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Cite this: DOI: 10.1039/c2gc35890e

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

Zhiyong Li,a Xinxin Liu,a Yuanchao Pei,a Jianji Wang*a and Mingyuan Heb

Received 12th June 2012, Accepted 2nd August 2012DOI: 10.1039/c2gc35890e

Ionic liquids (ILs) have numerous applications in industrial processes as a benign alternative toconventional volatile organic solvents. However, many of them are toxic to organisms and are poorlybiodegradable. In this work, a series of environmentally friendly cholinium ILs have been designed andsynthesized. It was found that these ILs could form aqueous two-phase systems (ATPSs) withpolypropylene glycol 400 (PPG400) which is thermo-sensitive, non-toxic and biodegradable. In order tounderstand 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 theeffects of anionic structure of the ionic liquids, nature of the proteins and difference in the concentrationof top- and bottom-phases on the partitioning behavior of some typical proteins were investigatedsystematically. It was shown that bovine serum albumin (BSA), trypsin, papain and lysozyme could beenriched effectively into the ionic liquid-rich phase of the ATPSs, and single-step extraction efficiencycould be as high as 86.4–99.9% under the optimized conditions. Furthermore, enzyme activity of thenative 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 monthsstorage. In addition, PPG400 has been recovered simply by heating and reused in the next extractionprocesses. This avoids the non-sustainable issue of highly salty water produced in the application of thepolyethylene glycol (PEG) + salt and ionic liquid + salt ATPSs.

Introduction

As an innovative class of solvents, ionic liquids (ILs) haveattracted much attention as greener replacements for traditionalvolatile organic solvents in a variety of fields such as chemicalsynthesis, catalytic chemistry, electrochemical devices, separ-ation and biomass dissolution due to their unique physico-chemical properties.1–5 ILs are composed solely of ions withnumerous combinations of anions and cations. The particularinterest is their highly tunable properties by varying the chemicalstructures of the ions comprised. Thus, by the rational design ofcation and anion, an IL with desired characteristics can becreated for a particular application.6,7

Despite being initially considered as environmentally benignsolvents,8 concerns about the toxicity of ILs have arisen over thelast few years. Actually, the reason that ILs are regarded as‘green’ solvents is because the volatility of ILs is extremely low,and atmospheric pollution is avoided. However, it is shown that

among a large number of ILs that have been investigated, the ILsconsisting of imidazolium or pyridinium cations and halide-containing anions have been demonstrated to be highly toxic andpoorly biodegradable.9 Therefore, widespread application of ILswould inevitably result in the loss of ILs into the water ecosys-tems, leading to aquatic environmental pollution. An alternativeapproach for overcoming these drawbacks is the development ofILs from their components, which have well characterized bio-degradable and toxicological properties. Therefore, the productionof non-toxic and environmentally benign ILs from renewablematerials has been proposed in recent years.10,11 Choline chlor-ide (also known as 2-hydroxyethyltrimethyl ammonium chlorideor vitamin B4), is known to be non-toxic, biodegradable and isalways used as an essential nutrient.12 In recent years, choli-nium-based ILs have been synthesized10,11,13–17 and found tohave excellent biodegradability and low toxicity.10 Carboxylicacids are the most common organic acids, and they are also low-toxic and biodegradable. Therefore, the ILs using cholinium asthe cation and carboxylates as the anions would be environmen-tally benign and biodegradable.10,11

In 2003, Rogers and co-workers18 demonstrated the ability ofILs to induce aqueous two-phase systems (ATPSs) in the pres-ence of inorganic salts. Due to their relatively high content ofwater and no use of volatile compounds, the IL-based ATPSs arefriendly to the environment, and therefore have been used to

aSchool of Chemistry and Chemical Engineering, Key Laboratory ofGreen Chemical Media and Reactions, Ministry of Education, HenanNormal University, Xinxiang, Henan 453007, P. R. China.E-mail: Jwang@henannu.edu.cnbShanghai Key Laboratory of Green Chemistry and Chemical Processes,Department of Chemistry, East China Normal University, Shanghai200062, P. R. China

This journal is © The Royal Society of Chemistry 2012 Green Chem.

