Influence of Different Phase-Forming Parameters on the Phase

Article pubs.acs.org/jced

Influence of Different Phase-Forming Parameters on the Phase Diagram of Several PEG−Salt Aqueous Two-Phase Systems Anna Glyk, Thomas Scheper, and Sascha Beutel* Institute of Technical Chemistry, Leibniz University of Hannover, Callinstrasse 5, 30167 Hannover, Germany S Supporting Information *

ABSTRACT: Different poly(ethylene glycol) (PEG) + potassium phosphate or sodium citrate aqueous two-phase systems (ATPS) were investigated at 23 °C, containing different PEG types (molecular weights 2000 g·mol−1 to 8000 g·mol−1) and pH values (5 to 9). Furthermore, the effect of the added salt NaCl (0 wt % to 8 wt %) on the PEG + potassium phosphate/sodium citrate ATPS was studied at 23 °C. The experimental binodal data were successfully correlated with the empirical nonlinear equation proposed by Hu. The effects of increasing molecular weight of PEG, pH, NaCl, and salt type on the obtained binodal curves were determined, resulting in a binodal curve shift toward the origin. Thus, an expansion of the two-phase region occurred by increasing molecular weight of the PEG, pH, and NaCl and due to the Gibbs free energy of hydration of ions of phosphate. Furthermore, the phase equilibrium compositions, tie-line lengths, slopes of tie-lines, critical points, and effective excluded volumes were obtained for all studied systems. Finally, the experimental tie-line compositions were successfully correlated by using the Othmer−Tobias and Bancroft equations, and linear dependency was confirmed.

1. INTRODUCTION

PEG, pH, NaCl concentration, and salt type for both mentioned ATPS has never been studied systematically. In the present work, the phase diagrams of different poly(ethylene glycol) (PEG) + potassium phosphate or sodium citrate ATPS were studied at 23 °C, containing different molecular weights of PEG, (2000, 4000, 6000, and 8000) g· mol−1, pH values (5, 6, 7, 8, and 9) as well as different NaCl concentrations, (0, 2, 4, 6, and 8) wt %. The binodal curves and several tie-lines were derived from the experiments. Additionally, the binodal data were successfully correlated with the Hu9 equation, and the experimental two-phase equilibrium data were fitted with Othmer−Tobias10 and Bancroft11 equations. The obtained results can be used for choosing suitable systems for the purification of individual biomolecules.

Aqueous two-phase systems (ATPS) are biphasic systems which are formed when either two incompatible water-soluble polymers, such as poly(ethylene glycol) (PEG) and dextran (polymer−polymer ATPS) or a polymer and a salt (polymer− salt ATPS) are dissolved in water beyond a critical concentration, resulting in two coexisting immiscible waterrich phases. These systems, introduced in 1965 by Albertsson, have been used in the separation of biomolecules, like proteins, enzymes, and nucleic acids due to their gentle aqueous environment.1−3 ATPS provide several advantages over conventional methods for the isolation and purification of biomolecules, such as biocompatibility, simplicity, nontoxicity, and easy scale-up potential. Among ATPS suitable for bioseparation, those formed by polymer and salt have been used predominantly due to their low cost and low viscosity, as well as short separation time.1,27 The selective distribution of ATPS components are influenced by different factors like molecular weight of polymer, type of salt, initial composition of the system, pH, and temperature.1,4 Therefore, information regarding the phase diagram as well as the physical properties of the phase-forming components in both phases is required for the development, design, simulation, optimization, and operation of separation processes using ATPS, thus predicting the separation behavior.5,6 Numerous polymer−salt ATPS containing PEG and potassium phosphate or sodium citrate have been investigated to understand the principles of aqueous twophase formation.2,7,8 However, the effect of molecular weight of © 2014 American Chemical Society

