Salt effects on the aqueous two-phase system of Gemini(12-3-12, 2Br -)/SDS/PEG

Article abstract of DOI:10.1007/s10953-009-9449-5

The effect of added salts (NaCl, KCl and NaBr) on the aqueous two-phase system (ATPS) formed in mixtures of Gemini(12-3-12, 2Br^-)/sodium dodecyl sulfate/polyethylene glycol has been investigated. Phase diagrams of the aqueous systems containing Gemini(12-3-12, 2Br^-), sodium dodecyl sulfate (SDS), polyethylene glycol(PEG) and a salt have been determined experimentally at 313.15 K. The results indicate that the addition of salts not only induces the appearance of ATPS-A (in which anionic surfactant is in excess), shortens the phase separation time, enlarges the regions of ATPS-C (in which cationic surfactant is in excess), and decreases the minimum concentration required for forming an ATPS, but also alters the matching between anionic and cationic surfactants. Extractive experiments also showed that these salts notably enhance the extraction ability of ATPS; the Gemini-rich phase exhibits prominent cohesive action with xylenol orange, regardless of whether or not it is the upper phase or the lower phase.

Full text of DOI:10.1007/s10953-009-9449-5

J Solution Chem (2009) 38: 1307–1313
DOI 10.1007/s10953-009-9449-5
Salt Effects on the Aqueous Two-Phase System
of Gemini(12-3-12, 2Br−)/SDS/PEG
Abdulaziz Mohammed Al-Hakimi · Yazhuo Shang ·
Honglai Liu · Ying Hu
Received: 27 February 2009 / Accepted: 14 April 2009 / Published online: 2 September 2009
© Springer Science+Business Media, LLC 2009
Abstract The effect of added salts (NaCl, KCl and NaBr) on the aqueous two-phase system
(ATPS) formed in mixtures of Gemini(12-3-12, 2Br−)/sodium dodecyl sulfate/polyethylene
glycol has been investigated. Phase diagrams of the aqueous systems containing Gemini(12-
3-12, 2Br−), sodium dodecyl sulfate (SDS), polyethylene glycol(PEG) and a salt have been
determined experimentally at 313.15 K. The results indicate that the addition of salts not
only induces the appearance of ATPS-A (in which anionic surfactant is in excess), shortens
the phase separation time, enlarges the regions of ATPS-C (in which cationic surfactant is
in excess), and decreases the minimum concentration required for forming an ATPS, but
also alters the matching between anionic and cationic surfactants. Extractive experiments
also showed that these salts notably enhance the extraction ability of ATPS; the Gemini-rich
phase exhibits prominent cohesive action with xylenol orange, regardless of whether or not
it is the upper phase or the lower phase.
Keywords Aqueous two-phase system · Salt · Gemini surfactant · SDS · Polyethylene
glycol
1 Introduction
A conventional aqueous two-phase system (ATPS) is composed of a water solution contain-
ing two structurally distinct hydrophilic polymers or one hydrophilic polymer and a salt. The
physicochemical properties of aqueous two-phase polymer–polymer or polymer–salt sys-
tems have been investigated extensively because they provide novel solvents for solubilizing
labile biological molecules in a large-scale purification process [1–6]. The ATPS containing
a hydroxypropyl derivative of starch (PPT) and poly(ethylene glycol) (PEG) is widely used
A.M. Al-Hakimi · Y. Shang ( ) · H. Liu · Y. Hu
ꢀ
Key Laboratory for Advanced Materials, East China University of Science and Technology, Shanghai,
200237, China
e-mail: shangyazhuo@ecust.edu.cn
H. Liu (
e-mail: hlliu@ecust.edu.cn
)
ꢀ