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extract/separate amino acids, drugs, low molecular mass com-pounds, radiological isotopes, proteins and enzymes.18–28

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 differentextents.24,25 On the other hand, high concentrations of inorganicsalts are always used in the IL-based ATPSs. This will produce agreat amount of highly salty water in the application, thuscausing environmental problems. Therefore, it is important todesign environmentally friendly IL-based ATPSs and to usethem 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(PPG400)29 have been developed for the high activity extractionof proteins. In this context, cholinium formate, choliniumacetate, cholinium propionate, cholinium butyrate, choliniumglycollate, cholinium lactate, cholinium benzoate, di-choliniumoxalate and tri-cholinium citrate have been synthesized andcharacterized. The liquid–liquid equilibria phase diagrams havebeen determined for these ATPSs, and used for the design ofaqueous 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, andthe effects of chemical structure of the ILs, size of the proteinsand 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 fromthe top polymer-rich phase simply by heating.

Results and discussion

Phase diagrams of the ATPSs

Binodal curves of the IL + PPG400 systems have been deter-mined through the cloud point titration method,18 and the resultsare shown in Fig. 1–3. It is interesting to find that these ILs,

Scheme 1 Chemical route for the synthesis of the cholinium ILs and chemical structure of the cation and anions used in this study.

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.

Green Chem. This journal is © The Royal Society of Chemistry 2012

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except for cholinium benzoate, are able to form ATPSs withPPG400, and the bottom phase is IL-rich while the top phase ispolymer-rich. For example, Fig. 1 shows the binodal curves forthe 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. Thesephase diagrams provide information about (i) the concentrationof phase-forming components required to form two phases; (ii)the concentration of phase components in the top and bottom

phases; and (iii) the ratio of phase volumes. It is known that thecloser to the origin of the binodal curve, the lower the IL con-centration required for the formation of two phases, and then thestronger the phase-forming ability of the IL. It can be seen fromFig. 1 that the phase-forming abilities of the ILs decrease in theorder: tri-cholinium citrate > di-cholinium oxalate > choliniumglycollate > cholinium propionate ≈ cholinium lactate ≈ choli-nium acetate > cholinium formate > cholinium butyrate. Con-sidering the fact that these ILs have the same cation but differentanions, the phase-forming ability of these ILs would be deter-mined by the nature of their anions. Actually, the salting-outaptitude of an anion is directly related to its hydrationcapacity.30–32 Anions with higher charge densities have strongerhydration capacities than those with lower charge densities,resulting in the decrease in the number of water molecules avail-able to hydrate the polymer in these systems.33,34 Thus, citrateand oxalate anions are stronger salting-out species, and are moreprone to induce liquid–liquid demixing of PPG from theaqueous media than, for example, acetate and formate anions.

In order to understand the effect of temperature on the phasebehaviour of these ATPSs, phase diagrams of cholinium propio-nate + PPG400, cholinium glycollate + PPG400 and choliniumlactate + PPG400 were also determined at 15, 25 and 35 °C. Theresults are presented in Fig. 3. It is obvious that an increase intemperature enhances the immiscibility region. This pattern is inclose agreement with that observed in the ATPSs composed ofPPG and inorganic salts,35–38 whereas the opposite trend hasbeen verified in the ATPSs of PEG + IL.30 This suggests that inour PPG + IL ATPS, PPG was salted-out by the IL; with increas-ing temperature, PPG becomes more hydrophobic,35–38 and thenwater is driven from the PPG-rich phase to the IL-rich phase.Therefore, the PPG concentration increases in the PPG-richphase, while the IL concentration decreases in the IL-rich phase.

Partitioning of the proteins

In the present work, BSA, trypsin, papain and lysozyme werechosen as model proteins, and their partition behavior in theATPSs of cholinium IL (cholinium acetate, cholinium propio-nate, cholinium butyrate, cholinium glycollate, cholinium lactate,di-cholinium oxalate, or tri-cholinium citrate) + PPG400 hasbeen examined at 25 °C. The obtained partition coefficients ofthe proteins in the studied ATPSs are given in Table 1. It can beseen that the values of the partition coefficients for BSA, trypsin,papain and lysozyme in the cholinium IL + PPG400 ATPSschange from 3.2 to 51.8. This demonstrates that these proteinsare highly preferentially partitioned in the IL-rich phase (hydro-philic phase), and these new ATPSs systems may be a cleanapproach for the separation and purification of proteins. As anexample, extraction efficiencies of the proteins in the choliniumlactate + PPG400 and tri-cholinium citrate + PPG400 ATPSs at25 °C are shown in Fig. 4. It is clearly indicated that86.4–99.9% of the proteins can be extracted into the IL-richphase of the ATPSs by a single-step extraction procedure, andthe extraction efficiency decreases in the order: lysozyme >papain > trypsin > BSA. This order can be understood from thesize effect of the proteins since the transfer of the protein to theIL-rich phase requires the breaking of the interactions between

Fig. 2 Phase diagram of cholinium IL + PPG400 ATPSs at 25 °C: (a)cholinium propionate; (b) cholinium glycollate; ■, binodal; and ●, totalcomposition.