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(ethylene glycol) (PEG) with an average molecular weight of 2000 g·mol−1 (PEG 2000) was purchased from Carl Roth GmbH & Co. KG (Germany). PEG with an average molecular weight of 4000 g·mol−1 (PEG 4000), 6000 g·mol−1 (PEG 6000), and 8000 g·mol−1 (PEG 8000) was obtained from Sigma-Aldrich (Germany). Potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), citric acid monohydrate (C6H6O7·H2O), trisodium citrate dihydrate (C6H5Na3O7·2H2O) and sodium chloride (NaCl) were supplied by Carl Roth GmbH & Co. KG Received: November 19, 2013 Accepted: February 11, 2014 Published: February 19, 2014 850

dx.doi.org/10.1021/je401002w | J. Chem. Eng. Data 2014, 59, 850−859

Journal of Chemical & Engineering Data

Article

citrate concentrations (0−3 wt % after dilution) were measured in both phases, at 25 °C, by electrical conductivity using a conductivity meter (InoLab Cond 730, WTW GmbH, Germany) with a standard conductivity cell (TetraCon 325, WTW GmbH, Germany) precise to within ± 0.0002 mS·cm−1. The constant temperature of 25 °C was maintained by placing the samples in a thermostatic bath (WGW Lauda M3, Lauda GmbH & Co. KG, Germany). The temperature uncertainty was about ± 0.05 °C. The PEG concentrations (0−10 wt % after dilution) in both phases were determined by refractive index measurements performed at 20 °C using a refractometer (RM40, Mettler-Toledo GmbH, Germany) with a precision of ± 0.0001. All measurements were performed in triplicate, and the average results are reported. The refractive index, nD, and the mass fraction of polymer, wp, and salt, ws, were determined by the following equation (eq 1) successfully applied by Cheluget12 nD = a0 + a1wp + a 2ws (1)

(Germany). All chemicals were of analytical grade with a minimum purity of 99 % and therefore used as received, without further purification. 2.2. Preparation of Phase-Forming Solutions. Phase systems were prepared from stock solutions of PEG (40 wt %), phosphate (30 wt %), citrate (30 wt %), and NaCl (20 wt %). All required solutions were prepared in deionized water (Arium 611, Sartorius Stedim Biotech, Germany). Phosphate stock solutions consisted of a mixture of KH2PO4 and K2HPO4 at appropriate pH (6, 7, 8, and 9) values. Citrate stock solutions were prepared by mixing C6H6O7·H2O and C6H5Na3O7·2H2O at different pH (5, 6, 7, and 8) values. The desired pH of the buffer solutions was obtained by changing the ratio of corresponding mono- and dibasic salts. All experiments were carried out at different pH (5, 6, 7, 8, and 9) values at constant temperature (23 °C) and different concentrations of NaCl (0, 2, 4, 6, and 8) wt %. The pH values of the solutions were measured precisely with a pH meter (Professional Meter PP-15, Sartorius Stedim Biotech, Germany) with a standard pH platinum electrode (PY-P11, Sartorius Stedim Biotech, Germany) containing a precision of ± 0.002. The constant temperature of 23 °C was maintained by placing the samples in a thermostatic bath (WGW Lauda M3, Lauda GmbH & Co. KG, Germany). The temperature uncertainty was about ± 0.05 °C. All weighing was carried out on an analytical weight balance with a precision of ± 0.0001 g (ED224S, Sartorius Stedim Biotech, Germany). Deionized water was used for all diluting purposes. 2.3. Determination of Binodal Curve. The binodal curves were determined experimentally by using the cloud point method. For this purpose, the concentrated (30 wt %) stock solution of salt (phosphate and citrate) was added dropwise to a known amount (5 g) of the concentrated (40 wt %) stock solution of PEG (2000, 4000, 6000, and 8000) in a conical flask by stirring for 1 min until turbidity appeared, indicating two-phase formation. Then, the biphasic system was diluted dropwise with water until turbidity disappeared and the solution became clear below the cloud point, which indicated the transition from two-phase to one-phase region. The composition of the mixture for each point on the binodal curve was calculated by measuring the weight of the added deionized water and salt using the above-mentioned analytical balance. The procedure of increasing the amount of water and salt was repeated in triplicate until enough data were obtained to form the binodal curve. For systems additionally containing NaCl, the same amount of the neutral salt was dissolved in each stock solution and in water, ensuring a constant concentration of NaCl during the performance of the cloud point method. 2.4. Determination of Tie-Line Length (TLL). For the determination of TLL, several ATPS of at least five different known total compositions were prepared in 15 mL tubes. Each mixture was prepared in triplicate. The solutions were vigorously mixed with a rotator-mixer (Multi Bio RS-24, Biosan, Latvia) for 15 min at 100 rpm. Then the samples were centrifuged at 4000g and 4 °C for 15 min (Megafuge 1.0 RS, Heraeus Instruments, Germany), accelerating the formation of ATPS. The systems were allowed to settle down for about 24 h at 23 °C in the thermostatic bath to ensure thermodynamic equilibrium. After the careful separation of the two transparent phases using a pipet and the conveniently dilution for analysis (see in the following), the compositions of the equilibrium phases were determined by analyzing the concentrations of the phase-forming compounds in both phases. The phosphate and