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J Solution Chem (2009) 38: 1307–1313
because of its low cost and the low viscosity of these systems. Among the polymer–salt
two-phase systems, the commonly used hydrophilic polyethylene glycol (PEG) molecule in
salt-containing solutions have acquired much research interest because they commonly form
large droplets that are useful for protein separation and purification on a large scale. The no-
table density difference between the two phases, as well as the lower viscosity of the whole
system, leads to a much faster separation speed than that found in aqueous PEG–dextran sys-
tems. Recently, in response to the growing demand for novel solvent systems for large-scale
purification and separation of biomolecules, many new ATPS have been proposed, includ-
ing ATPS in aqueous nonionic micellar solutions, ATPS formed by anionic and cationic
surfactants [7–11], as well as ATPS containing a polymer and a traditional cationic-anionic
surfactant [12].
Many studies have shown that aqueous two-phase systems are usually formed sponta-
neously when cationic and anionic surfactant solutions are mixed together under certain
conditions [13–15]. ATPS are regarded as characteristic of mixed solutions of anionic and
cationic surfactants where both phases are dilute and where the surfactant in one phase
is significantly different from that in the other. Therefore, biomaterials can be efficiently
isolated and purified in ATPS without a loss of activity. Moreover, the aqueous surfactant
two phase systems are proving to have many unique characteristics including high parti-
tion efficiency, high sensitivity, high partitioning selectivity, multistep partitioning, reuse of
surfactants, time-labor savings, etc.
It is well known that the addition of a salt often results in changes in the phase behavior
of aqueous mixtures of oppositely charged surfactants and that the polymer also can alter
the microstructure of micelles in mixed surfactant systems, thus leading to variation in the
phase behavior. ATPS can be formed both in anionic–cationic surfactant systems and in
polymer–salt systems.
What about mixed solutions containing anionic surfactant, cationic surfactant, polymer
and salt? What is the effect of salt on the properties of these mixed solutions? In this
work, a cationic Gemini surfactant (C12H25(CH3)2N–(CH2)3–N(CH3)2C12H25·2Br−, 12-
3-12, 2Br−), a conventional anionic surfactant SDS (C12H25SO4Na), polyethylene glycol
(PEG) and NaCl, KCl, or NaBr were used. Some properties of such mixed solutions are
described here. The effect of these added salts on the aqueous two-phase systems formed in
mixtures of Gemini (12-3-12, 2Br−)/sodium dodecyl sulfate/polyethylene glycol has been
investigated. Phase diagrams for such systems have been determined experimentally. The ex-
traction phenomena of aqueous two-phase systems on the anionic, water-soluble dye xylenol
orange have been studied.
2 Experimental Section
2.1 Materials
Sodium dodecyl sulfate (SDS) was purchased from FARCO (Hong Kong). Polyethylene
glycol (PEG) with an average molecular weight of 6000 was obtained from the Research In-
stitute of Xinhua (Shanghai, China), and sodium bromide (NaBr), sodium chloride (NaCl),
and potassium chloride (KCl) were analytical reagents provided by the Shanghai Chem-
ical Reagent Company. Xylenol orange (C31H28N2Na4O13S) was supplied by the Shang-
hai Reagent Factory. Gemini surfactant (12-12, 2Br−, the formula scheme is shown in
Scheme 1) was synthesized in our lab by the reaction of 1,3-dibromopropane with N,N,N-
dodecyldimethylamine [16]. All of the reagents were used as received. Redistilled water was
used in each experiment.

