Design of environmentally friendly ionic liquid aqueous two-phase systems for the efficient and high activity extraction of proteins

Article abstract of DOI:10.1039/c2gc35890e

Ionic liquids (ILs) have numerous applications in industrial processes as a benign alternative to conventional volatile organic solvents. However, many of them are toxic to organisms and are poorly biodegradable. In this work, a series of environmentally friendly cholinium ILs have been designed and synthesized. It was found that these ILs could form aqueous two-phase systems (ATPSs) with polypropylene glycol 400 (PPG400) which is thermo-sensitive, non-toxic and biodegradable. In order to understand the phase formation processes and possible application of these ATPSs for extraction/separation of proteins, the binodal curves and tie lines of these ATPSs were measured at 25 °C, and the effects of anionic structure of the ionic liquids, nature of the proteins and difference in the concentration of top- and bottom-phases on the partitioning behavior of some typical proteins were investigated systematically. It was shown that bovine serum albumin (BSA), trypsin, papain and lysozyme could be enriched effectively into the ionic liquid-rich phase of the ATPSs, and single-step extraction efficiency could be as high as 86.4-99.9% under the optimized conditions. Furthermore, enzyme activity of the native trypsin in water and in aqueous ionic liquid solutions was determined by using N-a-benzoyl-l-arginine ethyl ester as a substrate, and activity increases to about 127% was observed after 13 months storage. In addition, PPG400 has been recovered simply by heating and reused in the next extraction processes. This avoids the non-sustainable issue of highly salty water produced in the application of the polyethylene glycol (PEG) + salt and ionic liquid + salt ATPSs.

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PAPER
Design of environmentally friendly ionic liquid aqueous two-phase systems
for the efficient and high activity extraction of proteins
a
a
a
a
b
Zhiyong Li, Xinxin Liu, Yuanchao Pei, Jianji Wang* and Mingyuan He
Received 12th June 2012, Accepted 2nd August 2012
DOI: 10.1039/c2gc35890e
Ionic liquids (ILs) have numerous applications in industrial processes as a benign alternative to
conventional volatile organic solvents. However, many of them are toxic to organisms and are poorly
biodegradable. In this work, a series of environmentally friendly cholinium ILs have been designed and
synthesized. It was found that these ILs could form aqueous two-phase systems (ATPSs) with
polypropylene glycol 400 (PPG400) which is thermo-sensitive, non-toxic and biodegradable. In order to
understand the phase formation processes and possible application of these ATPSs for extraction/
separation of proteins, the binodal curves and tie lines of these ATPSs were measured at 25 °C, and the
effects of anionic structure of the ionic liquids, nature of the proteins and difference in the concentration
of top- and bottom-phases on the partitioning behavior of some typical proteins were investigated
systematically. It was shown that bovine serum albumin (BSA), trypsin, papain and lysozyme could be
enriched effectively into the ionic liquid-rich phase of the ATPSs, and single-step extraction efficiency
could be as high as 86.4–99.9% under the optimized conditions. Furthermore, enzyme activity of the
native trypsin in water and in aqueous ionic liquid solutions was determined by using N-a-benzoyl-L-
arginine ethyl ester as a substrate, and activity increases to about 127% was observed after 13 months
storage. In addition, PPG400 has been recovered simply by heating and reused in the next extraction
processes. This avoids the non-sustainable issue of highly salty water produced in the application of the
polyethylene glycol (PEG) + salt and ionic liquid + salt ATPSs.
Introduction
among a large number of ILs that have been investigated, the ILs
consisting of imidazolium or pyridinium cations and halide-
containing anions have been demonstrated to be highly toxic and
As an innovative class of solvents, ionic liquids (ILs) have
attracted much attention as greener replacements for traditional
volatile organic solvents in a variety of fields such as chemical
synthesis, catalytic chemistry, electrochemical devices, separ-
ation and biomass dissolution due to their unique physico-
9
poorly biodegradable. Therefore, widespread application of ILs
would inevitably result in the loss of ILs into the water ecosys-
tems, leading to aquatic environmental pollution. An alternative
approach for overcoming these drawbacks is the development of
ILs from their components, which have well characterized bio-
degradable and toxicological properties. Therefore, the production
of non-toxic and environmentally benign ILs from renewable
1
–5
chemical properties.
ILs are composed solely of ions with
numerous combinations of anions and cations. The particular
interest is their highly tunable properties by varying the chemical
structures of the ions comprised. Thus, by the rational design of
cation and anion, an IL with desired characteristics can be
10,11
materials has been proposed in recent years.
Choline chlor-
ide (also known as 2-hydroxyethyltrimethyl ammonium chloride
or vitamin B4), is known to be non-toxic, biodegradable and is
6
,7
created for a particular application.
12
Despite being initially considered as environmentally benign
always used as an essential nutrient. In recent years, choli-
8
1
0,11,13–17
solvents, concerns about the toxicity of ILs have arisen over the
nium-based ILs have been synthesized
and found to
10
last few years. Actually, the reason that ILs are regarded as
have excellent biodegradability and low toxicity. Carboxylic
acids are the most common organic acids, and they are also low-
toxic and biodegradable. Therefore, the ILs using cholinium as
the cation and carboxylates as the anions would be environmen-
‘
green’ solvents is because the volatility of ILs is extremely low,
and atmospheric pollution is avoided. However, it is shown that
1
0,11
a
tally benign and biodegradable.
School of Chemistry and Chemical Engineering, Key Laboratory of
1
8
In 2003, Rogers and co-workers demonstrated the ability of
ILs to induce aqueous two-phase systems (ATPSs) in the pres-
ence of inorganic salts. Due to their relatively high content of
water and no use of volatile compounds, the IL-based ATPSs are
friendly to the environment, and therefore have been used to
Green Chemical Media and Reactions, Ministry of Education, Henan
Normal University, Xinxiang, Henan 453007, P. R. China.
E-mail: Jwang@henannu.edu.cn
Shanghai Key Laboratory of Green Chemistry and Chemical Processes,
Department of Chemistry, East China Normal University, Shanghai
b
2
00062, P. R. China
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extract/separate amino acids, drugs, low molecular mass com-
pounds, radiological isotopes, proteins and enzymes.
