Article

pubs.acs.org/jced

Liquid−Liquid Equilibrium of Primary and Secondary Aqueous Two-

Phase Systems Composed of Sucrose + Triton X‑114 + Water at
Different Temperatures
Subbarayalu Ramalakshmi,† Ramakrishnan Nagasundara Ramanan,*,†,‡ Arbakariya B. Ariff,§
and Chien Wei Ooi*,†
†
Chemical Engineering Discipline, School of Engineering, and ‡Advanced Engineering Platform, School of Engineering, Monash
University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor Malaysia
§
Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, UPM
Serdang, Selangor Malaysia

ABSTRACT: Liquid−liquid equilibrium (LLE) data for aqueous two-

phase systems (ATPSs) composed of sucrose, Triton X-114, and water
were determined at 288 K, 293 K, and 298 K. At 293 K and 298 K, the
top and bottom phases of the ATPS were enriched with sucrose and
Triton X-114, respectively. However, phase inversion was observed in
the ATPS formed at a lower temperature (288 K). When the
temperature increased, the area of the biphasic region became larger,
whereas the slopes and the lengths of the tie lines decreased. Binodal
data obtained at different temperatures were correlated using a fourth-
order polynomial equation, while tie-line data were correlated using
the Othmer−Tobias and Bancroft equations. Moreover, the
Setschenow equation was used to investigate the sugaring-out effect of sucrose on the formation of the ATPS. The correlated
results and fitting parameters from these empirical equations confirmed the reliability and reproducibility of the experimental
results. Furthermore, both top- and bottom-phase solutions can be heated to 303 K to form the secondary ATPSs composed of
sucrose and Triton X-114, respectively. The LLE data for secondary ATPSs are presented in this paper.

1. INTRODUCTION CPT.9 Hence, hydrophobic proteins solubilized in surfactant

In the field of bioprocess engineering, the purification and
separation of the target product often involve conventional
downstream processes that are expensive and highly
complicated.1 The aqueous two-phase system (ATPS) has
been viewed as an alternative purification technique to
overcome these limitations. The applicability of ATPS in the
purification of bioproducts (e.g., enzymes, proteins, and nucleic
acids) without altering their native structures has been
successfully demonstrated.2,3 High water content, low inter-
facial tension, potential of scaling up, and chemical recycling are
some of the advantages offered by ATPS.4 The types of phase-
forming components include polymers,2 ionic liquids,5 salts,6
alcohols,7 and surfactants.8,9 The formation of a two-phase
system is influenced by the physical and chemical properties of
the ATPS, including its phase composition, temperature, pH,
and presence of any additives in the system.10,11
Polyethylene glycol tert-octylphenyl ether [O-
(CH2CH2O)xH, x = 8], commonly known as Triton X-114,
is a nonionic surfactant that has frequently been applied in the
extraction of hydrophobic membrane proteins because of its
low cloud point temperature (CPT), that is, 301 K to 310 K for
mass fraction ranging from 0.01 to 0.20.9,12 A nonionic-
surfactant-containing solution separates into two phases
(surfactant-rich and aqueous phases) when heated above its
were extracted to the surfactant-rich phase, whereas hydrophilic
proteins were present in the surfactant-depleted phase.9
Additionally, Triton X-114 can be used as a phase-forming
component along with polymers such as polyethylene glycol
(PEG) and dextran in ATPS formation.8 In addition to the
commonly used phase-forming components, the use of
carbohydrates in forming ATPSs with acetonitrile, ionic liquids
or polymers, has been reported.5,13,14 Unlike phase-forming
components such as PEG and polypropylene glycol (PPG),
carbohydrates are biodegradable, nontoxic, and inexpensive.5
Considering the advantages exhibited by both types of phase-
forming reagents, it is of interest to explore the formation of
ATPSs using a thermoseparating polymer in combination with
a disaccharide such as sucrose. This is the first report on the
liquid−liquid equilibrium of ATPSs composed of sucrose +
Triton X-114 + water. In this study, binodal curves and tie lines
were determined at different temperatures for an ATPS
composed of sucrose + Triton X-114 + water. The effect of
temperature on the LLE of the ATPS was studied, and several
empirical equations were used to correlate the binodal and tie-
Received: April 4, 2014
Accepted: August 4, 2014
Published: August 13, 2014