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.

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the phase components to create a cavity where the protein willbe included, energy for this process would be obtained from theinteractions between proteins and ILs.25 The larger the proteinsize, the greater the energy required. Thus, a larger sized proteinis more difficult to partition in the IL-rich phase than a smallerprotein. For the proteins studied in this work, lysozyme (MW =14 400) has a smaller molecular weight than papain (MW =21 000), trypsin (MW = 23 800) and BSA (MW = 65 000), thusthe reversed order for the extraction efficiency has beenobserved.

It is known that the partitioning degree of solutes depends onthe relative compositions of the top- and bottom-phases in theATPSs, and the tie line length (TLL) can be used to describe thedifference in composition of the two phases. As the TLLincreases, the top- and bottom-phases show increasing differencein compositions.18 Thus the difference in the composition of thetop- and bottom-phases could be regulated by the TLL. The TLLcan be calculated by the following equation:

TLL ¼ ½ðwt1 � wb

1Þ2 þ ðwt2 � wb

2Þ2�0:5 ð1Þ

where wt1, w

b1, w

t2 and wb

2 are the equilibrium mass fractions ofPPG400 (1) and IL (2) in the top (t) and bottom (b) phases.Table 2 shows the TLL dependence of the partition coefficientsof lysozyme in some typical cholinium IL + PPG400 ATPSs. Itis clearly indicated that the partition coefficients of lysozymedecrease with increasing TLL. As the TLL increases, the ILbecomes more concentrated in the bottom phase, and the bottomphase becomes more structured. The partition of proteins intothis 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 inATPSs by changing the TLL.

It is known that the pH is a crucial parameter to influence thepartitioning of solutes in IL ATPSs. In this work, an attempt hasbeen made to determine the partition coefficients of the proteinsin the pH range from 5 to 11. Unfortunately, we did not obtainreliable 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 enrichedinto the IL-rich phase. Therefore, the partitioning of proteins isnot sensitive to the change of pH values in the studied pH range.

Activity enhancement of trypsin in the aqueous ionic liquids

In the traditional purification processes of proteins, several stepsof manipulation were always needed, and enzyme activity is un-avoidably decreased in each step of the purification.39 In order toexamine how the enzyme activity of the proteins is affected bythe cholinium IL ATPSs, trypsin, one of the most widely usedenzymes in biocatalysis, was chosen as a model enzyme to studythe effect of cholinium ILs (cholinium acetate, cholinium propio-nate, cholinium butyrate, cholinium glycollate, cholinium lactateand tri-cholinium citrate) in aqueous solutions on the enzymeactivity of the proteins. The results were shown in Fig. 5, fromwhich it is surprising to find that the activity of trypsin was notdecreased, but rather increased by about 11% in aqueous IL solu-tions at an IL concentration of 1.237 mol L−1. Among the ILsinvestigated, cholinium lactate and tri-cholinium citrate are thebest stabilizer and/or promoter for the activity of trypsin inaqueous solutions.

It is also known that the content of water is another importantfactor that affects the enzyme activity.24,25 Thus, the effect of

Fig. 4 Extraction efficiency of the proteins by sample ATPSs at pH 7.0and 25 °C: cholinium lactate + PPG400 (black); tri-cholinium citrate +PPG400 (gray).

Table 1 Partition coefficients of BSA, trypsin, papain and lysozyme inthe ATPSs at pH 7.0 and 25 °C

System

Partition coefficient (K) of theproteins

Lysozyme Papain Trypsin BSA

Cholinium lactate + PPG400a 51.8 8.72 5.26 4.41Tri-cholinium citrate + PPG400a 37.9 7.83 6.96 5.55Cholinium acetate + PPG400a 15.5 16.4 14.0 6.16Cholinium propionate + PPG400a 37.7 9.58 4.28 9.31Cholinium butyrate + PPG400a 6.41 7.16 4.30 4.84Cholinium glycollate + PPG400a 46.5 3.23 7.40 3.38Di-cholinium oxalate + PPG400a 7.91 4.05 4.17 3.44

a The test concentration of the proteins was 4 g L−1; 0.4 g of IL, 1.0 g ofPPG400 and 1.3 ml of H2O were added for the formation of each ATPS.

