LiquidâLiquid Equilibrium of Various Aqueous Two-Phase Systems

Article pubs.acs.org/jced

Liquid−Liquid Equilibrium of Various Aqueous Two-Phase Systems: Experiment and Correlation Yang Liu,*,† Yuan-qi Feng,† and Yongjie Zhao‡ †

Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, P. R. China Department of Mechanical Engineering, College of Engineering, Shantou University, Shantou, Guangdong 515063, P. R. China

‡

ABSTRACT: Liquid−liquid equilibrium (LLE) data for aqueous two-phase systems (ATPS) were discussed through experiment and correlation at 25 °C, in polymer−organic salt ATPS, alcohol−salt ATPS, and polymer−surfactant ATPS, respectively. Various binodal curves empirical equations combined with simple experiments were applied to determine the LLE data with as wide composition concentrations as possible. The tie-line length (TLL) ranges of the investigated ATPS were also determined experimentally. Both the two-phase regions and the TLL ranges of polymer−organic salt ATPS or alcohol−salt ATPS were generally wider than polymer−surfactant ATPS, which would be caused by the solubility of two compositions in different ATPS. The parameters of tie-line equations including Othmer−Tobias and Bancroft equations were further obtained by LLE data, which can be used to predict the unknown tie-line data. The experimental volume ratios of various tie-lines in ATPS showed good agreement with the predicted data based on the small standard deviation (SD < 0.05). Compared to the complicated experimental method for the determination of ATPS LLE data, the binodal curves and tie-line empirical equations combined with simple experiments can provide the LLE data for various ATPS.

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atomic absorption spectrometry,23,24 and flame spectrometry.25 These measurements are accurate and reliable, but the experiments need some professional apparatus and careful operations. Moreover, the various concentration measurements of the compositions in ATPS lack uniformity, and their precisions are different in various ATPS. To obtain the ATPS LLE data directly, some empirical equations of LLE data in ATPS have been widely reported.20−23 The two kinds of empirical equations including binodal data equations and tieline data equations are used to correlate the LLE data of ATPS, respectively. The two kinds of empirical equations are usually applied based on the accurate compositions concentration in the top and bottom phases through the concentration measurements. There were few reports about the two kinds of empirical equations combination to determine the ATPS LLE data through the convenient experimental procedures and mathematical methods. In this paper, the method to determine LLE data, phase diagrams, and TLL ranges of the ATPS was discussed thoroughly, including the simple titration experiments and the mathematical fitting process by using the two kinds of empirical equations. Different kinds of ATPS LLE data have been investigated to test this method, including polymer− organic salt ATPS, alcohol−inorganic salt ATPS, and polymer− surfactant ATPS. The TLL ranges of various ATPS were further discussed based on the experiments and the different composition solubilities.

INTRODUCTION In 1950s, Albertson et al.1 reported the distribution of different biomolecules in aqueous two-phase systems (ATPS). From then on, aqueous two-phase extraction (ATPE) has become more and more popular in the biotechnology downstream process. ATPE has many advantages such as simple equipment and procedure, low energy, high yield, and mild conditions compared to the traditional separating technique. Hence, this technique has been widely used in the extraction of protein molecules,2,3 active enzyme,4,5 amino acid,6 antibiotics,7,8 nucleic acid,9 nanomaterials10 in biochemistry, cell biology, and biochemical engineering. Recently, polymer−organic salt ATPS were used to avoid environmental problems compared to traditional polymer−inorganic salt ATPS.11 Alcohol−salt ATPS were widely studied in separation of biological enzyme molecule,12 organic acids,13 and other important chemical products14 due to the low viscosity and rapid phase separating process of ATPS. Polymer−surfactant ATPS have the hydrophobic microstructure formed by the surfactant molecules in a micelle-rich phase,15−19 so these ATPS can provide the effective separation systems for the biomolecules based on the molecular hydrophobicity and steric hindrance. More and more researchers have paid attention to these three kinds of ATPS due to their own advantages. The LLE data of ATPS including binodal data and tie-line data are the basic applicable data for ATPE, which are usually shown as phase diagrams composed of a binodal curve and several tie-lines. The binodal data can be obtained by the turbidimetric titration method.20−23 The tie-line data were always determined by some concentration detection methods, such as the refractive index measurements,20 spectrophotometry,21,22 © XXXX American Chemical Society

Received: May 14, 2013 Accepted: September 5, 2013

A

dx.doi.org/10.1021/je400453b | J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