where a0 is the refractive index of pure water, while a1 and a2 are the fitting parameters corresponding to polymer and salt, respectively, obtained by regression analysis from linear calibration plots of refractive index measurements of solution. The uncertainty of mass fraction of PEG achieved using eq 1 was better than 0.002. The values of these coefficients and respective regression coefficient values, R2, are listed in Table 1. Table 1. Values of Coefficients of eq 1 for PEG,a Sodium Citrate,b and Potassium Phosphatec with Different pH Values, and Water at 23 °C, together with the Corresponding R2 Values system PEG−sodium citrate−H2O PEG−potassium phosphate−H2O

a0

a1

R2

a2 −3

1.3330

1.375 × 10

1.3330

1.375 × 10−3

1.745 × 10

−3

1.420 × 10−3

0.9999 0.9999

a

PEG of varying molecular weight: PEG 2000 to PEG 8000. bSodium citrate of varying pH value: pH 5 to pH 8. cPotassium phosphate of varying pH value: pH 6 to pH 9.

The coefficients a1 and a2 were independent of the polymers molecular weight, which was also reported for other PEG + salt systems.9 Furthermore, these coefficients were independent of the pH of phosphate and citrate. Equation 1 is valid only up to concentrations of 10 wt % polymer and 5 wt % phosphate/ citrate. Beyond these concentrations, linearity is not maintained because of the proximity to the two-phase region.9 Thus, before the refractive index measurements, it was necessary to dilute the samples to the above-mentioned mass fraction range.

3. RESULTS AND DISCUSSION 3.1. Correlation of Binodal Curve. The region below the binodal curve represents the single-phase and above it represents the two-phase region, thus a binodal curve is a boundary line between them. The binodal data were obtained by the cloud point method for PEG of varying molecular weight + potassium phosphate/sodium citrate + water system at various pH values and different amounts of NaCl. As an example, the experimental binodal data of PEG of varying molecular weight + potassium phosphate/sodium citrate + water system obtained at pH 7 and T = 23 °C are shown in Figures 1 and 2, as well as 5, and presented in the Supporting 851



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Figure 1. Tie-lines of PEG (p) + potassium phosphate (s) + water (w) system at different PEG molecular weights, pH 7, and T = 23 °C. (A) PEG 2000; (B) PEG 4000; (C) PEG 6000; (D) PEG 8000: ■, experimental binodal data; calculated binodal data using eq 2 (···); right pointing triangle, experimental tie-lines with overall compositions (middle points of TLL); calculated tie-line data using eqs 5 and 6 (); determined critical points, cp, by extrapolation (---). All compositions are in mass fraction.

3.2. Correlation of TLL. The equilibrium phase compositions of the two-phases were related by TLL which were calculated according to eq 3

Information in Tables S6 and S7, respectively. Furthermore, several binodal curves for PEG 4000 + potassium phosphate/ sodium citrate + water ATPS at different pH values, NaCl concentrations, different salt type, and T = 23 °C are shown exemplarily in Figures 6 to 8, as well as supplied in tabular form in the Supporting Information in Tables S8 to S11, respectively. It can be seen from these figures that each ATPS component had a particular influence on the binodal curve. These effects of each ATPS constituent are discussed separately ahead. There are several correlations available in the literature to correlate with the binodal data.11,13−16 However, the following empirical nonlinear equation (eq 2) proposed by Hu9 shows the best fitting for the presented experimental binodal data wp = a + bws 0.5 + cws + dws 2

