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Deep eutectic solvents as novel extraction media for protein partitioning† Qun Zeng, Yuzhi Wang,* Yanhua Huang, Xueqin Ding, Jing Chen and Kaijia Xu Four kinds of green deep eutectic solvent (DES) were synthesized, including choline chloride (ChCl)–urea, tetramethylammonium chloride (TMACl)–urea, tetrapropylammonium bromide (TPMBr)–urea and ChCl– methylurea. An aqueous two-phase system (ATPS) based ChCl–urea DES was studied for the first time for the extraction of bovine serum albumin (BSA). Single factor experiments proved that the extraction efficiency of BSA was influenced by the mass of the DES, concentration of K2HPO4 solution, separation time and extraction temperature. The optimum conditions were determined through an orthogonal experiment with the four factors described above. The results showed that under the optimum conditions, the average extraction efficiency could reach up to 99.94%, 99.72%, 100.05% and 100.05% (each measured three times). The relative standard deviations (RSD) of extraction efficiencies in precision, repeatability and stability experiments were 0.5533% (n ¼ 5), 0.8306% (n ¼ 5) and 0.9829% (n ¼ 5), respectively. UV-vis and FT-IR spectra confirmed that there were no chemical interactions between BSA and the DES in the extraction process, and the CD spectra proved that the conformation of BSA did not change after extraction. The conductivity, DLS and TEM were combined to investigate the microstructure of the top phase and the possible mechanism for the extraction. The results showed that
Received 3rd December 2013 Accepted 19th February 2014
hydrophobic interactions, hydrogen bonding interactions and the salting-out effect played important roles in the transfer process, and the aggregation and surrounding phenomenon were the main driving
DOI: 10.1039/c3an02235h
forces for the separation. All of these results proved that ionic liquid (IL)-based ATPSs could potentially be substituted with DES-based ATPSs to offer new possibilities in the extraction of proteins.
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1. Introduction In recent years, ionic liquids (ILs) have gained much attention in many elds such as separation technology,1,2 bio-catalysis,3 organic synthesis4,5 and electrochemistry6 because of their distinctive properties characterized by their low vapor pressure, non-ammability, excellent solubility and conductivity, wide electrochemical window and large liquid range. However, the synthesis process of ILs is complex and expensive and ILs are difficult to purify. In addition, pyridinium or imidazoliumbased ionic liquids are not completely “green”. Their toxicity is no less than traditional organic solvents, and they are sometimes even more toxic than some organic solvents.7,8 These shortcomings have limited their large-scale industrial applications and development. Therefore, nding a simple synthetic and greener alternative solvent has very important practical signicance. In 2003, Abbott rst found that a choline compound and urea could form a eutectic mixture, by hydrogen-bonding State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P.R. China. E-mail: [email protected]; Fax: +86-731-88821848; Tel: +86-731-88821903 † Electronic supplementary 10.1039/c3an02235h
information
(ESI)
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available.
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DOI:
interactions, which appeared as a liquid state at room temperature.9 This kind of mixture was dened as a deep eutectic solvent (DES), and its physical and chemical properties were systematically studied. It turned out that the DES had similar physical properties to ILs,10 so it was classied as a new class of IL or IL analogue.11,12 DESs are formed by a certain molar ratio of quaternary ammonium salts and hydrogen-bond donors (acid amides, carboxylic acids and polyhydric alcohols).13 For example, a molar ratio of 1/2 of choline chloride– urea14 deep eutectic solvent, can be expressed as HOCH2CH2N+(CH3)3Cl$2(NH2)2CO. Table 1 summarizes the quaternary ammonium salts and hydrogen-bond donors (HBDs) used as DESs in this work. As can be seen from this table, by adjusting the R group, X of the quaternary ammonium salt and HBD types, different kinds of DESs can be obtained. The DES is a new type of environmentally friendly solvent with excellent physical and chemical properties characterized by its non-toxicity, biodegradability, and atom utilization rate of 100% in the synthesis process. Compared with ILs, the synthetic process of DESs is relatively simple, and only needs a certain molar ratio of quaternary ammonium salts and hydrogen-bond donors mixed and stirred under heating until a homogeneous, colorless liquid is obtained without purication. In addition, because the synthetic materials of DESs are
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Analyst Table 1
Paper Quaternary ammonium salts and hydrogen-bond donors in deep eutectic solvents
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Quaternary ammonium salts (R1R2R3R4N+X)