Results and discussion
18–28
Phase diagrams of the ATPSs
However, highly toxic and poorly biodegradable imidazolium-
or pyridinium-based ILs are usually used in these applications,
resulting in the reduction of enzyme activity to different
Binodal curves of the IL + PPG400 systems have been deter-
mined through the cloud point titration method, and the results
are shown in Fig. 1–3. It is interesting to find that these ILs,
18
24,25
extents.
On the other hand, high concentrations of inorganic
salts are always used in the IL-based ATPSs. This will produce a
great amount of highly salty water in the application, thus
causing environmental problems. Therefore, it is important to
design environmentally friendly IL-based ATPSs and to use
them for the extraction/separation of proteins and enzymes.
In this work, environmentally friendly ATPSs formed by cho-
linium ILs (as depicted in Scheme 1) and non-toxic, biodegrad-
able and thermo-sensitive poly(propylene glycol) 400
29
(
PPG400) have been developed for the high activity extraction
of proteins. In this context, cholinium formate, cholinium
acetate, cholinium propionate, cholinium butyrate, cholinium
glycollate, cholinium lactate, cholinium benzoate, di-cholinium
oxalate and tri-cholinium citrate have been synthesized and
characterized. The liquid–liquid equilibria phase diagrams have
been determined for these ATPSs, and used for the design of
aqueous two-phase extraction process. Then, partition coeffi-
cients of bovine serum albumin (BSA), trypsin, papain and lyso-
zyme in the IL + PPG400 ATPSs are determined at 25 °C, and
the effects of chemical structure of the ILs, size of the proteins
and tie line length (TLL) of the ATPSs on the partitioning behav-
ior of these proteins have been investigated systematically.
Factors affecting enzyme activity of the proteins were also exam-
ined in detail. Because the polymer employed here is thermo-
sensitive, the attempt has been made to recycle the polymer from
the top polymer-rich phase simply by heating.
Fig. 1 Binodal curves of cholinium IL + PPG400 ATPSs at 25 °C: ○,
tri-cholinium citrate; ●, di-cholinium oxalate; △, cholinium glycollate;
▲
, cholinium lactate; ◆, cholinium butyrate; □, cholinium formate; ■,
cholinium propionate; and ▼, cholinium acetate.
Scheme 1 Chemical route for the synthesis of the cholinium ILs and chemical structure of the cation and anions used in this study.
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phases; and (iii) the ratio of phase volumes. It is known that the
closer to the origin of the binodal curve, the lower the IL con-
centration required for the formation of two phases, and then the
stronger the phase-forming ability of the IL. It can be seen from
Fig. 1 that the phase-forming abilities of the ILs decrease in the
order: tri-cholinium citrate > di-cholinium oxalate > cholinium
glycollate > cholinium propionate ≈ cholinium lactate ≈ choli-
nium acetate > cholinium formate > cholinium butyrate. Con-
sidering the fact that these ILs have the same cation but different
anions, the phase-forming ability of these ILs would be deter-
mined by the nature of their anions. Actually, the salting-out
aptitude of an anion is directly related to its hydration
30–32
capacity.
Anions with higher charge densities have stronger
hydration capacities than those with lower charge densities,
resulting in the decrease in the number of water molecules avail-
3
3,34
Fig. 2 Phase diagram of cholinium IL + PPG400 ATPSs at 25 °C: (a)
cholinium propionate; (b) cholinium glycollate; ■, binodal; and ●, total
composition.
able to hydrate the polymer in these systems.
Thus, citrate
and oxalate anions are stronger salting-out species, and are more
prone to induce liquid–liquid demixing of PPG from the
aqueous media than, for example, acetate and formate anions.
In order to understand the effect of temperature on the phase
behaviour of these ATPSs, phase diagrams of cholinium propio-
nate + PPG400, cholinium glycollate + PPG400 and cholinium
lactate + PPG400 were also determined at 15, 25 and 35 °C. The
results are presented in Fig. 3. It is obvious that an increase in
temperature enhances the immiscibility region. This pattern is in
close agreement with that observed in the ATPSs composed of
35–38
PPG and inorganic salts,
whereas the opposite trend has
been verified in the ATPSs of PEG + IL. This suggests that in
30
our PPG + IL ATPS, PPG was salted-out by the IL; with increas-
35–38
ing temperature, PPG becomes more hydrophobic,
and then
water is driven from the PPG-rich phase to the IL-rich phase.
Therefore, the PPG concentration increases in the PPG-rich
phase, while the IL concentration decreases in the IL-rich phase.
Partitioning of the proteins
In the present work, BSA, trypsin, papain and lysozyme were
chosen as model proteins, and their partition behavior in the
ATPSs of cholinium IL (cholinium acetate, cholinium propio-
nate, cholinium butyrate, cholinium glycollate, cholinium lactate,
di-cholinium oxalate, or tri-cholinium citrate) + PPG400 has
been examined at 25 °C. The obtained partition coefficients of
the proteins in the studied ATPSs are given in Table 1. It can be
seen that the values of the partition coefficients for BSA, trypsin,
papain and lysozyme in the cholinium IL + PPG400 ATPSs
change from 3.2 to 51.8. This demonstrates that these proteins
are highly preferentially partitioned in the IL-rich phase (hydro-
philic phase), and these new ATPSs systems may be a clean
approach for the separation and purification of proteins. As an
example, extraction efficiencies of the proteins in the cholinium
lactate + PPG400 and tri-cholinium citrate + PPG400 ATPSs at
25 °C are shown in Fig. 4. It is clearly indicated that
86.4–99.9% of the proteins can be extracted into the IL-rich
phase of the ATPSs by a single-step extraction procedure, and
the extraction efficiency decreases in the order: lysozyme >
papain > trypsin > BSA. This order can be understood from the
size effect of the proteins since the transfer of the protein to the
IL-rich phase requires the breaking of the interactions between
Fig. 3 Effect of temperature on the binodal curves of cholinium IL +
PPG400 ATPSs: (a), cholinium propionate; (b), cholinium glycollate;
(c), cholinium lactate; ▲, 15 °C; ●, 25 °C; ■, 35 °C.
