Formation of ether-functionalized ionic-liquid-based aqueous two-phase systems and their application in separation of protein and saccharides

Article abstract of DOI:10.1021/jp510984d

Ionic-liquid (IL)-based aqueous two-phase systems (ATPSs) have attracted much attention in the separation technology. In this work, we synthesized five novel ether-functionalized ILs and studied their applications in ATPS formation. The phase diagrams for

Full text of DOI:10.1021/jp510984d

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Article
Formation of Ether-Functionalized Ionic Liquid-Based Aqueous Two-Phase
Systems and Their Application in Separation of Protein and Saccharides
Zhijun Wang, Yuanchao Pei, Jing Zhao, Zhiyong Li, Yujuan Chen, and Kelei Zhuo
J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 03 Mar 2015
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Formation of Ether-Functionalized Ionic Liquid-Based Aqueous
Two-Phase Systems and Their Application in Separation of Protein
and Saccharides
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Zhijun Wang, Yuanchao Pei, Jing Zhao, Zhiyong Li, Yujuan Chen, Kelei Zhuo*
Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals,
Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of
Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, P. R.
China, E-mail: klzhuo@263.net, Tel.: +86 373 3329056, Fax: +86-373-3329056
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ABSTRACT
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Ionic liquid (IL)-based aqueous two-phase systems (ATPSs) have attracted much
attention in the separation technology. In this work, we synthesized five novel
ether-functionalized ILs and studied their applications in ATPS formation. The phase
diagrams for several systems were determined at 298.15 K. An as-prepared IL
(1-(2-butoxy-ethyl)-3-methylimidazolium chloride) was chosen as a representative to
construct ATPSs for separation of bovine serum albumin (BSA) from aqueous
saccharide solutions. Results showed that 76.1% − 94.3% of BSA was enriched into
the IL-rich top phase and almost all the saccharides were extracted into the salt-rich
bottom phase in one-step separation process. The main factors affecting the separation
process, such as kinds of saccharides and the amount of inorganic salts, were
discussed. Furthermore, the size distributions of aggregates in the IL-rich top phase
were determined by dynamic light scattering (DLS), and accordingly the possible
mechanism for the separation was investigated.
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KEYWORDS: aqueous two-phase system; ether-functionalized ionic liquid; bovine
serum albumin; saccharides; separation
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1. INTRODUCTION
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Proteins and saccharides are important components of living organism, and
coexist generally together. They are separated and purified difficultly. For example,
the crude saccharides often contain a large number of non-carbohydrate substances,
such as proteins.1 Furthermore, the existence of saccharides causes significant
deviation from the actual absorbance of proteins in the Bradford protein assay.2
Therefore, the separation of the complex mixtures has become a focus of the
researches. Several methods have been applied in the biological sample separation:
gas chromatography/mass spectrometry, liquid-liquid extraction, and solid-phase
extraction.3,4 As a kind of liquid-liquid extractive technique, aqueous two-phase
system (ATPS) is simple, low cost, and relatively reliable in scale-up, and has a great
potential in separation application. Kula and co-workers published a number of papers
describing the use of ATPS for protein purification.5,6 The practical application of
ATPSs to process development has been exploited for the recovery of biological
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products.7 9
−
Ionic liquids (ILs) as environment-friendly and designed solvents are attracting.