Table 2 Effect of the TLL on the partition coefficients of lysozyme atpH 7.0 and 25 °C

ATPS TLL K

Cholinium propionate–PPG400 66.07 37.776.46 11.177.47 10.889.00 3.17

Cholinium glycollate–PPG400 71.05 37.973.75 19.777.07 18.283.60 7.92

Tri-cholinium citrate–PPG400 69.35 46.571.03 33.778.40 15.482.48 7.52

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water content in aqueous tri-cholinium citrate solution on theactivity of trypsin was investigated in detail, and the results areshown in Fig. 6. It was found that compared with the activity(100%) in pure water, trypsin activity decreased and only80–94.8% of trypsin activity can be maintained when thecontent of water is below 30%. However, as the water contentwas greater than 40%, trypsin activity was increased by about10%. It has been reported that strong hydrogen bonding inter-actions between the IL and enzyme was the key factor thatcauses the unfolding of protein structures in ILs.40 Therefore, itseems likely that water present in the IL could protect theenzyme structure by forming hydrogen bonds with the IL, andaccordingly, the hydrogen bonding interactions between the ILand enzymes were weakened. The good activity of trypsinobserved in the presence of the cholinium ILs can be attributedto the hydroxylated choline cation.41 The ILs with hydroxylated

cations would facilitate the enzyme action because they hold theH2O-mimicking property and H-bonding functionality, so as tohelp the enzyme resume its flexible and active conformation.Therefore, it is not difficult to understand why cholinium ILscould improve the activity of trypsin.

The influence of temperature on the enzyme activity of trypsinwas also investigated. Here, the activity data of trypsin in a wideIL concentration range are given in Table 3 as a function oftemperature. It can be seen that when the temperature is below45 °C, the activity of trypsin increases by about 76% on averagewith increasing temperature in water and aqueous solutions ofcholinium propionate or cholinium lactate. However, when thetemperature is higher than 45 °C, the activity of trypsindecreased significantly with the increase of temperature. Inacti-vation of trypsin was observed at 65 °C in water, at 75–85 °C inaqueous cholinium propionate solutions, and at 85–95 °C inaqueous cholinium lactate solutions. At the same time, it wasfound that the higher the IL concentration, the stronger theability of the cholinium-based IL to maintain the activity oftrypsin. At a given IL concentration and a given temperature, theactivity of trypsin in aqueous cholinium lactate is higher thanthat in aqueous cholinium propionate. These experimental factsindicate that the addition of IL in water enhances the activity andthermal stability of trypsin, and cholinium lactate is a better ILto maintain the activity of trypsin in aqueous solutions, which isin agreement with the result obtained from Fig. 5.

In order to examine whether trypsin can be stored in theaqueous cholinium-based IL solutions for a long time withoutinactivation, we detected the activity of trypsin in aqueous choli-nium propionate and aqueous cholinium lactate solutions within13 months, and the results are given in Table 4. Surprisingly, itwas found that at the IL concentration of 1.237 mol L−1, theactivity of trypsin was generally increased in these aqueous ILsolutions within 13 months. On the contrary, only 60% and 30%of the activity of trypsin could be maintained in pure water after3 and 13 months, respectively. In addition, we determined theactivity of trypsin in the IL-rich phase of the cholinium IL-basedATPSs, and the results are shown in Fig. 7. It was found that theactivity of trypsin was also increased to a different content in theIL-rich phases of the studied cholinium IL ATPSs, except forcholinium acetate. However, only 88–90% and 0.46–93.1% oftrypsin activity were maintained in the imidazolium-based IL/salt ATPSs25 and in the PEG/salt ATPSs,42 respectively. There-fore, at the present stage it is appropriate to state that the aqueoussolutions of the cholinium-based ILs are excellent long-termstorage media of trypsin, and the cholinium IL + PPG400 ATPSsare promising clean systems for the efficient and high activityextraction and purification of proteins.

Recycling of the polymer

Inorganic salts are always used in IL + salt ATPSs. It is knownthat the concentration of the salt in the salt-rich phase is quitehigh.18,19 The wide application of such a separation techniquewould result in highly salty water, which cannot be consideredsustainable. In the present work, our novel APTSs are createdfrom PPG and green cholinium ILs. Here, PPG is used to replace

Fig. 5 Effect of IL nature on the trypsin activity at 25 °C: (a) buffersolution; (b) cholinium acetate; (c) cholinium glycollate; (d) choliniumpropionate; (e) cholinium butyrate; (f ) cholinium lactate; and (g) tri-cho-linium citrate. The concentration of the IL in the aqueous solution is1.237 mol L−1.