© 2014 American Chemical Society 2756 dx.doi.org/10.1021/je500571k | J. Chem. Eng. Data 2014, 59, 2756−2762
Journal of Chemical & Engineering Data
line data. The Setschenow equation15,16 was used to explain the
sugaring-out effect on the ATPS. Moreover, discrete top and
bottom phases from the primary ATPS were thermally
separated into secondary phases in the absence of any additives,
and the compositions of the secondary phases were obtained.
2. MATERIALS AND METHODS
2.1. Materials. Sucrose (ACS reagent, CAS No. 57-50-1;
molar mass, 342 g mol−1) and Triton X-114 (laboratory grade,
CAS No. 9036-19-5; molar mass, 537 g mol−1) were purchased
from Sigma-Aldrich and were used in the experiment without
further purification. Sulfuric acid (CAS No. 7664-93-9) and
phenol (CAS No. 108-95-2) were purchased from Merck.
Ultrapure water was used throughout the experiment. A water
bath (model WNB 22, Memmert, Germany) with a sensitivity
of ± 0.1 K was used for maintaining the ATPS at a constant
temperature. The removal of water from the samples was
carried out using a freeze drier (model 110-4 PRO, Scanvac,
Denmark).
2.2. Construction of Binodal Curves. Triton X-114 and
sucrose stock solution (2.92 mol·kg−1) were used to form the
ATPS. Binodal curves were constructed at different temper-
atures using the turbidometric titration method.17,18 In brief, an
ATPS with a final weight of 2 g was prepared by loading known
quantities of Triton X-114, sucrose, and ultrapure water to a
centrifuge tube. The turbid mixture that formed was maintained
at a specific temperature using a water bath. Water was then
added dropwise to the centrifuge tube until the solution turned
clear. The mass of water added to the mixture was calculated by
subtracting the final weight of the diluted solution from the
initial weight of the solution. The above-mentioned procedure
was repeated to obtain sufficient points for constructing the
binodal curve. All of the weight measurements were performed
using an analytical balance with a sensitivity of ± 0.001 g.
2.3. Determination of Tie Lines. For the determination of
tie lines, a series of ATPSs were first prepared using a sucrose
stock solution (2.92 mol·kg−1), Triton X-114, and ultrapure
water. The tie lines could be plotted after the concentrations of
phase-forming components in both phases were determined.
All of the ATPSs were prepared in a syringe, which allowed an
easier collection of samples from the bottom phase and avoided
disrupting the equilibriums of both phases during collection of
the bottom-phase samples. The bottom phase was observed to
be more viscous compared to the top phase; thus, collecting the
bottom phase would be difficult if a centrifuge tube was used. A
syringe (10 mL capacity) was used in this work, and the tip was
sealed with Parafilm prior to the loading of solution. Known
quantities of sucrose, Triton X-114 and ultrapure water were
added, and the final weight of the ATPS was set at 5 g. A
vortexer was used to mix the solution in the syringe, and it was
left undisturbed in a water bath at a constant temperature for
approximately 8 to 12 h to allow for the separation of phases by
gravitational settling. Once the phases were formed after the
incubation period, samples from both phases were collected for
quantitative analysis. The top-phase solution was collected
using a pipet. A needle was used to puncture the Parafilm
covering the tip opening of the syringe, and the bottom-phase
solution dripped through the created hole. Samples collected
from both phases were incubated in a chiller at 277 K to
prevent the Triton X-114 in the system from forming two
phases when it was left at room temperature. The
concentrations of sucrose in the top and bottom phases were
determined by using the phenol−sulfuric acid method
2757
Article
conducted at low temperature. This modified phenol−sulfuric
acid method allowed an accurate measurement of sucrose
concentration in the presence of thermosensitive polymers. In
brief, a 96-well microplate loaded with the prechilled samples
(3 μL sample per well) was maintained at an ice-cold
temperature (8 °C). A 75 μL aliquot of concentrated sulfuric
acid (18 M) was added to the well followed by the addition of
15 μL of 0.53 M phenol solution. The mixture was shaken for 5
min and the solution’s absorbance was then measured at 520
nm using a microplate reader (Sunrise, Tecan). The
concentration of Triton X-114 in the samples was determined
by subtracting the masses of water and sucrose from the total
mass of the system. A freeze-drier was used to remove water
from the samples. The difference in the mass of the samples
before and after freeze-drying corresponded to the weight of
water in the phase solution. All of the experiments were
conducted in triplicate. Moreover, the densities of the top phase
and of individual phase components (sucrose and Triton X-
114) present in that phase were calculated from their respective
masses and volumes. A similar procedure was repeated with the
bottom phase.
2.4. Secondary Two-Phase Formation. After the primary
two-phase separation, aliquots of the primary top phase (3.5
mL) and bottom phase (1.5 mL) were transferred to individual
syringes, which were maintained in a water bath at 303 K. After
8 h to 12 h of incubation, a clear interphase was observed in the
primary top phases and in the primary bottom phases in the
syringes. The secondary phases were collected, and the
concentrations of sucrose, water, and Triton X-114 were
determined using the methods described in section 2.3. The
volume ratio (Vr) of the secondary ATPS was calculated by
dividing the volume of the top phase by the volume of the
bottom phase.
3. RESULTS AND DISCUSSION
3.1. Binodal Curve Data and Correlation. Throughout
the study of ATPSs composed of sucrose + Triton X-114 +
water, it was crucial to maintain the ATPSs at a temperature
lower than the lower critical solution temperature (LCST) of
Triton X-114 to prevent interference from the thermosepara-
tion of Triton X-114. In addition, the presence of sucrose in the
Triton X-114-containing solution will reduce the cloud point
temperature (CPT) of Triton X-114.12 Because the LCST of
Triton X-114 (0.018 mol·kg−1 to 0.465 mol·kg−1) falls between
T = (301 to 310) K,12,19 a temperature lower than 301 K
should be used for the formation of the ATPS. On the other
hand, it was observed that the ATPS formed below 288 K was
very viscous because of the high content of Triton X-114 in the
system, thereby making the handling of the phases difficult.
Hence, a temperature range of T = (288 to 298) K was
employed in the formation of the ATPS containing Triton X-
114 at a molality range of 0.009 to 0.163 mol·kg−1.
The binodal curve data of ATPSs composed of sucrose +
Triton X-114 + water were determined by conducting
experiments at 288 K, 293 K, and 298 K, and the respective
binodal data are presented in Table 1. Binodal curves formed at
different temperatures are presented in Figures 1 and 2. Eqs 1
and 2 given below represent a variant of the Merchuk
equation20 and a fourth-order polynomial equation,21 respec-
tively.
w1 = a exp(bw2 0.5 − cw2 3) (1)
dx.doi.org/10.1021/je500571k | J. Chem. Eng. Data 2014, 59, 2756−2762
Journal of Chemical & Engineering Data Article