Hydrogen-bond donors
R1
R2
R3
R4
X
Acid amides
Carboxylic acids
Polyatomic alcohols
C2H5 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3
C2H5 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 PhCH2 CH3 CH3
C2H5 CH3 CH3 CH3 CH3 PhCH2 C2H5 CH3 CH3 CH3 C2H4OH CH3 CH3
C2H5 C2H4OH C2H4OH C2H4OH C2H4OH C2H4OH C2H4OH PhCH2 C2H4OAc C2H4Cl C2H4OH C2H4F C2H4OH
Br Cl BF4 NO3 F Cl Cl Cl Cl Cl Cl Br I
Urea 1-Methyl urea 1,3-Dimethyl urea 1,1-Dimethyl urea Thiourea Acetamide Benzamide Tetramethylurea Trichloroacetamide Ethylene urea N,N0 -Allyl urea
Adipic acid Benzoic acid Citric acid Malonic acid Oxalic acid Phenylacetic acid Phenylpropionic acid Succinic acid Tricarballylic acid
Ethanediol 1,4-Butanediol Propanetriol
abundant and inexpensive, DESs are expected to be applied successfully in large-scale industrial production. Proteins are the material basis of life and are involved in every cell and all of the other important parts of the body. It is particularly necessary to prepare pure proteins. But due to poor stability, proteins are easily denatured under acidic, alkaline or heating conditions. Therefore, the separation and purication of proteins become a bottleneck in the eld of biotechnology. Traditional protein purication methods include ammonium sulfate precipitation, salting-out, electrophoresis, ion-exchange chromatography and affinity chromatography. These methods are not only time consuming and costly, but also make proteins lose activity. The ATPS15,16 has emerged in recent years as a clean alternative for the traditional organic–water solvent extraction system. ATPSs are formed when two polymers, one polymer and one salt, or two salts are mixed at appropriate concentrations or at a particular temperature.17 The two phases are mostly composed of water and non volatile components, thus eliminating volatile organic compounds. Recently, ATPS, which is a special liquid–liquid extraction system involving the transfer of the solute from one aqueous phase to another, has been used in biotechnological applications involving proteins,18–22 enzymes,23 nucleic acids,24,25 antibodies26 and antibiotics27,28 as a nondenaturing and benign separation media. In this paper, the synthesis of four kinds of green deep eutectic solvent (DES) are reported, including choline chloride (ChCl)–urea, tetramethylammonium chloride (TMACl)–urea, tetrapropylammonium bromide (TPMBr)–urea and ChCl– methylurea. The extraction of proteins by an ATPS based on the synthesized deep eutectic solvent is reported for the rst time (as shown in Scheme 1). Aer phase separation, the proteins had transferred into the DES-rich top phase. The concentrations of the proteins in the top phases were determined by measuring the absorbance at 278 nm for bovine serum albumin (BSA), ovalbumin (OVA), and trypsin (Try) using a UV2450 UV-vis spectrophotometer. In addition, FTIR, CD, DLS, TEM and the conductivity were used to study the mechanism of the extraction process. It was suggested that the aggregation and surrounding phenomenon play a signicant role in the
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separation of proteins. All of these results showed that DESs are novel potential extraction media for the extraction of proteins.
2.
Experimental
2.1. Chemicals Choline chloride (ChCl) (98.0–101.0%, Shanghai Source Biological Technology Co., Ltd.) was recrystallized from absolute ethanol, ltered, and dried under vacuum. Urea ($99%, Sinopharm Chemical Reagent Co., Ltd.), tetramethylammonium chloride (TMACl) (>99%, Shanghai Source Biological Technology Co., Ltd.), tetrapropylammonium bromide (TPABr) (98%, Aladdin Chemical Reagent Co., Ltd.) and methylurea (>98%, Adamas Reagent Co., Ltd.) were all used without further purication. The inorganic salt K2HPO4 was purchased from Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin (BSA) was purchased from Sinopharm Chemical Reagent Co., Ltd., and ovalbumin (OVA) and trypsin (Try) were purchased from Shanghai Source Biological Technology Co., Ltd. 2.2. Apparatus UV-vis spectra were recorded using a UV2450 spectrophotometer (SHIMADZU, Japan). FTIR spectra were recorded on a Spectrum One FTIR spectrometer (Perkin Elmer, U.S.). The CD spectra were measured with a MOS-500 Circular Dichroism Spectrometer (Bio-Logic, France). The DLS measurements were carried out using a Zetasizer Nano-ZS90 (Malvern Instruments, U.K.). Aer being dried, the samples were imaged using a JEOL JEM-3010 transmission electron microscope. 2.3. Synthesis and characterization of DESs In this work, four kinds of deep eutectic solvent were synthesized by stirring two eutectic mixtures at 80 C until a homogeneous, colorless liquid was formed. The investigated deep eutectic solvents (as shown in Table 2) were based on quaternary ammonium salts (ChCl, TMACl and TPABr) and hydrogenbond donors (HBDs) (urea and methylurea) with a certain molar ratio (1 : 2) of quaternary ammonium salt to HBD. As an
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Scheme 1
DES-based ATPS for the extraction of proteins.