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EXPERIMENTAL SECTION Materials. PEG of different molar masses (2000, 4000, 6000, 8000, and 20000) (GR, ≥ 95 % mass purity) and Triton X-100 (GR, ≥ 95 % mass purity) were purchased from Merck Chemical Company (Shanghai, China). Sodium citrate (GR for analysis, ≥ 99.5 % mass purity) was purchased from Kaixin Chemical Industry Corporation Limited (Tianjin, China). Ethanol (GR for analysis, ≥ 99.7 % mass purity) and ammonium sulfate (GR for analysis, ≥ 99 % mass purity) were purchased from Xilong Chemical Corporation Limited, and dipotassium phosphate (GR for analysis, ≥ 99 % mass purity) was purchased from GuangCheng Chemical Reagent Corporation Limited (Tianjin, China). All of the chemicals were used without further purification. Apparatus and Procedure. The experimental binodal data of polymer−organic salt ATPS, alcohol−inorganic salt ATPS, and polymer−surfactant ATPS are determined by the previously reported titration method.20−23 The binodal data of various ATPS were determined by turbidimetric measurement in a DC-0506W thermostatic water bath (Shanghai) of 25 °C. The temperature was maintained with an uncertainty of ± 0.1 °C. Stock solutions of different compositions [50 % (w/w) polyethylene glycol (PEG) 2000, 4000, 6000 solution, 30 % (w/w) PEG 8000, 20000 solution, 60 % (w/w) ethanol solution] with high concentrations were prepared and were carefully added dropwise into the other composition solution [50 % (w/w) sodium citrate solution, 40 % (w/w) polyoxyethylene octyl phenyl solution (Trition X-100), 50 % (w/w) ammonium sulfate solution, and 30 % (w/w) dipotassium phosphate solution], respectively, until the two-phase region was reached (turbid samples). Then water was added dropwise until the one phase region was reached (transparent samples). The compositions where the change occurs from a two-phase to a monophase system lie on the binodal curve. A sample was regarded as monophasic when it was completely clear and no phase separation could be detected. The system was weighed repeatedly, and the phase compositions at the phase boundary were calculated. The composition of the mixture for each point on the binodal curve was calculated by mass using a BSM-120.4 analytical balance (Shanghai) with an uncertainty of ± 1·10−7 kg. The same operations were done under different solution conditions in three kinds of ATPS. For determination of tie-line data, the systems were prepared by different stock solutions as mentioned above. A known concentration stock solution of two compositions and water were weighted into a graduated test tube to the desired compositions; the overall mass of each sample was 10 g. The concentrations of the ATPS compositions in this work are given as the mass percent (w/w). After the ATPS mixtures were shaken at one round per minute for 60 min by a QB-128 mixer (Shanghai) at 25 °C, the top and bottom phases in ATPS were separated by a Universal 320R centrifuge (Germany) at 3420 × g, 25 °C for 30 min. The ATPS were left for 10 min at 25 °C, and then the top phase and bottom phase were drawn and measured the volumes, respectively, by the Dragonmed 720050 and 720060 pipettes (Shanghai) with an uncertainty of ± 0.01 mL. Empirical Equations. The reported empirical equations of binodal data21,26−32 fitted in ATPS have the following forms w1 = a + bw20.5 + cw2

(1)

w1 = aw2 3 + bw2 2 + cw2 + d

(2)

w1 = a exp(bw2 0.5 − cw2 3)

(3)

⎛ w ⎞ ⎛ w ⎞ w1 = a exp⎜ − 2 ⎟ + c exp⎜ − 2 ⎟ + e ⎝ b⎠ ⎝ d⎠

(4)

⎛ w ⎞ M * 2⎟ 1 w1 = −ln⎜V 213 * M 2 ⎠ V 213 ⎝

(5)

w1 = exp(a + bw2 0.5 + cw2 + dw2 2)

(6)

where w1 and w2 represent the mass fractions of the compositions enriched in top (1) and bottom (2) phases, respectively, and a, b, c, d, and e are the fitted parameters. In eq 5, the

Figure 1. Schematic diagram of ATPS phase diagram. The X-axis and Y-axis represent the mass fraction of the composition enriched in the bottom phase and in the top phase, respectively.

Figure 2. Flow diagram of ATPS LLE deduction and correlation. B



Article

Table 1. Experimental Binodal Data as the Mass Fraction of Polymer (1) + Organic Salt (2) + Water (3) ATPS at Temperature T = 25 °C and Pressure p = 0.1 MPaa 100 w1

100 w2

100 w1

PEG2000 (1) 29.2 5.7 25.0 7.7 19.5 9.8 16.3 12.3 14.8 13.6 PEG4000 (1) 16.8 10.0 16.0 10.8 15.2 11.4 14.3 12.2 13.8 12.6 13.3 13.3 12.6 13.8 12.2 14.2 11.6 14.8 PEG6000 (1) 18.0 7.0 17.2 7.4 15.5 7.8 13.6 8.2 10.2 9.1 10.1 9.5 10.0 10.3 9.9 11.2

1.3 1.5 2.0 2.2 3.9 1.6 2.4 3.4 4.5 5.2 6.0 7.3 8.0 9.2 0.3 0.4 0.7 1.2 5.8 6.1 6.5 6.6

100 w2

100 w1

100 w2

100 w1

+ C6H5Na3O7 (2) + Water (3) 13.7 15.0 9.3 24.5 12.6 16.6 8.6 27.8 11.6 18 8.1 31.0 10.5 19.5 7.3 38.5 9.9 22.6 6.5 + C6H5Na3O7 (2) + Water (3) 11.2 15.7 8.6 25.9 10.8 16.2 8.5 26.0 10.5 17.2 8.0 29.2 10.2 17.8 7.8 31.7 10.0 18.4 7.6 32.4 9.7 19.1 7.3 36.1 9.5 20.0 7.0 37.5 9.2 21.1 6.6 40.9 9.0 22.1 6.3 44 + C6H5Na3O7 (2) + Water (3) 9.7 12.4 7.4 35.5 9.6 13.8 6.8 39.4 9.4 14.6 6.7 41.9 9.3 17.0 6.0 44.3 8.9 20.1 5.2 49.3 8.6 24.0 4.3 8.4 28.9 3.5 7.8 33.2 2.8

Table 2. Experimental Binodal Data as the Mass Fraction of Alcohol (1) + Salt (2) + Water (3) ATPS at Temperature T = 25 °C and Pressure p = 0.1 MPaa