Table 1. Experimental LLE Mass Fractions w (Binodal Curve

Data) for Sucrose + Triton X-114 + Water ATPSs Formed at
Different Temperatures
T/K = 288 T/K = 293 T/K = 298
wsucrose wTriton X‑114 wsucrose wTriton X‑114 wsucrose wTriton X‑114
0.2994 0.0499 0.1389 0.0056 0.0502 0.0050
0.2500 0.0517 0.1314 0.0057 0.0459 0.0051
0.2538 0.0544 0.1250 0.0062 0.0411 0.0051
0.2514 0.0559 0.1262 0.0074 0.0384 0.0055
0.2345 0.0563 0.1053 0.0101 0.0324 0.0075
0.2159 0.0604 0.1050 0.0185 0.0274 0.0091
0.2099 0.0672 0.0995 0.0249 0.0270 0.0095
0.1956 0.0704 0.0999 0.0294 0.0261 0.0098
0.1934 0.0757 0.0968 0.0339 0.0245 0.0113
0.1927 0.0771 0.0948 0.0426 0.0246 0.0116
0.1872 0.0814 0.0919 0.0460 0.0225 0.0132
0.1855 0.0890 0.0219 0.0135 Figure 2. Experimental and correlated binodal curves for the sucrose +
0.0210 0.0140 Triton X-114 + water ATPS formed at 288 K: △, experimental binodal
0.0205 0.0154 curve; ---, binodal curve correlated using eq 2.
0.0200 0.0156
0.0196 0.0177 Table 2. Values of Fitting Parameters Using eq 1 for Sucrose
0.0206 0.0183 + Triton X-114 + Water ATPSs Formed at Different
0.0195 0.0195 Temperatures
0.0195 0.0216
T/K a b c R2 s.d.a
0.0190 0.0217
288 94.410 −0.569 −1.300 0.980 2.76
0.0195 0.0217
293 −11.967 −88.066 −0.004 0.876 12.96
1.998 2.180
298 −3.449 −1464.69 −0.088 0.897 8.86
2 − w2 ))/N)
a
s.d. = ((∑i=1 toN(wexp cal 0.5
where w2 represents the mass
fraction of the primary component in the bottom phase, N is the
number of binodal data, and the superscripts “exp” and “cal” represent
the experimental and calculated values, respectively.