example, the synthetic route of ChCl–urea DES is shown in Fig. 1. The structures of all of the synthetic DESs were conrmed by FT-IR, which is shown in ESI Fig. S1.† 2.4. Extraction and determination of proteins in both phases An appropriate mass (1.6 g) of the DES was put into a 10 mL centrifuge tube. Then, 2.0 mL K2HPO4 aqueous solution with a
Table 2
concentration of 0.60 g mL1 was added and 10 mg protein was mixed in. Aerwards, the mixture was shaken vigorously in a shaker for 10 min to ensure the transfer of the proteins into the DES-rich top phase. Aer extraction, the concentrations of the proteins in the top phases were determined by measuring the absorbance at 278 nm for BSA, OVA and Try using a UV2450 UVvis spectrophotometer. The data were measured three times to guarantee accuracy, and each standard curve was made at least
Structures of the quaternary ammonium salts and hydrogen-bond donors in the four investigated deep eutectic solvents Mole ratio of salt to HBD
Abbreviation
Choline chloride–urea
1:2
DES(a)
Tetramethylammonium chloride–urea
1:2
DES(b)
Tetrapropylammonium bromide–urera
1:2
DES(c)
Choline chloride– methylurea
1:2
DES(d)
DES
Quaternary ammonium salts
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Hydrogen-bond donors
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Fig. 1
Paper
Synthetic route of ChCl–urea deep eutectic solvent.
three times to reduce articial errors and guarantee good correlation coefficients. The standard curves were y ¼ 0.00291 + 0.61535x, R2 ¼ 0.99984 (BSA), y ¼ 0.01607 + 0.89953x, R2 ¼ 0.99971 (OVA), y ¼ 0.00154 + 0.13881x, R2 ¼ 0.99993 (Try). The linearity for analyzing BSA, OVA and Try was in the concentration range of 0.05–1.00 mg mL1, 0.10–1.00 mg mL1 and 0.05– 1.00 mg mL1 with the correlation coefficient 0.99984, 0.99971 and 0.99993, respectively. The extraction efficiency (E) was calculated using the following equation:29 E¼
Ct Vt Ct Vt þ Cb Vb
where Ct and Cb are concentrations of the proteins in the DESrich top phase and phosphate-rich bottom phase, respectively. Vt and Vb stand for the volume of the top phase and bottom phase, respectively. Vt and Vb were calculated by dividing the number of centimeters of top phase and bottom phase of 1 mL volume.
3.
Results and discussion
3.1. Phase behavior of DES-based ATPS In a system consisting of K2HPO4 aqueous solution (0.70 g mL1, 2.0 mL), the effect of the mass of the DES on the formation of an ATPS was studied at room temperature. The results are shown in Fig. 2a. A further increase in the mass of the DES guarantees an increase in the volume of the DES-rich top phase, and a decrease in the volume of the bottom phase. Moreover, the volume ratio of top phase to bottom phase linearly increases with a slope of 1.11. In order to investigate the effect of K2HPO4 on the formation of the two-phase system and its capability for phase separation, various concentrations of K2HPO4 (2.0 mL) were added at room temperature to the system with a certain amount of ChCl–urea DES (1.4 g). The results are shown in Fig. 2b. The experiments indicated that further increases in the concentration of K2HPO4 ensure a slight increase in the volume of the bottom phase, and a decrease in the volume of the top phase. Moreover, the volume ratio of top phase to bottom phase linearly decreases with a slope of 1.96. 3.2. Selection of DES for the extraction of proteins Four kinds of DES have been investigated for the extraction of three proteins (BSA, OVA and Try). The extraction efficiencies are shown in ESI Fig. S2.† It is obvious that different DESs have different abilities to extract various proteins. According to Fig. S2,† BSA was chosen as the protein for this research, and
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ChCl–urea DES was the best extraction solvent. We can also improve the extraction efficiency of OVA and Try by changing the type of DES, mass of DES, concentration of K2HPO4, temperature, separation time, pH value or the other conditions. However, in this study, we aimed for BSA partitioning. Maybe in future work, we can also apply this method to other proteins. 3.3. Single factor experiments 3.3.1. Effect of the mass of DES. Using BAS as an example, the effect of the mass of ChCl–urea DES on the protein distribution in the two phases was studied, and the results are illustrated in Fig. 3a. A ChCl–urea/K2HPO4 (0.7 g mL1, 2.0 mL) ATPS was used. 10 mg BSA was added. As shown in Fig. 3a, 85.88–99.91% of BSA was transferred into the DES-rich top phase. When the mass of the DES varied from 0.8 g to 1.4 g, the extraction efficiency increased rapidly. The reason is that, with a higher concentration of DES, the number of formed DES micelles gradually increased, so that more BSA could be aggregated by the DES. However, when the DES mass was increased to 1.6 g, the extraction efficiency did not obviously increase any more. So the phenomenon indicated