TLL =

Δwp2 + Δws 2

(3)

where Δwp and Δws are the differences between the concentrations of PEG and salt in the top and bottom phases expressed in mass fractions, and the slope of tie-line (STL) was determined from the concentration difference of PEG and salt between the equilibrium phases using eq 4 STL =

Δwp Δws

(4)

A series of TLL in the two-phase region of the binodal curve were investigated and are given exemplarily in the Supporting Information in Table S1 and shown in Figures 1 and 2 for the PEG + potassium phosphate/sodium citrate + water system at different PEG molecular weights, pH 7, and T = 23 °C, together with the overall composition, TLL, STL, and cp. Additionally, Tables S2 and S3 in the Supporting Information provide the corresponding experimental phase equilibrium compositions of ATPS containing the PEG 4000 + potassium phosphate/sodium citrate + water system at different pH values, different NaCl concentrations, and T = 23 °C, respectively. It is apparent from these tables that the higher is the salt concentration in the bottom phase and the lower the PEG concentration is, and vice versa, the higher is the TLL. The TLL increases due to an increase in hydrophobicity of the

(2)

where a, b, c, and d are the fitting parameters. These coefficients were determined by a least-squares regression of the binodal data. The fitting coefficients along with the corresponding R2 values are shown in the Supporting Information in Tables S12 and S13 for all investigated binodal curves, respectively. On the basis of the obtained high R2 values eq 2 can be successfully used for the correlation of the present experimental binodal data, ensuring that the binodal data points are well fitted with the correlation. Therefore, eq 2 can be taken as a widely suitable equation for the binodal data fitting of PEG−salt ATPS. 852



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Figure 2. Tie-lines of PEG (p) + sodium citrate (s) + water (w) system at different PEG molecular weights, pH 7, and T = 23 °C. (A) PEG 2000; (B), PEG 4000; (C), PEG 6000; (D), PEG 8000: ■, experimental binodal data; calculated binodal data using eq 2 (···); right pointing triangle, experimental tie-lines with overall compositions (middle points of TLL); calculated tie-line data using eqs 5 and 6 (); determined critical points, cp, by extrapolation (---). All compositions are in mass fraction.

Figure 3. Linear dependency of Othmer−Tobias equation (eq 5) for (A) PEG (p) + potassium phosphate (s) + water (w) system, and (B) PEG (p) + sodium citrate (s) + water (w) System at pH 7, T = 23 °C, and different PEG molecular weights: ■, PEG 2000; ●, PEG 4000; ▲, PEG 6000; ◆, PEG 8000.

phases.17 The TLL were obtained by a linear regression of each corresponding set of overall, top, and bottom phase composition. As an example, the tie-lines for the systems containing PEG + potassium phosphate/sodium citrate + water at pH 7, T = 23 °C and different PEG molecular weights are shown in Figures 1 and 2, respectively. For all of the investigated systems, the overall system composition has no significant effect upon the STL, which implies that tie-lines are parallel to each other, thus allowing us to know the coexisting phase compositions for any given overall polymer−salt composition.18 In each binodal

curve there is a critical point, cp, in which the composition and volume of the two liquid phases become almost equal (TLL = 0).19,20 As illustrated in Figures 1 and 2, cp divides a binodal curve into a one- and two-phase region. The location of cp for the studied systems was estimated by extrapolation through the middle points of a number of tie-lines.20 The tie-line compositions of the studied ATPS were correlated by using the following equations proposed by Othmer−Tobias10 (eq 5) and Bancroft11 (eq 6), which were earlier successfully applied for other polymer−salt systems:21,22 853



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Figure 4. Linear dependency of Bancroft equation (eq 6) for (A) PEG (p) + sodium citrate (s) + water (w) system, and (B) PEG (p) + sodium citrate (s) + water (w) system at pH 7, T = 23 °C, and different PEG molecular weights: ■, PEG 2000; ●, PEG 4000; ▲, PEG 6000; ⧫, PEG 8000.

