- Email: [email protected]
Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
Biochemical Engineering Journal xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems Xiayun Li, Dr. Yang Liu ∗ , Feifei Li Department of Biology & Guangdong Provincial Key Laboratory of Marine Biotechnology, College of Science, Shantou University, Shantou, Guangdong 515063, PR China
a r t i c l e
i n f o
Article history: Received 30 June 2016 Received in revised form 12 September 2016 Accepted 15 September 2016 Available online xxx Keywords: ILATPS Electric field TLL DC voltage Partitioning
a b s t r a c t The liquid–liquid equilibrium and partitioning of ionic liquid-based aqueous two phase systems (ILATPS) including [Bmim]Cl-K2 HPO4 ATPS, [Bmim]BF4 -NaH2 PO4 ATPS and [Bmim]BF4 -Na3 C6 H5 O7 ATPS in a DC electric field were extensively studied. The demixing rates of three ILATPSs with different tie-line lengths (TLL) were measured at different DC voltage levels with normal polarity (NP) or reverse polarity (RP), respectively, and the partitioning of ILs and model proteins (including lysozyme and bovine serum albumin) were investigated simultaneously. [Bmim]Cl-K2 HPO4 ATPS had higher overall salt concentrations (>15 wt%) and lower overall IL concentrations (<20 wt%) compared with [Bmin]BF4 -based ATPSs, which led to faster two phase separation rates and higher efficiency of electrokinetic demixing mainly due to the salting-out effect. The effects of TLL and DC voltage on the dispersing and gathering of IL molecules in the three ILATPS collectively gave the electrokinetic promotion (accelerate ILATPS demixing rate) and electrokinetic inhibition (decelerate ILATPS demixing rate) for the ILATPS demixing. In [Bmim]BF4 NaH2 PO4 ATPS, the highest promotion demixing were at TLL = 55, 90 V (RP), while the highest inhibition demixing were at TLL = 64, 150 V (RP). The movement and type of organic cations or inorganic anions of IL molecules played an important role in IL partitioning in the three ILATPSs, which generally caused the partition coefficient of IL to decrease in NP and increase in RP to different degrees. There were two different protein partitioning characteristics in the three ILATPS that most model proteins distributed in the IL-rich phase in [Bmim]Cl-K2 HPO4 ATPS due to the salting-out effect and distributed in the salt-rich phase in [Bmim]BF4 -based ATPS due to hydrophobic repulsion. For the model protein partitioning in the electric field, the DC voltage and electric polarity had no significant effect on the protein partitioning trend. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In recent years, a novel kind of green solvent-ionic liquid (IL) has attracted more and more attention. ILs is completely composed of ions and generally maintains a liquid state at room temperature. They are suitable for a variety of applications involving varying their appropriate combination of cations and anions, such as organic synthesis, biphasic catalysis, and the separation and extraction process [1,2]. Due to ILs’ mild dissolution, low toxicity, negligible volatility and chemical stability [3–5], they have been used as an effective extractant in the separation of proteins, amino acids and other biomaterials [6–8].
In 2003, Rogers et al. reported that 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) with its high structure unsymmetry and low melting point could form an aqueous two phase system (ATPS) that contained a [Bmim]Cl-rich upper phase and a salt-rich bottom phase in the presence of an inorganic salt like K3 PO4 [9]. They proposed that the salting-out effect promoted the formation of the ILATPS. Since then, many ionic liquid-inorganic salt ATPSs have been widely studied [10,11] due to their advantages of lower viscosity, little emulsion formation, quick phase separation, and mild biocompatible environment. ILATPSs were gradually applied in biomacromolecules including proteins [12–16], antibiotics [17,18], polysaccharose [19,20] and enzymes [21] separation and purification, in which the extraction efficiency was often higher than that of traditional ATPSs such as polymer-salt ATPSs. The inorganic salts would cause pollution because they are non-degradable, while the environmentally friendly organic salts would have better
∗ Corresponding author. E-mail address: [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.bej.2016.09.008 1369-703X/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model BEJ-6550; No. of Pages 9
ARTICLE IN PRESS X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
2
application prospects for ILATPSs. Therefore, IL-organic salts ATPSs have been increasingly investigated [22–27]. Though ILATPSs have wide application in biomacromolecule separation, the phase demixing rate should be further increased for their industrial application, like the traditional ATPSs. To address the comparatively slow demixing time of the phases, Naveen et al. have applied an electric field to an ATPS and raised the demixing rate of the ATPS [28–32]. Electrokinetic demixing times have been shortened effectively in polymer–polymer ATPSs and polymer-salt ATPSs, which was caused by the effect of electroosmotic and hydrodynamic circulation flows on the polymer droplet during two phase separation in the electric field. In the electric field, the movement of two phase droplets and the partitioning of biological substances in traditional ATPS have been discussed, except that the effects of an electric field on ILATPSs have not been mentioned. ILATPSs have better electrical conductivity than polymers, so in theory the change in phase demixing time would be greater. Electrokinetic demixing of ILATPSs and the effects of the electric field on partitioning of ILs and biomolecules in ILATPSs are worthy of further research. In this paper, three ILATPSs were selected to investigate the effects of the electric field on phase equilibrium and partitioning. The ILATPSs included IL-inorganic salt ATPSs and IL-organic salt ATPSs, namely [Bmim]Cl-Dipotassium Phosphate (K2 HPO4 ), [Bmim]BF4 -Sodium Dihydrogen Phosphate (NaH2 PO4 ), and [Bmim]BF4 -Sodium Citrate (Na3 C6 H5 O7 ). ILATPS phase diagrams including binodal curves and tie-lines were investigated. The demixing experiments of three ILATPSs were determined under different DC voltages, mechanisms of electrokinetic demixing in ILATPSs were discussed. ILs distribution in top phase and bottom phase under different DC voltages were detected. Bovine serum albumin (BSA) and lysozyme (LYS) were chosen to investigate the protein partitioning in three ILATPSs in the electric field. 2. Materials and methods 2.1. Materials [Bmim]Cl (1-butyl-3-methylimidazolium chloride) and [Bmim]BF4 (1-butyl-3- methylimidazolium tetrafluoroborate) were purchased from ChengJie Chemical Co., Ltd. (Shanghai, China) with a purity >99% and used without further purification. Bovine serum albumin (BSA) and lysozyme (LYS) were purchased from Sigma-Aldrich (China). KH2 PO4 , K2 HPO4 , NaCl, Na3 C6 H5 O7 and NaH2 PO4 were analytical grade and purchased from Xilong Chemical Co., Ltd. (Guangdong, China). Glucose, sucrose and agarose were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China), the BCA Protein Quantitation Kit was purchased from Gen Star Biosolutions Co., Ltd. (Beijing, China). 2.2. Phase diagram of ILATPS The binodal curves of [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS were carried out by the cloud point titration method. The titrations were carried out in a DC-0506 low constant temperature bath (Shanghai, China), and the temperature was maintained at 25 ◦ C with an uncertainty of ±0.1 ◦ C. Stock solutions of different compositions with high concentrations (50% (w/w) [Bmim]Cl solution, 80% (w/w) [Bmim]BF4 solution) were prepared, and were carefully added drop-wise into the other composition solutions (50% (w/w) K2 HPO4 solution, 20% (w/w) NaH2 PO4 solution, 30% (w/w) Na3 C6 H5 O7 solution,), respectively, until the two-phase region was reached (turbid samples). Then water was added drop-wise until the one phase region was reached (transparent samples). The compositions where the change occurs
Fig. 1. Schematic representation of experimental setup for electrokinetic demixing of ILATPSs. A: aqueous two-phase dispersion region, B: cooling jacket (with circulating water), C: 1.5 w/v% agarose, D: platinum electrodes, E: DC electrophoresis apparatus, H: circulating water, a: potassium phosphate buffer, b: saturated sodium chloride.
from a two-phase to a monophase system lie on the binodal curve. The titration system was weighed repeatedly using a BSM120.4 analytical balance (Shanghai, China) with an uncertainty of ±1 × 10−4 g, and the composition of the mixture for each point on the binodal curve was calculated. The same operations were operated under different solution conditions in three ILATPSs. The experimental titration data was deduced by a fitted binodal curve equation using ATPS-LLE software developed by our laboratory [33,34]. To determine tie-line data including some feed samples with a known composition concentration, several ILATPSs were prepared with different stock solutions of salt and IL. A known concentration stock solution of two compositions and water was mixed into a graduated test tube, and then volume ratios (Vr ) of the ILATPSs were measured at 25 ◦ C. The composition concentration in the top phase (w2 t , w1 t ) and the composition concentration in the bottom phase (w2 b , w1 b ) were calculated based on the known overall concentrations (w2 f , w1 f ) and Vr of the feed samples based on the fitted binodal curve equation using ATPS-LLE software. In general for ATPS, the composition enriched in the top phase was labeled composition 1 (the ordinate in phase diagram), while the composition enriched in the bottom phase was labeled composition 2 (the abscissa in phase diagram). In the three ILATPSs, IL was enriched in the top phase in [Bmim]Cl(1)-K2 HPO4 (2) ATPS, while IL was enriched in the bottom phase in [Bmim]BF4 (2)-NaH2 PO4 (1) and [Bmim]BF4 (2)- Na3 C6 H5 O7 (1) ATPS. 2.3. Electrokinetic demixing of ILATPS Different tie-line lengths (TLL, roughly TLL = 42, TLL = 55, TLL = 64) of these three ILATPSs were chosen for electrokinetic demixing by adding different masses of stock solutions and water, respectively, then thoroughly stirring the mixture. The electrokinetic demixing apparatus was assembled by common instruments in this research (Fig. 1) as reported [31]. The water-jacketed elec-
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
0.15
(a)
Mass fraction of Na3C6H5O7 (w1)
Mass fraction of [Bmim]Cl (w 1)
0.4
3
0.3
0.2
0.1
0
(b)
0.12 0.09 0.06 0.03 0
0
0.1
0.2
0.3
0.4
0.5
0.6
0
Mass fraction of NaH2PO4 (w1)
Mass fraction of K2HPO4 (w2) 0.2
0.1
0.2 0.3 0.4 0.5 0.6 Mass fraction of [Bmim]BF4 (w2)
0.7
0.8
(c)
0.15
0.1
0.05
0 0
0.1
0.2 0.3 0.4 0.5 0.6 0.7 Mass fraction of [Bmim]BF4 (w2)
0.8
0.9
Fig. 2. Binodal curves () and tie lines () of ILATPS phase diagrams. (a): [Bmim]Cl- K2 HPO4 ATPS, (b): [Bmim]BF4 -NaH2 PO4 ATPS, (c): [Bmim]BF4 -Na3 C6 H5 O7 ATPS.
trophoresis column (30 cm in height and 1.6 cm in diameter) was filled with 60 g ILATPS through 1.5 w/v% agarose gel plugs. These plugs were made in 0.01 M phosphate buffer. Two ends of the column were connected by the electrode buffer, which was prepared by the saturated sodium chloride solution in the bottom phase, and 0.01 M phosphate buffer including 0.05 M glucose and 0.2 M sucrose in the top phase. The demixing experiments were carried out under different output DC voltages (0 V, 30 V, 90 V, 150 V) at normal polarity (NP, anode at the top of the column, electric field going in the same direction with gravitation settling) and reverse polarity (RP, cathode at the top of the column). After the demixing experiments, the ILATPSs reached phase equilibrium under different DC voltages. The top phase and bottom phase in various systems were separated and collected, respectively.