Fig. 6 The activity of trypsin in aqueous tri-cholinium citrate solutionswith different water contents at 25 °C.

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the inorganic salt. This polymer is thermo-sensitive and can berecovered conveniently by heating.

In order to investigate the recycling performance of PPG fromthe aqueous solutions, the cloud point curve of aqueousPPG400 has been determined experimentally and is illustrated inFig. 8. It can be seen that aqueous PPG solutions exhibit a lowercritical solution temperature at around 46 °C. When the temp-erature of an aqueous PPG solution is increased to the critical

value, the solution becomes highly turbid, and a phase separ-ation takes place from homogeneous to two liquid phases. Thisphenomenon is attributed to the reduction of hydrophilicity ofthe PO chain of PPG, caused by the rise in temperature.

In the cholinium IL + PPG400 ATPSs, the top phase is com-posed of the PPG400, water and small amount of IL, in whichthe mass fraction of PPG400 is about 65–85% and that of the ILis about 2–7%. In order to study the recycling performance ofthe polymer from the top phase, the cloud point temperatures ofthe aqueous PPG400 solutions have been determined in the pre-sence of 2 wt% of IL, the result is shown in Fig. 9. It can beseen that the addition of an IL decreases the cloud point

Table 3 Effect of temperature on the activity of trypsin at varied concentrations of the ILsa

System CIL(mol L−1)

Ur

15 °C 25 °C 35 °C 45 °C 55 °C 65 °C 75 °C 85 °C 95 °C

Water 80.2 100 127 124 17.3 — — — —Cholinium propionate 0.5 80.3 103 126 105 19.0 7.7 — — —

1.0 81.9 104 137 134 52.4 14.4 — — —1.5 83.1 108 139 151 65.9 22.9 3.7 — —2.0 84.2 110 140 154 97.7 36.9 19.5 — —2.5 84.4 110 139 155 107 49.8 21.3 — —

Cholinium lactate 0.5 82.8 108 136 135 45.4 19.0 9.5 — —1.0 83.3 108 137 153 78.9 32.7 19.3 15.1 —1.5 84.0 111 140 164 118 32.7 22.7 21.2 —2.0 81.6 108 140 162 142 54.1 24.0 22.2 —2.5 83.4 106 138 164 168 54.1 33.7 33.2 —

a— , inactivation of trypsin at that temperature.

Table 4 Effect of storage time on the activity of trypsin at 25 °C

System

Ur

1 d 1 month 2 month 3 month 5 month 12 month 13 month

Water 100 94.8 81.0 60.0 35.5 33.1 28.7Cholinium propionatea 100 124 125 110 82.2 136 129Cholinium lactatea 100 120 118 108 76.7 129 124

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) choliniumpropionate; (d) cholinium glycollate; (e) cholinium lactate; (f ) tri-choli-nium citrate; and (g) cholinium butyrate. 0.4 g of IL, 0.8 g of PPG400and 1.3 ml of H2O were added for the formation of each ATPS.

Fig. 8 The cloud point curve for aqueous PPG400 solutions.

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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 thepolymer, and the effect of the ILs on the cloud point temperatureof the aqueous PPG400 is in agreement with the order of theirphase-forming ability.

On the basis of the results mentioned above, the aqueous solu-tion of cholinium propionate (2 wt%) + PPG400 (70 wt%) wasselected as a model system of the PPG400-rich phase to investi-gate the recycling of PPG400 from the aqueous solution. It isevident from Fig. 8 and 9 that the addition of cholinium propio-nate (2 wt%) into the aqueous PPG400 solution reduces thecloud point temperature of the system significantly from 55 °Cto 26.6 °C. From Fig. 10, it can be seen that the system becomescloudy at 26.6 °C. Then, PPG400 is driven from the aqueoussolution of cholinium propionate + PPG400 as the temperature isincreased to 35 °C, giving rise to an improved concentration ofPPG400 in the polymer-rich phase (about 78 wt%). Furthermore,as the temperature goes up to 45 °C, more water is driven fromthe PPG-rich top phase to the IL-rich bottom phase, and the con-centration of PPG400 in the top phase increases to about90 wt%. In this case, almost all of the IL is driven into thebottom phase, and the PPG400 can be effectively recovered fromthe polymer-rich phase. Therefore, the polymer can be recoveredsimply by heating and can be reused in the next extractionprocess.