that 1.4 g was the optimum mass of DES for the ATPS, which was adopted in subsequent work. 3.3.2. Effect of the concentration of K2HPO4. As an example, Fig. 3b shows the effect of the amount of K2HPO4 on BSA distribution. ChCl–urea DES (1.4 g)/K2HPO4 (2.0 mL) ATPS was used and 10 mg BSA was added. As indicated in this gure, the extraction efficiency changes with various concentrations of K2HPO4 over the range of 0.5–1.0 g mL1. It is clear that the extraction efficiency increases with an increasing concentration of K2HPO4 solution below 0.6 g mL1, but it decreases when the concentration of K2HPO4 is higher than 0.6 g mL1. The protein structure is dependent on the hydrophobic–hydrophilic balance. K2HPO4 is a strong salting-out salt and large amounts of K2HPO4 in a protein solution can increase hydrophobic interactions, leading to the reduction of the solubility of protein in water. The reason for the decrease in extraction efficiency when the K2HPO4 concentration was higher than 0.6 g mL1 might be because the molecular structure of the protein is also controlled by the water molecules at the protein surface. When the bottom phase is highly hydrophilic, hydrogen bonding interactions between the water molecules at the protein surface and the amino acid residue drive the protein to transfer into the DES-rich top phase. Therefore, it is likely that the hydrophobic interactions, salting-out effect and hydrogen bonding interactions act as the important driving force for the protein to transfer into the DES-rich top phase. Therefore, the optimum
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(a) Effect of the amount of the DES on the formation of an ATPS at 25 C. 2 mL K2HPO4 solution (0.70 g mL1) was added into the ATPS. (b) Effect of the amount of K2HPO4 on the formation of an ATPS at 25 C. 1.4 g DES was added into the ATPS. Fig. 2
salt concentration was determined as 0.6 g mL1 and was used in the following experiments. 3.3.3. Effect of the separation time. Separation time is an obvious inuencing factor on the extraction efficiency. The extraction of BSA was carried out over a time range of 2–30 min, and the time dependence of the extraction efficiency is illustrated in Fig. 3c. ChCl–urea (1.4 g)/K2HPO4 (0.6 g mL1, 2.0 mL) ATPS was adopted and 10 mg BSA was added. It is shown that within 8 minutes, the amount of BSA transferred into the DESbased top phase grows linearly with an increase in separation time. When the separation time was increased to 8 min, almost
99.49% of BSA was extracted into the top phase, and the extraction efficiency was no longer increased with increasing separation time. Therefore, the DES-based ATPS in this work has a high-efficiency and saves time. 3.3.4. Effect of the temperature. As an example, the extraction efficiency of BSA was studied over a temperature range of 10–50 C, and the results are presented in Fig. 3d. ChCl–urea (1.4 g)/K2HPO4 (0.6 g mL1, 2.0 mL) ATPS was used and 10 mg BSA was added. It is obvious that at temperatures below 40 C, the BSA that was transferred into the DES-based top phase remained virtually unchanged, keeping a high
Fig. 3 Effect of mass of DES (a), concentration of K2HPO4 (b), separation time (c), and temperature (d) on the extraction efficiency in the DES/ K2HPO4 ATPS.
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extraction efficiency. When the temperature was kept at 50 C or higher, BSA was denaturated because of heat treatment. Therefore, the temperature was controlled to not exceed 40 C to ensure that the protein remained unchanged in the separation process. The extraction efficiency maintained a slow growth when the temperature was increased slowly. A possible reason for this phenomenon is that the hydrophobic interactions are enhanced with increasing temperature, but when the temperature is too high the hydrogen bonding interactions between the water molecules at the protein surface and the amino acid residue are destroyed, the formation of hydrogen bonds are less inuenced by hydrophobic interactions so the hydrophobic effect is weakened. It is worth mentioning that incremental increases in temperature facilitated protein extraction but this was not obvious, so room temperature makes the separation process more convenient.
Table 4. Apparatus precision was investigated by measuring the top phase solution ve times, in parallel, under the same conditions. The RSD of extraction efficiency is 0.55% which indicates that the precision of the UV-vis spectrometer is excellent (taking into account ve copies of the same sample measured under the same conditions). The value of RSD is 0.83%, indicating that the detection method has good repeatability. The stability experiment was performed by taking a sample detected continuously over ve days under the same conditions. The results show the RSD of extraction efficiency is 0.98%, proving that the sample is recoverable within ve days.