0.5 mg g−1 . Concentrations of BSA and LYS were measured using the BCA Protein Quantitation Kit. Protein yield (Y) is calculated using the equation:
2.4. Determination of ILs and proteins concentration in ILATPSs
Fig. 2 shows the phase diagrams of the three ILATPSs including binodal curves and the tie lines with various TLLs. To obtain bestfit results, the experimental binodal data of various ILATPSs were correlated by different binodal empirical equations, respectively [33,34] by ATPS-LLE software, and the best-fit empirical equation of the binodal curve was expressed as follows:
The concentrations of IL in both the top phase and the bottom phase were determined quantitatively by UV–vis spectrophotometry at 240 nm [10,17], respectively, where the IL molecules had the high absorption peaks. The partition coefficient (K) of IL is calculated using the equation: K=
Ct,IL Cb,IL
Y (%) =
Ct × Vt × 100% Ct × Vt + Cb × Vb
where Ct and Cb are the equilibrium concentrations of protein in the top phase and the bottom protein concentration (the initial protein concentration), respectively. Vtop and Vb represent the top phase volume and the bottom volume, respectively. 3. Results and discussion 3.1. Phase diagram of ILATPS
w 2
w1 = a1 exp − (1)
where Ct,IL and Cb,IL are the equilibrium concentrations of IL in the top phase and the bottom phase, respectively. The BSA and LYS were chosen as the model proteins. Protein yield (Y) in the ILATPS was determined under different output DC voltages, respectively. The initial protein concentration was
(2)
b1
w 2
+ a2 exp −
b2
+c
(3)
Table 1 shows the values of coefficients in the empirical equations of binodal data for different ILATPSs and the lowest standard deviation (SD) values of the best-fit empirical equation. It shows that the empirical equations can be satisfactorily applied to correlate the binodal data of the investigated ILATPSs based on all SD < 0.05. Table 2 and Fig. 2 show the calculated tie-lines data
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
4
Table 1 Coefficients of Different Equations for the Correlation Binodal Data of ILATPS at Temperature T = 25 ◦ C and Pressure p = 0.1 MPaa . ATPS
[Bmim]Cl(1)- K2 HPO4 (2) [Bmim]BF4 (2)- NaH2 PO4 (1) [Bmim]BF4 (2)- Na3 C6 H5 O7 (1) a b
c
coefficientsb
SDc
a1
b1
a2
b2
c
0.5246 0.2938 0.3762
0.2984 0.1222 0.0545
0.2148 0.3603 0.1052
0.0260 0.0334 0.2934
−0.0800 0.0159 −0.0054
Standard uncertainties u are u(T) = 0.1 ◦ C, u(w) = 0.001, and u(p) = 10 kPa. The empirical equation of binodal curve is w1 = a1 exp −
W2 b1
+ a2 exp −
SD represents the standard deviations and can be calculated by SD =
W2 b2
+ c.
ni=1 wl,cal − wl,exp
2 0.5 /n
0.0019 0.0017 0.0010
, where n, w1,cal and w1,exp are the number of binodal data, the calculated
and experimental composition 1 mass fraction, respectively. Table 2 Experimental Volume Ratio (Vr,exp ), 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(mass fraction)
Vr,exp
w1 t
w2 t
w1 b
w2 b
TLL
[Bmim]Cl (1) + K2 HPO4 (2) + water (3) (0.318,0.182) (0.306,0.170) (0.296,0.160) (0.286,0.150) (0.276,0.140)
0.993 1.046 1.020 1.056 1.162
0.355 0.331 0.305 0.270 0.220
0.067 0.082 0.098 0.124 0.166
0.006 0.009 0.015 0.036 0.073
0.569 0.533 0.496 0.444 0.370
0.61 0.55 0.49 0.40 0.25
NaH2 PO4 (1) + [Bmim]BF4 (2) + water (3) (0.438, 0.090) (0.433, 0.080) (0.428, 0.070) (0.423, 0.060) (0.408, 0.050)
1.110 1.068 1.154 1.070 1.230
0.1023 0.1149 0.1288 0.155 0.174
0.1589 0.1422 0.1268 0.104 0.091
0.8106 0.7737 0.688 0.674 0.586
0.01436 0.01423 0.0178 0.0180 0.0188
0.72 0.67 0.57 0.53 0.42
Sodium Citrate (1) + [Bmim]BF4 (2) + water (3) 1.056 (0.430, 0.065) 1.039 (0.430, 0.055) 1.006 (0.430, 0.430) (0.430, 0.045) 0.995 (0.430, 0.035) 0.995 (0.430, 0.030) 0.990 0.921 (0.430, 0.025)
0.1035 0.108 0.117 0.144 0.176 0.201 0.254
0.126 0.118 0.108 0.086 0.068 0.056 0.043
0.759 0.753 0.742 0.718 0.686 0.659 0.609
0.0027 0.0030 0.0035 0.0042 0.0047 0.0050 0.0070
0.67 0.65 0.62 0.58 0.51 0.46 0.36
a
Standard uncertainties u are u(T) = 0.1 ◦ C, u(w) = 0.001, u(Vr ) = 0.01, and u(p) = 10 kPa.
including the two composition concentrations in the top or bottom phase across the experimental feed samples, which indicate the accurate fitted result. It can be seen that the [Bmim]BF4 concentration of IL-rich phases in [Bmim]BF4 -based ILATPSs were generally higher (>60 wt%) than the [Bmim]Cl concentration of IL-rich phases in [Bmim]Cl-K2 HPO4 ATPS, which was consistent with the reported research results [23,27]. As mentioned above, the top phase was the [Bmim]Cl-rich phase in [Bmim]Cl-K2 HPO4 ATPS, while the bottom phase was the [Bmim]BF4 -rich phase in the bottom phase in [Bmim]BF4 -NaH2 PO4 or [Bmim]BF4 -Na3 C6 H5 O7 ATPS. Each 10 g ILATPS with different TLL was adequately shaken for more than 5 min in a 10 mL sealed tube and settled for demixing. Fig. 3 shows the phase separation processing photographs during settling at different moments including 30 s, 60 s and 90 s. It can clearly be seen that the two phase separation rate in [Bmim]Cl-K2 HPO4 ATPS was faster than the rate in [Bmim]BF4 -based ATPS. [Bmim]Cl-K2 HPO4 ATPS can reach complete phase separation within 90 s, while the two [Bmim]BF4 -based ATPSs can’t. During the phase separation of the three ILATPS, it should be clear that the droplets turned to bigger and fewer with the droplets coalesced. In [Bmim]Cl-K2 HPO4 ATPS (Fig. 3a), the droplets became large and sparse from the bottom phase to the top phase, which can be obviously observed at 60 s (close to complete phase separation). It was indicated that the droplets of coalescence layer of [Bmim]Cl-K2 HPO4 ATPS ascended during demixing process. On the other hand, in [Bmim]BF4 -based ATPS (Fig. 3b and c), the droplets became large and sparse from the top phase to the bottom phase clearly observed at 90s, which indicated that the droplets of coalescence layer of [Bmim]BF4 -based
ATPS descended during demixing process. The top phase was saltrich phase in [Bmim]Cl-K2 HPO4 ATPS, while the top phase was IL-rich phase in [Bmim]BF4 -based ATPS. It had been generally recognized that the coalescence layer of ATPS dispersed from the dispersed phase to the continuous phase [35,36] during demixing process. The above experiment phenomena in Fig. 3 displayed that the salt-rich phase was the continuous phase and the IL-rich phase was the dispersed phase in the demixing process of the three ILATPS. It had been found that the demixing kinetic behavior was more dependent on the physical property (especially on viscosity) of continuous phase because the continuous phase strongly influence the movement of the drops of the dispersed phase [36,37]. For the three ILATPS, the IL type and concentration in the salt-rich phase would play an important role in the demixing rate. Compared with [Bmim]Cl-K2 HPO4 ATPS, the higher IL concentration of salt-rich phase in [Bmim]BF4 -based ATPS (in Fig. 2) caused slower demixing rate. 3.2. Electrokinetic demixing of ILATPS The electrokinetic demixing experiment for the three ILATPSs with various TLLs was carried out by varying the output DC voltage (0 V, 30 V, 90 V, 150 V) at normal polarity and reverse polarity, respectively. Fig. 4 displays the effect of output DC voltage and polarity on the demixing rate of the three ILATPSs, which is displayed as the emerged clear liquor height in the top phase per unit of time. It can be seen that the demixing rate of the three ILATPSs generally increased with an increase in output DC voltage
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model BEJ-6550; No. of Pages 9
ARTICLE IN PRESS X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
80 70 60 50 40
5
TLL=42,NP TLL=42,RP TLL=55,NP TLL=55,RP TLL=64,NP TLL=64,RP
[Bmim]Cl-K2HPO4 ATPS
30 20 10
Demixing rate (cm/min)
0
[Bmim]BF4-NaH2PO4 ATPS
12 10 8 6 4 2 0
[Bmim]BF4-Na3C6H5O7 ATPS
16 12 8 4 0 0
30
90
150
Voltage (V) Fig. 4. Effect of the output DC voltage including normal polarity (NP) and reverse polarity (RP) on demixing rate of [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 - Na3 C6 H5 O7 ATPS with different TLL, respectively.
Fig. 3. The phase separation processing photographs at different momemts (30 s, 60 s and 90 s) during settling of [Bmim]Cl-K2 HPO4 ATPS (a), [Bmim]BF4 -NaH2 PO4 ATPS (b) and [Bmim]BF4 -Na3 C6 H5 O7 ATPS (c) with TLL = 64.
or TLL based on hydrodynamic flow-electroosmotic flow (HEF). HEF caused the charged droplets move to an electrode, which was reaction flow or circulation flow (resistance to droplets) subjected to gravity, buoyant and electric field force [31]. This HEF model can explain more clearly electrokinetic demixing of ATPSs in a column, which was mainly influenced by the two phase solutions respective viscosity. Thus, the droplets would be subjected to more resistance in the continuous phase with higher IL concentration (such as the salt-rich phase in [Bmim]BF4 -based ATPS with higher viscosity). Three special rules will be discussed in detail below. Firstly, the demixing rate of [Bmim]Cl-K2 HPO4 ATPS was faster (nearly 20 cm/min) than the other two [Bmim]BF4 -based ILATPSs as mentioned above, and the efficiency of electrokinetic demixing of [Bmim]Cl-K2 HPO4 ATPS was also higher (the demixing rate increased more than 2.5 fold) than that of the other two ILATPSs. The overall concentrations of salt are much higher (>15 wt%) in [Bmim]Cl-K2 HPO4 ATPS than in [Bmim]BF4 -
based ATPSs (<8–10 wt%), and the overall [Bmim]Cl concentrations are lower (<20 wt%) than the overall [Bmim]BF4 concentrations (>40 wt%) for the three ILATPSs mentioned above. The salting-out effect led to a higher demixing rate in [Bmim]Cl-K2 HPO4 ATPS compared with the [Bmim]BF4 -based ATPS. The high concentration of salts also enhanced the conductivity and salting-out effect in the electric field, which caused the demixing rate of [Bmim]Cl-K2 HPO4 ATPS to increase from 18.75 cm/min to 48.98 cm/min in TLL = 64 using the output DC voltage of 150 V (RP). Moreover, the lower IL concentration with lower viscosity would likely enhance the disperse rate of IL-rich phase in the corresponding ILATPS, which would likely further increase the demixing rate. Secondly, TLLs played an important role in electrokinetic demixing. In [Bmim]BF4 -NaH2 PO4 ILATPS, the efficiency of electrokinetic demixing decreased with an increase in output DC voltage, and the demixing rate even decreased at TLL = 64. In [Bmim]BF4 based ILATPS, these three tie-lines had higher concentrations of IL (>60 wt% in the top phase, >10 wt% in the bottom phase) and lower concentration of salts (<15–20 wt%) than [Bmim]Cl-K2 HPO4 ATPS, so the salting-out effect was weaker and IL concentration played a more important role in the demixing process. There were obviously two opposite effects of the higher IL concentrations on the demixing rate of ILATPS in the electric field. One was the dispersion of IL molecules that increased the demixing rate, similar to the
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model BEJ-6550; No. of Pages 9
ARTICLE IN PRESS X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
6
80
TLL=42,NP
TLL=42,RP
TLL=55,NP
TLL=55,RP
TLL=64,NP
TLL=64,RP
[Bmim]Cl-K2HPO4 ATPS
60
40
20
Fig. 5. The most electrokinetic promotion (a–c) and inhibition (d) processing photographs of [Bmim]Cl-K2 HPO4 ATPS with TLL = 64 at 25 s, V = 150 V (a), [Bmim]BF4 -NaH2 PO4 ATPS with TLL = 55 at 60 s, V = 90 V (b), [Bmim]BF4 - Na3 C6 H5 O7 ATPS with TLL = 64 at 60 s, V = 150 V (c) and [Bmim]BF4 -NaH2 PO4 ATPS with TLL = 64 at 180 s, V = 150 V (d) under different output DC voltage of reverse polarity.