Conclusions

From the above discussion, the following conclusions can bedrawn: (i) both the cholinium ionic liquid and PPG400 are non-toxic and biodegradable, therefore the ATPSs developed in thepresent work are green ATPSs; (ii) the cholinium IL + PPG400ATPSs can be used to extract proteins efficiently, and the extrac-tion efficiency decreases with the increase of the size of proteinsand the tie line length of the ATPSs; (iii) in these IL ATPSs,thermo-sensitive PPG400 is used to replace the inorganic saltscommonly used in IL + inorganic salt ATPSs, thus the polymer

can be recovered conveniently by heating; (iv) the activity oftrypsin was increased to a different extent in the IL-rich phases,except for the cholinium acetate-rich phase, of the studiedATPSs, indicating that aqueous solutions of the cholinium-basedILs are a type of excellent long-term storage media for proteinsat least for trypsin.

To the best of our knowledge, this is the first study on thenon-toxic and biodegradable ATPSs and their application in theefficient and high activity extraction and purification of proteins.The work reported here may bring the IL-based ATPSs a greennature and open a new way to recycle the phase component inthe extraction–separation processes by using IL-based ATPSs.However, it should be emphasized that effective and economicaltechniques for the recovery and reuse of the ILs need to bedeveloped in future work.

Experimental section

Chemicals

Cholinium chloride (>98%), PPG400 (>99%), glycolic acid(>99%), ion exchange resin of Amberlite IRA 400-Cl and N-a-benzoyl-L-arginine ethyl ester (BAEE) were obtained from AlfaAesar. Sodium hydroxide, formic acid, acetic acid, propionicacid, butyric acid, lactic acid, benzoic acid, oxalic acid, citricacid, methanol and acetonitrile were purchased from ShanghaiChem. Co. and used without further purification. BSA, trypsin,papain and lysozyme were obtained from Sigma-Aldrich, andused without further purification. The stock solution of trypsin(4 g L−1) was prepared in water and stored at 4 °C. Doubly dis-tilled deionized water was used throughout the experiments.

Synthesis of the cholinium ILs. Nine types of cholinium ILs,including cholinium formate, cholinium acetate, cholinium pro-pionate, cholinium butyrate, cholinium glycollate, choliniumlactate, cholinium benzoate, di-cholinium oxalate and tri-choli-nium citrate, have been synthesized by the modification of theOhno method.43 First, cholinium hydroxides were prepared bypassing the aqueous cholinium chloride (5 wt%) through acolumn of anion exchange resin (Amberlite IRA 400-Cl). For

Fig. 9 The cloud point curves of aqueous PPG400 in the presence ofcholinium IL (2 wt%): ▲, cholinium lactate; ●, cholinium acetate; ■,cholinium propionate.

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)45 °C; 0.1 g of cholinium propionate, 3.5 g of PPG400 and 1.4 g ofH2O were added for the preparation of the mixture; the top phases of (c)and (d) were colored with malachite green.

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this purpose, a glass column was packed with the resin, followedby washing with plenty of water. The column was charged withan aqueous NaOH solution (2 M) to modify the resin beadswith OH− anions. Then the column was washed thoroughly withwater to remove any traces of unexchanged OH− anions (testedwith pH paper), and an aqueous solution of cholinium chloridewas passed through the column to exchange the Cl− anions withOH− anions. Upon complete substitution of chloride, no precipi-tation of AgCl can be found by addition of a few drops ofAgNO3 solution. Next, the as-prepared aqueous solutions of cho-linium hydroxide were neutralized with stoichiometric car-boxylic acids and the reaction mixture was stirred magneticallyfor 12 h at room temperature. The water in the systems was thenremoved by rotary evaporation, and the resulting products weredissolved in a mixed solvent of methanol–acetonitrile (1 : 9) andfiltered to remove any residual solid impurities. Finally, the sol-vents were removed again, and all the cholinium ILs were driedat 70 °C for 72 h under vacuum before use. The water content inthe ILs was determined by Karl–Fisher titration. It was foundthat less than 0.02 wt% of water still remained in the choliniumILs.