In 2003, for the first time, Rogers and co-workers10 reported on a new ATPS
composed of a hydrophilic IL, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl),
and a inorganic salt (K3PO4). The inorganic salt at a high concentration increased the
hydrophobicity of the bottom phase containing rich-salt.11 In rich-salt phase, the
solubility of protein was markedly reduced because of the competition for water
molecules between the protein and inorganic ions. Consequently, the addition of
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inorganic salts enhances the transfer of protein from the bottom phase to the top
phase.12 Moreover, in the process of forming ATPSs, the negatively charged amino
acids on the surface of protein interact with the positively charged IL-cation.13 These
factors promote the transfer of protein to the top phase. Therefore, we can change the
structures of ILs and kinds of inorganic salts to improve the extraction efficiency of
proteins using IL-based ATPSs. Recently, IL-based ATPSs are widely used for the
separation and purification of various biomolecules,14,15 based on their unique
properties such as low viscosity, quick phase separation, high extraction efficiency,
and gentle biocompatible environment.16,17 A lot of researches were restricted to the
extraction of the single component from one phase to another. However, IL-ATPS for
separation of bovine serum albumin (BSA) from aqueous saccharide solutions was
rarely reported. In previous work, we reported on the separation of BSA from aqueous
saccharides solutions using an ATPS constructed by 1-butyl-3-methylimidazolium
dicyanamide [C4mim][N(CN)2], K2HPO4, and H2O.18 Results showed that more than
82% of BSA could be enriched into the IL-rich top phase and almost all the
saccharides were extracted into the salt-rich bottom phase of ATPS. It is highly
desired to construct new IL-based ATPSs for separations.
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To form the IL-water biphasic systems for the extraction, hydrophobic ILs have
to be used. Hydrophobic ILs are more expensive than hydrophilic ILs and the number
of hydrophobic ILs is limited. In addition, using the simple IL-water biphasic systems
for separation of biomacromolecules, their denaturation would happen possibly in the
separation process.18 Therefore, hydrophilic ILs are more likely used in the extraction
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process. In recent decades, a lot of hydrophilic ILs were synthesized and applied to
form ATPSs for extraction separation. For example, [C6mim]Cl was used to construct
ATPSs for extraction of BSA.19 The ether-functionalized ILs also are hydrophilic. But
compared with the alkyl-substituted ILs, they have a variety of unique properties,
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such as lower viscosity and lower melting point.20 22 In addition, the ether group
−
reduces the toxicity of ILs.23 Therefore, it is significant to use the
environment-friendly ether-based ILs for separation processes.
Herein, we synthesized five novel (except [C2OC2mim]Cl) hydrophilic
ether-functionalized ionic liquids: 1-(2-alkoxy-ethyl)-3-methylimidazolium chloride
([CnOC2mim]Cl (n
=
2, 3, 4)), 1-(2-propoxy-ethyl)-3-methylimidazolium
p-toluenesulfonic
acid
([C3OC2mim][p-TSA]),
p-toluenesulfonic
and
1-(2-benzyloxy-ethyl)-3-methylimidazolium
acid
([PhCOC2mim][p-TSA]). The ability of the ether-functionalized ILs for the formation
of IL-based ATPSs was discussed. For such a purpose, phase diagrams for systems
composed of different ether-functionalized ILs, inorganic salts (K3PO4/K2HPO4), and
water at 298.15 K were determined. Then an as-prepared IL [C4OC2mim]Cl was used
to form IL-based ATPSs for separating BSA from aqueous saccharide (glucose,
sucrose, and dextran) solutions. Besides, dynamic light scattering (DLS) was applied
to determine the size distribution of aggregates in the top IL-rich phase and to explore
the possible mechanism for the separation of BSA.
2. EXPERIMENTAL SECTION
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2.1 Materials. 1-Methylimidazole (CP) was purchased from HWRK Chemical
Company (Beijing, China), 2-chloroethyl ethyl ether, 2-chloroethyl propyl ether, and
2-chloroethyl butyl ether were purchased from Tokyo Chemical Industry (TCI, Tokyo,
Japan), respectively. They were used without further purification. Ethylene glycol
propyl ether, p-toluenesulfonyl chloride, 4-dimethylaminopyridine, and 2-(benzyloxy)
ethanol were purchased from Aladdin Chemistry (Los Angeles, USA). Triethylamine
was purchased from Sinopharm Chemical Reagent Co (Shanghai, China). K3PO4 from
TCI (Tokyo, Japan), K2HPO4 from Alfa Aesar (Ward Hill, MA, USA), bovine serum
albumin (BSA), glucose, sucrose, and dextran (Mr~1500) from Sigma (St. Loius, MO,
USA) were used without further purification. Ultrapure water was used to prepare
solutions. All chemicals were of analytical grade.