salting-out effect, and the other was the gathering of IL molecules with higher IL concentration that decreased the demixing rate with an increase in output DC voltage. The more rapid demixing rates (9.6 cm/min and 11.16 cm/min) of the two [Bmim]BF4 -based ILATPSs consistently appeared with the intermediate TLL = 55 at the output DC voltage of 90 V (RP). Thirdly, in the three ILATPSs, reverse polarity had generally higher efficiency of electrokinetic demixing than normal polarity, especially in the [Bmim]BF4 -based ILATPS. Fig. 5 shows the highest electrokinetic promotion (a, b, c) and inhibition (d) processing photographs of the three IL-based ATPS under different output DC voltages (150 V, 90 V and 150 V) of reverse polarity at different moments, which had the highest or the lowest demixing rates of various ILATPSs according to Fig. 4. It has been reported that the droplets motion can be interpreted during ATPS demixing process [38] by Stokes law. The Stokes resistance of droplets during moving can decrease with the decrease of droplet size. It can be seen that the droplet size in the three ILATPSs’ (in Fig. 5a–c) coalescence layer generally shrink under the electric field with reverse polarity, which decrease the Stokes resistance of droplets. It would lead to accelerate the demixing ratio of the ILATPS. On the other hand, the reverse polarity (90 V and 150 V) had an adverse effect on the demixing rate at TLL = 42 in [Bmim]Cl-K2 HPO4 ATPS and TLL = 64 in [Bmim]BF4 -NaH2 PO4 ATPS. Fig. 5d shows the electrokinetic inhibition (TLL = 64, 150 V) for [Bmim]BF4 -NaH2 PO4 ATPS demixing processing photographs. It can be seen that the more concentrated and smaller droplets at 150 V (RP) in Fig. 5d impeded the coalescence rate of the top and bottom phase, which caused a decrease in the demixing rate. 3.3. Partitioning of ILs in ILATPS The partitioning of [Bmim]Cl or [Bmim]BF4 was studied through quantitative determination of IL at different DC voltages (0 V, 30 V, 90 V, 150 V) in these three ILATPSs with different TLLs. The values of the partition coefficient (K) of IL could be obtained by Eq. (1) at various conditions. K > 1 means the IL distributed more in the top phase, as seen in [Bmim]Cl-based ATPS, while K < 1 means the IL partitioning occurred more in the bottom phase, such as in [Bmim]BF4 -based ATPS. Table 3 shows the theoretical partition coefficient by ATPS-LLE soft and experimental partition coefficient of different TLLs in these three ILATPSs, which were broadly con-
Patition coefficient of IL
0
[Bmim]BF4-NaH2PO4 ATPS
0.25 0.2 0.15 0.1 0.05 0
[Bmim]BF4-Na3C6H5O7 ATPS
0.5 0.4 0.3 0.2 0.1 0 0
30 Voltage (V)
90
150
Fig. 6. Effect of DC voltage and field polarity on partition coefficient of ILs in [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLL.
sistent. This spectrophotometric method was proved reliable for IL concentration measurement. Fig. 6 displays the effect of DC voltage and field polarity on the partition coefficient of ILs in [Bmim]Cl-K2 HPO4 , [Bmim]BF4 NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLLs, respectively. In the three ILATPSs, due to the differences in ILs-rich phase, the K values of IL increased with increasing TLL in [Bmim]Clbased ILATPS, while the K values of IL decreased with increasing TLL in [Bmim]BF4 -based ILATPS. Regarding the effect of electric field polarity, it can be seen that the K values of ILs decreased in the NP field and increased in the RP field in the three ILATPSs. Thus, ILs distributed more to the bottom phase in the NP field, while ILs distributed more to the top phase in the RP field regardless of IL type. The cations and anions of ILs partially decomposed under the electric field [39,40], and the bigger cations moved faster towards the cathode (bottom phase in NP and top phase in RP). Moreover, the smaller anions of [Bmim]Cl caused the above trend more obviously in the [Bmim]Cl-based ILATPS. This indicated that the ion’s type and movement in the electric field of different ILs played a key role in the IL partitioning in ILATPSs. Fig. 6 displayed that the electric voltage had no significant effect on the IL partitioning in ILATPS under the electric field. Based on the above experimental results, it could
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
7
Table 3 The Theoretical Partition Coefficient (Ktheory ) and Experimental Partition Coefficient (Kexp ) of ILs with Different TLL. ILATPS
[Bmim]Cl-K2 HPO4
TLL
0.64 0.55 0.42
110
[Bmim]BF4 -NaH2 PO4 Ktheory
Kexp
Ktheory
Kexp
Ktheory
56.991 29.760 8.517
60.402 32.777 9.590
0.150 0.201 0.259
0.167 0.208 0.276
0.172 0.296 0.376
0.153 0.284 0.347
TLL=42,NP
TLL=42,RP
TLL=55,NP
TLL=42,NP
TLL=42,RP
TLL=55,NP
TLL=55,RP
TLL=64,NP
TLL=64,RP
TLL=55,RP
TLL=64,NP
TLL=64,RP
[Bmim]Cl-K2HPO4 ATPS
110
100
100
90
90
80
80
70
[Bmim]Cl-K2HPO4 ATPS
70
60
60
[Bmim]BF4-NaH2PO4 ATPS
80
[Bmim]BF4-NaH2PO4 ATPS
100
LYS Yield (%)
BSA Yield (%)
[Bmim]BF4 -Na3 C6 H5 O7
Kexp
60 40 20
80 60 40
0
[Bmim]BF4-Na3C6H5O7 ATPS
20
[Bmim]BF4-Na3C6H5O7 ATPS
100
60
90
40
80 20
70 0 0
30 90 Voltage (V)
150
Fig. 7. Effect of DC voltage and field polarity on BSA yield in [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLL.
be speculated that the presence of electric field would regulate the binodal curves of the same ILATPS. Further, the two phase areas of [Bmim]Cl-based ILATPS would increase in RP electric field, while the two phase areas of [Bmim]BF4 -based ILATPS would increase in NP electric field. 3.4. Distribution of proteins in ILATPS Two model proteins (BSA and LYS) were selected to investigate the partitioning in the three ILATPSs at different output DC voltages. Most of the proteins generally distributed in the top phase in each ILATPS, while few proteins distributed in the bottom phase. Figs. 7 and 8 display the effect of DC voltage and field polarity on yields of BSA and LYS in [Bmim]Cl-K2 HPO4 ,
60 0
30 Voltage (V)
90
150
Fig. 8. Effect of DC voltage and field polarity on LYS yield in [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLL.
[Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLLs, respectively. Comparing the two protein yields shown in the two figures, it displayed that the LYS yield was generally higher than BSA yield due to LYS’s lower molecular weight. For instance, the protein yield of BSA and LYS reached 78–94% and 93–99% at different DC voltages, respectively, in [Bmim]ClK2 HPO4 ATPS with TLL = 42. In [Bmim]Cl-K2 HPO4 ATPS, there was quite a strong salting-out effect due to the high concentration of salts as mentioned above. Thus, salting-out effect played a critical role in the protein distribution of [Bmim]Cl-based ATPS. Higher inorganic salt concentration ([K2 HPO4 ] > 40 wt%) in the top phase resulted in stronger salting-out effect for proteins, which drove protein partitioning in the IL-rich phase (top phase). The protein
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
8
Table 4 The pH Values of Various ILATPS and Electrostatic Charges (E.C.)a of Proteins in ILATPS.
to the salting-out effect and partitioned in the salt-rich phase in [Bmim]BF4 -based ATPS due to hydrophobic repulsion.
ILATPS
[Bmim]ClK2 HPO4
[Bmim]BF4 NaH2 PO4
[Bmim]BF4 Na3 C6 H5 O7
Acknowledgements
pH E.C. of BSA (pI = 4.7) E.C. of LYS (pI = 11.1)
9.6–9.8 −− +
3.8−4.0 + +++
6.4–6.7 − ++
This work is supported by the National Natural Science Foundation of China (No. 21476135), Outstanding Young Teachers Training Program in Guangdong Higher Education Institutions (No. Yq2013076).
a The symbols of “+” and “−” denotes the positive and negative E.C. of proteins, respectively, and the numbers of written symbols visually displays the amount of proteins E.C. in ILATPS.