because the standard deviation (s.d.) values shown in Table 2

Figure 1. Experimental and correlated binodal curves for sucrose +
Triton X-114 + water ATPSs formed at 293 K and 298 K: △,
experimental binodal curve obtained at 293 K; ◊, experimental binodal
curve obtained at 298 K; ---, binodal curve correlated using eq 2.
w1 = aw2 4 + bw2 3 + cw2 2 + dw2 + e
(2)
where w1 and w2 are the mass fractions of the primary
component in the top and bottom phases, respectively. The a,
b, c, d, and e coefficients are fitting parameters. The fitting
parameters and coefficients of determination (R2) for the ATPS
at different temperatures using eqs 1 and 2 are listed in Tables 2
and 3, respectively. In general, eq 1 has been used to correlate
the binodal data for ATPSs containing polymer + salt,17 ionic
liquid + salt18 and alcohol + salt.7 However, eq 1 was not
reliable in correlating the experimental binodal curve data of
sucrose + Triton X-114 + water ATPS obtained in this study
2758
are considerably high. Moreover, Figures 1 and 2 did not follow
an exponential pattern, and the Merchuk equation, which best
fits such patterns, could not be applied in this case. Therefore,
an attempt was made to use eq 2 to correlate the experimental
binodal curve data, and the results of the correlation (Table 3)
are promising. All of the R2 values in Table 3 are close to unity,
which shows that eq 2 could be successfully used for correlating
the experimental binodal curve data.
3.2. Effect of Temperature on the Binodal Curve.
Triton X-114 at a molality range of (0.018 to 0.465) mol·kg−1
existed as a homogeneous solution below its LCST (301 K)
because the micelles were solubilized in water due to the
hydrogen bonding between the oxygen atoms of the detergent
and the solvent.9 At a temperature below the LCST of Triton
X-114, the system was less entropic because ethylene oxide
(EO) groups of Triton X-114 were highly ordered and
hydrated.12 When sucrose was added to the solution containing
Triton X-114, the hydroxyl groups (hydrogen bond breakers)22
present in the sucrose molecule disrupted the hydrogen
bonding. The solubility of Triton X-114 was reduced because
water molecules were attracted by sucrose which is more
hydrophilic than Triton X-114. Interactions among the EO
groups of Triton X-114 molecules would be promoted, thereby
leading to the aggregation of micelles followed by the two-
phase formation. In the sucrose + Triton X-114 + water ATPS,
the concentration of sucrose required to induce the phase
formation is inversely proportional to the temperature. At the
higher temperature condition, hydrogen bonding was weaker,
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Journal of Chemical & Engineering Data Article

Table 3. Values of Fitting Parameters Using eq 2 for Sucrose + Triton X-114 + Water ATPSs Formed at Different Temperatures
T/K a b c d e R2 s.d.a
288 0.001 −0.094 3.610 −62.080 407.310 1.000 0.46
293 0.066 −0.862 4.091 −8.664 17.042 0.979 0.66
298 1.349 −8.477 20.054 −21.718 11.367 0.973 0.27
2 − w2 ))/N)
a
s.d. = ((∑i=1 to N(wexp cal 0.5
where w2 represents the mass fraction of the primary component in the bottom phase, N is the number of
binodal data, and the superscripts “exp” and “cal” represent the experimental and calculated values, respectively.