100 w2

100 w1

5.9 5.0 4.2 2.9

6.1 11.8 15.0 17.9 21.4 22.7

5.8 5.7 5.0 4.4 4.1 3.4 3.2 2.6 2.2

8.1 9.1 10.0 10.9 11.9 12.5 13.5

2.4 2.0 1.4 1.1 0.5

w2b

=

100 w1

100 w2

100 w1

CH3CH2OH (1) + (NH4)2SO4 (2) + Water (3) 41.1 26.0 15.9 36.3 8.9 45.4 33.3 28.3 14.1 38.0 8.0 46.0 25.9 29.8 13.0 40.5 7.0 48.6 21.8 31.5 11.9 42.6 6.0 52.3 19.0 32.9 10.7 43.0 5.9 54.4 17.9 35.0 9.7 44.1 5.1 CH3CH2OH (1) + K2HPO4 (2) + Water (3) 27.4 15.1 19.3 21.4 13.2 31.5 26.0 15.8 18.6 22.3 12.5 33.3 24.9 17.0 17.4 23.1 11.9 34.8 23.8 18.2 16.3 24.6 10.8 36.6 22.7 18.8 15.9 26.0 9.9 21.9 20.0 14.8 27.9 8.7 21.0 20.5 13.9 29.4 7.8

100 w2 4.9 4.5 3.8 3.1 2.7

6.9 6.0 5.5 5.0

Table 3. Experimental Binodal Data as the Mass Fraction of Polymer (1) + Surfactant (2) + Water (3) ATPS at Temperature T = 25 °C and Pressure p = 0.1 MPaa 100 w1 13.1 14.0 15.0 15.4 15.8 15.9 16.3 16.5

parameters V*213, M1, and M2 are the effective excluded volume of salt, and the molar mass of polymer and salt, respectively. These equations have been used to fit the binodal data of different ATPS through titration method, respectively. Here, the various empirical equations of binodal data were compared and selected for various ATPS based on the corresponding standard deviations. The empirical equations of tie-line, including Othmer−Tobias and Bancroft equations, have been widely reported to correlate the tie-line data25,27,29,33 in ATPS and can be expressed as follows

w3b

100 w2

Standard uncertainties u are u(T) = 0.1 °C, u(w) = 0.001, and u(p) = 10 kPa.

Standard uncertainties u are u(T) = 0.1 °C, u(w) = 0.001, and u(p) = 10 kPa.

⎛ w3t ⎞r k1⎜ t ⎟ ⎝ w1 ⎠

100 w1

a

a

⎛ 1 − w b ⎞n ⎛ 1 − w1t ⎞ 2 ⎜⎜ ⎟⎟ k = ⎟ ⎜ t b ⎝ w1 ⎠ ⎝ w2 ⎠

100 w2

7.3 7.4 7.7 7.9 8.2 8.5 8.7 9.0 9.3

(7) 0.2 0.5 0.9 1.27 1.7 2.0 2.9 3.3 3.5

(8)

where the superscripts t and b represent the compositions in top phase and in bottom phase, respectively. w3 is the mass fraction of water. The parameters k, k1, n, and r along with the corresponding standard deviations for the investigated systems can be obtained by tie-line data in ATPS. Deduction Equations. Figure 1 shows the schematic diagram of ATPS phase diagram. In Figure 1, the points A, B, and M represent the top phase, the bottom phase, and the overall composition, respectively. The curve ACB is the binodal curve, and the line segment AB is the tie-line of the ATPS. The ratio of the mass in point M of the top phase to the bottom phase equals the ratio of lengths of MB to MA, which follows

100 w2

100 w1

100 w2

100 w1

100 w2

100 w1

PEG4000 (1) + Triton X-100 (2) + Water (3) 18.5 17.0 8.7 20.0 3.5 23.4 15.9 17.3 8.0 20.8 2.5 23.8 13.3 17.6 7.5 21.1 1.9 24.7 12.4 18.0 6.8 21.8 1.4 25.3 11.8 18.4 6.1 22.6 1.1 26.0 11.1 18.8 5.3 22.8 1.0 10.5 19.2 4.7 22.8 0.9 9.8 19.5 4.2 23.3 0.8 PEG8000 (1) + Triton X-100 (2) + Water (3) 15.9 9.5 9.6 11.6 4.8 12.6 15.4 9.7 9.0 11.7 4.6 12.7 14.7 9.9 8.5 11.8 4.4 12.9 14.0 10.1 8.1 11.9 4.2 12.1 13.1 10.3 7.7 12.0 4.0 12.3 12.3 10.8 6.6 12.1 3.8 13.1 11.7 11.2 5.7 12.2 3.7 13.3 11.0 11.4 5.2 12.4 3.5 13.7 10.3 11.5 5.0 12.5 3.4 PEG20000 (1) + Triton X-100 (2) + Water (3) 39.1 3.8 14.1 6.4 5.1 8.6 34.0 3.9 14.0 6.6 4.9 8.8 27.3 4.3 12.4 6.8 4.1 9.0 25.9 4.5 11.7 6.9 3.9 9.7 23.2 4.6 10.9 7.2 3.4 9.8 19.4 4.8 9.5 7.8 2.4 9.9 17.1 5.1 8.7 8.2 2.2 10.1 16.1 6.0 6.3 8.2 1.9 10.2 15.2 6.1 6.0 8.5 1.7 10.4

100 w2 0.7 0.6 0.5 0.4 0.3

3.2 3.0 2.7 2.4 2.1 1.5 1.3 1.0

1.6 1.4 1.3 1.1 1.0 0.9 0.7 0.6 0.5

a Standard uncertainties u are u(T) = 0.1 °C, u(w) = 0.001, and u(p) = 10 kPa.

the inverse lever rule. When the density is very similar in both phases, volume ratios (Vr) in point M approximately can be expressed as follows C