except for cholinium benzoate, are able to form ATPSs with
PPG400, and the bottom phase is IL-rich while the top phase is
polymer-rich. For example, Fig. 1 shows the binodal curves for
the cholinium IL + PPG400 systems at 25 °C, and Fig. 2 illus-
trates the phase diagrams determined for the cholinium propio-
nate/cholinium glycollate + PPG400 ATPSs at 25 °C. These
phase diagrams provide information about (i) the concentration
of phase-forming components required to form two phases; (ii)
the concentration of phase components in the top and bottom
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Table 1 Partition coefficients of BSA, trypsin, papain and lysozyme in
the ATPSs at pH 7.0 and 25 °C
Table 2 Effect of the TLL on the partition coefficients of lysozyme at
pH 7.0 and 25 °C
Partition coefficient (K) of the
proteins
ATPS
TLL
K
System
Lysozyme Papain Trypsin BSA
Cholinium propionate–PPG400
66.07
37.7
11.1
10.8
7
6.46
a
Cholinium lactate + PPG400
Tri-cholinium citrate + PPG400
Cholinium acetate + PPG400
Cholinium propionate + PPG400 37.7
Cholinium butyrate + PPG400
Cholinium glycollate + PPG400
Di-cholinium oxalate + PPG400
51.8
37.9
15.5
8.72
7.83
16.4
9.58
7.16
3.23
4.05
5.26
6.96
14.0
4.28
4.30
7.40
4.17
4.41
5.55
6.16
9.31
4.84
3.38
3.44
77.47
89.00
71.05
73.75
77.07
83.60
69.35
a
3.17
a
Cholinium glycollate–PPG400
Tri-cholinium citrate–PPG400
37.9
19.7
18.2
a
a
6.41
46.5
7.91
a
a
7.92
46.5
33.7
15.4
7
7
8
1.03
8.40
2.48
a
The test concentration of the proteins was 4 g L−1; 0.4 g of IL, 1.0 g of
O were added for the formation of each ATPS.
PPG400 and 1.3 ml of H
2
7.52
t
1
b
1
t
2
b
2
where w , w , w and w are the equilibrium mass fractions of
PPG400 (1) and IL (2) in the top (t) and bottom (b) phases.
Table 2 shows the TLL dependence of the partition coefficients
of lysozyme in some typical cholinium IL + PPG400 ATPSs. It
is clearly indicated that the partition coefficients of lysozyme
decrease with increasing TLL. As the TLL increases, the IL
becomes more concentrated in the bottom phase, and the bottom
phase becomes more structured. The partition of proteins into
this phase needs more energy to destroy the structure of the IL–
water network, thus reducing the partition coefficients of the pro-
teins. Therefore, we can regulate the partition of proteins in
ATPSs by changing the TLL.
It is known that the pH is a crucial parameter to influence the
partitioning of solutes in IL ATPSs. In this work, an attempt has
been made to determine the partition coefficients of the proteins
in the pH range from 5 to 11. Unfortunately, we did not obtain
reliable results. The reason for this is that the proteins investi-
gated here have high partition coefficients in the cholinium IL +
PPG400 ATPSs, more than 86% proteins have been enriched
into the IL-rich phase. Therefore, the partitioning of proteins is
not sensitive to the change of pH values in the studied pH range.
Fig. 4 Extraction efficiency of the proteins by sample ATPSs at pH 7.0
and 25 °C: cholinium lactate + PPG400 (black); tri-cholinium citrate +
PPG400 (gray).
the phase components to create a cavity where the protein will
be included, energy for this process would be obtained from the
interactions between proteins and ILs. The larger the protein
size, the greater the energy required. Thus, a larger sized protein
is more difficult to partition in the IL-rich phase than a smaller
protein. For the proteins studied in this work, lysozyme (MW =
Activity enhancement of trypsin in the aqueous ionic liquids
2
5
In the traditional purification processes of proteins, several steps
of manipulation were always needed, and enzyme activity is un-
39
avoidably decreased in each step of the purification. In order to
examine how the enzyme activity of the proteins is affected by
the cholinium IL ATPSs, trypsin, one of the most widely used
enzymes in biocatalysis, was chosen as a model enzyme to study
the effect of cholinium ILs (cholinium acetate, cholinium propio-
nate, cholinium butyrate, cholinium glycollate, cholinium lactate
and tri-cholinium citrate) in aqueous solutions on the enzyme
activity of the proteins. The results were shown in Fig. 5, from
which it is surprising to find that the activity of trypsin was not
decreased, but rather increased by about 11% in aqueous IL solu-
1
2
4 400) has a smaller molecular weight than papain (MW =
1 000), trypsin (MW = 23 800) and BSA (MW = 65 000), thus
the reversed order for the extraction efficiency has been
observed.
It is known that the partitioning degree of solutes depends on
the relative compositions of the top- and bottom-phases in the
ATPSs, and the tie line length (TLL) can be used to describe the
difference in composition of the two phases. As the TLL
increases, the top- and bottom-phases show increasing difference
18
−1
in compositions. Thus the difference in the composition of the
top- and bottom-phases could be regulated by the TLL. The TLL
can be calculated by the following equation:
tions at an IL concentration of 1.237 mol L . Among the ILs
investigated, cholinium lactate and tri-cholinium citrate are the
best stabilizer and/or promoter for the activity of trypsin in
aqueous solutions.
It is also known that the content of water is another important
t
1
b
1
2
t
2
b
2 0:5
24,25
TLL ¼ ½ðw ꢀ w Þ þ ðw ꢀ w Þ ꢁ
ð1Þ
2
factor that affects the enzyme activity.
Thus, the effect of
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cations would facilitate the enzyme action because they hold the
H O-mimicking property and H-bonding functionality, so as to
2
help the enzyme resume its flexible and active conformation.
Therefore, it is not difficult to understand why cholinium ILs
could improve the activity of trypsin.