J Solution Chem (2009) 38: 1307–1313
CH3
1309
CH3
CH3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢁ
ꢂ
CH CH OH
3
2
C12H25N + BrC3H6Br−−−−−−−→ C12H25N+—C3H6— +NC12H25 2Br−
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
CH3
CH3
CH3
−
Scheme 1 The formula of Gemini surfactant 12-3-12, 2Br
3 Experimental Methods
We first prepared solutions containing PEG and different salts. We then used these solutions
as solvents to prepare solutions containing 0.1 mol·L−1 Gemini and 0.1 mol·L−1 SDS, re-
spectively. We mixed these two solutions as well as the corresponding PEG–saline solution
at different mixing ratios, placed them into test tubes. In order to make the viscous surfactant
solutions disperse uniformly in the test tubes, we placed the test tubes containing solutions
in an ultrasonic bath for half an hour, before immersed them in a temperature-controlled bath
at 313 1 K for 48 h or longer until equilibrium was attained. Then, the ATPS results were
plotted on a pseudo-ternary phase diagram with the three vertices representing 0.1 mol·L−1
Gemini, 0.1 mol·L−1 SDS, and 1.0% PEG + 0.1 mol·kg−1 salt. The criterion for determin-
ing the presence of an ATPS is the appearance of a clear interface between the two phases.
When the interface is perpendicular to the tube wall, the ATPS has reached equilibrium.
4 Results and Discussion
4.1 Effect of Salts on the Phase Diagram of ATPS
Figure 1(a) shows the ternary phase diagram of ATPS for the (12-3-12), 2Br−/SDS/PEG
system at 313.15 K; the studied composition region is 0.04 mol·L−1 ≤ c ≤ 0.10 mol·L−1
where c represents the total concentration of surfactants. The narrow region of the single
ATPS-C (in which cationic surfactant is in excess) is located on the right-hand side of the
phase diagram. Compared with a traditional cationic-anionic surfactants system, ATPS-A
(in which anionic surfactant is in excess) disappears from the Gemini-SDS-PEG aqueous
system [15]. The total concentration of surfactants needed for ATPS-C formation is above
0.05 mol·L−1. Figure 1(a) also shows that a lower total concentration of surfactants causes
ATPS formation (above 0.05 mol·L−1) compared to that needed for the traditional ternary
system. This lower total concentration contributes to the lower CMC of mixed surfactants,
leading to more aggregates in solution. When PEG is mixed with surfactants, the aggregates
are more easily formed due to the combinational contribution of the long polymer chain and
the peculiar molecular structure of Gemini’s, as well as the strong coulombic interaction be-
tween positive and negative charges, which leads to a separate phase even in dilute solutions
above the CMC.
Figure 1(b), (c) and (d) show the effect of the different salts NaCl, KCl and NaBr on the
phase diagrams of ATPS for aqueous solution containing 12-3-12, 2Br−, SDS and 1 wt-%
PEG. Compared with the salt-free system, the addition of salts induces the appearance of
ATPS-A and the regions of ATPS-C are all moved towards increasing Gemini concentration.
The ranges for ATPS-C become wider. Furthermore, the minimum concentration needed for
forming an ATPS is much lowered. This implies that the salts participate in the formation of
micelles in the mixed solutions and lead to variations of the matching between anionic and

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−
Figure 1 Ternary phase diagrams of the 12-3-12, 2Br /SDS/PEG system at 313.15 K: (a) without salt;
(b) with NaBr; (c) with NaCl; and (d) with KCl
cationic surfactants. The addition of salts induces the formation of surfactant aggregation at
lower concentrations and thus leads to a phase separation in dilute solutions.
In ATPS-A, the anionic surfactant is in excess and the aggregates formed by Gemini and
SDS mixtures are negatively charged. When replacing a pure PEG solution with a PEG and
NaBr mixed solution, the ATPS-A region becomes wider than those of the corresponding
NaCl- and KCl-containing system as presented in Fig. 1(b)–(d). Figure 1(c) and (d) shows
the effect of the different salts NaCl and KCl on the phase diagrams of ATPS for aqueous
solutions containing 12-3-12, 2Br−, SDS and PEG. Because the two chloride salts contain
different cations, the position of ATPS-A region is slightly different in their phase diagrams.
With decreasing radius of the positive ions (Na+ < K+), the region of ATPS-A not only
becomes wider but also moves away from the equimolar line towards higher SDS concen-
trations. In the SDS/Gemini/PEG/salt system, the K+ cation with its larger radius exhibits
less freedom of movement than Na+. Therefore, addition of KCl results in a weaker salt
effect on the ATPS-A region than addition of NaCl.
Figure 1(b) and (c) shows the effect of NaCl and NaBr on the phase diagrams of ATPS
for aqueous solutions containing 12-3-12, 2Br−, SDS and PEG. With increasing radius of
the negative ion, the ATPS-C region becomes wider. The region of ATPS-C in the PEG–
NaBr system is so wide that it stretches up to the PEG–NaBr apex. It is obvious that the
cationic surfactant (Gemini) is in excess in ATPS-C. The surfactant aggregates are most