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Table 4. Coefficients of Different Equations for the Correlation Binodal Data of Investigated ATPS at Temperature T = 25 °C and Pressure p = 0.1 MPaa coefficients ATPS

a

b

c

d

0.821 −0.039 −0.667 0.771 0.738 0.1407 0.191 0.132

−15.969 −5.992 0.779 −1.598 −3.218 0.168 0.0146 −3.231

36.681 6.285 −19.479 0.771 25.37 0.079 723.86 36.163

−147.08 −80.354 −35.216

best-fit empirical equations for binodal curve w1 = exp(a +

PEG200−C6H5Na3O7 PEG4000−C6H5Na3O7 PEG6000−C6H5Na3O7 CH3CH2OH−(NH4)2SO4 CH3CH2OH−K2HPO4 PEG4000−TritonX-100 PEG8000−TritonX-100 PEG20000−TritonX-100

bw20.5

+ cw2 +

dw22)

w2 = a + bw10.5 + cw1 w2 = aexp(bw10.5 − cw13) w2 = a exp(−w1/b) + c exp(−w1/d) + e w2 = a exp(bw10.5 − cw13)

0.0039 2473.2

e

0.086 −723.7

SDb 0.0056 0.0037 0.0037 0.0061 0.0022 0.0029 0.0067 0.0018

Standard uncertainties u are u(T) = 0.1 °C, u(w) = 0.001, and u(p) = 10 kPa. bSD represents the standard deviations and can be calculated by SD = 1/n(∑ni=1(w1,cal − w1,exp)2)1/2, where n, w1,cal, and w1,exp are the number of binodal data and the calculated and experimental composition 1 mass fraction, respectively.

a

Figure 3. Correlated LLE data of polymer−organic salt ATPS at 25 °C. bottom phases with different TLL.

Vr =

|MB| |MA|

▲,

to combine with eq 9. For instance, eqs 1 and 9 can be expressed as follows

(9)

⎧(1 + V )w f − V w t − w b = 0 r 1 r 1 1 ⎪ ⎪(1 + V )w f − V w t − w b = 0 ⎪ r 2 r 2 2 ⎨ t t 0.5 ⎪ w1 = a + b(w2) + cw2t ⎪ ⎪ w b = a + b(w b)0.5 + cw b ⎩ 1 2 2

For the ATPS phase diagram, LLE data would be obtained at first for ATPE. The tie-line length (TLL) can be regarded as a measurement for the relative difference between the top and the bottom phase. Namely, TLL reflects the influence of the composition concentration on the distribution of the target biomolecules in ATPS. TLL is expressed as follows TLL =

(w1b

−

w1t)2

+

(w2b

−

w2t)2

experimental feed samples; ■, the calculated compositions in top and

(11)

where superscript f represents the overall mass fractions of the composition in TLL, namely, it represents the feed sample of ATPS at M point in Figure 1. Thus, the composition concentration in the top phase (wt2,wt1) and the composition concentration in the bottom phase (wb2,wb1) can be calculated based on the known the overall concentrations (wf2,wt1) and volume ratio (Vr) of the feed samples by eq 11. Several feed samples (> 6) in the two phase region can be found, and the

(10)

First, experimental binodal data of ATPS obtained by titration were collected to fit the empirical equations of binodal data from eqs 1 to 6; the equations can be compared and selected. The coefficients of the empirical equations can be determined. Second, the best fitted empirical equation of binodal data with the lowest standard deviation will be selected D



Figure 4. Correlated LLE data of alcohol−salt ATPS at 25 °C. phases with different TLL.

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▲,

experimental feed samples; ■, the calculated compositions in top and bottom

Figure 5. Correlated LLE data of polymer−surfactant ATPS at 25 °C. bottom phases with different TLL.

▲,

experimental feed samples; ■, the calculated compositions in top and

(eqs 1 to 6) for different ATPS with the lowest fitted standard deviations. The standard deviations of various empirical equations for each investigated ATPS are less than 10−2 according to Table 4. It shows that the empirical equations can be satisfactorily applied to correlate the binodal data of the investigated ATPS. In Figures 3 to 5, the correlated binodal curves of polymer−organic salt ATPS, alcohol−salt ATPS, and polymer−surfactant ATPS with the lowest fitted standard deviations are shown, respectively. Polymer−organic salt ATPS and alcohol−salt ATPS obviously had the better separating phase ability and wider two phase regions than polymer− surfactant ATPS due to the subtle solubility of nonionic surfactants. Specifically, the binodal data fitted of polymer− surfactant ATPS have quite a difference with PEG molar mass increase on the basis of the complex empirical equations form and the clear coefficient value differences in Table 4. As mentioned above, the sensitive solubility of nonionic surfactants would obviously interfere in APTS forming with PEG molar mass increase. On the other hand, the stable and high solubility of ethanol and inorganic salt in alcohol−salt

corresponding tie-lines can be determined. Third, the Othmer− Tobias (eq 7) and Bancroft (eq 8) equations would be correlated by several tie-line data, and the corresponding correlation parameters in two equations could be determined. Last, for various ATPS, any unknown tie-lines can be predicted using eqs 7 and 8 with the known parameters. The process of ATPS LLE data deduction and correlation were shown as the flow diagram in Figure 2.