Characterization of the cholinium ILs. All the purified choli-nium ILs were analyzed by 1H NMR (Bruker, AV-400) toconfirm the absence of any major impurities, and the purity ofthese ILs is found to be greater than 99% in mass fraction. The1H NMR data of cholinium acetate, cholinium propionate, choli-nium butyrate, cholinium benzoate and cholinium glycollatewere in good agreement with those reported in literature.10,11

The 1H NMR data of all the ILs and the 13C NMR data of choli-nium lactate, di-cholinium oxalate and tri-cholinium citrate arereported as follows:

Cholinium formate: 1H NMR (400 MHz, [d6] DMSO, TMS):δ = 3.12 (s, 9H, –NCH3), 3.42 (t, 2H, –NCH2), 3.83 (m, 2H,–OCH2), 8.50 (s, 1H, –OOCH) ppm.

Cholinium acetate: 1H NMR (400 MHz, [d6] DMSO, TMS):δ = 1.58 (s, 3H, –OOCCH3), 3.13 (s, 9H, –NCH3), 3.43 (t, 2H,–NCH2), 3.82 (d, 2H, –OCH2) ppm.

Cholinium propionate: 1H NMR (400 MHz, [d6] DMSO,TMS): δ = 0.88 (t, 3H, –CH3), 1.85 (q, 2H, –CH2COO), 3.14 (s,9H, –NCH3), 3.44 (t, 2H, –NCH2), 3.84 (m, 2H, –OCH2) ppm.

Cholinium butyrate: 1H NMR (400 MHz, D2O): δ = 0.76 (t,3H, –CH3), 1.45 (m, 2H, –CH2CH2COO), 2.02 (t, 2H,–CH2COO), 3.05 (s, 9H, –NCH3), 3.38 (q, 2H, –NCH2), 3.93(m, 2H, –OCH2) ppm.

Cholinium glycollate: 1H NMR (400 MHz, [d6] DMSO,TMS): δ = 3.12 (s, 9H, –NCH3), 3.37 (s, 2H, –CH2(OH)COO),3.44 (t, 2H, –NCH2), 3.85 (m, 2H, –OCH2) ppm.

Cholinium benzoate: 1H NMR (400 MHz, [d6] DMSO, TMS):δ = 3.16 (s, 9H, –NCH3), 3.49 (t, 2H, –NCH2), 3.91 (d,2H,–OCH2), 7.28 (t, 3H, benzene ring H), 7.87 (d, 2H, benzene ringH) ppm.

Cholinium lactate: 1H NMR (400 MHz, [d6] DMSO, TMS):δ = 1.08 (d, 3H, –CH3), 3.12 (s, 10H, –NCH3, –OH), 3.44(t, 2H, –NCH2), 3.56 (m, 1H,–CH), 3.85 (m, 2H, –OCH2) ppm.

13C NMR (100 MHz, [d6] DMSO), δ = 21.9, 53.4, 55.4, 67.5,178.0 ppm.

Di-cholinium oxalate: 1H NMR (400 MHz, D2O): δ = 3.93(m, 4H, –OCH2), 3.38 (t, 4H, –NCH2), 3.05 (s, 18H, –NCH3)ppm.

13C NMR (100 MHz, D2O): δ = 53.9, 55.6, 67.5, 173.6 ppm.Tri-cholinium citrate: 1H NMR (400 MHz, D2O): δ =

2.41–2.53 (m, 4H, –CH2–), 3.06 (s, 27H, –NCH3), 3.37–3.39(m, 6H, –NCH2), 3.90–3.94 (m, 6H, –CH2O) ppm.

13C NMR (100 MHz, D2O): δ = 45.7, 53.9, 55.6, 67.5, 75.2,178.9, 181.8 ppm.

The melting temperature (Tm) and the glass transition temp-erature (Tg) for the cholinium ILs were measured using a Perkin-Elmer Diamond DSC equipped with an intercooler at a scan rateof 10 °C min−1. For all the samples, at least one cycle of heatingand cooling were performed before taking the final heating scan.The obtained results are given in Table 5, together with thosereported in literature.10,11

Measurements of the binodal curves and tie lines. Thebinodal curves were determined by the cloud-point method.18 Ina test tube, a polymer solution of known concentration wasadded, and an IL solution of known mass fraction was thenadded dropwise until the mixture became turbid or cloudy, thena known mass of water was added to make the mixture clearagain. This procedure was repeated to obtain sufficient data forthe construction of a liquid–liquid equilibrium binodal curve.The temperature of the systems was controlled to 25 ± 0.1 °C bya DC-2006 water thermostat (Shanghai Hengping InstrumentFactory, China). Concentration of the phase components wasdetermined by weight quantification of all the components addedwithin an uncertainty of ±10−4 g. The tie lines, which describethe concentrations of polymer and IL in the two phases, weremeasured with the procedure outlined in our previous work.44