References yield generally decreased with increasing TLL, which was tested by the proteins loss in the interphase (the insoluble solid content between the top phase and the bottom phase) due to the decrease of protein solubility in ATPS [41], and this trend was particularly apparent for BSA in [Bmim]BF4 -based ATPS. Moreover, in [Bmim]BF4 -NaH2 PO4 and [Bmim]BF4 -Na3 C6 H5 O7 ATPSs, the proteins distributed in the salt-rich phase (top phase) when [Bmim]BF4 enriched in the bottom phase. Compared with the bottom phase in [Bmim]Cl-K2 HPO4 ATPS, the high concentration of hydrophobic [Bmim]BF4 would lead to the lower dissolving capacity for the water-soluble proteins in the [Bmim]BF4 -rich phase. It indicated that the hydrophobic repulsion played a leading role in protein partitioning in [Bmim]BF4 -based ATPS, which was in accord with reports on the other IL ATPS [16,42]. In the top phase of [Bmim]BF4 -based ATPS, there were still some [Bmim]BF4 with low concentration (about 10–20 wt%). The decrease of protein yield with increasing TLL can be observed in Figs. 7 and 8, which was due to the increase of hydrophobic repulsion with increasing [Bmim]BF4 concentration in the top phase. Table 4 displayed the solutions pH values of three ILATPS and the amount of electrostatic charges of BSA and LYS in the three ILATPSs. Though the electrical property and the charge amount of BSA and LYS were obviously different in the three ILATPSs, the variation trends of two proteins yield were almost same with DC voltage or the field polarity. Figs. 7 and 8 and 8 showed the increase of DC voltage or the difference of field polarity had weak effect on proteins yield in the two ILATPSs, which indicated that the various electrostatic charges of proteins did not play a key role in proteins partitioning in the three ILATPSs. 4. Conclusions The liquid–liquid equilibrium of three ILATPSs consisting of [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 and [Bmim]BF4 Na3 C6 H5 O7 under an electric field were extensively studied. The eletrokinetic demixing of the three ILATPSs experiments were operated at different DC voltage levels under NP or RP electric fields, and the partitioning of ILs and model proteins (including LYS and BSA) under electric fields were investigated, respectively. The results indicated that [Bmim]Cl-K2 HPO4 ATPS with higher salt concentrations and lower IL concentrations had a faster two phase separation rate and a higher efficiency of electrokinetic demixing due to the salting-out effect. IL molecules dispersing and gathering changed with TLL and DC voltage, which led to the highest electrokinetic promotion and the highest inhibition demixing in [Bmim]BF4 -NaH2 PO4 ATPS at TLL = 55, 90 V (RP) and TLL = 64, 150 V (RP), respectively. The movement of bigger organic cations of IL molecules under an electric field generally caused the K values of IL in the three ILATPSs to decrease under an NP electric field and to increase under an RP electric field. For the larger model protein partitioning in the three ILATPSs, the electric field had no significant effect on the protein distribution trend. Most model proteins partitioned in the IL-rich phase in [Bmim]Cl-K2 HPO4 ATPS due
[1] R. Kenneth, J. Seddon, Ionic liquids for clean technology, J. Chem. Technol. Biotechnol. 68 (1997) 351–356. [2] W.L. Hough, M. Smiglak, H. Rodríguez, R.P. Swatloski, S.K. Spear, D.T. Daly, et al., The third evolution of ionic liquids: active pharmaceutical ingredients, New J. Chem. 31 (2007) 1429–1436. [3] T. Welton, Room-temperature ionic liquids: solvents for synthesis and catalysis, Chem. Rev. 99 (1999) 2071–2084. [4] P. Wasserscheid, W. Keim, Ionic liquids-new solutions for transition metal catalysis, Angew. Chem. Int. Ed. 39 (2000) 3772–3789. [5] J. Dupont, R.F.D. Souza, P.A.Z. Suarez, Ionic liquid (molten salt) phase organometallic catalysis, Cheminform 34 (3) (2003) 3667–3692. [6] S. Pandey, Analytical applications of room-temperature ionic liquids: a review of recent efforts, Anal. Chim. Acta 556 (2006) 38–45. [7] X.X. Han, D.W. Armstrong, Ionic liquids in separations, Acc. Chem. Res. 40 (2007) 1079–1086. [8] K.N.K. Shimojo, N. Kamiya, M. Goto, Crown ether-mediated extraction and functional conversion of cytochrome c in ionic liquids, Biomacromolecules 7 (2006) 2–5. [9] K.E. Gutowski, G.A. Broker, H.D. Willauer, J.G. Huddleston, R.P. Swaloski, J.D. Holbrey, R.D. Rogers, Controlling the aqueous miscibility of ionic liquids: aqueous biphasic systems of water-miscible ionic liquids and water-structuring salts for recycle, metathesis, and separations, J. Am. Chem. Soc. 125 (22) (2003) 6632–6633. [10] Y. Pei, J. Wang, L. Liu, Liquid-liquid equilibria of aqueous biphasic systems containing selected imidazolium ionic liquids and salts, J. Chem. Eng. Data 52 (5) (2007) 2026–2031. [11] J. Han, Y. Wang, W. Kang, C. Li, Y. Yan, J. Pan, X. Xie, Phase equilibrium and macrolide antibiotics partitioning in real water samples using a two-phase system composed of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and an aqueous solution of an inorganic salt, Microchim. Acta 169 (1–2) (2010) 15–22. [12] Y.C. Pei, Z.Y. Li, L. Li, J.J. Wang, H.Y. Wang, Selective separation of protein and saccharides by ionic liquids aqueous two-phase systems, Sci. China 53 (7) (2010) 1554–1560. [13] S. Dreyer, P. Salim, U. Kragl, Driving forces of protein partitioning in an ionic liquid-based aqueous two-phase system, Biochem. Eng. J. 46 (2) (2009) 176–185. [14] Iyyaswami Regupathi, Shanon L. Monteiro, 1-Hexyl-3-methylimidazolium chloride-potassium carbonate aqueous two phase system: equilibrium characteristics and BSA partitioning behavior, J. Dispersion Sci. Technol. 35 (3) (2013) 418–427. [15] D. Zhuo, Y.L. Yu, J.H. Wang, Extraction of proteins from biological fluids by use of an ionic liquid/aqueous two-phase system, Chemistry 13 (7) (2007) 2130–2137. [16] Y. Pei, J. Wang, K. Wu, X. Xuan, X. Lu, Ionic liquid-based aqueous two-phase extraction of selected proteins, Sep. Purif. Technol. 64 (3) (2009) 288–295. [17] Y. Jiang, H. Xia, C. Guo, I. Mahmood, H. Liu, Enzymatic hydrolysis of penicillin in mixed ionic liquids/water two-phase system, Biotechnol. Prog. 23 (4) (2007) 829–835. [18] Y.F. Li, J. Han, Y. Wang, J.J. Ma, Y.S. Yan, Partitioning of cephalexin in ionic liquid aqueous two-phase system composed of 1-butyl-3-methylimidazolium tetrafluoroborate and ZnSO4 , J. Chem. (2013) 1–5. [19] J.K. Yan, H.L. Ma, J.J. Pei, Z.B. Wang, J.Y. Wu, Facile and effective separation of polysaccharides and proteins from Cordyceps sinensis mycelia by ionic liquid aqueous two-phase system, Sep. Purif. Technol. 135 (2014) 278–284. [20] Z.J. Tan, F.F. Li, X.L. Xu, J.M. Xing, Z.J. Tan, F.F. Li, et al., Simultaneous extraction and purification of aloe polysaccharides and proteins using ionic liquid based aqueous two-phase system coupled with dialysis membrane, Desalination 286 (1) (2012) 389–393. [21] B. Jiang, Z. Feng, C. Liu, Y. Xu, D. Li, J. Guo, Extraction and purification of wheat-esterase using aqueous two-phase systems of ionic liquid and salt, J. Food Sci. Technol. 52 (5) (2015) 2878–2885. [22] J. Han, R. Pan, X. Xie, Y. Wang, Y. Yan, G. Yin, et al., Liquid-liquid equilibria of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate + sodium and ammonium citrate aqueous two-phase systems at (298.15, 308.15, and 323.15) K, J. Chem. Eng. Data 55 (9) (2010) 3749–3754. [23] J. Han, C. Yu, W. Yun, X. Xie, Y. Yan, G. Yin, et al., Liquid–liquid equilibria of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and sodium citrate/tartrate/acetate aqueous two-phase systems at 298. 15 K: experiment and correlation, Fluid Phase Equilib. 295 (1) (2010) 98–103.
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model BEJ-6550; No. of Pages 9
ARTICLE IN PRESS X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
[24] J. Han, Y. Wang, C. Yu, Y. Li, W. Kang, Y. Yan, (Liquid + liquid) equilibrium of (imidazolium ionic liquids + organic salts) aqueous two-phase systems at T = 298.15 K and the influence of salts and ionic liquids on the phase separation, J. Chem. Thermodyn. 45 (1) (2012) 59–67. [25] J. Han, Y. Wang, C. Chen, W. Kang, Y. Liu, K. Xu, et al., (Liquid + liquid) equilibria and extraction capacity of (imidazolium ionic liquids + potassium tartrate) aqueous two-phase systems, J. Mol. Liq. 193 (2014) 23–28. [26] M.T. Zafarani-Moattar, S. Hamzehzadeh, Effect of pH on the phase separation in the ternary aqueous system containing the hydrophilic ionic liquid 1-butyl-3-methylimidazolium bromide and the kosmotropic salt potassium citrate at T = 298.15 K, Fluid Phase Equilib. 304 (1) (2011) 110–120. [27] M.T. Zafarani-Moattar, S. Hamzehzadeh, Salting-out effect, preferential exclusion, and phase separation in aqueous solutions of chaotropic water-miscible ionic liquids and kosmotropic salts: effects of temperature, anions, and cations, J. Chem. Eng. Data 55 (4) (2010) 1598–1610. [28] K.S.M.S. Raghavarao, R.M. Stewart, P. Todd, Electrokinetic demixing of two-phase aqueous polymer systems. I. Separation rates of polyethylene glycol-dextran mixtures, Sep. Sci. Technol. 25 (9–10) (1990) 985–996. [29] K.S.M.S. Raghavarao, R.M. Stewart, P. Todd, Electrokinetic demixing of two-phase aqueous polymer systems II. Separation rates of polyethylene glycol-maltodextrin mixtures, Sep. Sci. Technol. 26 (2) (1991) 257–267. [30] K.S.M.S. Raghavarao, R.M. Stewart, S.R. Rudge, P. Todd, Electrokinetic demixing of aqueous two-phase systems 3. Drop electrophoretic mobilities and demixing rates, Biotechnol. Prog. 14 (6) (1998) 922–930. [31] N. Naveen, C. Sampangi, K.S.M.S. Raghavarao, Electrokinetic demixing of aqueous two-phase polymer-salt systems, Electrophoresis 26 (1) (2005) 10–17. [32] M.C. Madhusudhan, S. Chethana, K.S.M.S. Raghavarao, Electrokinetic demixing of polymer-salt systems containing biomolecules, Sep. Sci. Technol. 46 (5) (2011) 727–733.
9
[33] Y. Liu, Y. Feng, Y. Zhao, Liquid-liquid equilibrium of various aqueous two-phase systems: experiment and correlation, J. Chem. Eng. Data 58 (10) (2013) 2775–2784. [34] L. Liu, Y. Liu, L. Du, Y. Zhao, H. Du, (Liquid + liquid) phase equilibrium of aqueous two-phase system containing (surfactant + sodium sulfate + water) at different temperatures, Fluid Phase Equilib. 415 (15) (2016) 25–33. [35] V.H. Nagaraja, R. Iyyaswami, Phase demixing studies in aqueous two-phase system with polyethylene glycol (PEG) and sodium citrate, Chem. Eng. Commun. 200 (10) (2013) 1293–1308. [36] H. Yuan, Y. Liu, W. Wei, Y. Zhao, Phase separation behavior and system properties of aqueous two-phase systems with polyethylene glycol and different salts: experiment and correlation, J. Fluids (2015) 1–10. [37] J.A. Asenjo, S.L. Mistry, B.A. Andrews, J.C. Merchuk, Phase separation rates of aqueous two-phase systems: correlation with system properties, Biotechnol. Bioeng. 79 (2) (2002) 217–223. [38] J.C. Merchuk, B.A. Andrews, J.A. Asenjo, Aqueous two-phase systems for protein separation: studies on phase inversion, J. Chromatogr. B: Biomed. Sci. Appl. 711 (1–2) (1998) 285–293. [39] A.P. Abbott, Application of hole theory to the viscosity of ionic and molecular liquids, Chemphyschem 5 (2004) 1242–1246. [40] A.P. Abbott, Model for the conductivity of ionic liquids based on an infinite dilution of holes, Chemphyschem 6 (2005) 2502–2505. [41] K.C. Ross, C. Zhang, Separation of recombinant -glucuronidase from transgenic tobacco by aqueous two-phase extraction, Biochem. Eng. J. 49 (3) (2010) 343–350. [42] C. Wu, J. Wang, Z. Li, J. Jing, H. Wang, Relative hydrophobicity between the phases and partition of cytochrome-c in glycine ionic liquids aqueous two-phase systems, J. Chromatogr. A 1305 (2013) 1–6.