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2.2 Synthesis and Characterization of ILs. 1-(2-Alkoxy-ethyl)-3-methylimidazo-
lium chlorides ([CnOC2mim]Cl (n = 2, 3, 4)) were synthesized according to the
procedure (Figure 1) described in the literature.24 Chloroethyl alkylether was added
dropwise to 1-methylimidazole under stirring at 0 °C. The reaction temperature
increased to 80 °C. The mixture was stirred at 80 °C for 48 h, and then cooled to room
temperature, washed three times with ethyl acetate. The ethyl acetate in produce was
removed by rotary evaporation. The obtained ether-functionalized ILs products were
dried under reduced pressure for 48 h at 50 °C in the presence of P2O5. The chemical
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structures of the ether-functionalized ILs were confirmed by H NMR spectroscopy.
Their 1H NMR spectral characteristics were shown as follows:
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[C2OC2mim]Cl: H NMR (400 MHz, CDCl3): δ 10.49 (s, 1H), 7.57 (d, 2H),
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4.63-4.55 (t, 2H), 4.11 (s, 3H), 3.82-3.79 (t, 2H), 3.52 (q, 2H), 1.17 (t, 3H).
[C3OC2mim]Cl: 1H NMR (400 MHz, CDCl3): δ 10.49 (s, 1H), 7.47 (s, 1H), 7.46
(s, 1H), 4.57-4.53 (t, 2H), 4.06 (s, 3H), 3.77-3.71 (t, 2H), 3.36 (t, 2H), 1.51 (q, 2H),
0.82 (t, 3H).
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[C4OC2mim]Cl: 1H NMR (400 MHz, CDCl3): δ 10.37 (s, 1H), 7.52 (s, 1H), 7.46
(s, 1H), 4.55-4.47 (t, 2H), 4.03 (s, 3H), 3.73-3.69 (t, 2H), 3.37 (t, 2H), 1.49 (m, 2H),
1.28-1.17 (m, 2H), 0.81 (t, 3H).
OCnH2n+1
H2n+1CnO
N
N
N
N
+
Cl
Cl
Figure 1. Synthesis process for [CnOC2mim]Cl (n = 2, 3, 4).
1-(2-Propoxy-ethyl)-3-methylimidazolium
p-toluenesulfonic
acid
([C3OC2mim][p-TSA]) and 1-(2-benzyloxy-ethyl)-3-methylimidazolium
p-toluenesulfonic acid ([PhCOC2mim][p-TSA]) were synthesized according to the
procedure (Figures 2 and 3) described in the literature.25,26 Ethylene glycol propyl
ether was added to a dichloromethane solution of p-toluenesulfonyl chloride under
stirring at 0 °C. Then triethylamine addition was followed by a catalytic amount of
4-dimethylaminopyridine. After 4 h the solution was washed with ultrapure water,
brine, and sodium bicarbonate. After drying with sodium sulfate, dichloromethane
was removed by rotary evaporation. Then p-toluenesulfonate was reacted with
1-methylimidazole under stirring at room temperature. After 48 h the mixture was
washed three times with ethyl acetate. The ethyl acetate was removed by rotary
evaporation. The synthesis of ionic liquid [PhCOC2mim][p-TSA] followed the same
procedure as the preparation of [C3OC2mim][p-TSA]. The obtained
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ether-functionalized ILs products were dried under reduced pressure for 48 h at 50 °C
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in the presence of P2O5. Their H NMR spectral characteristics were shown as
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follows:
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[C3OC2mim][p-TSA]: 1H NMR (400 MHz, CDCl3): δ 9.61 (s, 1H), 7.73 (s, 1H),
7.71 (s, 1H), 7.37 (m, 2H), 7.11 (d, 2H), 4.37-4.34 (t, 2H), 3.88 (s, 3H), 3.65-3.61 (t,
2H), 3.30 (t, 2H), 2.30 (s, 3H), 1.52-1.43 (m, 2H), 0.80 (t, 3H).