The influence of temperature on the enzyme activity of trypsin
was also investigated. Here, the activity data of trypsin in a wide
IL concentration range are given in Table 3 as a function of
temperature. It can be seen that when the temperature is below
4
5 °C, the activity of trypsin increases by about 76% on average
with increasing temperature in water and aqueous solutions of
cholinium propionate or cholinium lactate. However, when the
temperature is higher than 45 °C, the activity of trypsin
decreased significantly with the increase of temperature. Inacti-
vation of trypsin was observed at 65 °C in water, at 75–85 °C in
aqueous cholinium propionate solutions, and at 85–95 °C in
aqueous cholinium lactate solutions. At the same time, it was
found that the higher the IL concentration, the stronger the
ability of the cholinium-based IL to maintain the activity of
trypsin. At a given IL concentration and a given temperature, the
activity of trypsin in aqueous cholinium lactate is higher than
that in aqueous cholinium propionate. These experimental facts
indicate that the addition of IL in water enhances the activity and
thermal stability of trypsin, and cholinium lactate is a better IL
to maintain the activity of trypsin in aqueous solutions, which is
in agreement with the result obtained from Fig. 5.
Fig. 5 Effect of IL nature on the trypsin activity at 25 °C: (a) buffer
solution; (b) cholinium acetate; (c) cholinium glycollate; (d) cholinium
propionate; (e) cholinium butyrate; (f) cholinium lactate; and (g) tri-cho-
linium citrate. The concentration of the IL in the aqueous solution is
−1
1
.237 mol L .
In order to examine whether trypsin can be stored in the
aqueous cholinium-based IL solutions for a long time without
inactivation, we detected the activity of trypsin in aqueous choli-
nium propionate and aqueous cholinium lactate solutions within
1
3 months, and the results are given in Table 4. Surprisingly, it
−1
was found that at the IL concentration of 1.237 mol L , the
activity of trypsin was generally increased in these aqueous IL
solutions within 13 months. On the contrary, only 60% and 30%
of the activity of trypsin could be maintained in pure water after
3
and 13 months, respectively. In addition, we determined the
activity of trypsin in the IL-rich phase of the cholinium IL-based
ATPSs, and the results are shown in Fig. 7. It was found that the
activity of trypsin was also increased to a different content in the
IL-rich phases of the studied cholinium IL ATPSs, except for
cholinium acetate. However, only 88–90% and 0.46–93.1% of
trypsin activity were maintained in the imidazolium-based IL/
Fig. 6 The activity of trypsin in aqueous tri-cholinium citrate solutions
with different water contents at 25 °C.
25
42
salt ATPSs and in the PEG/salt ATPSs, respectively. There-
fore, at the present stage it is appropriate to state that the aqueous
solutions of the cholinium-based ILs are excellent long-term
storage media of trypsin, and the cholinium IL + PPG400 ATPSs
are promising clean systems for the efficient and high activity
extraction and purification of proteins.
water content in aqueous tri-cholinium citrate solution on the
activity of trypsin was investigated in detail, and the results are
shown in Fig. 6. It was found that compared with the activity
(100%) in pure water, trypsin activity decreased and only
8
0–94.8% of trypsin activity can be maintained when the
content of water is below 30%. However, as the water content
was greater than 40%, trypsin activity was increased by about
1
0%. It has been reported that strong hydrogen bonding inter-
actions between the IL and enzyme was the key factor that
Recycling of the polymer
4
0
causes the unfolding of protein structures in ILs. Therefore, it
seems likely that water present in the IL could protect the
enzyme structure by forming hydrogen bonds with the IL, and
accordingly, the hydrogen bonding interactions between the IL
and enzymes were weakened. The good activity of trypsin
observed in the presence of the cholinium ILs can be attributed
Inorganic salts are always used in IL + salt ATPSs. It is known
that the concentration of the salt in the salt-rich phase is quite
18,19
high.
The wide application of such a separation technique
would result in highly salty water, which cannot be considered
sustainable. In the present work, our novel APTSs are created
from PPG and green cholinium ILs. Here, PPG is used to replace
4
1
to the hydroxylated choline cation. The ILs with hydroxylated
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a
Table 3 Effect of temperature on the activity of trypsin at varied concentrations of the ILs
U
r
IL(mol L−
1
)
15 °C
25 °C
35 °C
45 °C
55 °C
65 °C
75 °C
85 °C
95 °C
System
C
Water
Cholinium propionate
80.2
80.3
81.9
83.1
84.2
84.4
82.8
83.3
84.0
81.6
83.4
100
103
104
108
110
110
108
108
111
108
106
127
126
137
139
140
139
136
137
140
140
138
124
105
134
151
154
155
135
153
164
162
164
17.3
19.0
52.4
65.9
97.7
107
45.4
78.9
118
—
7.7
—
—
—
—
—
—
—
—
—
—
15.1
21.2
22.2
33.2
—
—
—
—
—
—
—
—
—
—
—
0.5
1
1
2
2
.0
.5
.0
.5
14.4
22.9
36.9
49.8
19.0
32.7
32.7
54.1
54.1
3.7
19.5
21.3
9.5
19.3
22.7
24.0
33.7
Cholinium lactate
0.5
1.0
1.5
2.0
2.5
142
168
a
—
, inactivation of trypsin at that temperature.
Table 4 Effect of storage time on the activity of trypsin at 25 °C
U
r
System
1 d
1 month
2 month
3 month
5 month
12 month
13 month
Water
Cholinium propionate
Cholinium lactate
100
100
100
94.8
124
120
81.0
125
118
60.0
110
108
35.5
82.2
76.7
33.1
136
129
28.7
129
124
a
a
a
The concentration of the IL is 1.237 mol L−1
.
Fig. 7 The activity of trypsin in the IL-rich phase of cholinium IL +
PPG400 ATPSs: (a) buffer solution; (b) cholinium acetate; (c) cholinium
propionate; (d) cholinium glycollate; (e) cholinium lactate; (f) tri-choli-
nium citrate; and (g) cholinium butyrate. 0.4 g of IL, 0.8 g of PPG400
Fig. 8 The cloud point curve for aqueous PPG400 solutions.
value, the solution becomes highly turbid, and a phase separ-
ation takes place from homogeneous to two liquid phases. This
phenomenon is attributed to the reduction of hydrophilicity of
the PO chain of PPG, caused by the rise in temperature.