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
Biochemical Engineering Journal xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems Xiayun Li, Dr. Yang Liu ∗ , Feifei Li Department of Biology & Guangdong Provincial Key Laboratory of Marine Biotechnology, College of Science, Shantou University, Shantou, Guangdong 515063, PR China
a r t i c l e
i n f o
Article history: Received 30 June 2016 Received in revised form 12 September 2016 Accepted 15 September 2016 Available online xxx Keywords: ILATPS Electric field TLL DC voltage Partitioning
a b s t r a c t The liquid–liquid equilibrium and partitioning of ionic liquid-based aqueous two phase systems (ILATPS) including [Bmim]Cl-K2 HPO4 ATPS, [Bmim]BF4 -NaH2 PO4 ATPS and [Bmim]BF4 -Na3 C6 H5 O7 ATPS in a DC electric field were extensively studied. The demixing rates of three ILATPSs with different tie-line lengths (TLL) were measured at different DC voltage levels with normal polarity (NP) or reverse polarity (RP), respectively, and the partitioning of ILs and model proteins (including lysozyme and bovine serum albumin) were investigated simultaneously. [Bmim]Cl-K2 HPO4 ATPS had higher overall salt concentrations (>15 wt%) and lower overall IL concentrations (<20 wt%) compared with [Bmin]BF4 -based ATPSs, which led to faster two phase separation rates and higher efficiency of electrokinetic demixing mainly due to the salting-out effect. The effects of TLL and DC voltage on the dispersing and gathering of IL molecules in the three ILATPS collectively gave the electrokinetic promotion (accelerate ILATPS demixing rate) and electrokinetic inhibition (decelerate ILATPS demixing rate) for the ILATPS demixing. In [Bmim]BF4 NaH2 PO4 ATPS, the highest promotion demixing were at TLL = 55, 90 V (RP), while the highest inhibition demixing were at TLL = 64, 150 V (RP). The movement and type of organic cations or inorganic anions of IL molecules played an important role in IL partitioning in the three ILATPSs, which generally caused the partition coefficient of IL to decrease in NP and increase in RP to different degrees. There were two different protein partitioning characteristics in the three ILATPS that most model proteins distributed in the IL-rich phase in [Bmim]Cl-K2 HPO4 ATPS due to the salting-out effect and distributed in the salt-rich phase in [Bmim]BF4 -based ATPS due to hydrophobic repulsion. For the model protein partitioning in the electric field, the DC voltage and electric polarity had no significant effect on the protein partitioning trend. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In recent years, a novel kind of green solvent-ionic liquid (IL) has attracted more and more attention. ILs is completely composed of ions and generally maintains a liquid state at room temperature. They are suitable for a variety of applications involving varying their appropriate combination of cations and anions, such as organic synthesis, biphasic catalysis, and the separation and extraction process [1,2]. Due to ILs’ mild dissolution, low toxicity, negligible volatility and chemical stability [3–5], they have been used as an effective extractant in the separation of proteins, amino acids and other biomaterials [6–8].
In 2003, Rogers et al. reported that 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) with its high structure unsymmetry and low melting point could form an aqueous two phase system (ATPS) that contained a [Bmim]Cl-rich upper phase and a salt-rich bottom phase in the presence of an inorganic salt like K3 PO4 [9]. They proposed that the salting-out effect promoted the formation of the ILATPS. Since then, many ionic liquid-inorganic salt ATPSs have been widely studied [10,11] due to their advantages of lower viscosity, little emulsion formation, quick phase separation, and mild biocompatible environment. ILATPSs were gradually applied in biomacromolecules including proteins [12–16], antibiotics [17,18], polysaccharose [19,20] and enzymes [21] separation and purification, in which the extraction efficiency was often higher than that of traditional ATPSs such as polymer-salt ATPSs. The inorganic salts would cause pollution because they are non-degradable, while the environmentally friendly organic salts would have better
∗ Corresponding author. E-mail address: [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.bej.2016.09.008 1369-703X/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model BEJ-6550; No. of Pages 9
ARTICLE IN PRESS X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
2
application prospects for ILATPSs. Therefore, IL-organic salts ATPSs have been increasingly investigated [22–27]. Though ILATPSs have wide application in biomacromolecule separation, the phase demixing rate should be further increased for their industrial application, like the traditional ATPSs. To address the comparatively slow demixing time of the phases, Naveen et al. have applied an electric field to an ATPS and raised the demixing rate of the ATPS [28–32]. Electrokinetic demixing times have been shortened effectively in polymer–polymer ATPSs and polymer-salt ATPSs, which was caused by the effect of electroosmotic and hydrodynamic circulation flows on the polymer droplet during two phase separation in the electric field. In the electric field, the movement of two phase droplets and the partitioning of biological substances in traditional ATPS have been discussed, except that the effects of an electric field on ILATPSs have not been mentioned. ILATPSs have better electrical conductivity than polymers, so in theory the change in phase demixing time would be greater. Electrokinetic demixing of ILATPSs and the effects of the electric field on partitioning of ILs and biomolecules in ILATPSs are worthy of further research. In this paper, three ILATPSs were selected to investigate the effects of the electric field on phase equilibrium and partitioning. The ILATPSs included IL-inorganic salt ATPSs and IL-organic salt ATPSs, namely [Bmim]Cl-Dipotassium Phosphate (K2 HPO4 ), [Bmim]BF4 -Sodium Dihydrogen Phosphate (NaH2 PO4 ), and [Bmim]BF4 -Sodium Citrate (Na3 C6 H5 O7 ). ILATPS phase diagrams including binodal curves and tie-lines were investigated. The demixing experiments of three ILATPSs were determined under different DC voltages, mechanisms of electrokinetic demixing in ILATPSs were discussed. ILs distribution in top phase and bottom phase under different DC voltages were detected. Bovine serum albumin (BSA) and lysozyme (LYS) were chosen to investigate the protein partitioning in three ILATPSs in the electric field. 2. Materials and methods 2.1. Materials [Bmim]Cl (1-butyl-3-methylimidazolium chloride) and [Bmim]BF4 (1-butyl-3- methylimidazolium tetrafluoroborate) were purchased from ChengJie Chemical Co., Ltd. (Shanghai, China) with a purity >99% and used without further purification. Bovine serum albumin (BSA) and lysozyme (LYS) were purchased from Sigma-Aldrich (China). KH2 PO4 , K2 HPO4 , NaCl, Na3 C6 H5 O7 and NaH2 PO4 were analytical grade and purchased from Xilong Chemical Co., Ltd. (Guangdong, China). Glucose, sucrose and agarose were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China), the BCA Protein Quantitation Kit was purchased from Gen Star Biosolutions Co., Ltd. (Beijing, China). 2.2. Phase diagram of ILATPS The binodal curves of [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS were carried out by the cloud point titration method. The titrations were carried out in a DC-0506 low constant temperature bath (Shanghai, China), and the temperature was maintained at 25 ◦ C with an uncertainty of ±0.1 ◦ C. Stock solutions of different compositions with high concentrations (50% (w/w) [Bmim]Cl solution, 80% (w/w) [Bmim]BF4 solution) were prepared, and were carefully added drop-wise into the other composition solutions (50% (w/w) K2 HPO4 solution, 20% (w/w) NaH2 PO4 solution, 30% (w/w) Na3 C6 H5 O7 solution,), respectively, until the two-phase region was reached (turbid samples). Then water was added drop-wise until the one phase region was reached (transparent samples). The compositions where the change occurs
Fig. 1. Schematic representation of experimental setup for electrokinetic demixing of ILATPSs. A: aqueous two-phase dispersion region, B: cooling jacket (with circulating water), C: 1.5 w/v% agarose, D: platinum electrodes, E: DC electrophoresis apparatus, H: circulating water, a: potassium phosphate buffer, b: saturated sodium chloride.
from a two-phase to a monophase system lie on the binodal curve. The titration system was weighed repeatedly using a BSM120.4 analytical balance (Shanghai, China) with an uncertainty of ±1 × 10−4 g, and the composition of the mixture for each point on the binodal curve was calculated. The same operations were operated under different solution conditions in three ILATPSs. The experimental titration data was deduced by a fitted binodal curve equation using ATPS-LLE software developed by our laboratory [33,34]. To determine tie-line data including some feed samples with a known composition concentration, several ILATPSs were prepared with different stock solutions of salt and IL. A known concentration stock solution of two compositions and water was mixed into a graduated test tube, and then volume ratios (Vr ) of the ILATPSs were measured at 25 ◦ C. The composition concentration in the top phase (w2 t , w1 t ) and the composition concentration in the bottom phase (w2 b , w1 b ) were calculated based on the known overall concentrations (w2 f , w1 f ) and Vr of the feed samples based on the fitted binodal curve equation using ATPS-LLE software. In general for ATPS, the composition enriched in the top phase was labeled composition 1 (the ordinate in phase diagram), while the composition enriched in the bottom phase was labeled composition 2 (the abscissa in phase diagram). In the three ILATPSs, IL was enriched in the top phase in [Bmim]Cl(1)-K2 HPO4 (2) ATPS, while IL was enriched in the bottom phase in [Bmim]BF4 (2)-NaH2 PO4 (1) and [Bmim]BF4 (2)- Na3 C6 H5 O7 (1) ATPS. 2.3. Electrokinetic demixing of ILATPS Different tie-line lengths (TLL, roughly TLL = 42, TLL = 55, TLL = 64) of these three ILATPSs were chosen for electrokinetic demixing by adding different masses of stock solutions and water, respectively, then thoroughly stirring the mixture. The electrokinetic demixing apparatus was assembled by common instruments in this research (Fig. 1) as reported [31]. The water-jacketed elec-
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
0.15
(a)
Mass fraction of Na3C6H5O7 (w1)
Mass fraction of [Bmim]Cl (w 1)
0.4
3
0.3
0.2
0.1
0
(b)
0.12 0.09 0.06 0.03 0
0
0.1
0.2
0.3
0.4
0.5
0.6
0
Mass fraction of NaH2PO4 (w1)
Mass fraction of K2HPO4 (w2) 0.2
0.1
0.2 0.3 0.4 0.5 0.6 Mass fraction of [Bmim]BF4 (w2)
0.7
0.8
(c)
0.15
0.1
0.05
0 0
0.1
0.2 0.3 0.4 0.5 0.6 0.7 Mass fraction of [Bmim]BF4 (w2)
0.8
0.9
Fig. 2. Binodal curves () and tie lines () of ILATPS phase diagrams. (a): [Bmim]Cl- K2 HPO4 ATPS, (b): [Bmim]BF4 -NaH2 PO4 ATPS, (c): [Bmim]BF4 -Na3 C6 H5 O7 ATPS.
trophoresis column (30 cm in height and 1.6 cm in diameter) was filled with 60 g ILATPS through 1.5 w/v% agarose gel plugs. These plugs were made in 0.01 M phosphate buffer. Two ends of the column were connected by the electrode buffer, which was prepared by the saturated sodium chloride solution in the bottom phase, and 0.01 M phosphate buffer including 0.05 M glucose and 0.2 M sucrose in the top phase. The demixing experiments were carried out under different output DC voltages (0 V, 30 V, 90 V, 150 V) at normal polarity (NP, anode at the top of the column, electric field going in the same direction with gravitation settling) and reverse polarity (RP, cathode at the top of the column). After the demixing experiments, the ILATPSs reached phase equilibrium under different DC voltages. The top phase and bottom phase in various systems were separated and collected, respectively.