Figure 5. Binodal curves of (A) PEG (p) + potassium phosphate (s) + water (w) system, and (B) PEG (p) + sodium citrate (s) + water (w) system at different PEG molecular weights, pH 7, and T = 23 °C. Experimental data: ■, PEG 2000; ●, PEG 4000; ▲, PEG 6000; ⧫, PEG 8000. Calculated data using eq 2 (). All compositions are in mass fraction.

⎛ 100 − w T ⎞ ⎛ 100 − w B ⎞n p s ⎟ = k OT⎜ ⎜ ⎟ ⎟ ⎜ B T w w ⎝ ⎠ ⎠ ⎝ s p

(5)

⎛ w T ⎞r ⎛w B⎞ w ⎜ B ⎟ = kB⎜⎜ wT ⎟⎟ ⎝ ws ⎠ ⎝ wp ⎠

(6)

T

wwT,

wsB,

results. The R2 values of both correlations that are closer to one are the result of the degree of consistency of the experimental tie-line data. The R2 values reported in the Supporting Information in Tables S4 and S5 are always greater than 0.999. On the basis of these values, eqs 5 and 6 can be successfully used to correlate the tie-line data of the investigated systems. Furthermore, the results proved the reliability of the calculation method and the corresponding tieline data. 3.3. Effect of Molecular Weight of PEG on the Binodal Curve. The effect of PEG molecular weight on binodal curve or rather phase-forming ability of PEG + potassium phosphate/ sodium citrate + water systems at pH 7, T = 23 °C, and different PEG molecular weights is shown as an example in Figure 5. It can be seen that the binodal curves have similar shapes for the different PEG molecular weights, respectively.23,24 However, the two-phase region is expanded with an increase of PEG molecular weight, which is in good agreement with the reported experimental results for other ATPS in literature.25−29 Thus, a small shift of the binodal curves to the origin to lower PEG and salt concentrations is indicated for all investigated systems as the PEG molecular weight increases, requiring lower concentrations for phase separation or rather aqueous two-phase formation. In a similar way the salt concentration is removed in the PEG-rich top phase as the molecular weight of PEG increases. A similar

wwB,

where wp , and are the mass fractions of the phase-forming components. Superscripts T and B represent the polymer-rich phase (top phase) and the salt-rich phase (bottom phase), respectively. Subscripts p, s, and w stand for PEG, salt, and water, respectively; kOT, n, kB, and r represent the fitting parameters to be determined. The determination of the fitting coefficients of eqs 5 and 6 is performed by linearization of both sides of the equations. The values of the estimated fitting coefficients, as well as the corresponding R2 values are reported in the Supporting Information in Table S1 for all systems presented in Figures 1 and 2. The efficiency of the Othmer−Tobias as well as the Bancroft correlation is measured through the linearity of the experimental tie-line data. Using the tie-line data reported in Supporting Information, Table S1, a linear dependency of the plots log [(100 − wpT)/ wpT] against log [(100 − wsB)/wsB], as well as log [wwB/wsB] against log [wwT/wpT], is obtained and shown exemplarily in Figures 3 and 4, indicating an acceptable consistency of the 854