and hence, a lower concentration of sucrose was required for

breaking the bond. Therefore, the area of the biphasic region
increased when the temperature increased from 293 K to 298
K. A similar trend was observed in previously reported
studies.15,16 In comparison with the binodal curves formed at
293 K and 298 K (Figure 1), the binodal curve formed at 288 K
(Figure 2) displaced further from the origin in the phase
diagram, indicating that a higher amount of sucrose (molality >
0.641 mol·kg−1) was required to induce ATPS formation as the
temperature decreased. Furthermore, phase inversion occurred
in the ATPS formed at 288 K, in which the top and bottom
phases of the ATPS were predominantly composed of Triton
X-114 and sucrose, respectively. Phase inversion behavior was
also noted in glycerol + Triton X-114 systems formed at 277 K
when the glycerol concentration exceeded 4.657 mol·kg−1 and
became denser than the other phase enriched with detergent.22
3.3. Tie-Line Data and Correlation. Experimental tie lines
for the sucrose + Triton X-114 + water ATPS are shown in Figure 4. Tie lines for the sucrose + Triton X-114 + water ATPS
Figures 3 to 5. The Othmer−Tobias (eq 3) and Bancroft formed at 293 K: △, experimental binodal curve; , binodal curve
correlated using eq 2; ---, experimental tie-line; ●, selected total
composition.

Figure 3. Tie lines for the sucrose + Triton X-114 + water ATPS
formed at 298 K: △, experimental binodal curve; , binodal curve Figure 5. Tie lines for the sucrose + Triton X-114 + water ATPS at
correlated using eq 2; ---, experimental tie-line; ●, selected total 288 K: △, experimental binodal curve; , binodal curve correlated
composition. using eq 2; ---, experimental tie-line; ●, selected total composition.

where wt1 and wb2 represent the mass fractions of the primary
17,18
equations (eq 4) were used to correlate the experimental component in the top and bottom phases, respectively; wt3 and
tie-line data listed in Table 4. The R2 values (greater than 0.96) wb3 represent the mass fractions of water in the top and bottom
shown in Table 5 reflect the reliability of the experimental phases, respectively; and K1, K2, n, and r are fitting parameters.
results. Furthermore, the experimental tie-line data were also correlated
⎛ 1 − W b ⎞η using the Setschenow equation (eq 5), and the s.d. values
⎛ 1 − W1t ⎞
⎜ ⎟ = K1⎜⎜ 2
⎟⎟ presented in Table 6 show that the experimental results were
⎝ W1 ⎠
t
⎝ W2 ⎠
b
(3) fitted satisfactorily.
t
⎛Wb ⎞ msucrose
⎛ W t ⎞r ln b
= β + K sucrose(m Triton t
X − 114 − m Triton X − 114 )
⎜⎜ 3b ⎟⎟ = K 2⎜ 3t ⎟ b
msucrose
⎝ W2 ⎠ ⎝ W1 ⎠ (4) (5)

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Journal of Chemical & Engineering Data Article

Table 4. Tie-Line Data for Sucrose + Triton X-114 + Water ATPSs Formed at Different Temperaturesa
total composition top-phase composition bottom-phase composition
T/K wsucrose wTriton X‑114 wsucrose wTriton X‑114 wsucrose wTriton X‑114 LTL STL
298 0.0300 0.0160 0.0360 0.0072 0.0146 0.0407 0.04 −0.65
0.0320 0.0180 0.0390 0.0069 0.0154 0.0446 0.05 −0.45
0.0340 0.0190 0.0410 0.0061 0.0140 0.0674 0.06 −0.50
0.0370 0.0210 0.0450 0.0058 0.0155 0.0564 0.07 −0.52
293 0.1100 0.0270 0.1220 0.0081 0.0841 0.0849 0.08 −0.49
0.1150 0.0280 0.1250 0.0070 0.0840 0.0850 0.09 −0.53
0.1200 0.0290 0.1345 0.0065 0.0850 0.0948 0.10 −0.57
0.1250 0.0300 0.1420 0.0060 0.0854 0.1060 0.11 −0.57
288 0.2050 0.0750 0.1407 0.1418 0.2177 0.0590 0.11 −1.08
0.2100 0.0770 0.1406 0.1493 0.2275 0.0596 0.12 −1.03
0.2150 0.0790 0.1384 0.1638 0.2378 0.0589 0.14 −1.06
0.2200 0.0810 0.1358 0.1813 0.2484 0.0586 0.16 −1.09
a
Mass fractions of sucrose and Triton X-114 are represented by wsucrose and wTriton X‑114, respectively. Expanded uncertainty Uc are Uc(wsucrose) =
Uc(wTriton X‑114) = 0.02 (95 % level of confidence).