0.5 mg g−1 . Concentrations of BSA and LYS were measured using the BCA Protein Quantitation Kit. Protein yield (Y) is calculated using the equation:
2.4. Determination of ILs and proteins concentration in ILATPSs
Fig. 2 shows the phase diagrams of the three ILATPSs including binodal curves and the tie lines with various TLLs. To obtain bestfit results, the experimental binodal data of various ILATPSs were correlated by different binodal empirical equations, respectively [33,34] by ATPS-LLE software, and the best-fit empirical equation of the binodal curve was expressed as follows:
The concentrations of IL in both the top phase and the bottom phase were determined quantitatively by UV–vis spectrophotometry at 240 nm [10,17], respectively, where the IL molecules had the high absorption peaks. The partition coefficient (K) of IL is calculated using the equation: K=
Ct,IL Cb,IL
Y (%) =
Ct × Vt × 100% Ct × Vt + Cb × Vb
where Ct and Cb are the equilibrium concentrations of protein in the top phase and the bottom protein concentration (the initial protein concentration), respectively. Vtop and Vb represent the top phase volume and the bottom volume, respectively. 3. Results and discussion 3.1. Phase diagram of ILATPS
w 2
w1 = a1 exp − (1)
where Ct,IL and Cb,IL are the equilibrium concentrations of IL in the top phase and the bottom phase, respectively. The BSA and LYS were chosen as the model proteins. Protein yield (Y) in the ILATPS was determined under different output DC voltages, respectively. The initial protein concentration was
(2)
b1
w 2
+ a2 exp −
b2
+c
(3)
Table 1 shows the values of coefficients in the empirical equations of binodal data for different ILATPSs and the lowest standard deviation (SD) values of the best-fit empirical equation. It shows that the empirical equations can be satisfactorily applied to correlate the binodal data of the investigated ILATPSs based on all SD < 0.05. Table 2 and Fig. 2 show the calculated tie-lines data
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
4
Table 1 Coefficients of Different Equations for the Correlation Binodal Data of ILATPS at Temperature T = 25 ◦ C and Pressure p = 0.1 MPaa . ATPS
[Bmim]Cl(1)- K2 HPO4 (2) [Bmim]BF4 (2)- NaH2 PO4 (1) [Bmim]BF4 (2)- Na3 C6 H5 O7 (1) a b
c
coefficientsb
SDc
a1
b1
a2
b2
c
0.5246 0.2938 0.3762
0.2984 0.1222 0.0545
0.2148 0.3603 0.1052
0.0260 0.0334 0.2934
−0.0800 0.0159 −0.0054
Standard uncertainties u are u(T) = 0.1 ◦ C, u(w) = 0.001, and u(p) = 10 kPa. The empirical equation of binodal curve is w1 = a1 exp −
W2 b1
+ a2 exp −
SD represents the standard deviations and can be calculated by SD =
W2 b2
+ c.
ni=1 wl,cal − wl,exp
2 0.5 /n
0.0019 0.0017 0.0010
, where n, w1,cal and w1,exp are the number of binodal data, the calculated
and experimental composition 1 mass fraction, respectively. Table 2 Experimental Volume Ratio (Vr,exp ), 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(mass fraction)
Vr,exp
w1 t
w2 t
w1 b
w2 b
TLL
[Bmim]Cl (1) + K2 HPO4 (2) + water (3) (0.318,0.182) (0.306,0.170) (0.296,0.160) (0.286,0.150) (0.276,0.140)
0.993 1.046 1.020 1.056 1.162
0.355 0.331 0.305 0.270 0.220
0.067 0.082 0.098 0.124 0.166
0.006 0.009 0.015 0.036 0.073
0.569 0.533 0.496 0.444 0.370
0.61 0.55 0.49 0.40 0.25
NaH2 PO4 (1) + [Bmim]BF4 (2) + water (3) (0.438, 0.090) (0.433, 0.080) (0.428, 0.070) (0.423, 0.060) (0.408, 0.050)
1.110 1.068 1.154 1.070 1.230
0.1023 0.1149 0.1288 0.155 0.174
0.1589 0.1422 0.1268 0.104 0.091
0.8106 0.7737 0.688 0.674 0.586
0.01436 0.01423 0.0178 0.0180 0.0188
0.72 0.67 0.57 0.53 0.42
Sodium Citrate (1) + [Bmim]BF4 (2) + water (3) 1.056 (0.430, 0.065) 1.039 (0.430, 0.055) 1.006 (0.430, 0.430) (0.430, 0.045) 0.995 (0.430, 0.035) 0.995 (0.430, 0.030) 0.990 0.921 (0.430, 0.025)
0.1035 0.108 0.117 0.144 0.176 0.201 0.254
0.126 0.118 0.108 0.086 0.068 0.056 0.043
0.759 0.753 0.742 0.718 0.686 0.659 0.609
0.0027 0.0030 0.0035 0.0042 0.0047 0.0050 0.0070
0.67 0.65 0.62 0.58 0.51 0.46 0.36
a
Standard uncertainties u are u(T) = 0.1 ◦ C, u(w) = 0.001, u(Vr ) = 0.01, and u(p) = 10 kPa.
including the two composition concentrations in the top or bottom phase across the experimental feed samples, which indicate the accurate fitted result. It can be seen that the [Bmim]BF4 concentration of IL-rich phases in [Bmim]BF4 -based ILATPSs were generally higher (>60 wt%) than the [Bmim]Cl concentration of IL-rich phases in [Bmim]Cl-K2 HPO4 ATPS, which was consistent with the reported research results [23,27]. As mentioned above, the top phase was the [Bmim]Cl-rich phase in [Bmim]Cl-K2 HPO4 ATPS, while the bottom phase was the [Bmim]BF4 -rich phase in the bottom phase in [Bmim]BF4 -NaH2 PO4 or [Bmim]BF4 -Na3 C6 H5 O7 ATPS. Each 10 g ILATPS with different TLL was adequately shaken for more than 5 min in a 10 mL sealed tube and settled for demixing. Fig. 3 shows the phase separation processing photographs during settling at different moments including 30 s, 60 s and 90 s. It can clearly be seen that the two phase separation rate in [Bmim]Cl-K2 HPO4 ATPS was faster than the rate in [Bmim]BF4 -based ATPS. [Bmim]Cl-K2 HPO4 ATPS can reach complete phase separation within 90 s, while the two [Bmim]BF4 -based ATPSs can’t. During the phase separation of the three ILATPS, it should be clear that the droplets turned to bigger and fewer with the droplets coalesced. In [Bmim]Cl-K2 HPO4 ATPS (Fig. 3a), the droplets became large and sparse from the bottom phase to the top phase, which can be obviously observed at 60 s (close to complete phase separation). It was indicated that the droplets of coalescence layer of [Bmim]Cl-K2 HPO4 ATPS ascended during demixing process. On the other hand, in [Bmim]BF4 -based ATPS (Fig. 3b and c), the droplets became large and sparse from the top phase to the bottom phase clearly observed at 90s, which indicated that the droplets of coalescence layer of [Bmim]BF4 -based
ATPS descended during demixing process. The top phase was saltrich phase in [Bmim]Cl-K2 HPO4 ATPS, while the top phase was IL-rich phase in [Bmim]BF4 -based ATPS. It had been generally recognized that the coalescence layer of ATPS dispersed from the dispersed phase to the continuous phase [35,36] during demixing process. The above experiment phenomena in Fig. 3 displayed that the salt-rich phase was the continuous phase and the IL-rich phase was the dispersed phase in the demixing process of the three ILATPS. It had been found that the demixing kinetic behavior was more dependent on the physical property (especially on viscosity) of continuous phase because the continuous phase strongly influence the movement of the drops of the dispersed phase [36,37]. For the three ILATPS, the IL type and concentration in the salt-rich phase would play an important role in the demixing rate. Compared with [Bmim]Cl-K2 HPO4 ATPS, the higher IL concentration of salt-rich phase in [Bmim]BF4 -based ATPS (in Fig. 2) caused slower demixing rate. 3.2. Electrokinetic demixing of ILATPS The electrokinetic demixing experiment for the three ILATPSs with various TLLs was carried out by varying the output DC voltage (0 V, 30 V, 90 V, 150 V) at normal polarity and reverse polarity, respectively. Fig. 4 displays the effect of output DC voltage and polarity on the demixing rate of the three ILATPSs, which is displayed as the emerged clear liquor height in the top phase per unit of time. It can be seen that the demixing rate of the three ILATPSs generally increased with an increase in output DC voltage
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model BEJ-6550; No. of Pages 9
ARTICLE IN PRESS X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
80 70 60 50 40
5
TLL=42,NP TLL=42,RP TLL=55,NP TLL=55,RP TLL=64,NP TLL=64,RP
[Bmim]Cl-K2HPO4 ATPS
30 20 10
Demixing rate (cm/min)
0
[Bmim]BF4-NaH2PO4 ATPS
12 10 8 6 4 2 0
[Bmim]BF4-Na3C6H5O7 ATPS
16 12 8 4 0 0
30
90
150
Voltage (V) Fig. 4. Effect of the output DC voltage including normal polarity (NP) and reverse polarity (RP) on demixing rate of [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 - Na3 C6 H5 O7 ATPS with different TLL, respectively.
Fig. 3. The phase separation processing photographs at different momemts (30 s, 60 s and 90 s) during settling of [Bmim]Cl-K2 HPO4 ATPS (a), [Bmim]BF4 -NaH2 PO4 ATPS (b) and [Bmim]BF4 -Na3 C6 H5 O7 ATPS (c) with TLL = 64.
or TLL based on hydrodynamic flow-electroosmotic flow (HEF). HEF caused the charged droplets move to an electrode, which was reaction flow or circulation flow (resistance to droplets) subjected to gravity, buoyant and electric field force [31]. This HEF model can explain more clearly electrokinetic demixing of ATPSs in a column, which was mainly influenced by the two phase solutions respective viscosity. Thus, the droplets would be subjected to more resistance in the continuous phase with higher IL concentration (such as the salt-rich phase in [Bmim]BF4 -based ATPS with higher viscosity). Three special rules will be discussed in detail below. Firstly, the demixing rate of [Bmim]Cl-K2 HPO4 ATPS was faster (nearly 20 cm/min) than the other two [Bmim]BF4 -based ILATPSs as mentioned above, and the efficiency of electrokinetic demixing of [Bmim]Cl-K2 HPO4 ATPS was also higher (the demixing rate increased more than 2.5 fold) than that of the other two ILATPSs. The overall concentrations of salt are much higher (>15 wt%) in [Bmim]Cl-K2 HPO4 ATPS than in [Bmim]BF4 -
based ATPSs (<8–10 wt%), and the overall [Bmim]Cl concentrations are lower (<20 wt%) than the overall [Bmim]BF4 concentrations (>40 wt%) for the three ILATPSs mentioned above. The salting-out effect led to a higher demixing rate in [Bmim]Cl-K2 HPO4 ATPS compared with the [Bmim]BF4 -based ATPS. The high concentration of salts also enhanced the conductivity and salting-out effect in the electric field, which caused the demixing rate of [Bmim]Cl-K2 HPO4 ATPS to increase from 18.75 cm/min to 48.98 cm/min in TLL = 64 using the output DC voltage of 150 V (RP). Moreover, the lower IL concentration with lower viscosity would likely enhance the disperse rate of IL-rich phase in the corresponding ILATPS, which would likely further increase the demixing rate. Secondly, TLLs played an important role in electrokinetic demixing. In [Bmim]BF4 -NaH2 PO4 ILATPS, the efficiency of electrokinetic demixing decreased with an increase in output DC voltage, and the demixing rate even decreased at TLL = 64. In [Bmim]BF4 based ILATPS, these three tie-lines had higher concentrations of IL (>60 wt% in the top phase, >10 wt% in the bottom phase) and lower concentration of salts (<15–20 wt%) than [Bmim]Cl-K2 HPO4 ATPS, so the salting-out effect was weaker and IL concentration played a more important role in the demixing process. There were obviously two opposite effects of the higher IL concentrations on the demixing rate of ILATPS in the electric field. One was the dispersion of IL molecules that increased the demixing rate, similar to the
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model BEJ-6550; No. of Pages 9
ARTICLE IN PRESS X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
6
80
TLL=42,NP
TLL=42,RP
TLL=55,NP
TLL=55,RP
TLL=64,NP
TLL=64,RP
[Bmim]Cl-K2HPO4 ATPS
60
40
20
Fig. 5. The most electrokinetic promotion (a–c) and inhibition (d) processing photographs of [Bmim]Cl-K2 HPO4 ATPS with TLL = 64 at 25 s, V = 150 V (a), [Bmim]BF4 -NaH2 PO4 ATPS with TLL = 55 at 60 s, V = 90 V (b), [Bmim]BF4 - Na3 C6 H5 O7 ATPS with TLL = 64 at 60 s, V = 150 V (c) and [Bmim]BF4 -NaH2 PO4 ATPS with TLL = 64 at 180 s, V = 150 V (d) under different output DC voltage of reverse polarity.