2
and 1.3 ml of H O were added for the formation of each ATPS.
the inorganic salt. This polymer is thermo-sensitive and can be
recovered conveniently by heating.
In the cholinium IL + PPG400 ATPSs, the top phase is com-
posed of the PPG400, water and small amount of IL, in which
the mass fraction of PPG400 is about 65–85% and that of the IL
is about 2–7%. In order to study the recycling performance of
the polymer from the top phase, the cloud point temperatures of
the aqueous PPG400 solutions have been determined in the pre-
sence of 2 wt% of IL, the result is shown in Fig. 9. It can be
seen that the addition of an IL decreases the cloud point
In order to investigate the recycling performance of PPG from
the aqueous solutions, the cloud point curve of aqueous
PPG400 has been determined experimentally and is illustrated in
Fig. 8. It can be seen that aqueous PPG solutions exhibit a lower
critical solution temperature at around 46 °C. When the temp-
erature of an aqueous PPG solution is increased to the critical
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Fig. 10 Photos of the aqueous cholinium propionate + PPG400 sol-
utions at different temperatures: (a) 25 °C; (b) 26.6 °C; (c) 35 °C; (d)
4
H
5 °C; 0.1 g of cholinium propionate, 3.5 g of PPG400 and 1.4 g of
O were added for the preparation of the mixture; the top phases of (c)
Fig. 9 The cloud point curves of aqueous PPG400 in the presence of
cholinium IL (2 wt%): ▲, cholinium lactate; ●, cholinium acetate; ■,
cholinium propionate.
2
and (d) were colored with malachite green.
can be recovered conveniently by heating; (iv) the activity of
trypsin was increased to a different extent in the IL-rich phases,
except for the cholinium acetate-rich phase, of the studied
ATPSs, indicating that aqueous solutions of the cholinium-based
ILs are a type of excellent long-term storage media for proteins
at least for trypsin.
To the best of our knowledge, this is the first study on the
non-toxic and biodegradable ATPSs and their application in the
efficient and high activity extraction and purification of proteins.
The work reported here may bring the IL-based ATPSs a green
nature and open a new way to recycle the phase component in
the extraction–separation processes by using IL-based ATPSs.
However, it should be emphasized that effective and economical
techniques for the recovery and reuse of the ILs need to be
developed in future work.
temperature of aqueous PPG400 solutions. For a given choli-
nium IL, the cloud point temperature of the aqueous PPG400 +
IL solutions decreases with increasing concentration of the
polymer, and the effect of the ILs on the cloud point temperature
of the aqueous PPG400 is in agreement with the order of their
phase-forming ability.
On the basis of the results mentioned above, the aqueous solu-
tion of cholinium propionate (2 wt%) + PPG400 (70 wt%) was
selected as a model system of the PPG400-rich phase to investi-
gate the recycling of PPG400 from the aqueous solution. It is
evident from Fig. 8 and 9 that the addition of cholinium propio-
nate (2 wt%) into the aqueous PPG400 solution reduces the
cloud point temperature of the system significantly from 55 °C
to 26.6 °C. From Fig. 10, it can be seen that the system becomes
cloudy at 26.6 °C. Then, PPG400 is driven from the aqueous
solution of cholinium propionate + PPG400 as the temperature is
increased to 35 °C, giving rise to an improved concentration of
PPG400 in the polymer-rich phase (about 78 wt%). Furthermore,
as the temperature goes up to 45 °C, more water is driven from
the PPG-rich top phase to the IL-rich bottom phase, and the con-
centration of PPG400 in the top phase increases to about
Experimental section
Chemicals
Cholinium chloride (>98%), PPG400 (>99%), glycolic acid
(>99%), ion exchange resin of Amberlite IRA 400-Cl and N-a-
9
0 wt%. In this case, almost all of the IL is driven into the
benzoyl-L-arginine ethyl ester (BAEE) were obtained from Alfa
Aesar. Sodium hydroxide, formic acid, acetic acid, propionic
acid, butyric acid, lactic acid, benzoic acid, oxalic acid, citric
acid, methanol and acetonitrile were purchased from Shanghai
Chem. Co. and used without further purification. BSA, trypsin,
papain and lysozyme were obtained from Sigma-Aldrich, and
used without further purification. The stock solution of trypsin
(4 g L− ) was prepared in water and stored at 4 °C. Doubly dis-
tilled deionized water was used throughout the experiments.
bottom phase, and the PPG400 can be effectively recovered from
the polymer-rich phase. Therefore, the polymer can be recovered
simply by heating and can be reused in the next extraction
process.
1
Conclusions
From the above discussion, the following conclusions can be
drawn: (i) both the cholinium ionic liquid and PPG400 are non-
toxic and biodegradable, therefore the ATPSs developed in the
present work are green ATPSs; (ii) the cholinium IL + PPG400
ATPSs can be used to extract proteins efficiently, and the extrac-
tion efficiency decreases with the increase of the size of proteins
and the tie line length of the ATPSs; (iii) in these IL ATPSs,
thermo-sensitive PPG400 is used to replace the inorganic salts
commonly used in IL + inorganic salt ATPSs, thus the polymer
Synthesis of the cholinium ILs. Nine types of cholinium ILs,
including cholinium formate, cholinium acetate, cholinium pro-
pionate, cholinium butyrate, cholinium glycollate, cholinium
lactate, cholinium benzoate, di-cholinium oxalate and tri-choli-
nium citrate, have been synthesized by the modification of the
Ohno method. First, cholinium hydroxides were prepared by
passing the aqueous cholinium chloride (5 wt%) through a
column of anion exchange resin (Amberlite IRA 400-Cl). For
43
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Green Chem.

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1
this purpose, a glass column was packed with the resin, followed
by washing with plenty of water. The column was charged with
an aqueous NaOH solution (2 M) to modify the resin beads
Di-cholinium oxalate: H NMR (400 MHz, D O): δ = 3.93
2
(m, 4H, –OCH ), 3.38 (t, 4H, –NCH ), 3.05 (s, 18H, –NCH )
2
2
3
ppm.