J Solution Chem (2009) 38: 1307–1313
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probably positively charged. The counter ion layer is then negatively charged. The negative
ions of the added salts are preferentially adsorbed and directly participate in the formation
of a counter ion layer that enhances the negative charge density. As a result, because more
positive charges are needed to counter balance the negative charges in the ordered config-
urations, the Geminis increase accordingly. For the negative ion, the degrees of freedom
decrease with increasing radius, raising the stability of the counter ion layer. Consequently,
the more positive charges that are needed at equilibrium, the larger is the increase in Gem-
inis [14].
4.2 Effect of Salts on the Phase Separation Time
The ATPS in the 12-3-12, 2Br−/SDS/PEG system do not reach equilibrium until two days
because of the higher viscosity of the mixed solutions. Addition of a salt shortens the phase-
separation time drastically. This should be attributed to the increase in counter ion concen-
tration that leads to a decrease of the average thickness of the repulsive electric double-layer
of the ionic micelles, thus shortening the time needed for formation of stable micelles. Cor-
respondingly, this shortening quickens the phase separation of ATPS. Among the salts used,
NaBr has the most prominent effect and phase separation starts as soon as the samples are
put into the temperature-controlled bath. It was seen that part of the liquid ascends and the
rest descends clearly, and a clear interface appears within 5 minutes.
4.3 Extraction of Xylenol Orange in ATPS of Systems Containing Salts
Xylenol orange (C31H28N2Na4O13S) was selected to investigate the extraction efficiency of
ATPS. Attempted extraction of xylenol orange in the system without salts shows no evident
results. However, in a salt-containing system, the salt causes a strong impact on the extrac-
tion of xylenol orange. Whether changing the total surfactant concentration c(a) or the molar
ratio R of Gemini to SDS (b), almost all xylenol orange is extracted into the oil-like phase
no matter whether the oil-like phases are upper phases or lower phases, as shown in Figs. 2,
3 and 4.
The ATPS-C formed in 12-3-12, 2Br−/SDS/PEG/salt systems exhibits an outstanding
cohesive interaction with the dye. Regarding the ATPS-C of this novel system, the cationic
surfactant (Gemini) is always in excess. For this surfactant-abundant solution, the extrac-
tive efficiency for xylenol orange is mainly attributed to the electrostatic attraction between
the positively-charged surfactant aggregates and the negatively-charged dye, as well as hy-
drophobic interaction between the long hydrophobic chains of Gemini and xylenol orange.
−
Figure 2 Partitioning behavior of xylenol orange in a ATPS-C of 12-3-12, 2Br /SDS/PEG/NaBr system at
313 K with: (a) different surfactant concentrations and (b) different molar ratios of Gemini to SDS

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Figure 3 Partitioning behavior for xylenol orange in ATPS-C of the 12-3-12, 2Br /SDS/PEG/NaCl system
at 313 K with: (a) various total surfactant concentrations and (b) different molar ratios of Gemini to SDS
−
Figure 4 Partitioning behavior of xylenol orange in ATPS-C of the 12-3-12, 2Br /SDS/PEG/KCl system at
313 K with: (a) different total surfactant concentrations and (b) various molar ratios of Gemini to SDS
Xylenol orange is negatively charged when it is ionized in aqueous solution. Therefore, it
is much easier for it to be adsorbed by a cationic-rich phase (the Gemini-containing phase).
Furthermore, the hydrophobic region of xylenol orange tends to favor the surfactant-rich
phase, because the oil-like phase contains much more Gemini with two cationic head groups
and two long hydrophobic tails, which provides a strong driving-force to absorb xylenol or-
ange.
5 Conclusion
Inorganic salts have a significant effect on the properties of ATPS of the Gemini/SDS/PEG
system. The addition of salts induces the appearance of ATPS-A, shortens the phase sep-
aration time, enlarges the regions of ATPS-C, decreases the minimum concentration for
forming an ATPS and alters the matching between anionic and cationic surfactants in ATPS.
The extraction experiments also showed that the ATPS formed in systems containing salts
have a strong ability for extraction; the Gemini-rich phase exhibits prominent cohesive ac-
tion with xylenol orange, regardless of whether it is the upper phase or the lower phase. In
extraction, the lower surfactant concentration of ATPS keeps the biomaterials from losing
their activities, thereby raising the efficiency of a separation process based on partition-
ing. The stronger extraction power of ATPS in saline aqueous systems of Gemini(12-3-12,
2Br−)/SDS/polymer may provide a more economical way to separate biomaterials.

J Solution Chem (2009) 38: 1307–1313
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Acknowledgements This work was supported by the National Natural Science Foundation of China
(Projects Nos. 20706013, 20736002), Program for Changjiang Scholars and Innovative Research Team in
University (No. IRT0721), and the 111 Project of Ministry of Education of China (No. B08021).
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