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[PhCOC2mim][p-TSA]: H NMR (400 MHz, CDCl3): δ 9.69 (s, 1H), 7.76 (s,
1H), 7.74 (s, 1H), 7.32-7.25 (m, 5H), 7.22-7.19 (m, 2H), 7.11 (d, 2H), 4.47-4.39 (m,
4H), 3.90 (s, 3H), 3.74 (t, 2H), 2.31 (s, 3H).
O
S Cl
O
O
O S
O
O
O
+
OH
O
O
O S
O
N
N
O
N
N
+
O
S O
O
Figure 2. Synthesis process for [C3OC2mim][p-TSA].
O
O S
O
O
S Cl
O
O
O
+
OH
O
N
N
O
O S
O
O
N
N
+
O
S O
O
Figure 3. Synthesis process for [PhCOC2mim][p-TSA].
At the same time, based on the graphs of thermogravimetric analysis (TGA) and
differential scanning calorimetry (DSC), the thermal decomposition temperature and
glass transition temperature were obtained, and are given in Table 1. Both
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measurements were carried out under N2 with a temperature ramp of 10 K/min.
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Table 1. Melting Point (Tm) and Thermal Decomposition Temperature (Td) of ILs.
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Ionic liquid
Tm/°C
Td/°C
336.0
328.8
257.8
257.0
254.3
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[C3OC2mim][p-TSA]
50.79
[PhCOC2mim][p-TSA] −23.87a
[C2OC2mim]Cl
–35.98a
−40.66a
–45.95a
[C3OC2mim]Cl
[C4OC2mim]Cl
a glass transition temperature
2.3 Preparation of Phase Diagrams. The phase diagrams were determined by the
turbid titration method.10,18 In short, a few grams of ether-based IL were weighed into
a test tube. Then a small amount of water was added to the tube for dissolving the IL.
A known-concentration solution of salt was added dropwise to the test tube until the
mixture became turbid. The added volume of the salt solution was recorded and the
composition of this mixture was calculated. Then a few drops of water was added to
make the two-phase system clear again. The above procedure was repeated to gain
enough data for constructing a liquid-liquid equilibrium binodal curve. The
concentration of the phase components was determined by weight quantification of all
the components with a standard uncertainty of 10–4 g. The tie lines, which depict the
concentrations of IL and salt in the top/bottom phases, were determined with the
process outlined in our previous work.27
2.4 Separation of Protein from Saccharides. Method for the separation of protein
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from saccharides is similar to our previous work.18,28 The details are given in the
Supporting Information. The dynamic light scattering (DLS) measurements were
carried out using Zeta-Meter (Nano-ZS 90) laser light scattering photometer (Malvern,
U.K.).18 Also see the Supporting Information.
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3. RESULTS AND DISCUSSION
3.1 Phase Diagram of IL-Based ATPS. The binodal curves and tie lines for the
ether-functionalized IL + salt systems were determined at 298.15 K, and shown in
Figures 4–6 and Figures S1–S3 in the Supporting Information, respectively.
The binodal data were correlated by the following equation:29
w1 = aln(w2 + c) + b
(3)
where w1 and w2 are the mass fractions of ILs and salts, respectively, and a, b, and c,
are fitting parameters. The fitting parameters gained from the correlation of
experimental binodal data are given in Table 2, along with the correlation coefficients
(R2) and the corresponding standard deviations (s) of eq 3. Based on the obtained R2
and s, we can conclude that eq 3 has more satisfactory accuracy in binodal data fitting
for the studied systems.29
Table 2. Parameters (a, b, and c) Obtained by the Regression of the Experimental Binodal Data
Through the Application of Eq 3 for the IL(1) + Salt(2) + H2O Systems at 298.15 K.