−
13
with OH anions. Then the column was washed thoroughly with
C NMR (100 MHz, D O): δ = 53.9, 55.6, 67.5, 173.6 ppm.
2
−
1
water to remove any traces of unexchanged OH anions (tested
Tri-cholinium citrate: H NMR (400 MHz, D O): δ =
2
with pH paper), and an aqueous solution of cholinium chloride
2.41–2.53 (m, 4H, –CH –), 3.06 (s, 27H, –NCH ), 3.37–3.39
2
3
−
was passed through the column to exchange the Cl anions with
(m, 6H, –NCH ), 3.90–3.94 (m, 6H, –CH O) ppm.
2
2
−
13
OH anions. Upon complete substitution of chloride, no precipi-
C NMR (100 MHz, D O): δ = 45.7, 53.9, 55.6, 67.5, 75.2,
2
tation of AgCl can be found by addition of a few drops of
178.9, 181.8 ppm.
AgNO solution. Next, the as-prepared aqueous solutions of cho-
The melting temperature (T ) and the glass transition temp-
3
m
linium hydroxide were neutralized with stoichiometric car-
boxylic acids and the reaction mixture was stirred magnetically
for 12 h at room temperature. The water in the systems was then
removed by rotary evaporation, and the resulting products were
dissolved in a mixed solvent of methanol–acetonitrile (1 : 9) and
filtered to remove any residual solid impurities. Finally, the sol-
vents were removed again, and all the cholinium ILs were dried
at 70 °C for 72 h under vacuum before use. The water content in
the ILs was determined by Karl–Fisher titration. It was found
that less than 0.02 wt% of water still remained in the cholinium
ILs.
erature (T ) for the cholinium ILs were measured using a Perkin-
g
Elmer Diamond DSC equipped with an intercooler at a scan rate
of 10 °C min− . For all the samples, at least one cycle of heating
and cooling were performed before taking the final heating scan.
The obtained results are given in Table 5, together with those
1
10,11
reported in literature.
Measurements of the binodal curves and tie lines. The
18
binodal curves were determined by the cloud-point method. In
a test tube, a polymer solution of known concentration was
added, and an IL solution of known mass fraction was then
added dropwise until the mixture became turbid or cloudy, then
a known mass of water was added to make the mixture clear
again. This procedure was repeated to obtain sufficient data for
the construction of a liquid–liquid equilibrium binodal curve.
The temperature of the systems was controlled to 25 ± 0.1 °C by
a DC-2006 water thermostat (Shanghai Hengping Instrument
Factory, China). Concentration of the phase components was
determined by weight quantification of all the components added
within an uncertainty of ±10− g. The tie lines, which describe
the concentrations of polymer and IL in the two phases, were
Characterization of the cholinium ILs. All the purified choli-
1
nium ILs were analyzed by H NMR (Bruker, AV-400) to
confirm the absence of any major impurities, and the purity of
these ILs is found to be greater than 99% in mass fraction. The
1
H NMR data of cholinium acetate, cholinium propionate, choli-
4
nium butyrate, cholinium benzoate and cholinium glycollate
1
0,11
were in good agreement with those reported in literature.
44
1
13
measured with the procedure outlined in our previous work.
The H NMR data of all the ILs and the C NMR data of choli-
nium lactate, di-cholinium oxalate and tri-cholinium citrate are
Extraction of the proteins. A given amount of IL, PPG400
and aqueous protein solution were added into a graduated glass
tube. The volume of the glass tube was calibrated before use.
The mixture was diluted by the addition of a given volume of
water, and then shaken vigorously for 30 min to attain equili-
brium by a desktop thermostated oscillator (Jinghong Instrument
Factory, Shanghai, China), and the temperature of the system
was controlled at 25 ± 0.1 °C with a DC-2006 low temperature
thermostat. The phase separation quickly occurred after cessation
of the shaking process. Then, a XYJ-802 centrifuge (Jiangsu
Medical Instrument Factory, Jiangsu, China) operated at
reported as follows:
Cholinium formate: H NMR (400 MHz, [d ] DMSO, TMS):
δ = 3.12 (s, 9H, –NCH ), 3.42 (t, 2H, –NCH ), 3.83 (m, 2H,
1
6
3
2
–
OCH ), 8.50 (s, 1H, –OOCH) ppm.
2
1
Cholinium acetate: H NMR (400 MHz, [d ] DMSO, TMS):
δ = 1.58 (s, 3H, –OOCCH ), 3.13 (s, 9H, –NCH ), 3.43 (t, 2H,
6
3
3
–
NCH ), 3.82 (d, 2H, –OCH ) ppm.
2
2
1
Cholinium propionate: H NMR (400 MHz, [d ] DMSO,
TMS): δ = 0.88 (t, 3H, –CH ), 1.85 (q, 2H, –CH COO), 3.14 (s,
6
3
2
9
H, –NCH ), 3.44 (t, 2H, –NCH ), 3.84 (m, 2H, –OCH ) ppm.
3 2 2
1
Cholinium butyrate: H NMR (400 MHz, D O): δ = 0.76 (t,
2
4
000 rpm was used to run for a period of 2 min in each test to
3
H, –CH ), 1.45 (m, 2H, –CH CH COO), 2.02 (t, 2H,
3
2
2
–
CH COO), 3.05 (s, 9H, –NCH ), 3.38 (q, 2H, –NCH ), 3.93
2 3 2
(
m, 2H, –OCH ) ppm.
Cholinium glycollate: H NMR (400 MHz, [d ] DMSO,
a
2
Table 5 Thermal properties of the cholinium-based ILs
1
6
TMS): δ = 3.12 (s, 9H, –NCH ), 3.37 (s, 2H, –CH (OH)COO),
IL
T
m
/°C
g
T /°C
3
2
3
.44 (t, 2H, –NCH ), 3.85 (m, 2H, –OCH ) ppm.