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the PEG molecular weight as above-mentioned. This means that higher EEV values correspond to a higher salting-out ability of polymer in polymer + salt + water systems.34,35 The higher EEV values are reflected in the phase diagrams in Figure 5 by a shift in the position of the binodal curve toward the origin, corresponding to an increase of the two-phase region, and thus a lower concentration of polymer is needed to form two-phase systems. This is in accordance with the reported literature for other systems.37 3.4. Effect of pH on the Binodal Curve. The effect of pH on the binodal curve or rather phase-forming ability of ATPS containing PEG 4000 + potassium phosphate/sodium citrate + water systems at T = 23 °C, and different pH values is shown as an example in Figure 6. It can be seen that the binodal curves have similar shapes for the different pH values, respectively.18 However, the two-phase region is expanded with increasing pH value, which is in good agreement with the reported experimental results for other ATPS in literature.18,38 Hence, the binodal curves shift to the origin, downward to higher pH values, or rather lower PEG and salt concentrations, requiring lower concentrations for phase separation at basic pH values with higher ratios between di- and trivalent salt ions. This is in agreement with the fact that trivalent ions are more efficient than divalent ions in promoting two-phase formation.40 Furthermore, the binodal curve shift at increasing pH indicates that either the volume exclusion or the salting-out effect predominate in phase separation.18 A similar behavior was also observed for ATPS at the other studied PEG molecular weights (data not shown). A similar behavior was also noticed by authors for other ATPS.16,38 The effect of the pH value on the location of the binodal curve may be explained by a change of the solute charge or the ratio of the charged salt ions present in the aqueous solution.40 It may be that the hydrogen-bond interactions of PEG are weakened at higher pH values like those in ATPS containing poly(propylene glycol), PPG, which is structurally closely related to PEG as reported in literature.18,38 Here, the expansion of the two-phase region by increase of the pH value may be related to the salting-out effect mentioned above, resulting from the weakening of the PEG−solvent interactions. This may be explained based on the salt’s ability of promoting the water structure.36 On the basis of the effect of kosmotropic salts to promote interactions between water molecules, when a kosmotropic salt like potassium phosphate or sodium citrate is dissolved in an aqueous solution, the hydration effect of salt will occur, in which the salt ions are surrounded by a layer of water molecules. This hydration process results in a structuring and immobility of water molecules, in such a way that there is a reduction of their function as a solvent for other molecules. By addition of a kosmotropic salt to an aqueous solution containing hydrophilic PEG a competition for water molecules takes place between salt and PEG. Because of the stronger salt ion affinity for water in comparison to PEG, the solubility and hydration of PEG is decreased. Therefore, a salting-out and exclusion of PEG is observed at certain concentrations in accordance with the reported literature for other systems.38 This may occur because the protonation state of the salt anions is changed by the changing pH value of the aqueous solution.39 There is less protonation of anions, and therefore a higher valence, as the pH of the aqueous medium increases. Anions with a higher valence are better salting-out agents than anions with a lower valence. For this reason anions with higher

behavior was also observed for these ATPS at the other investigated pH values, as well as NaCl concentrations (data not shown). Furthermore, a similar behavior was also reported earlier in literature by other authors.18,29 This behavior may be caused by the decrease of the PEG solubility in water with increasing PEG molecular weight. In fact, for PEG with high molecular weight there is a saturation of the PEG-rich top phase at relatively low PEG concentrations, because the size of the PEG molecules is quite large. This trend is in agreement with other experimental results.1,24,30−32 Owing to the more hydrophobic character of PEG with higher molecular weight, the incompatibility between the phaseforming components is increased, leading to the polymer salting-out. Similar conclusions were previously found by other authors.1,2,8,15,33 Furthermore, the effect of PEG molar weight on the obtained binodal curves can be explained by using the model proposed by Guan.34,35 This model is based on statistical geometry from which the effective excluded volume (EEV) can be determined. The space available in a network of one component (PEG) to occupy the other component (salt) is denoted by EEV. This value is affected by the size, shape, and molecular interactions of the phase-forming components in ATPS.34,35 In the present work, the EEV values were calculated by the following equation (eq 7) developed by Guan:34,35 ⎛ w ⎞ w * p ⎟⎟ + V123 * s =0 ln⎜⎜V123 Mp ⎠ Ms ⎝

(7)

where V123 * is the EEV and Mp and Ms represent the corresponding molecular weights of polymer and salt, respectively. The EEV values obtained by regression analysis of eq 7 from the correlation of binodal data of some PEG + potassium phosphate/sodium citrate + water systems at different polymer molecular weights, pH 7, and T = 23 °C are reported exemplarily in Table 2 along with the corresponding standard Table 2. EEV Values Calculated by eq 7 for PEG (p) + Potassium Phosphate/Sodium Citrate (s) + Water (w) System at Different PEG Molecular Weights, pH 7, and T = 23 °C, together with the Corresponding SD Values system PEG PEG PEG PEG PEG PEG PEG PEG

2000−potassium phosphate 4000−potassium phosphate 6000−potassium phosphate 8000−potassium phosphate 2000−sodium citrate 4000−sodium citrate 6000−sodium citrate 8000−sodium citrate