systems
a
b
c
R2
103s
[C2OC2mim]Cl + K3PO4 + H2O
[C2OC2mim]Cl + K2HPO4 + H2O
−0.31488 −0.22952 0.09645 0.9987 2.1
−0.20134 −0.18407 −0.02090 0.9975 1.3
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[C3OC2mim]Cl + K3PO4 + H2O
−0.22889 −0.20371 0.02362 0.9995 1.8
−0.20764 −0.20321 −0.02012 0.9954 1.3
−0.37854 −0.51823 0.03649 0.9961 4.0
−0.29155 −0.20758 0.11986 0.9997 1.1
−0.25214 −0.21485 −0.03914 0.9985 2.2
[C3OC2mim]Cl + K2HPO4 + H2O
[C3OC2mim][p-TSA] + K3PO4 + H2O
[C4OC2mim]Cl + K3PO4 + H2O
[C4OC2mim]Cl + K2HPO4 + H2O
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[PhCOC2mim][p-TSA] + K3PO4 + H2O −0.12733 −0.27418 −0.04208 0.9930 1.8
3.1.1 Effect of the structure of ILs. ILs composed of different cations and anions
have different hydrophobicity. ATPSs can be constructed by adding a certain amount
of K2HPO4 or K3PO4 to aqueous IL solutions. Figure 4 indicates the influence of the
alkyl-chain length of ILs on the phase diagrams. Obviously, the phase-forming ability
of these ILs is observed: [C4OC2mim]Cl > [C3OC2mim]Cl > [C2OC2mim]Cl. This
order is in agreement with chaotropic ability of IL-cations. The difference in
chaotropic ability results from the structural difference of the cations.30 With the
increase of the alkyl-chain length ([CnOC2mim]+ (n = 2, 3, 4)), the binodal curves are
more closer to the origin,31 showing that the phase-forming ability of ILs increases
with increasing alkyl-chain length. That is to say, [C4OC2mim]Cl is the most
chaotropic salt and this binodal is furthest to the left (it is the easiest to salt-out).
Obviously, the phase-forming ability of [C4OC2mim]Cl is the best among the three
ILs.
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Figure 4. Phase diagrams for the aqueous two-phase systems composed by [C2OC2mim]Cl /
[C3OC2mim]Cl / [C4OC2mim]Cl + K3PO4 / K2HPO4 at 298.15 K: w, mass fraction.
Figure 5 indicates the effect of ions of ether-based ILs on the phase diagram. At
a certain amount of K3PO4, the phase-forming ability of these ILs is observed:
[PhCOC2mim][p-TSA] > [C3OC2mim][p-TSA] > [C3OC2mim]Cl. This can be
interpreted by the facts that the addition of the electro-rich aromatic π system
(hydrophobic) to the cation and anion increases the hydrophobicity of the IL,32,33 and
then increase the phase-forming ability. Therefore, we suggest that the hydrophobicity
of the ILs play a critical role in aqueous two-phase extraction process.
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Figure 5. Phase diagrams for the aqueous two-phase systems composed by [PhCOC2mim][p-TSA]
/ [C3OC2mim][p-TSA] / [C3OC2mim]Cl + K3PO4 at 298.15 K: w, mass fraction.
3.1.2 Effect of salt category. Figure 6 shows the phase diagrams for the ATPSs of
[C4OC2mim]Cl with K2HPO4/K3PO4. It can be seen that the binodal curve for K3PO4
is closer to the origin than that for K2HPO4. Consequently, the phase-forming ability
of both salts is: K3PO4 > K2HPO4. This order is the same as the previous researches
on the ATPSs of [C4min]X (X=Cl or Br) and [Cnmin]Br (n = 6 or 8).30,34 Since cation
of both salts is the same K+, but anions are different, the phase-forming ability is
controlled by different anions. Results showed that anion with higher valence was
used for constructing ATPSs, a lower concentration of salt was needed. It may be
explained by their different salting-out abilities (kosmotropicity): the higher valent
anions show bigger salting-out ability, because the anions with high charge density
can hydrate more water molecules, and thus the less water is available to hydrate ILs.