Extraction of the proteins. A given amount of IL, PPG400and aqueous protein solution were added into a graduated glasstube. The volume of the glass tube was calibrated before use.The mixture was diluted by the addition of a given volume ofwater, and then shaken vigorously for 30 min to attain equili-brium by a desktop thermostated oscillator (Jinghong InstrumentFactory, Shanghai, China), and the temperature of the systemwas controlled at 25 ± 0.1 °C with a DC-2006 low temperaturethermostat. The phase separation quickly occurred after cessationof the shaking process. Then, a XYJ-802 centrifuge (JiangsuMedical Instrument Factory, Jiangsu, China) operated at4000 rpm was used to run for a period of 2 min in each test to

Table 5 Thermal properties of the cholinium-based ILsa

IL Tm/°C Tg/°C

Cholinium formate — —Cholinium acetate10 80 —Cholinium propionate10 — −74Cholinium butyrate10 45 —Cholinium glycollate11 38 −67Cholinium benzoate11 47 −51Cholinium lactate — −52Di-choline oxalate 99 —Tri-choline citrate 72 —

a—, not detected

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ensure complete phase separation. After the volume of the topand bottom phases was recorded, the sample was collected fromthe bottom IL-rich phase for analysis. A mass balance check wasmade between the initial mass of the protein and the amounts inthe lower and upper phases on the basis of equilibrium compo-sitions. The relative error in the mass balance is within 3%. Toavoid interference from the phase components, the samples werediluted and analyzed against the blanks containing the samephase components but without protein. The protein concentrationin both phases were determined by measuring the absorbance at280 nm using a Shanghai 752N UV-vis spectrophotometer.Partition coefficients (K) and extraction efficiencies (E%) of theproteins were calculated by using the equations:

K ¼ Cb=Ct ð2Þ

E% ¼ CbV b=C0V 0 � 100% ð3Þwhere Cb and Ct are the equilibrium concentrations of the parti-tioned proteins in the IL-rich phase and in the polymer-richphase, respectively. Vb is the volume of the IL-rich phase, C0 isthe initial concentration of the proteins in water, and V0 is thevolume of the aqueous phase added into the test tube before theseparation process.

Determination of trypsin activity in the aqueous IL solutions.Trypsin activity in aqueous IL solutions was measured with theBAEE method45 at pH 7.6 and 25 °C. The experimental appar-atus employed is essentially the same as that used previously byus.25 The spectrophotometrical measurements were carried out at253 nm with a Shanghai 752N UV-vis spectrophotometer usinga 1 cm cell. Aqueous solutions of the ILs were prepared at differ-ent concentrations by dissolving the pure ILs in water, and thentrypsin was added to the ILs solutions. 0.2 ml of trypsin solutionwas added to 3.0 ml of 0.5 mM BAEE solution containing100 mM Tris buffer and 10 mM CaCl2, followed by immediatemixing by inversion. The A253 nm was recorded every 0.5 min at25 °C for about 5 min. The measurements were performed in tri-plicate, and then an average was obtained. Trypsin activity wascalculated by the literature method.46 Here, the activity of theextracted trypsin or trypsin in aqueous IL solution was expressedby the relative activity (Ur) which is defined by the followingequation:

U r ¼ U e=U n � 100% ð4Þwhere Ue (U mg−1) is the activity of trypsin in the tested media(IL-rich phase and aqueous IL solutions), and Un (U mg−1) isthe activity of trypsin dissolved in water with the same trypsinconcentration.

Determination of the cloud point temperatures. The cloudpoint temperature (Tc) was determined by visual observation.47

A glass tube containing the sample solution was immersed in awater bath, the temperature of which was maintained using aDC-2006 water thermostat. The temperature was raised at a rateof approximately 0.5 °C min−1. As soon as the temperature washigher than the cloud point temperature, the sample was cooledbelow Tc, and then it was heated again to check the reproduci-bility of the measurements. The heating–cooling cycle was

repeated three times for a given sample, and good reproducibilitywas obtained for the Tc values.

Acknowledgements

This work was supported financially by the National NaturalScience Foundation of China (no. 21133009), the InnovationScientists and Technicians Troop Construction Projects of HenanProvince (no. 092101510300), and the foundation of ShanghaiKey Laboratory of Green Chemistry and Chemical Processes.

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