Table 5. Values of Fitting Parameters Using eqs 3 and 4 for molalities of Triton X-114 in the top and bottom phases,
Sucrose + Triton X-114 + Water ATPSs Formed at Different respectively.
Temperatures The slope of the tie line (STL) and the length of the tie line
(LTL) were determined using eqs 6 and 7, respectively.
T/K K1 n R2 K2 r R2
288 −0.169 1.726 0.980 0.156 0.502 0.999 STL = ΔY /ΔX (6)
293 0.149 0.679 0.955 0.193 0.875 0.961
298 0.306 0.308 0.970 0.876 0.394 0.960 LTL = (ΔX2 + ΔY 2) (7)

Table 6. Values of Fitting Parameters Using eq 5 for Sucrose where ΔX and ΔY represent the difference in mass fractions of
+ Triton X-114 + Water ATPSs Formed at Different the bottom and top phase-forming components (ΔX = wt2 −
Temperatures wb2; ΔY = wt1 − wb1).
Previous studies reported that the LTL and STL of polymer
T/K β Ksucrose s.d.a R2 + salt ATPSs increased with the increase of temperature.16,23
288 0.695 1.610 0.030 0.959 However, it was noticed that the LTL and STL of sucrose +
293 −0.002 2.206 0.001 1.000 Triton X-114 + water ATPS obtained in this study increased as
298 0.380 3.209 0.001 0.996 the temperature decreased from 298 K to 293 K. Phase
a
sucrose − Msucrose))/N)
s.d. = ((∑i = 1 to N(Mexp cal 0.5
where Msucrose represents inversion occurred when the temperature of ATPS further
the molalities of sucrose, N is the number of tie-line data, and the reduced to 288 K and the values of LTL and STL obtained at
superscripts “exp” and “cal” represent the experimental and calculated this temperature were greater than that at 298 K.
values, respectively. 3.4. Effect of Sugaring-Out on ATPS. The phenomenon
of two-phase formation in Triton X-114 + sucrose + water
ATPS was associated with the entropy change in the system
where Ksucrose is sugaring-out coefficient; β is a constant; mtsucrose and the enthalpic interaction between the phase-forming
and mbsucrose are the molalities of sucrose in the top and bottom components. As reported in the past studies,13,24 polymer +
phases, respectively; and mtTriton X−114 and mbTriton X−114 are the saccharide + water ATPS formation was endothermic and the

Table 7. LLE Data of the Secondary ATPS Composed of Sucrose + Triton X-114 + Water ATPS Formed at 303 K. The
Secondary ATPS Was Prepared from Both Phases of a Primary ATPS Formed at 298 Ka
total composition secondary top phase secondary bottom phase
wsucrose wTriton X‑114 wsucrose wTriton X‑114 wsucrose wTriton X‑114 Vr
Primary Top Phase
0.0360 0.0072 0.0365 0.0008 0.0206 0.0535 10.11
0.0390 0.0069 0.0396 0.0006 0.0241 0.0592 10.75
0.0410 0.0061 0.0419 0.0021 0.0235 0.0538 12.31
0.0450 0.0058 0.0459 0.0001 0.0243 0.0591 14.35
Primary Bottom Phase
0.0146 0.0407 0.0322 0.0386 0.0113 0.0533 0.17
0.0154 0.0446 0.0334 0.0228 0.0110 0.0499 0.56
0.0155 0.0674 0.0326 0.0272 0.0077 0.0578 1.00
0.0149 0.0564 0.0336 0.0250 0.0061 0.0680 0.67

a
wsucrose and wTriton X‑114 represent the mass fractions of sucrose and Triton X-114, respectively. Expanded uncertainty Uc are Uc(wsucrose) =
Uc(wTriton X‑114) = 0.02 (95 % level of confidence).