salting-out effect, and the other was the gathering of IL molecules with higher IL concentration that decreased the demixing rate with an increase in output DC voltage. The more rapid demixing rates (9.6 cm/min and 11.16 cm/min) of the two [Bmim]BF4 -based ILATPSs consistently appeared with the intermediate TLL = 55 at the output DC voltage of 90 V (RP). Thirdly, in the three ILATPSs, reverse polarity had generally higher efficiency of electrokinetic demixing than normal polarity, especially in the [Bmim]BF4 -based ILATPS. Fig. 5 shows the highest electrokinetic promotion (a, b, c) and inhibition (d) processing photographs of the three IL-based ATPS under different output DC voltages (150 V, 90 V and 150 V) of reverse polarity at different moments, which had the highest or the lowest demixing rates of various ILATPSs according to Fig. 4. It has been reported that the droplets motion can be interpreted during ATPS demixing process [38] by Stokes law. The Stokes resistance of droplets during moving can decrease with the decrease of droplet size. It can be seen that the droplet size in the three ILATPSs’ (in Fig. 5a–c) coalescence layer generally shrink under the electric field with reverse polarity, which decrease the Stokes resistance of droplets. It would lead to accelerate the demixing ratio of the ILATPS. On the other hand, the reverse polarity (90 V and 150 V) had an adverse effect on the demixing rate at TLL = 42 in [Bmim]Cl-K2 HPO4 ATPS and TLL = 64 in [Bmim]BF4 -NaH2 PO4 ATPS. Fig. 5d shows the electrokinetic inhibition (TLL = 64, 150 V) for [Bmim]BF4 -NaH2 PO4 ATPS demixing processing photographs. It can be seen that the more concentrated and smaller droplets at 150 V (RP) in Fig. 5d impeded the coalescence rate of the top and bottom phase, which caused a decrease in the demixing rate. 3.3. Partitioning of ILs in ILATPS The partitioning of [Bmim]Cl or [Bmim]BF4 was studied through quantitative determination of IL at different DC voltages (0 V, 30 V, 90 V, 150 V) in these three ILATPSs with different TLLs. The values of the partition coefficient (K) of IL could be obtained by Eq. (1) at various conditions. K > 1 means the IL distributed more in the top phase, as seen in [Bmim]Cl-based ATPS, while K < 1 means the IL partitioning occurred more in the bottom phase, such as in [Bmim]BF4 -based ATPS. Table 3 shows the theoretical partition coefficient by ATPS-LLE soft and experimental partition coefficient of different TLLs in these three ILATPSs, which were broadly con-
Patition coefficient of IL
0
[Bmim]BF4-NaH2PO4 ATPS
0.25 0.2 0.15 0.1 0.05 0
[Bmim]BF4-Na3C6H5O7 ATPS
0.5 0.4 0.3 0.2 0.1 0 0
30 Voltage (V)
90
150
Fig. 6. Effect of DC voltage and field polarity on partition coefficient of ILs in [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLL.
sistent. This spectrophotometric method was proved reliable for IL concentration measurement. Fig. 6 displays the effect of DC voltage and field polarity on the partition coefficient of ILs in [Bmim]Cl-K2 HPO4 , [Bmim]BF4 NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLLs, respectively. In the three ILATPSs, due to the differences in ILs-rich phase, the K values of IL increased with increasing TLL in [Bmim]Clbased ILATPS, while the K values of IL decreased with increasing TLL in [Bmim]BF4 -based ILATPS. Regarding the effect of electric field polarity, it can be seen that the K values of ILs decreased in the NP field and increased in the RP field in the three ILATPSs. Thus, ILs distributed more to the bottom phase in the NP field, while ILs distributed more to the top phase in the RP field regardless of IL type. The cations and anions of ILs partially decomposed under the electric field [39,40], and the bigger cations moved faster towards the cathode (bottom phase in NP and top phase in RP). Moreover, the smaller anions of [Bmim]Cl caused the above trend more obviously in the [Bmim]Cl-based ILATPS. This indicated that the ion’s type and movement in the electric field of different ILs played a key role in the IL partitioning in ILATPSs. Fig. 6 displayed that the electric voltage had no significant effect on the IL partitioning in ILATPS under the electric field. Based on the above experimental results, it could
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
7
Table 3 The Theoretical Partition Coefficient (Ktheory ) and Experimental Partition Coefficient (Kexp ) of ILs with Different TLL. ILATPS
[Bmim]Cl-K2 HPO4
TLL
0.64 0.55 0.42
110
[Bmim]BF4 -NaH2 PO4 Ktheory
Kexp
Ktheory
Kexp
Ktheory
56.991 29.760 8.517
60.402 32.777 9.590
0.150 0.201 0.259
0.167 0.208 0.276
0.172 0.296 0.376
0.153 0.284 0.347
TLL=42,NP
TLL=42,RP
TLL=55,NP
TLL=42,NP
TLL=42,RP
TLL=55,NP
TLL=55,RP
TLL=64,NP
TLL=64,RP
TLL=55,RP
TLL=64,NP
TLL=64,RP
[Bmim]Cl-K2HPO4 ATPS
110
100
100
90
90
80
80
70
[Bmim]Cl-K2HPO4 ATPS
70
60
60
[Bmim]BF4-NaH2PO4 ATPS
80
[Bmim]BF4-NaH2PO4 ATPS
100
LYS Yield (%)
BSA Yield (%)
[Bmim]BF4 -Na3 C6 H5 O7
Kexp
60 40 20
80 60 40
0
[Bmim]BF4-Na3C6H5O7 ATPS
20
[Bmim]BF4-Na3C6H5O7 ATPS
100
60
90
40
80 20
70 0 0
30 90 Voltage (V)
150
Fig. 7. Effect of DC voltage and field polarity on BSA yield in [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLL.
be speculated that the presence of electric field would regulate the binodal curves of the same ILATPS. Further, the two phase areas of [Bmim]Cl-based ILATPS would increase in RP electric field, while the two phase areas of [Bmim]BF4 -based ILATPS would increase in NP electric field. 3.4. Distribution of proteins in ILATPS Two model proteins (BSA and LYS) were selected to investigate the partitioning in the three ILATPSs at different output DC voltages. Most of the proteins generally distributed in the top phase in each ILATPS, while few proteins distributed in the bottom phase. Figs. 7 and 8 display the effect of DC voltage and field polarity on yields of BSA and LYS in [Bmim]Cl-K2 HPO4 ,
60 0
30 Voltage (V)
90
150
Fig. 8. Effect of DC voltage and field polarity on LYS yield in [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLL.
[Bmim]BF4 -NaH2 PO4 , and [Bmim]BF4 -Na3 C6 H5 O7 ATPS with different TLLs, respectively. Comparing the two protein yields shown in the two figures, it displayed that the LYS yield was generally higher than BSA yield due to LYS’s lower molecular weight. For instance, the protein yield of BSA and LYS reached 78–94% and 93–99% at different DC voltages, respectively, in [Bmim]ClK2 HPO4 ATPS with TLL = 42. In [Bmim]Cl-K2 HPO4 ATPS, there was quite a strong salting-out effect due to the high concentration of salts as mentioned above. Thus, salting-out effect played a critical role in the protein distribution of [Bmim]Cl-based ATPS. Higher inorganic salt concentration ([K2 HPO4 ] > 40 wt%) in the top phase resulted in stronger salting-out effect for proteins, which drove protein partitioning in the IL-rich phase (top phase). The protein
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model
ARTICLE IN PRESS
BEJ-6550; No. of Pages 9
X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
8
Table 4 The pH Values of Various ILATPS and Electrostatic Charges (E.C.)a of Proteins in ILATPS.
to the salting-out effect and partitioned in the salt-rich phase in [Bmim]BF4 -based ATPS due to hydrophobic repulsion.
ILATPS
[Bmim]ClK2 HPO4
[Bmim]BF4 NaH2 PO4
[Bmim]BF4 Na3 C6 H5 O7
Acknowledgements
pH E.C. of BSA (pI = 4.7) E.C. of LYS (pI = 11.1)
9.6–9.8 −− +
3.8−4.0 + +++
6.4–6.7 − ++
This work is supported by the National Natural Science Foundation of China (No. 21476135), Outstanding Young Teachers Training Program in Guangdong Higher Education Institutions (No. Yq2013076).
a The symbols of “+” and “−” denotes the positive and negative E.C. of proteins, respectively, and the numbers of written symbols visually displays the amount of proteins E.C. in ILATPS.