Table 4 The results of the precision, repeatability and stability experiments
Precision measurement results (n ¼ 5)
3.4. Orthogonal experiment The optimized conditions were determined through an orthogonal experiment using the mass of DES (factor A), concentration of K2HPO4 (factor B), temperature (factor C) and separation time (factor D). The results of the orthogonal experiment on the four factors and three levels are shown in Table 3. K1, K2 and K3 are the average extraction efficiencies of each factor in each of the levels. R is the difference value between the highest and the lowest values of K. The bigger the difference of R, the more inuence the factor has on the extraction efficiency. Table 3 shows that the order of importance of each factor is ADBC and the optimized conditions are A3B1C2D3, when the average extraction efficiency reaches up to 99.94%, 99.72%, 100.05% and 100.05% (each measured three times). 3.5. Methodological study
Repeats
1
2
3
4
5
Absorbance Extraction efficiency (%) RSD (%)
0.326 99.78 0.55
0.324 99.17
0.327 100.08
0.323 98.86
0.327 100.08
Repeatability measurement results (n ¼ 5) Sample number
1
2
3
4
5
Absorbance Extraction efficiency (%) RSD (%)
0.326 99.78 0.83
0.321 98.26
0.324 99.17
0.319 97.65
0.322 98.56
Stability measurement results (n ¼ 5) Day number
1
2
3
4
5
Absorbance Extraction efficiency (%) RSD (%)
0.326 99.78 0.98
0.319 97.65
0.322 98.56
0.327 100.08
0.324 99.17
The method used in this study was validated by precision, repeatability and stability experiments. The results are shown in Table 3
Results of orthogonal experiment L9 (34)
Experiment
A DES (g)
B K2HPO4 (g mL1)
C T ( C)
D t (min)
E (%)
1 2 3 4 5 6 7 8 9 K1
1(1.2) 1 1 2(1.4) 2 2 3(1.6) 3 3 94.07
1(0.6) 2(0.7) 3(0.8) 1 2 3 1 2 3 96.68
1(20) 2(25) 3(30) 2 3 1 3 1 2 95.35
1(4) 2(8) 3(10) 3 1 2 2 3 1 93.69
93.34 95.08 93.80 97.05 90.95 93.38 99.64 99.32 96.77 Major and minor order: A > D > B > C optimized conditions: A3B1C2D3
K2 K3 R Optimal level
93.79 98.58 4.78 A3
95.12 94.65 2.03 B1
96.30 94.80 1.50 C2
96.03 96.72 3.04 D3
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3.6. Extraction mechanism
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3.6.1. UV-vis spectroscopy. In order to examine the protein conformation before and aer extraction, UV-vis spectra were investigated for BSA. ESI Fig. S3† shows the UV-vis spectra of BSA in pure water and in the DES-rich top phase aer
extraction. It is clear that the maximum absorption peak of BSA in the DES-rich top phase still existed at 278 nm, and the conformations of BSA before and aer extraction were similar, suggesting that there are no chemical bonds between BSA molecules and DESs.
Fig. 4
The size distribution of the DES aggregates in aqueous solution at different DES concentrations. (a) 0.20; (b) 0.30; (c) 0.40; (d) 0.50 g mL1.
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3.6.2. The microscopic structure of the DES-rich top phase. Examination of the microscopic structure of the DES-rich top phase furthers our understanding of the separation process. As shown in Fig. 4, the DES solution is not microscopically homogeneous, but polydisperse. The average sizes are 574, 619.2, 687.5 and 968.6 nm, when the concentrations are 0.20, 0.30, 0.40, 0.50 g mL1, respectively. The size of the DES aggregates increases with an increasing concentration of DES. Then, TEM images of the DES (0.5 g mL1), BSA (5.0 mg mL1) and a mixture (0.5 g mL1 DES + 5.0 mg mL1 BSA) were taken as shown in Fig. 5. Fig. 5a shows the conformation of DES aggregates, Fig. 5b shows the appearance of BSA aggregates, and Fig. 5c showed the distribution of the mixture aggregates (scale bars 0.2 mm, 0.2 mm and 0.5 mm, respectively). From Fig. 5c, it can be seen that the DES aggregates encircle the BSA aggregate, which is the main driving force in the uptake of protein by the DES-based ATPS.