2760 dx.doi.org/10.1021/je500571k | J. Chem. Eng. Data 2014, 59, 2756−2762

Journal of Chemical & Engineering Data
phase separation was dominantly driven by an entropy increase
because the contribution of enthalpy interaction to the phase
separation was weak. The formation of Triton X-114 + sucrose
+ water ATPS requires a strong entropy dominance. Sucrose
acting as a sugaring-out agent could attract water molecules
from Triton X-114 and hence increase the entropy of system.
At a lower temperature (i.e., 288 K), a relatively high
concentration of sucrose was required to achieve two-phase
formation. In this case, the density of sucrose is higher than that
of Triton X-114. Thus, phase inversion was observed when a
high concentration of sucrose was present in the ATPS. The
sugaring-out effect was also observed in ATPSs containing
carbohydrates and phase-forming components such as acetoni-
trile25 and ionic liquids.5,20 The sugaring-out phenomenon
could be studied using the Setschenow equation (eq 5). The
values of Ksucrose and β obtained from eq 5 are given in Table 6.
It could be observed that all values of Ksucrose are positive and
increase with the increase of temperature. This kind of trend
can be explained by the kosmotropicity of sugars, in which
water molecules are readily attracted by sucrose at a higher
temperature and the sugaring-out effect is greatly pronounced.
This is similar to the salting-out effect of salts on ATPS
formation.26−28
3.5. Secondary Two-Phase Data. As shown in Table 4,
the top and bottom phases of ATPSs formed at 288 K, 293 K,
and 298 K contain a sufficient amount of Triton X-114 that can
be thermally separated to form secondary ATPSs. The
feasibility of generating a secondary ATPS using a phase
derived from primary ATPS was demonstrated using an ATPS
formed at 298 K because the primary top phase has a minimal
concentration of Triton X-114 (molality < 0.018 mol·kg−1) that
permits the induction of two-phase formation by heating above
the LCST of Triton X-114. The generation of the secondary
ATPS was conducted at 303 K because this temperature is
slightly higher than the LCST of Triton X-114 (301 K for 0.018
mol·kg−1 to 0.465 mol·kg−1). Table 7 presents the LLE data for
the secondary ATPS composed of sucrose + Triton X-114 +
water formed at 303 K. The volume ratio (Vr) of the secondary
phases formed from a primary top phase was found to be
greater than the one formed from a primary bottom phase. In
the secondary ATPS formed from the primary top phase,
Triton X-114 was predominantly concentrated into a compact
secondary bottom phase, whereas sucrose was not one-sidedly
partitioned to any of the secondary phases formed from a
primary top phase. In contrast, not all of the Triton X-114 from
the primary bottom phase completely aggregated to form a
Triton X-114-enriched secondary bottom phase. Because the
primary bottom phase contained a greater amount of Triton X-
114 than the primary top phase, it is hypothesized that the heat
supplied at 303 K was insufficient to aggregate all of the Triton
X-114, and therefore, a noticeable amount of Triton X-114 was
present in the secondary top phase formed from the primary
bottom phase. Overall, the feasibility of thermoseparation of
the primary phase solution into secondary ATPS holds great
potential in recycling the phase-forming components. Fur-
thermore, this would allow further fractionation and separation
of biomolecules in the formed secondary ATPS.
4. CONCLUSION
Phase diagrams of ATPSs composed of sucrose + Triton X-114
+ water at 288 K, 293 K, and 298 K were determined in this
study. The experimental binodal curve data were successfully
correlated using the Merchuk equation, but satisfactory
2761
Article
correlation results were obtained using a fourth-order
polynomial equation. Tie-line data were correlated using the
Othmer−Tobias and Bancroft equations. The correlated results
confirmed the consistency and reliability of the tie-line
experimental data. The sugaring-out effect of sucrose on
ATPS formation was studied using the Setschenow equation,
and it was noted that the values of Ksucrose increased with
increasing temperature. It was also observed that as the
temperature increased, the area of the two-phase region became
wider, whereas the LTL and STL decreased. Phase inversion
was observed in the ATPS formed at 288 K. The effect of
temperature on secondary ATPS formation and the distribution
of sucrose and Triton X-114 in the secondary phases provide
directions for future investigations.
■
AUTHOR INFORMATION
Corresponding Authors
*Tel: +60 3 55146201. Fax: +60 3 55146207. E-mail: ooi.chien.
wei@monash.edu.
*Tel.: + 603 5514 6235. Fax: + 603 5514 6207. E-mail:
ramanan@monash.edu.
Funding
The authors would like to acknowledge Ministry of Science,
Technology and Innovation (MOSTI) for the e-Science
funding (02-02-10-SF088) provided. Ramalakshmi Subbarayalu
is a recipient of the Higher Degree by Research Scholarship
(HDR) awarded by Monash University, Malaysia. Also, the
authors are grateful to Monash University Malaysia for
providing the research support needed for this work.
Notes
The authors declare no competing financial interest.
■
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