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RESULTS AND DISCUSSION Polymer−organic salt ATPS including PEG2000, PEG4000, PEG6000, and sodium citrate (C6H5Na3O7), alcohol−salt ATPS including ethanol (CH3CH2OH) and ammonium sulfate ((NH4)2SO4), dipotassium phosphate (K2HPO4), and polymer−surfactant ATPS including PEG4000, PEG8000, PEG20000, and Triton X-100 were chosen to investigate the LLE data. Binodal Curve of ATPS. Tables 1 to 3 show the titrated binodal data of various investigated ATPS. Table 4 shows the values of coefficients in the empirical equations of binodal data E



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Table 5. Experimental Volume Ratio (Vr) and Concentrations of Feed Samples and Calculated Compositions Mass Fraction in Top Phase (wt) and Bottom Phase (wb) at Temperature T = 25 °C and Pressure p = 0.1 MPaa feed samples (wt %) PEG2000 (12, 13) (12.5, 13.5) (13, 14) (13.5, 14.5) (14, 15) (14.5, 15.5) (15, 16) (15.5, 16.5) (17, 18) (17.5, 18.5) (18, 19) (18.5, 19.5) (19, 20) (19.5, 20.5) (20, 21) (20.5, 21.5) (21, 22) PEG4000 (10.5, 16) (11, 17) (11.5, 18) (12, 19) (12.5, 20) (13, 21) (14, 22) (15, 23) (16, 24) (17, 25) (18, 26) (19, 27) PEG6000 (9, 11) (9.5, 11.5) (10, 12) (10.5, 12.5) (11, 13) (11.5, 13.5) (12, 14) (12.5, 14.5) (13, 15) (13.5, 15.5) (14, 16) (15, 17) (16, 18) (17, 19) (18, 20) (19, 21) a

Vr

100 wt2

100 wt1

(1) + C6H5Na3O7 (2) + Water 0.80 5.3 26.7 0.80 4.7 28.8 0.78 4.2 30.9 0.78 3.9 32.4 0.78 3.6 33.8 0.76 3.3 35.6 0.78 3.2 36.4 0.76 2.9 38.1 0.7 2.3 43.7 0.7 2.2 44.8 0.7 2.1 46.0 0.7 2.0 47.2 0.7 1.9 48.5 0.69 1.8 49.9 0.69 1.7 51.2 0.69 1.6 52.4 0.69 1.5 53.5 (1) + C6H5Na3O7 (2) + Water 1.12 5.1 28.0 1.09 4.4 31.1 1.12 3.9 33.3 1.12 3.5 35.5 1.09 3.1 38.0 1.10 2.7 40.0 1.05 2.3 42.9 1.05 2.0 44.9 1.02 1.8 47.4 1.02 1.6 49.3 1.02 1.4 51.3 1.02 1.2 53.3 (1) + C6H5Na3O7 (2) + Water 1.11 5.8 17.9 1.00 5.1 20.7 0.94 4.6 23.0 0.80 4.0 26.1 0.86 3.8 27.1 0.78 3.3 29.9 0.78 3.1 31.2 0.76 2.8 33.0 0.75 2.6 34.5 0.75 2.4 35.9 0.73 2.1 37.8 0.73 1.7 40.3 0.68 1.2 44.4 0.7 0.9 46.1 0.7 0.6 48.5 0.7 0.2 50.9

100 wb2 (3) 17.4 18.7 19.8 21.0 22.1 23.0 24.2 25.1 27.3 28.2 29.2 30.1 31.0 31.8 32.7 33.6 34.5 (3) 16.6 18.3 20.0 21.5 22.8 24.3 26.2 28.6 30.6 32.8 35.0 37.2 (3) 12.6 13.9 15.1 15.9 17.2 17.9 19.0 19.9 20.9 21.9 22.7 24.6 26.1 28.3 30.2 32.1

100 wb1

feed samples (wt %)

Vr

100 wt2

100 wt1

100 wb2

CH3CH2OH (1) + (NH4)2SO4 (2) + Water (3) (15, 30) 1.56 7.2 39.8 27.2 (15.5, 30.5) 1.63 6.4 41.6 30.3 (16, 31) 1.68 5.4 44.0 31.9 (16.5, 31.5) 1.50 4.8 45.8 34.1 (17, 32) 1.50 4.3 47.4 36.1 (17.5, 32.5) 1.50 3.8 49.0 38.1 (18, 33) 1.50 3.4 50.4 40.0 CH3CH2OH (1) + K2HPO4 (2) + Water (3) (16.2, 20.4) 1.57 8.4 28.7 28.5 (16.4, 20.8) 1.57 7.6 30.0 30.2 (16.6, 21.2) 1.57 7.0 31.3 31.7 (16.8, 21.6) 1.57 6.5 32.4 33.1 (17, 22) 1.57 6.0 33.4 34.3 (17.2, 22.4) 1.65 5.6 34.4 35.5 (17.4, 22.8) 1.57 5.2 35.4 36.6 PEG4000 (1) + Triton X-100 (2) + Water (3) (14, 16) 0.86 2.7 20.6 23.8 (14.2, 16.2) 0.92 2.1 21.1 25.4 (14.4, 16.4) 0.94 1.5 21.6 26.5 (14.6, 16.6) 0.94 1.1 22.2 27.2 (14.8, 16.8) 0.98 1.0 22.5 28.3 (15, 17) 1.00 0.8 22.9 29.2 (15.2, 17.2) 1.02 0.7 23.3 30.0 PEG8000 (1) + Triton X-100 (2) + Water (3) (13.4, 8.4) 1.16 6.1 10.7 21.9 (13.6, 8.6) 1.16 4.7 11.6 23.9 (13.8, 8.8) 1.26 4.0 12.2 26.1 (14, 9) 1.24 3.6 12.8 26.9 (14.2, 9.2) 1.27 3.2 13.4 27.8 (14.4, 9.4) 1.24 2.9 13.9 28.6 (14.6, 9.6) 1.32 2.7 14.4 30.3 (14.8, 9.8) 1.32 2.5 14.9 31.0 (15, 10) 1.32 2.3 15.4 31.7 (15.2, 10.2) 1.32 2.2 15.9 32.4 (15.4, 10.4) 1.34 2.0 16.4 33.4 (15.6, 10.6) 1.34 1.9 16.8 34.0 (15.8, 10.8) 1.34 1.8 17.3 34.6 (16, 11) 1.34 1.7 17.8 35.2 PEG20000 (1) + Triton X-100 (2) + Water (3) (13, 4.6) 0.83 2.9 7.6 21.4 (14, 4.8) 0.83 1.8 8.6 24.2 (15, 5) 0.83 1.1 9.5 26.5 (16, 5.2) 0.78 0.5 10.5 28.0 (17, 5.4) 0.80 0.3 11.1 30.3 (18, 5.6) 0.80 0.1 11.8 32.2