EEV/g·mol−1

SDa

27.75 34.99 41.17 47.84 36.25 46.68 55.70 64.20

0.34 0.45 0.77 1.07 0.70 0.47 0.66 1.04

exp 2 0.5 SD = (∑i N= 1(100wcal s − 100ws ) /N) , where N represents the number of binodal data, respectively. a

deviations (SD). Here, V*123 as the single fitted parameter of the investigated systems is significant which should, at constant salt molar mass, be related to the salting-out strength of the polymer, as pointed out by Huddleston and Rogers.36 As shown in Table 2, the EEV values are greater at higher PEG molecular weight. The EEV values are ranked in the order PEG 8000 > PEG 6000 > PEG 4000 > PEG 2000, indicating that the salting-out strength of the polymer is increased by increasing 855



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Figure 6. Binodal curves of (A) PEG 4000 (p) + potassium phosphate (s) + water (w) system, and (B) PEG 4000 (p) + sodium citrate (s) + water (w) system at different pH values, and T = 23 °C. Experimental data: (B) ▼, pH 5; (A, B) ■, pH 6; (A, B) ●, pH 7; (A, B) ▲, pH 8; (A) ◆, pH 9. Calculated data using eq 2 (). All compositions are in mass fraction.

Figure 7. Binodal curves of (A) PEG 4000 (p) + potassium phosphate (s) + water (w) system, and (B) PEG 4000 (p) + sodium citrate (s) + water (w) system at different NaCl concentrations, pH 7, and T = 23 °C. Experimental data: ■, 0 wt % NaCl; ●, 2 wt % NaCl; ▲, 4 wt % NaCl; ◆, 6 wt % NaCl; ▼, 8 wt % NaCl. Calculated data using eq 2 (). All compositions are in mass fraction.

out effect due to the presence of NaCl.33,41 Hence, NaCl produces a shift of the binodal curves toward the origin side by an increase of NaCl concentration; less PEG and salt concentrations are required for aqueous two-phase formation at higher NaCl concentrations. Similar observations were previously found by other authors.33,42,43 The effect of NaCl on the position of the binodal curve may be explained by the kosmotropic effect of salts mentioned above. NaCl demonstrated to be an effective salt in aqueous two-phase formation, facilitating a large two-phase region of ATPS.43,44 The salting-out ability of NaCl follows the wellknown Hofmeister series or lyotropic series, in which salts are ordered from kosmotropic to chaotropic, based on their capacity to induce salting-out.45 Furthermore, the salting-out ability can be related, by using a thermodynamic approach, to the Gibbs free energy of hydration of ions, ΔGhyd, which has been used to quantify the Hofmeister series and explain the salting-out phenomenon.46 Because of this series and the large negative ΔGhyd, −365 kJ·mol−1 for sodium ion, Na+,47 it is confirmed as a kosmotropic ion. The high negative ΔGhyd results from the structured water lattice, and consequently there is a great salting-out effect of Na+.43 In other words, kosmotropic cations strengthen the hydrogen bonds which are donated by the inner shell of water molecules, resulting in a high salting-out ability of NaCl or to be precise Na+. In addition, the negative ΔGhyd

valences have a higher hydration ability compared to those with lower valences. Also in the case of anions with higher valences, the repulsive interaction between the anions and the anioniclike polyether functionality of PEG is larger. Consequently, the anions with higher valences in solutions with a basic pH value are more efficient in promoting the two-phase formation compared to those with minor valences in solutions with an acidic pH value. This observation, that an increase of pH in aqueous solution leads to an increase of the water-structurepromoting capability of the salt ions, is in agreement with the fact that the structuring of water is accompanied by decreasing of the water acidity in pure water.36 Thus, the hydronium ions which are present in solutions with an acidic pH value, lead to a blocking of the water structure.38 3.5. Effect of NaCl on the Binodal Curve. The effect of NaCl on the binodal curve or rather phase-forming ability of ATPS containing PEG 4000 + potassium phosphate/sodium citrate + water systems at T = 23 °C, and different pH values, is shown in Figure 7. It can be seen that the addition of NaCl does not change the shape of the binodal curves of the corresponding systems. However, the NaCl induces a displacement of the binodal curves, thus expanding the two-phase region with increasing NaCl concentration. A similar behavior was also observed for these ATPS at the other investigated PEG molecular weights (data not shown). This behavior was also noticed for other ATPS which is a consequence of the salting856