4. Conclusions This is the rst report for the extraction of proteins with a deep eutectic solvent-based ATPS. The greatest benet of the proposed method is that the adapted extraction solvent is green and environmentally friendly. In comparison with ionic liquids, deep eutectic solvents display more superiority as they are nontoxic, biodegradable, and have an atom utilization rate of 100% in the synthesis process. By adjusting the types of quaternary ammonium salts and HBDs, different kinds of DESs can be obtained. In addition, since the synthetic materials of DESs are abundant and inexpensive, DESs are expected to be applied successfully in large-scale industrial production. As a result, the present method uses innovative extraction media (DESs) that can replace traditional ILs, and demonstrates higher extraction efficiencies in DES-based ATPSs than in ILATPSs. Besides the single factor experiments, the orthogonal experiment was performed to acquire the optimum conditions for the extraction of proteins with this DES-based ATPS. These data fully illustrated the choices of factors used in this system. It is worth mentioning that the aggregation and surrounding phenomenon plays an important role in the extraction process. Along with the other driving forces involved in the partitioning of the protein between the DES-rich phase and phosphate phase, the aggregation and surrounding phenomenon could be applied to a variety of different samples and exhibit potential value. But much to our regret, it is hard to back-extract the targeted protein free from the DES in this work. We chose a low concentration of SDS aqueous solution as a stripping reagent but we failed because DES is hydrophilic and so we were unable to separate the DES phase from the aqueous solution. Maybe it will be the direction of our future research. Fig. 5 TEM images of the aggregates for DES (0.5 g mL1), BSA (5.0 mg mL1) and DES (0.5 g mL1) + BSA (5.0 mg mL1). (a) DES; (b) BSA; (c) DES + BSA.
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Acknowledgements The authors greatly appreciate the nancial support of the National Natural Science Foundation of China (no. 21175040;
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no. 21375035; no. J1210040) and the Foundation for Innovative Research Groups of NSFC (Grant 21221003).
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References 1 G. T. Wei, Z. S. Yang and C. J. Chen, Anal. Chim. Acta, 2003, 488, 183–188. 2 M. Matsumoto, K. Mochiduki, K. Fukunishi and K. Kondo, Sep. Purif. Technol., 2004, 40, 97–103. 3 S. Shah and M. N. Gupta, Bioorg. Med. Chem. Lett., 2007, 17, 921–928. 4 C. E. Song and E. J. Roh, Chem. Commun., 2000, 837–842. 5 F. Favre, H. Olivier-Bourbigou, D. Commereuc and L. Saussine, Chem. Commun., 2001, 1360–1363. 6 D. R. MacFarlane, M. Forsyth, P. C. Howlett, J. M. Pringle, J. Sun, G. Annat, W. Neil and E. I. Izgorodina, Acc. Chem. Res., 2007, 40, 1165–1173. 7 D. J. Couling, R. J. Bernot, K. M. Docherty, et al., Green Chem., 2006, 8, 82–90. 8 A. S. Wells and V. T. Coombe, Org. Process Res. Dev., 2006, 10, 794–798. 9 A. P. Abbott, G. Capper and D. L. Davies, Chem. Commun., 2003, 70–71. 10 A. P. Abbott, D. Boothby, G. Capper, D. L. Davies and R. K. Rasheed, J. Am. Chem. Soc., 2004, 126, 9142–9147. 11 A. P. Abbott and K. J. McKenzie, Phys. Chem. Chem. Phys., 2006, 8, 4265–4279. 12 K. Haerens, E. Matthijs and K. Binnemansc, Green Chem., 2009, 11, 1357–1365. 13 L. Wei and Y. Fan, Chemistry, 2011, 74, 333–338. 14 A. P. Abbott, D. Boothby, G. Capper, D. L. Davies and R. K. Rasheed, J. Am. Chem. Soc., 2004, 126, 9142–9147.