2.0 1.3 0.8 0.5 0.3 0.2 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.6 1.6 0.9 0.6 0.4 0.2 0.1 0.0 0.0 0.0 0.0 0.0 3.3 2.3 1.7 1.3 0.9 0.7 0.5 0.4 0.3 0.2 0.1 0.1 0.0 0.0 0.0 0.0

100 wb1 14.7 12.5 11.4 10.1 8.9 7.8 6.9 7.4 6.3 5.4 4.7 4.0 3.5 3.0 12.0 11.7 11.5 11.4 11.2 11.1 11.0 5.8 5.2 4.5 4.3 4.0 3.8 3.3 3.1 2.9 2.7 2.4 2.2 2.0 1.8 2.1 1.6 1.3 1.0 0.8 0.6

Standard uncertainties u are u(T) = 0.1 °C, u(w) = 0.001, u(Vr) = 0.01, and u(p) = 10 kPa.

ATPS simplifies the binodal data fitted with eq 1 or 3 only containing three unknown coefficients. Hence, empirical equations of binodal data for various ATPS become more complicated with more unknown coefficients due to the solubility differences of the composition in ATPS. Tie-Lines of ATPS. Several biphase feed samples (> 6) and their volume ratios in different ATPS were experimentally determined as widely as possible. Table 5 and Figures 3 to 5 show the calculated tie-lines data including the two composition concentrations in the top or bottom phase across the experimental feed samples. The ranges of TLL and

compositions concentrations in various ATPS are summarized in Table 6, and the ranges of TLL cannot be enlargened by the experiments. The TLL ranges of polymer−organic salt ATPS or alcohol−salt ATPS are generally wider than polymer− surfactant ATPS, which would be caused by the solubility of two compositions in different ATPS. For example, the TLL range of CH3CH2OH-(NH4)2SO4 ATPS is larger than that of CH 3 CH 2 OH-K 2 HPO 4 ATPS because the solubility of (NH4)2SO4 is higher than that of K2HPO4 in aqueous solution at 25 °C. Moreover, the white and granuliform sediments will appear in the two alcohol−salt ATPS with high concentrations F



Article

Table 6. Range of Calculated Composition Mass Fractions in Top and Bottom Phases and Range of TLL in Various ATPS ATPS

range of 100 wt2

PEG2000−C6H5Na3O7 PEG4000−C6H5Na3O7 PEG6000−C6H5Na3O7

1.51−5.3 1.2−5.1 0.2−5.8

CH3CH2OH−(NH4)2SO4 CH3CH2OH−K2HPO4

3.4−7.2 4.9−8.4

PEG4000−Triton X-100 PEG8000−Triton X-100 PEG20000−Triton X-100

0.1−1.7 1.7−6.1 0.1−2.9

range of 100 wt1

range of 100 wb2

Polymer−Organic Salt 26.7−53.5 28.0−53.3 17.9−50.9 Alcohol−Salt 39.8−50.4 28.7−36.3 Polymer−Surfactant 21.8−25.3 10.7−17.8 7.6−11.8

range of 100 wb1

range of TLL

17.4−34.5 16.6−37.2 12.6−32.1

0−2.0 0−2.6 0−3.3

27.5−62.9 27.9−64.4 16.1−60.1

27.2−40.0 28.5−37.6

6.9−14.7 2.7−7.4

32.1−57.0 29.4−47.0

24.6−30.6 21.9−35.2 21.4−32.2

8.9−11.0 1.9−5.8 0.6−2.1

25.3−34.7 16.5−37.2 19.2−34.0

Figure 6. Linear dependency of Othmer−Tobias (2.2.7) and Bancroft (2.2.8) equations for PEG−C6H5Na3O7 ATPS.

Figure 7. Linear dependency of Othmer−Tobias (2.2.7) and Bancroft (2.2.8) equations for CH3CH2OH−salt ATPS.

Figure 8. Linear dependency of Othmer−Tobias (2.2.7) and Bancroft (2.2.8) equations for PEG−Triton X-100 ATPS.

sediment at higher surfactant concentrations in PEG4000Triton X-100 ATPS. For polymer−surfactant ATPS, the twophase regions and Triton X-100 concentrations in the top phase (PEG enriched) increase with the increase in PEG molar mass.

of two inorganic salts. The polymer−organic salt ATPS also have a wider TLL range due to the higher solubility of sodium citrate (720 g/L, 25 °C). As a nonionic surfactant, the solubility of Trition X-100 is lower than the salt solubility and varies with the polymer molecules.34 There was white and viscous G



Article

Table 7. Values of Parameters in Othmer−Tobias (eq 7) and Bancroft (eq 8) Equations for Various ATPS k