2–
This is identical to the hydration Gibbs free energy of the anions: HPO4 (−1125
kJ·mol−1), PO43− (–2765 kJ·mol−1).35 The order is also in agreement with the strength
of the kosmotropic salts (Hofmeister series). Since K2HPO4 is able to make a suitable
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pH range for the gentle separation of BSA, it was selected for the further researches in
the present work.
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Figure 6. Phase diagrams for the aqueous two-phase systems composed by [C4OC2mim]Cl+
K3PO4 / K2HPO4 at 298.15 K: w, mass fraction.
3.2 Separation of BSA and Saccharides in [C4OC2mim]Cl + K2HPO4 ATPSs
3.2.1 Effect of inorganic salt amount. Amount of salts is a key factor to control the
extraction efficiency.36 As shown in Figure 7, as the concentration of salts (K2HPO4)
increases, the extraction yield for BSA increases at first and decreases afterwards. The
possible reason is that K2HPO4 has a strong salting-out effect.37 When kosmotropic
ions were added into aqueous solutions of ILs, the molecules of water surrounding the
ions would become electro-constriction states. Hence, the hydrogen bonding of water
was increased in the phase, and thus more energy is required to form a cavity in this
solution for the organic cation [C4OC2mim]+.10 As the concentration of salt increases,
the salt-rich bottom phase became more hydrophobic38 and more structured, and
consequently the solubility of protein was markedly reduced because of the
competition for water molecules between the protein molecules and inorganic ions.12
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Moreover, in the process of forming ATPSs, the negatively charged amino acids on
the surface of protein interact with the positively charged IL-cation.13 These factors
lead to a larger transfer of protein to the top IL-rich phase. At a given content of
inorganic salt, the ILs with the more hydrophobic cations and the less
water-structuring anions would be separated from the inorganic salts. However,
extraction efficiency decreases when the content of inorganic salt is too high. The
possible reason is the denaturation of protein to some extent under the circumstance.
Furthermore, these ATPSs could be used repeatedly or used to enrich hydrophilic ILs
from water solutions.39,40 Therefore, these ATPSs would be more appropriate for the
extraction of biomolecules.
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Figure 7. Effect of the concentration of salt on the extraction efficiency of BSA and saccharides
.
in [C4OC2mim]Cl + K2HPO4 3H2O ATPSs at 298.15 K: the concentrations of BSA and
saccharides were at 4.0 mg·mL−1 and 1.0 mg·mL–1, respectively; 0.4 g IL, 1.2 mL H2O were
.
added for the construction of ATPSs; m (g) is the total mass of K2HPO4 3H2O added. The
deviations of extraction efficiency of BSA and saccharides were 2% and 5%, respectively.
3.2.2 Effect of different saccharides. Table 3 shows that the extraction yields of
different saccharides and BSA in [C4OC2mim]Cl + K2HPO4 ATPSs. From this table,
we observed that the extraction yields for BSA from the saccharide solutions increase
with the increase of the number of hydroxyl groups in a saccharide molecule
(dextran > sucrose > glucose). The extraction yields of BSA from different saccharide
solutions are different. Most of the sugars were separated into the salt-rich phase.
Because of the positive values of viscosity B coefficients of saccharides in aqueous
solutions, the saccharide molecules act overall as kosmotropes (to form stronger
hydrogen bond networks with water).39,41,42 Moreover, the greater the number of
hydroxyl groups on a sugar molecule, the more kosmotropic the sugar. Therefore, the
presence of sugar in the [C4OC2mim]Cl + K2HPO4 ATPS would be benefited to the
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separation of kosmotropic salt K2HPO4. Because of the formation of hydrogen bonds
between water and saccharide molecules, the content of free water in the bottom
salt-rich phase will decrease, which forces more chaotropic IL and BSA to be
transferred into the top IL-rich phase, compared with the ATPSs without saccharides.
Consequently, the number of hydroxyl groups on saccharide molecules is a key factor
influencing the distribution of saccharides and BSA in these ATPSs.