2
2
Cholinium formate
Cholinium acetate
Cholinium propionate
Cholinium butyrate
—
80
—
45
38
47
—
99
72
—
—
−74
—
−67
−51
−52
—
1
Cholinium benzoate: H NMR (400 MHz, [d ] DMSO, TMS):
10
6
1
0
δ = 3.16 (s, 9H, –NCH ), 3.49 (t, 2H, –NCH ), 3.91 (d,2H,
–
3
2
1
0
OCH ), 7.28 (t, 3H, benzene ring H), 7.87 (d, 2H, benzene ring
2
11
Cholinium glycollate
H) ppm.
11
Cholinium benzoate
Cholinium lactate
Di-choline oxalate
Tri-choline citrate
1
Cholinium lactate: H NMR (400 MHz, [d ] DMSO, TMS):
6
δ = 1.08 (d, 3H, –CH ), 3.12 (s, 10H, –NCH , –OH), 3.44
3
3
—
(t, 2H, –NCH ), 3.56 (m, 1H,–CH), 3.85 (m, 2H, –OCH ) ppm.
2
2
1
3
C NMR (100 MHz, [d ] DMSO), δ = 21.9, 53.4, 55.4, 67.5,
a
6
—, not detected
1
78.0 ppm.
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ensure complete phase separation. After the volume of the top
and bottom phases was recorded, the sample was collected from
the bottom IL-rich phase for analysis. A mass balance check was
made between the initial mass of the protein and the amounts in
the lower and upper phases on the basis of equilibrium compo-
sitions. The relative error in the mass balance is within 3%. To
avoid interference from the phase components, the samples were
diluted and analyzed against the blanks containing the same
phase components but without protein. The protein concentration
in both phases were determined by measuring the absorbance at
repeated three times for a given sample, and good reproducibility
was obtained for the T values.
c
Acknowledgements
This work was supported financially by the National Natural
Science Foundation of China (no. 21133009), the Innovation
Scientists and Technicians Troop Construction Projects of Henan
Province (no. 092101510300), and the foundation of Shanghai
Key Laboratory of Green Chemistry and Chemical Processes.
2
80 nm using a Shanghai 752N UV-vis spectrophotometer.
Partition coefficients (K) and extraction efficiencies (E%) of the
proteins were calculated by using the equations:
References
K ¼ Cb=Ct
ð2Þ
ð3Þ
1
M. A. P. Martins, C. P. Frizzo, D. N. Moreira, N. Zanatta and
H. G. Bonacorso, Chem. Rev., 2008, 108, 2015–2050.
2 N. V. Plechkova and K. R. Seddon, Chem. Soc. Rev., 2008, 37, 123–150.
E% ¼ CbVb=C0V0 ꢂ 100%
3
P. Wasserscheid and W. Keim, Angew. Chem., Int. Ed., 2000, 39,
3772–3789.
where C and C are the equilibrium concentrations of the parti-
b
t
tioned proteins in the IL-rich phase and in the polymer-rich
4 X. Han and D. W. Armstrong, Acc. Chem. Res., 2007, 40, 1079–1086.
A. Pinkert, K. N. Marsh, S. Pang and M. P. Staiger, Chem. Rev., 2009,
09, 6712–6728.
6 K. R. Seddon, J. Chem. Technol. Biotechnol., 1997, 68, 351–356.
7 W. L. Hough, M. Smiglak, H. Rodriguez, R. P. Swatloski, S. K. Spear,
D. T. Daly, J. Pernak, J. E. Grisel, R. D. Carliss, M. D. Soutullo,
J. H. Davis Jr. and R. D. Rogers, New J. Chem., 2007, 31, 1429–1436.
5
phase, respectively. V is the volume of the IL-rich phase, C is
b
0
1
the initial concentration of the proteins in water, and V is the
0
volume of the aqueous phase added into the test tube before the
separation process.
8
P. Anastas and J. C. Warner, Green Chemistry: Theory and Practice,
Oxford University Press, USA, 2000.
Determination of trypsin activity in the aqueous IL solutions.
9
M. Petkovic, K. R. Seddon, L. P. N. Rebelo and C. Silva Pereira, Chem.
Soc. Rev., 2011, 40, 1383–1403.
Trypsin activity in aqueous IL solutions was measured with the
45
BAEE method at pH 7.6 and 25 °C. The experimental appar-
atus employed is essentially the same as that used previously by
1
0 M. Petkovic, J. L. Ferguson, H. Q. N. Gunaratne, R. Ferreira,
M. C. Leitão, K. R. Seddon, L. P. N. Rebelo and C. S. Pereira, Green
Chem., 2010, 12, 643–649.
2
5
us. The spectrophotometrical measurements were carried out at
53 nm with a Shanghai 752N UV-vis spectrophotometer using
11 Y. Fukaya, Y. Iizuka, K. Sekikawa and H. Ohno, Green Chem., 2007, 9,
2
1155–1157.
a 1 cm cell. Aqueous solutions of the ILs were prepared at differ-
ent concentrations by dissolving the pure ILs in water, and then
trypsin was added to the ILs solutions. 0.2 ml of trypsin solution
was added to 3.0 ml of 0.5 mM BAEE solution containing
12 J. K. Blusztajn, Science, 1998, 281, 794.
13 S. Hu, T. Jiang, Z. Zhang, A. Zhu, B. Han, J. Song, Y. Xie and W. Li,
Tetrahedron Lett., 2007, 48, 5613–5617.
1
4 P. Moriel, E. J. García-Suárez, M. Martínez, A. B. García, M. A. Montes-
Morán, V. Calvino-Casilda and M. A. Bãnares, Tetrahedron Lett., 2010,
51, 4877–4881.
1
00 mM Tris buffer and 10 mM CaCl , followed by immediate
2
15 Y. H. Yu, X. M. Lu, Q. Zhou, K. Dong, H. W. Yao and S. J. Zhang,
Chem.–Eur. J., 2008, 14, 11174–11182.
mixing by inversion. The A253 nm was recorded every 0.5 min at
5 °C for about 5 min. The measurements were performed in tri-
plicate, and then an average was obtained. Trypsin activity was
2
16 K. D. Weaver, H. J. Kim, J. Sun, D. R. MacFarlane and G. D. Elliott,
Green Chem., 2010, 12, 507–513.