ATPS PEG2000−C6H5Na3O7 PEG4000−C6H5Na3O7 PEG6000−C6H5Na3O7

0.3799 0.504 0.3423

CH3CH2OH−(NH4)2SO4 CH3CH2OH−K2HPO4

0.7166 1.1555

PEG4000−Triton X-100 PEG8000−Triton X-100 PEG20000−Triton-X-100

2.1483 2.7367 3.8183

R2

n

Polymer−Organic Salt ATPS 1.284 0.998 0.9851 0.996 1.3018 0.996 Alcohol−Salt ATPS 0.7719 0.995 0.8359 0.999 Polymer−Surfactant ATPS 0.5067 0.995 0.9 0.995 0.8865 0.996

k1

r

R2

2.1898 2.0765 2.3229

0.7881 1.0296 0.7724

0.998 0.997 0.995

1.4838 0.9018

1.2126 1.1609

0.995 0.999

0.129 0.3301 0.2066

2.2979 1.0993 0.8865

0.995 0.996 0.996

Table 8. Predicted Feed Samples (Feedpre) Mass Fraction of Midpoints with Different TLL and Experimental Data of Volume Ratio (Vr,exp) of Various ATPS at Temperature T = 25 °C and Pressure p = 0.1 MPaa feedpre (wt %)

TLL

Vr,exp

feedpre (wt %)

PEG2000−C6H5Na3O7 (14.91, 21.8) 52.2 (14.55, 21.1) 50.0 (13.89, 19.77) 45.7 (13.4, 18.7) 42.3 (12.94, 17.68) 39.1 (12.53, 16.71) 35.9 (12.15, 15.79) 33.0 (11.8, 14.91) 30.1

1.02 1.05 1.00 1.00 1.00 1.00 1.00 1.00

(18.21, (17.32, (16.55, (15.57, (14.27, (13.25, (12.41, (11.71, (11.11,

SDb

0.0169

SDb

CH3CH2OH−(NH4)2SO4 (21.36, 28.51) 55.7 (20.58, 28.21) 51.9 (19.9, 27.97) 48.4 (19.3, 27.76) 45.1 (18.76, 27.59) 41.9 (18.28, 27.45) 38.9 SDb PEG4000−Triton X-100 (13.39, 16.52) 23.4 (13.59, 16.44) 25.0 (13.89, 16.44) 26.9 (14.58, 16.74) 29.5

SDb

1.00 0.98 1.02 1.07 1.02 1.02 0.0290 1.04 1.04 1.04 1.04

0.0141

(21.11, (20.78, (20.39, (19.94, (19.54, (21.11, SDb

TLL

PEG4000−C6H5Na3O7 25.82) 62.4 24.78) 59.3 23.87) 56.5 22.65) 52.8 20.93) 47.4 19.49) 42.8 18.23) 38.7 17.11) 35.0 16.1) 31.6

Vr,exp

feedpre (wt %)

1.02 1.00 1.05 0.96 1.00 0.98 0.98 1.00 1.00 0.0242

CH3CH2OH−K2HPO4 19.83) 46.1 19.12) 44.5 18.88) 42.3 18.64) 39.7 18.43) 37.2 19.83) 46.1

PEG8000−Triton X-100 (18.18, 9.63) 36.1 (17.56, 9.39) 32.7 (16.6, 8.97) 30.6 (16.86, 8.72) 27.6 (15.34, 8.6) 25.2 (15, 8.569) 23.2 SDb

TLL

PEG6000−C6H5Na3O7 (15.23, 24.17) 58.0 (14.32, 22.55) 53.1 (13.45, 20.9) 48.2 (12.66, 19.3) 43.5 (11.94, 17.78) 39.1 (11.29, 16.35) 34.9 (10.72, 15.03) 31.0 (10.21, 13.81) 27.4 (9.77, 12.68) 24.0 (9.38, 11.66) 20.9 SDb

Vr,exp 1.00 1.02 0.96 0.91 1.00 1.00 0.91 0.96 0.98 1.07 0.0464

0.98 1.02 1.00 1.05 1.07 0.98 0.0362 0.98 0.98 1.07 1.07 1.02 1.00 0.0379

(12.37, (12.71, (13.15, (13.76, (14.08, (14.96, SDb

PEG20000−Triton X-100 5.05) 20.6 5.08) 22.4 5.17) 24.5 5.34) 26.9 5.45) 28.0 5.78) 30.8

0.96 1.00 0.98 0.96 0.98 0.98 0.0150

Standard uncertainties u are u(T) = 0.1 °C, u(Vr) = 0.01, and u(p) = 10 kPa. bSD represents the standard deviations and can be calculated by SD = 1/n(∑ni=1(1 − Vr,exp,i)2)1/2, where n is the number of predicted feed samples. a

Application of Tie-Line Empirical Equations. The above tie-line data in various ATPS can be used to determine the parameters of eqs 7 and 8. The linear dependencies of the plots ln((1 − w1t)/w1t) against ln((1 − w2b)/w2b), and ln(w3b/w2b) against ln(w3t/w1t) can indicate the consistency of the calculated tie-line compositions. For the three kinds of ATPS, the correlated linear dependencies are shown in Figures 6, 7, and 8, respectively. Table 7 shows the fitted results by eqs 7 and 8 for each investigated ATPS. The high fit degrees (R2 > 0.99) were obtained though the method combining the simple titration experiments and empirical equation correlations. The result is no less accurate than that by the concentration detection methods.22,23,25 Moreover, the feed samples of

predicted tie-lines midpoints were examined, of which the volume ratios (Vr) values should equal 1. Table 8 shows the experimental Vr values of feed samples in predicted tie-lines midpoints with different TLL for various ATPS. An acceptable consistency (SD < 0.05) between the experimental and the predicted tie-line data was obtained. Though this method, almost parallel tie-lines with different TLL and several feed samples with different Vr values can be predicted by the Othmer−Tobias (eq 7) and Bancroft (eq 8) equations.