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15 A. M. Azevedo, P. A. J. Rosa and I. F. Ferreira, Sep. Purif. Technol., 2009, 65, 31–33. 16 J. S. Becker, O. R. T. Thomas and M. Franzreb, Sep. Purif. Technol., 2009, 65, 46–49. 17 L. Li, F. Liu and X. X. Kong, Anal. Chim. Acta, 2002, 452, 321– 327. 18 D. H. Chen, X. W. Chen, Y. Shu and J. H. Wang, Talanta, 2008, 75, 1270–1278. 19 Z. Du, Y.-L. Yu and J.-H. Wang, Chem.–Eur. J., 2007, 13, 2130– 2137. 20 Z. Li, X. Liu, Y. Pei, J. Wang and M. He, Green Chem., 2012, 14, 2941–2950. 21 Y. Pei, J. Wanga, K. Wua, X. Xuana and X. Lu, Sep. Purif. Technol., 2009, 64, 288–295. 22 P. YuanChao, L. ZhiYong, L. Li, W. JianJi and W. HuiYong, Sci. China: Chem., 2010, 53, 1554–1560. 23 C. K. Su and B. H. Chiang, Process Biochem., 2006, 41, 257– 261. 24 G. A. Gomes, A. M. Azevedo, M. R. Aires-Barros and D. M. F. Prazeres, Sep. Purif. Technol., 2009, 65, 22–27. 25 J. H. Wang, D. H. Cheng, X. W. Chen, Z. Du and Z. L. Fang, Anal. Chem., 2007, 79, 620–624. 26 A. M. Azevedo, A. G. Gomes, P. A. J. Rosa, I. F. Ferreira, A. M. M. M. O. Pisco and M. R. Aires-Barros, Sep. Purif. Technol., 2009, 65, 14–18. 27 B. Mokhtarani, R. Karimzadeh, M. Amini and S. D. Manesh, Biochem. Eng. J., 2008, 38, 241–248. 28 M. M. Bora, S. Borthakur, P. C. Rao and N. N. Dutta, Sep. Purif. Technol., 2005, 45, 153–156. 29 S. Saravanan, J. R. Rao, T. Murugesan, B. U. Nair and T. Ramasami, Chem. Eng. Sci., 2007, 62, 969–978.
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Deep eutectic solvents as novel extraction media for protein partitioning† Qun Zeng, Yuzhi Wang,* Yanhua Huang, Xueqin Ding, Jing Chen and Kaijia Xu Four kinds of green deep eutectic solvent (DES) were synthesized, including choline chloride (ChCl)–urea, tetramethylammonium chloride (TMACl)–urea, tetrapropylammonium bromide (TPMBr)–urea and ChCl– methylurea. An aqueous two-phase system (ATPS) based ChCl–urea DES was studied for the first time for the extraction of bovine serum albumin (BSA). Single factor experiments proved that the extraction efficiency of BSA was influenced by the mass of the DES, concentration of K2HPO4 solution, separation time and extraction temperature. The optimum conditions were determined through an orthogonal experiment with the four factors described above. The results showed that under the optimum conditions, the average extraction efficiency could reach up to 99.94%, 99.72%, 100.05% and 100.05% (each measured three times). The relative standard deviations (RSD) of extraction efficiencies in precision, repeatability and stability experiments were 0.5533% (n ¼ 5), 0.8306% (n ¼ 5) and 0.9829% (n ¼ 5), respectively. UV-vis and FT-IR spectra confirmed that there were no chemical interactions between BSA and the DES in the extraction process, and the CD spectra proved that the conformation of BSA did not change after extraction. The conductivity, DLS and TEM were combined to investigate the microstructure of the top phase and the possible mechanism for the extraction. The results showed that
Received 3rd December 2013 Accepted 19th February 2014
hydrophobic interactions, hydrogen bonding interactions and the salting-out effect played important roles in the transfer process, and the aggregation and surrounding phenomenon were the main driving
DOI: 10.1039/c3an02235h
forces for the separation. All of these results proved that ionic liquid (IL)-based ATPSs could potentially be substituted with DES-based ATPSs to offer new possibilities in the extraction of proteins.
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1. Introduction In recent years, ionic liquids (ILs) have gained much attention in many elds such as separation technology,1,2 bio-catalysis,3 organic synthesis4,5 and electrochemistry6 because of their distinctive properties characterized by their low vapor pressure, non-ammability, excellent solubility and conductivity, wide electrochemical window and large liquid range. However, the synthesis process of ILs is complex and expensive and ILs are difficult to purify. In addition, pyridinium or imidazoliumbased ionic liquids are not completely “green”. Their toxicity is no less than traditional organic solvents, and they are sometimes even more toxic than some organic solvents.7,8 These shortcomings have limited their large-scale industrial applications and development. Therefore, nding a simple synthetic and greener alternative solvent has very important practical signicance. In 2003, Abbott rst found that a choline compound and urea could form a eutectic mixture, by hydrogen-bonding State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P.R. China. E-mail: [email protected]; Fax: +86-731-88821848; Tel: +86-731-88821903 † Electronic supplementary 10.1039/c3an02235h
information
(ESI)
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available.