References yield generally decreased with increasing TLL, which was tested by the proteins loss in the interphase (the insoluble solid content between the top phase and the bottom phase) due to the decrease of protein solubility in ATPS [41], and this trend was particularly apparent for BSA in [Bmim]BF4 -based ATPS. Moreover, in [Bmim]BF4 -NaH2 PO4 and [Bmim]BF4 -Na3 C6 H5 O7 ATPSs, the proteins distributed in the salt-rich phase (top phase) when [Bmim]BF4 enriched in the bottom phase. Compared with the bottom phase in [Bmim]Cl-K2 HPO4 ATPS, the high concentration of hydrophobic [Bmim]BF4 would lead to the lower dissolving capacity for the water-soluble proteins in the [Bmim]BF4 -rich phase. It indicated that the hydrophobic repulsion played a leading role in protein partitioning in [Bmim]BF4 -based ATPS, which was in accord with reports on the other IL ATPS [16,42]. In the top phase of [Bmim]BF4 -based ATPS, there were still some [Bmim]BF4 with low concentration (about 10–20 wt%). The decrease of protein yield with increasing TLL can be observed in Figs. 7 and 8, which was due to the increase of hydrophobic repulsion with increasing [Bmim]BF4 concentration in the top phase. Table 4 displayed the solutions pH values of three ILATPS and the amount of electrostatic charges of BSA and LYS in the three ILATPSs. Though the electrical property and the charge amount of BSA and LYS were obviously different in the three ILATPSs, the variation trends of two proteins yield were almost same with DC voltage or the field polarity. Figs. 7 and 8 and 8 showed the increase of DC voltage or the difference of field polarity had weak effect on proteins yield in the two ILATPSs, which indicated that the various electrostatic charges of proteins did not play a key role in proteins partitioning in the three ILATPSs. 4. Conclusions The liquid–liquid equilibrium of three ILATPSs consisting of [Bmim]Cl-K2 HPO4 , [Bmim]BF4 -NaH2 PO4 and [Bmim]BF4 Na3 C6 H5 O7 under an electric field were extensively studied. The eletrokinetic demixing of the three ILATPSs experiments were operated at different DC voltage levels under NP or RP electric fields, and the partitioning of ILs and model proteins (including LYS and BSA) under electric fields were investigated, respectively. The results indicated that [Bmim]Cl-K2 HPO4 ATPS with higher salt concentrations and lower IL concentrations had a faster two phase separation rate and a higher efficiency of electrokinetic demixing due to the salting-out effect. IL molecules dispersing and gathering changed with TLL and DC voltage, which led to the highest electrokinetic promotion and the highest inhibition demixing in [Bmim]BF4 -NaH2 PO4 ATPS at TLL = 55, 90 V (RP) and TLL = 64, 150 V (RP), respectively. The movement of bigger organic cations of IL molecules under an electric field generally caused the K values of IL in the three ILATPSs to decrease under an NP electric field and to increase under an RP electric field. For the larger model protein partitioning in the three ILATPSs, the electric field had no significant effect on the protein distribution trend. Most model proteins partitioned in the IL-rich phase in [Bmim]Cl-K2 HPO4 ATPS due
[1] R. Kenneth, J. Seddon, Ionic liquids for clean technology, J. Chem. Technol. Biotechnol. 68 (1997) 351–356. [2] W.L. Hough, M. Smiglak, H. Rodríguez, R.P. Swatloski, S.K. Spear, D.T. Daly, et al., The third evolution of ionic liquids: active pharmaceutical ingredients, New J. Chem. 31 (2007) 1429–1436. [3] T. Welton, Room-temperature ionic liquids: solvents for synthesis and catalysis, Chem. Rev. 99 (1999) 2071–2084. [4] P. Wasserscheid, W. Keim, Ionic liquids-new solutions for transition metal catalysis, Angew. Chem. Int. Ed. 39 (2000) 3772–3789. [5] J. Dupont, R.F.D. Souza, P.A.Z. Suarez, Ionic liquid (molten salt) phase organometallic catalysis, Cheminform 34 (3) (2003) 3667–3692. [6] S. Pandey, Analytical applications of room-temperature ionic liquids: a review of recent efforts, Anal. Chim. Acta 556 (2006) 38–45. [7] X.X. Han, D.W. Armstrong, Ionic liquids in separations, Acc. Chem. Res. 40 (2007) 1079–1086. [8] K.N.K. Shimojo, N. Kamiya, M. Goto, Crown ether-mediated extraction and functional conversion of cytochrome c in ionic liquids, Biomacromolecules 7 (2006) 2–5. [9] K.E. Gutowski, G.A. Broker, H.D. Willauer, J.G. Huddleston, R.P. Swaloski, J.D. Holbrey, R.D. Rogers, Controlling the aqueous miscibility of ionic liquids: aqueous biphasic systems of water-miscible ionic liquids and water-structuring salts for recycle, metathesis, and separations, J. Am. Chem. Soc. 125 (22) (2003) 6632–6633. [10] Y. Pei, J. Wang, L. Liu, Liquid-liquid equilibria of aqueous biphasic systems containing selected imidazolium ionic liquids and salts, J. Chem. Eng. Data 52 (5) (2007) 2026–2031. [11] J. Han, Y. Wang, W. Kang, C. Li, Y. Yan, J. Pan, X. Xie, Phase equilibrium and macrolide antibiotics partitioning in real water samples using a two-phase system composed of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and an aqueous solution of an inorganic salt, Microchim. Acta 169 (1–2) (2010) 15–22. [12] Y.C. Pei, Z.Y. Li, L. Li, J.J. Wang, H.Y. Wang, Selective separation of protein and saccharides by ionic liquids aqueous two-phase systems, Sci. China 53 (7) (2010) 1554–1560. [13] S. Dreyer, P. Salim, U. Kragl, Driving forces of protein partitioning in an ionic liquid-based aqueous two-phase system, Biochem. Eng. J. 46 (2) (2009) 176–185. [14] Iyyaswami Regupathi, Shanon L. Monteiro, 1-Hexyl-3-methylimidazolium chloride-potassium carbonate aqueous two phase system: equilibrium characteristics and BSA partitioning behavior, J. Dispersion Sci. Technol. 35 (3) (2013) 418–427. [15] D. Zhuo, Y.L. Yu, J.H. Wang, Extraction of proteins from biological fluids by use of an ionic liquid/aqueous two-phase system, Chemistry 13 (7) (2007) 2130–2137. [16] Y. Pei, J. Wang, K. Wu, X. Xuan, X. Lu, Ionic liquid-based aqueous two-phase extraction of selected proteins, Sep. Purif. Technol. 64 (3) (2009) 288–295. [17] Y. Jiang, H. Xia, C. Guo, I. Mahmood, H. Liu, Enzymatic hydrolysis of penicillin in mixed ionic liquids/water two-phase system, Biotechnol. Prog. 23 (4) (2007) 829–835. [18] Y.F. Li, J. Han, Y. Wang, J.J. Ma, Y.S. Yan, Partitioning of cephalexin in ionic liquid aqueous two-phase system composed of 1-butyl-3-methylimidazolium tetrafluoroborate and ZnSO4 , J. Chem. (2013) 1–5. [19] J.K. Yan, H.L. Ma, J.J. Pei, Z.B. Wang, J.Y. Wu, Facile and effective separation of polysaccharides and proteins from Cordyceps sinensis mycelia by ionic liquid aqueous two-phase system, Sep. Purif. Technol. 135 (2014) 278–284. [20] Z.J. Tan, F.F. Li, X.L. Xu, J.M. Xing, Z.J. Tan, F.F. Li, et al., Simultaneous extraction and purification of aloe polysaccharides and proteins using ionic liquid based aqueous two-phase system coupled with dialysis membrane, Desalination 286 (1) (2012) 389–393. [21] B. Jiang, Z. Feng, C. Liu, Y. Xu, D. Li, J. Guo, Extraction and purification of wheat-esterase using aqueous two-phase systems of ionic liquid and salt, J. Food Sci. Technol. 52 (5) (2015) 2878–2885. [22] J. Han, R. Pan, X. Xie, Y. Wang, Y. Yan, G. Yin, et al., Liquid-liquid equilibria of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate + sodium and ammonium citrate aqueous two-phase systems at (298.15, 308.15, and 323.15) K, J. Chem. Eng. Data 55 (9) (2010) 3749–3754. [23] J. Han, C. Yu, W. Yun, X. Xie, Y. Yan, G. Yin, et al., Liquid–liquid equilibria of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and sodium citrate/tartrate/acetate aqueous two-phase systems at 298. 15 K: experiment and correlation, Fluid Phase Equilib. 295 (1) (2010) 98–103.
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008
G Model BEJ-6550; No. of Pages 9
ARTICLE IN PRESS X. Li et al. / Biochemical Engineering Journal xxx (2016) xxx–xxx
[24] J. Han, Y. Wang, C. Yu, Y. Li, W. Kang, Y. Yan, (Liquid + liquid) equilibrium of (imidazolium ionic liquids + organic salts) aqueous two-phase systems at T = 298.15 K and the influence of salts and ionic liquids on the phase separation, J. Chem. Thermodyn. 45 (1) (2012) 59–67. [25] J. Han, Y. Wang, C. Chen, W. Kang, Y. Liu, K. Xu, et al., (Liquid + liquid) equilibria and extraction capacity of (imidazolium ionic liquids + potassium tartrate) aqueous two-phase systems, J. Mol. Liq. 193 (2014) 23–28. [26] M.T. Zafarani-Moattar, S. Hamzehzadeh, Effect of pH on the phase separation in the ternary aqueous system containing the hydrophilic ionic liquid 1-butyl-3-methylimidazolium bromide and the kosmotropic salt potassium citrate at T = 298.15 K, Fluid Phase Equilib. 304 (1) (2011) 110–120. [27] M.T. Zafarani-Moattar, S. Hamzehzadeh, Salting-out effect, preferential exclusion, and phase separation in aqueous solutions of chaotropic water-miscible ionic liquids and kosmotropic salts: effects of temperature, anions, and cations, J. Chem. Eng. Data 55 (4) (2010) 1598–1610. [28] K.S.M.S. Raghavarao, R.M. Stewart, P. Todd, Electrokinetic demixing of two-phase aqueous polymer systems. I. Separation rates of polyethylene glycol-dextran mixtures, Sep. Sci. Technol. 25 (9–10) (1990) 985–996. [29] K.S.M.S. Raghavarao, R.M. Stewart, P. Todd, Electrokinetic demixing of two-phase aqueous polymer systems II. Separation rates of polyethylene glycol-maltodextrin mixtures, Sep. Sci. Technol. 26 (2) (1991) 257–267. [30] K.S.M.S. Raghavarao, R.M. Stewart, S.R. Rudge, P. Todd, Electrokinetic demixing of aqueous two-phase systems 3. Drop electrophoretic mobilities and demixing rates, Biotechnol. Prog. 14 (6) (1998) 922–930. [31] N. Naveen, C. Sampangi, K.S.M.S. Raghavarao, Electrokinetic demixing of aqueous two-phase polymer-salt systems, Electrophoresis 26 (1) (2005) 10–17. [32] M.C. Madhusudhan, S. Chethana, K.S.M.S. Raghavarao, Electrokinetic demixing of polymer-salt systems containing biomolecules, Sep. Sci. Technol. 46 (5) (2011) 727–733.
9
[33] Y. Liu, Y. Feng, Y. Zhao, Liquid-liquid equilibrium of various aqueous two-phase systems: experiment and correlation, J. Chem. Eng. Data 58 (10) (2013) 2775–2784. [34] L. Liu, Y. Liu, L. Du, Y. Zhao, H. Du, (Liquid + liquid) phase equilibrium of aqueous two-phase system containing (surfactant + sodium sulfate + water) at different temperatures, Fluid Phase Equilib. 415 (15) (2016) 25–33. [35] V.H. Nagaraja, R. Iyyaswami, Phase demixing studies in aqueous two-phase system with polyethylene glycol (PEG) and sodium citrate, Chem. Eng. Commun. 200 (10) (2013) 1293–1308. [36] H. Yuan, Y. Liu, W. Wei, Y. Zhao, Phase separation behavior and system properties of aqueous two-phase systems with polyethylene glycol and different salts: experiment and correlation, J. Fluids (2015) 1–10. [37] J.A. Asenjo, S.L. Mistry, B.A. Andrews, J.C. Merchuk, Phase separation rates of aqueous two-phase systems: correlation with system properties, Biotechnol. Bioeng. 79 (2) (2002) 217–223. [38] J.C. Merchuk, B.A. Andrews, J.A. Asenjo, Aqueous two-phase systems for protein separation: studies on phase inversion, J. Chromatogr. B: Biomed. Sci. Appl. 711 (1–2) (1998) 285–293. [39] A.P. Abbott, Application of hole theory to the viscosity of ionic and molecular liquids, Chemphyschem 5 (2004) 1242–1246. [40] A.P. Abbott, Model for the conductivity of ionic liquids based on an infinite dilution of holes, Chemphyschem 6 (2005) 2502–2505. [41] K.C. Ross, C. Zhang, Separation of recombinant -glucuronidase from transgenic tobacco by aqueous two-phase extraction, Biochem. Eng. J. 49 (3) (2010) 343–350. [42] C. Wu, J. Wang, Z. Li, J. Jing, H. Wang, Relative hydrophobicity between the phases and partition of cytochrome-c in glycine ionic liquids aqueous two-phase systems, J. Chromatogr. A 1305 (2013) 1–6.
Please cite this article in press as: X. Li, et al., Effects of DC electric field on phase equilibrium and partitioning of ionic liquid-based aqueous two-phase systems, Biochem. Eng. J. (2016), http://dx.doi.org/10.1016/j.bej.2016.09.008