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CONCLUSIONS ATPS LLE data determination was discussed in various ATPS including polymer−organic salt ATPS, alcohol−salt ATPS, and H



Article

(12) Amid, M.; Shuhaimi, M.; Islam Sarker, M. Z.; Abdul Manap, M. Y. Purification of serine protease from mango (Mangifera Indica Cv. Chokanan) peel using an alcohol/salt aqueous two phase system. Food Chem. 2012, 132 (3), 1382−1386. (13) Guo, Y. X.; Han, J.; Zhang, D. Y.; Wang, L. H.; Zhou, L. L. An ammonium sulfate/ethanol aqueous two-phase system combined with ultrasonication for the separation and purification of lithospermic acid B from Salvia miltiorrhiza Bunge. Ultrason. Sonochem. 2012, 19 (4), 719−724. (14) Li, Z.; Teng, H.; Xiu, Z. Aqueous two-phase extraction of 2,3butanediol from fermentation broths using an ethanol/ammonium sulfate system. Process Biochem. 2010, 45 (5), 731−737. (15) Everberg, H.; Leiding, T.; Schiöth, A.; Tjerneld, F.; Gustavsson, N. Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/ polymer aqueous two-phase partitioning for proteome analysis. J. Chromatogr., A 2006, 1122 (1−2), 35−46. (16) Sivars, U.; Tjerneld, F. Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins. BBA - Gen. Subjects 2000, 1474 (2), 133−146. (17) Deng, G.; Lin, D.-Q.; Yao, S.-J.; Mei, L.-H. Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation. J. Chromatogr., B 2007, 852 (1−2), 167−173. (18) Mazzola, P. G.; Lopes, A. M.; Hasmann, F. A.; Jozala, A. F.; Penna, T. C. V.; Magalhaes, P. O.; Rangel-Yagui, C. O.; Pessoa, A., Jr. Liquid−liquid extraction of biomolecules: an overview and update of the main techniques. J. Chem. Technol. Biottechnol. 2008, 83 (2), 143− 157. (19) Wang, Z.; Dai, Z. Extractive microbial fermentation in cloud point system. Enzyme Microb. Technol. 2010, 46 (6), 407−418. (20) Zafarani-Moattar, M. T.; Hamzehzadeh, S. Liquid−liquid equilibria of aqueous two-phase systems containing polyethylene glycol and sodium succinate or sodium formate. CALPHAD: Comput. Coupling Phase Diagrams Thermochem. 2005, 29 (1), 1−6. (21) Liu, Y.; Wu, Z.; Dai, J. Phase equilibrium and protein partitioning in aqueous micellar two-phase system composed of surfactant and polymer. Fluid Phase Equilib. 2012, 320, 60−64. (22) Murugesan, T.; Perumalsamy, M. Liquid−liquid equilibria of poly(ethylene glycol) 2000 + sodium citrate + water at (25, 30, 35, 40, and 45) °C. J. Chem. Eng. Data 2005, 50 (4), 1392−1395. (23) Hu, M.; Zhai, Q.; Jiang, Y.; Jin, L.; Liu, Z. Liquid−liquid and liquid−liquid−solid equilibrium in PEG + Cs2SO4 + H2O. J. Chem. Eng. Data 2004, 49 (5), 1440−1443. (24) de Oliveira, R. M.; Coimbra, J. S. d. R.; Francisco, K. R.; Minim, L. A.; da Silva, L. H. M.; Pereira, J. A. M. Liquid−liquid equilibrium of aqueous two-phase systems containing poly(ethylene) glycol 4000 and zinc sulfate at different temperatures. J. Chem. Eng. Data 2008, 53 (4), 919−922. (25) Zafarani-Moattar, M. T.; Hamzehzadeh, S.; Hosseinzadeh, S. Phase diagrams for liquid−liquid equilibrium of ternary poly(ethylene glycol)+di-sodium tartrate aqueous system and vapor−liquid equilibrium of constituting binary aqueous systems at T = (298.15, 308.15, and 318.15)K: Experiment and correlation. Fluid Phase Equilib. 2008, 268, 142−152. (26) Chen, Y.; Meng, Y.; Zhang, S.; Zhang, Y.; Liu, X.; Yang, J. Liquid−liquid equilibria of aqueous biphasic systems composed of 1butyl-3-methyl imidazolium tetrafluoroborate + sucrose/maltose + water. J. Chem. Eng. Data 2010, 55 (9), 3612−3616. (27) Feng, Z.; Li, J. Q.; Sun, X.; Sun, L.; Chen, J. Liquid−liquid equilibria of aqueous systems containing alcohol and ammonium sulfate. Fluid Phase Equilib. 2012, 317, 1−8. (28) Shekaari, H.; Sadeghi, R.; Jafari, S. A. Liquid−liquid equilibria for aliphatic alcohols + dipotassium oxalate + water. J. Chem. Eng. Data 2010, 55 (11), 4586−4591. (29) Xie, X.; Yan, Y.; Han, J.; Wang, Y.; Yin, G.; Guan, W. Liquid− liquid equilibrium of aqueous two-phase systems of PPG400 and

polymer−surfactant ATPS. The method can correlate and predict binodal curve data and tie-line data of different ATPS by combining titration experiments and empirical equations correlations quite accurately. The results showed that both the two-phase regions and the TLL ranges of polymer-organic salt ATPS or alcohol−salt ATPS were generally wider than polymer−surfactant ATPS due to the solubility of two compositions in different ATPS. Othmer−Tobias and Bancroft equations fitted by LLE data can be used to predict the unknown tie-line data, and the linear dependency of Othmer− Tobias and Bancroft equations showed good agreement with experimental data.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 754 86502749. Fax: +86 754 86502726. E-mail: [email protected]. Funding

This work is supported by the National Natural Science Foundation of China (No. 21006062), Science and Technology Planning Project of Guangdong Province, China (No. 2012B060400006), Educational Commission of Guangdong Province, China (No. 2012KJCX0052). Notes

The authors declare no competing financial interest.

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