4
6
calculated by the literature method. Here, the activity of the
extracted trypsin or trypsin in aqueous IL solution was expressed
17 Q. Liu, X. Hou, N. Li and M. Zong, Green Chem., 2012, 14, 304–307.
18 K. E. Gutowski, G. A. Broker, H. D. Willauer, J. G. Huddleston,
R. P. Swatloski, J. D. Holbrey and R. D. Rogers, J. Am. Chem. Soc.,
by the relative activity (U ) which is defined by the following
r
2
003, 125, 6632–6633.
19 Z. Li, Y. Pei, H. Wang, J. Fan and J. Wang, TrAC, Trends Anal. Chem.,
010, 29, 1336–1346.
equation:
2
Ur ¼ Ue=Un ꢂ 100%
ð4Þ
20 M. G. Freire, C. M. S. S. Neves, I. M. Marrucho, J. N. C. Lopes,
L. P. N. Rebelo and J. A. P. Coutinho, Green Chem., 2010, 12,
1715–1718.
where U (U mg− ) is the activity of trypsin in the tested media
1
e
−1
2
1 F. J. Deive, A. Rodríguez, A. B. Pereiro, J. M. M. Araújo, M. A. Longo,
M. A. Z. Coelho, J. N. C. Lopes, J. M. S. S. Esperanc, L. P. N. Rebelo
and I. M. Marrucho, Green Chem., 2011, 13, 390–396.
(
IL-rich phase and aqueous IL solutions), and U (U mg ) is
the activity of trypsin dissolved in water with the same trypsin
concentration.
n
22 J. F. B. Pereira, Á. S. Lima, M. G. Freire and J. A. P. Coutinho, Green
Chem., 2010, 12, 1661–1669.
2
2
3 Y. Pei, Z. Li, L. Liu and J. Wang, J. Chromatogr., A, 2012, 1231, 2–7.
4 Q. Cao, L. Quan, C. He, N. Li, K. Li and F. Liu, Talanta, 2008, 77,
160–165.
Determination of the cloud point temperatures. The cloud
47
point temperature (T ) was determined by visual observation.
c
2
5 Y. Pei, J. Wang, K. Wu, X. Xuan and X. Lu, Sep. Purif. Technol., 2009,
A glass tube containing the sample solution was immersed in a
water bath, the temperature of which was maintained using a
DC-2006 water thermostat. The temperature was raised at a rate
64, 288–295.
2
6 S. P. M. Ventura, S. G. Sousa, M. G. Freire, L. S. Serafim, Á. S. Lima
and J. A. P. Coutinho, J. Chromatogr., B, 2011, 879, 2679–2687.
−1
of approximately 0.5 °C min . As soon as the temperature was
higher than the cloud point temperature, the sample was cooled
27 Y. Lu, W. Lu, W. Wang, Q. Guo and Y. Yang, Talanta, 2011, 85,
621–1626.
1
2
8 M. G. Freire, A. F. M. Cláudio, J. M. M. Araújo, J. A. P. Coutinho,
I. M. Marrucho, J. N. C. Lopes and L. P. N. Rebelo, Chem. Soc. Rev.,
2012, 41, 4966–4995.
below T , and then it was heated again to check the reproduci-
c
bility of the measurements. The heating–cooling cycle was
This journal is © The Royal Society of Chemistry 2012
Green Chem.

View Online
2
9 S. Tachibana, N. Kuba, F. Kawai, J. A. Duine and M. Yasuda, FEMS
Microbiol. Lett., 2003, 218, 345–349.
37 M. T. Zafarani-Moattar, F. Samadi and R. Sadeghi, J. Chem. Thermodyn.,
2004, 36, 871–875.
3
0 M. G. Freire, J. F. B. Pereira, M. Francisco, H. Rodriquze,
L. P. N. Rebelo, R. D. Rogers and J. A. P. Coutinho, Chem.–Eur. J.,
38 R. Sadeghi and F. Ziamajidi, Fluid Phase Equilib., 2006, 249, 165–172.
39 I. Pan and Y. Li, J. Chromatogr., B, 2001, 754, 179–184.
40 H. Weingärtner, C. Cabrele and C. Herrmann, Phys. Chem. Chem. Phys.,
2012, 14, 415–426.
41 J. Lai, Z. Li, Y. Lü and Z. Yang, Green Chem., 2011, 13, 1860–1868.
42 L. A. Oliveira, L. A. Sarubbo, A. L. F. Porto, G. M. Campos-Takaki and
E. B. Tambourgi, Process Biochem., 2002, 38, 693–699.
43 K. Fukumoto, M. Yoshizawa and H. Ohno, J. Am. Chem. Soc., 2005,
127, 2398–2399.
44 Y. Pei, J. Wang, L. Liu, K. Wu and Y. Zhao, J. Chem. Eng. Data, 2007,
52, 2026–2031.
45 G. W. Schwert and Y. Takenaka, Biochim. Biophys. Acta, 1955, 16, 570–575.
46 X. Zeng and E. Ruckenstein, Ind. Eng. Chem. Res., 1998, 37, 159–165.
47 T. Inoue and T. Misono, J. Colloid Interface Sci., 2008, 326, 483–489.
2
012, 18, 1831–1839.
3
3
1 Y. Marcus, J. Chem. Soc., Faraday Trans., 1991, 87, 2995–2999.
2 S. Shahriari, C. M. S. S. Neves, M. G. Freire and J. A. P. Coutinho,
J. Phys. Chem. B, 2012, 116, 7252–7258.
3
3
3
3
3 N. L. Abbot, D. Blankschtein and T. A. Hatton, Bioseparation, 1990, 1,
1
91–225.
4 J. Huddleston, A. Veide, K. Köhler, J. Flanagan, S. Enfors and
A. Lyddiatt, Trends Biotechnol., 1991, 9, 381–388.
5 R. Sadeghi and B. Jamehbozorg, Fluid Phase Equilib., 2009, 280,
6
8–75.
6 M. T. Zafarani-Moattar, S. Emamian and S. Hamzehzadeh, J. Chem. Eng.
Data, 2008, 53, 456–461.
Green Chem.
This journal is © The Royal Society of Chemistry 2012