the order SO42− >C6H5O73− > HC6H5O72− with ammonium as cation. The tie-line compositions were determined and the effect of temperature on the tie-line length and slope of tie-line were discussed. As the temperature increases, the water molecules move from the top phase to bottom phase resulting in the increase in TLL and STL. The accuracy of experimental tie-lines was verified by correlating it with the Othmer−Tobias and Bancroft equations. The saltingout capacity at different temperatures was further confirmed by the determined salting-out coefficients from the experimental tie-lines.

Bancroft equation (eq 11)

K

n

K

R2

r

K1

R2

298.15 308.15 318.15

1.7947 1.7228 1.6081

0.2787 0.2757 0.2543

0.992 0.985 0.993

0.5867 0.5999 0.6367

2.1024 2.1867 2.4235

0.992 0.985 0.992


Further, the salting-out coefficient of the salt was determined using the Setschenow-type equation20,28 for the present experimental tie-lines and is depicted in Figure 8. The

Figure 8. Setschenow-type plot for the tie-line data of PEG 2000 + triammonium citrate + water system. Single and double prime denotes the top and bottom phase concentration, respectively. Δ, 298.15 K; ○, 308.15 K; □, 318.15 K.

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salting-out coefficients for PEG 2000 + triammonium citrate + water system at different temperatures (298.15, 308.15, and 318.15) K were determined from eq 12 and are given along

Corresponding Author

*E-mail: [email protected]. Tel.: +91-0431-2503112. Fax: +910431-2500133.

Table 6. Parameter Values of Setschenow-Type Equation for PEG 2000 + Triammonium Citrate + Water System at Different Temperaturesa (eq 12) T/K 298.15 308.15 318.15 a

−1

Kca/kg·mol

R

2.2029 2.2279 2.1967

0.994 0.991 0.991

1.2400 1.4636 2.0643

Funding

This work was financially supported (Sanction No: SR/S3/CE/ 074/2009) by the Department of Science and Technology (DST), New Delhi, India. The authors gratefully acknowledge the grant and the SRF.

2

intercept

Notes


The authors declare no competing financial interest.

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with R2 values in Table 6. The high R2 values (> 0.99) indicate the best fit of the Setschenow-type equation. ⎛ mp′ ⎞ ⎟⎟ = k p(mp″ − mp′ ) + kca(mca ″ − mca ′) ln⎜⎜ ⎝ mp″ ⎠

AUTHOR INFORMATION

REFERENCES

(1) Pietruszka, N.; Galaev, I. Y.; Kumar, A.; Brzozowski, Z. K.; Mattiasson, B. New Polymers Forming Aqueous Two-Phase Polymer Systems. Biotechnol. Prog. 2000, 16 (3), 408−415. (2) Albertsson, P.-Å. Partition of Cell Particles and Macromolecules, 3rd ed.; John Wiley and Sons: New York, 1986. (3) Asenjo, J. A.; Andrews, B. A. Aqueous two-phase systems for protein separation: A perspective. J. Chromatogr. A 2011, 1218 (49), 8826−35. (4) Platis, D.; Labrou, N. E. Application of a PEG/salt aqueous twophase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract. Biotechnol. J. 2009, 4 (9), 1320− 7.

(12)

where mp and mca denotes the molality of PEG and salt, respectively. The single and double prime represents the upper and lower phase respectively. kp and kca designates the parameter relating the activity coefficient of PEG to its concentration and the salting-out coefficient, respectively. The salting-out coefficient signifies the salting-out strength of the salt at the given temperature. It is evident from Table 6 that the F



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Phase Equilibrium of PEG 2000 + Triammonium Citrate + Water

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