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Experimental results showed that 76.1% − 94.3% of the BSA were extracted into
the top IL-rich phase and sugars were separated into the bottom salt-rich phase in
one-step separation process. Compared with our previous work,18 the ATPSs based on
ether-functionalized ILs also exhibit excellent extraction efficiency for BSA from
aqueous saccharides. Furthermore, the ether-based ILs possess low viscosity, low
melting point, and low toxicity, and thus have wide application in the extraction
separation by using IL-based ATPSs.
Table 3. Extraction efficiencies (E) of BSA and saccharides in [C4OC2mim]Cl + K2HPO4 ATPSs
at 298.15 Ka
Saccharide
glucose
EBSA
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93.5
94.3
%
Esaccharide
68.9
%
sucrose
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dextran
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a
Test concentrations of BSA and saccharides were at 4.0 mg·mL–1 and
1.0 mg·mL−1, respectively; 0.4 g of IL, 0.9 g of K2HPO4·3H2O, and 1.2
mL of H2O were mixed for the construction of ATPSs.
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3.3 Size Distribution of Aggregates. In order to understand the separation process,
we measured the size distribution of aggregates in the IL-ATPSs. In the
[C4OC2mim]Cl + K2HPO4 ATPS, the IL-rich top phase consists of IL, less amount of
inorganic salt, and water, and the salt-rich bottom phase consists of inorganic salt, less
amount of IL, and water. In the top IL-rich phase, the presence of high IL
concentration causes a remarkable aggregation of cations of the IL due to their
amphiphilic characteristics.43 To confirm the state of [C4OC2mim]Cl in the IL-rich
phase, we measured the size distribution of aggregates for [C4OC2mim]Cl and BSA in
the aqueous solution by DLS at 298.15 K, and the results are shown in Figure 8.
Obviously, when BSA was added to the ATPS, a novel aggregate was generated at
260 nm. The novel aggregate is larger than both BSA and IL aggregates. The similar
results were observed for the protein and surfactant systems.13,44,45 We could conclude
that the separation process of BSA was drived by the generation of novel IL-protein
complex aggregates.
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Figure 8. Size distribution of the aggregates of BSA, IL, and BSA + IL in aqueous solution: (a)
BSA (1.23 g·L–1); (b) IL (1.2 mol·kg−1); (c) BSA (1.23 g·L–1) + IL (1.2 mol·kg−1).
4. CONCLUSIONS
In this work, we synthesized five novel hydrophilic ether-functionalized ionic
liquids and studied their applications in ATPS formation. The phase diagrams for
several systems were determined at 298.15 K. The results showed that BSA was
extracted into the IL-rich top phase but most of saccharides were transferred into the
salt-rich bottom phase. Extraction yield of BSA enhanced with increasing the number
of hydroxyl groups in a saccharide molecule (dextran > sucrose > glucose). The
generation of the novel IL-protein complex aggregates is a key factor for separation of
BSA from the aqueous solutions of saccharides. We expect that [C4OC2mim]Cl +
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K2HPO4 ATPSs have a potential to separate protein from saccharide solutions, and
therefore highlight a new opportunity of ether-functionalized IL-ATPSs to separate
biomolecules from complex systems.
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ASSOCIATED CONTENT
Supporting Information
Experiments for preparation of phase diagrams, separation of protein from
saccharides, and dynamic light scattering measurements; Phase diagrams for
[CnOC2mim]Cl (n = 2, 3, 4) + K2HPO4 aqueous two-phase systems at 298.15 K;
binodal data for the ether-functionalized ILs (1) + K3PO4 / K2HPO4 (2) + H2O (3)
ATPSs at T = 298.15 K.
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-373-3329056; fax: +86-373-3329056; e-mail: klzhuo@263.net
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial supports from the National Natural Science Foundation of China (Nos.
21173070，21303044) and the Plan for Scientific Innovation Talent of Henan
Province (No. 124200510014) are gratefully acknowledged.
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