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forming salt, requiring less concentrations of PEG and salt for aqueous two-phase formation. Furthermore, it was observed that ΔGhyd is a driving force for the formation of the investigated ATPS. Additionally, the EEV were calculated from the binodal data for different PEG molecular weights, resulting in a greater salting-out effect at a higher PEG molecular weight, as the EEV are larger at higher molecular weights of PEG. Owing to the excluded volume effect by an increase of PEG, as well as the salting-out ability by an enhancement of PEG, pH, NaCl, and ΔGhyd of phosphate, a displacement of the coexistence curve occurred. Furthermore, the phase equilibrium compositions, TLL, STL, and critical points were determined for all studied systems. Finally, the tie-line compositions were successfully correlated by using the Othmer−Tobias and Bancroft equations, resulting in good agreement of the experimental data with the linear dependency of Othmer−Tobias and Bancroft. On the basis of the obtained results, it can be concluded that all the equations used in this study have a good performance in the correlation and prediction of the tie-line compositions of the investigated systems. These characteristics and several additional advantages, such as fast phase separation and low cost, make the examined PEG−salt ATPS a versatile, promising, and attractive purification and extraction technology in the field of bioseparation.

indicates a large hydration shell, and thus the amount of water available for the hydration of PEG is reduced, contributing to a salting-out of PEG.46 3.6. Effect of Salt Type on the Binodal Curve. The effect of the phase-forming salt type on the binodal curve or the ability to form ATPS consisting of PEG 4000 + potassium phosphate/sodium citrate + water systems at pH 7, and T = 23 °C is illustrated exemplarily in Figure 8. It can be seen that the

Figure 8. Binodal curves of PEG 4000 (p) + salt (s) + water (w) system at pH 7, and T = 23 °C. Experimental data: ●, potassium phosphate; ▲, sodium citrate. Calculated data using eq 2 (). All compositions are in mass fraction.

■

ASSOCIATED CONTENT

S Supporting Information *

Data shown in the figures are available in tabulated form. This material is available free of charge via the Internet at http:// pubs.acs.org.

shapes of both binodal curves are similar. However, the binodal curve of the PEG−potassium phosphate ATPS is shifted to the origin compared to that one of PEG−sodium citrate ATPS. This means that a lower concentration of phosphate is required to form two-phase systems, providing a bigger two-phase region of PEG−phosphate systems in comparison to PEG−citrate systems. This behavior can be explained as in the case of NaCl by means of the salting-out effect or more precisely by the kosmotropic effect with the help of ΔGhyd described above. As for the investigated salts, the influence of anions is more dominant than of cations, as previously reported by other authors.46,48−50 The salting-out ability of cations is in the order phosphate, PO43− > citrate, C6H5O73‑, according to their ΔGhyd values previously determined: ΔG hyd = −2,835 kJ·mol −1 for phosphate,47 and ΔGhyd = −2,793 kJ·mol−1 for citrate.39 Thus, a more negative ΔGhyd value reflects a higher ability for aqueous two-phase formation, and a lower salt concentration is needed to obtain ATPS.46,50 Therefore, phosphates are more effective in ATPS formation than citrates.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: + 49-511-762-2868. Fax: + 49-511-762-3004. Funding

We thank the Leibniz University of Hannover for support within the framework of the “Wege in die Forschung” Program. Notes

The authors declare no competing financial interest.

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REFERENCES

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4. CONCLUSIONS In the present work, the binodal curves and TLL were obtained for different PEG−salt ATPS at 23 °C. The experimental binodal data of all investigated systems were successfully correlated with the empirical nonlinear equation proposed by Hu. The effect of PEG molecular weight, pH value, salt type, and furthermore additional NaCl concentration on these binodal curves has been studied. It was found that a considerably shift of the binodal curves occurred, thus the two-phase region was expanded by an increase of PEG molecular weight, pH, and NaCl concentration, and by using potassium phosphate instead of sodium citrate as the phase857



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