See
DOI:
interactions, which appeared as a liquid state at room temperature.9 This kind of mixture was dened as a deep eutectic solvent (DES), and its physical and chemical properties were systematically studied. It turned out that the DES had similar physical properties to ILs,10 so it was classied as a new class of IL or IL analogue.11,12 DESs are formed by a certain molar ratio of quaternary ammonium salts and hydrogen-bond donors (acid amides, carboxylic acids and polyhydric alcohols).13 For example, a molar ratio of 1/2 of choline chloride– urea14 deep eutectic solvent, can be expressed as HOCH2CH2N+(CH3)3Cl$2(NH2)2CO. Table 1 summarizes the quaternary ammonium salts and hydrogen-bond donors (HBDs) used as DESs in this work. As can be seen from this table, by adjusting the R group, X of the quaternary ammonium salt and HBD types, different kinds of DESs can be obtained. The DES is a new type of environmentally friendly solvent with excellent physical and chemical properties characterized by its non-toxicity, biodegradability, and atom utilization rate of 100% in the synthesis process. Compared with ILs, the synthetic process of DESs is relatively simple, and only needs a certain molar ratio of quaternary ammonium salts and hydrogen-bond donors mixed and stirred under heating until a homogeneous, colorless liquid is obtained without purication. In addition, because the synthetic materials of DESs are
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Analyst Table 1
Paper Quaternary ammonium salts and hydrogen-bond donors in deep eutectic solvents
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Quaternary ammonium salts (R1R2R3R4N+X)
Hydrogen-bond donors
R1
R2
R3
R4
X
Acid amides
Carboxylic acids
Polyatomic alcohols
C2H5 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3
C2H5 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 PhCH2 CH3 CH3
C2H5 CH3 CH3 CH3 CH3 PhCH2 C2H5 CH3 CH3 CH3 C2H4OH CH3 CH3
C2H5 C2H4OH C2H4OH C2H4OH C2H4OH C2H4OH C2H4OH PhCH2 C2H4OAc C2H4Cl C2H4OH C2H4F C2H4OH
Br Cl BF4 NO3 F Cl Cl Cl Cl Cl Cl Br I
Urea 1-Methyl urea 1,3-Dimethyl urea 1,1-Dimethyl urea Thiourea Acetamide Benzamide Tetramethylurea Trichloroacetamide Ethylene urea N,N0 -Allyl urea
Adipic acid Benzoic acid Citric acid Malonic acid Oxalic acid Phenylacetic acid Phenylpropionic acid Succinic acid Tricarballylic acid
Ethanediol 1,4-Butanediol Propanetriol
abundant and inexpensive, DESs are expected to be applied successfully in large-scale industrial production. Proteins are the material basis of life and are involved in every cell and all of the other important parts of the body. It is particularly necessary to prepare pure proteins. But due to poor stability, proteins are easily denatured under acidic, alkaline or heating conditions. Therefore, the separation and purication of proteins become a bottleneck in the eld of biotechnology. Traditional protein purication methods include ammonium sulfate precipitation, salting-out, electrophoresis, ion-exchange chromatography and affinity chromatography. These methods are not only time consuming and costly, but also make proteins lose activity. The ATPS15,16 has emerged in recent years as a clean alternative for the traditional organic–water solvent extraction system. ATPSs are formed when two polymers, one polymer and one salt, or two salts are mixed at appropriate concentrations or at a particular temperature.17 The two phases are mostly composed of water and non volatile components, thus eliminating volatile organic compounds. Recently, ATPS, which is a special liquid–liquid extraction system involving the transfer of the solute from one aqueous phase to another, has been used in biotechnological applications involving proteins,18–22 enzymes,23 nucleic acids,24,25 antibodies26 and antibiotics27,28 as a nondenaturing and benign separation media. In this paper, the synthesis of four kinds of green deep eutectic solvent (DES) are reported, including choline chloride (ChCl)–urea, tetramethylammonium chloride (TMACl)–urea, tetrapropylammonium bromide (TPMBr)–urea and ChCl– methylurea. The extraction of proteins by an ATPS based on the synthesized deep eutectic solvent is reported for the rst time (as shown in Scheme 1). Aer phase separation, the proteins had transferred into the DES-rich top phase. The concentrations of the proteins in the top phases were determined by measuring the absorbance at 278 nm for bovine serum albumin (BSA), ovalbumin (OVA), and trypsin (Try) using a UV2450 UV-vis spectrophotometer. In addition, FTIR, CD, DLS, TEM and the conductivity were used to study the mechanism of the extraction process. It was suggested that the aggregation and surrounding phenomenon play a signicant role in the
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separation of proteins. All of these results showed that DESs are novel potential extraction media for the extraction of proteins.
2.
Experimental
2.1. Chemicals Choline chloride (ChCl) (98.0–101.0%, Shanghai Source Biological Technology Co., Ltd.) was recrystallized from absolute ethanol, ltered, and dried under vacuum. Urea ($99%, Sinopharm Chemical Reagent Co., Ltd.), tetramethylammonium chloride (TMACl) (>99%, Shanghai Source Biological Technology Co., Ltd.), tetrapropylammonium bromide (TPABr) (98%, Aladdin Chemical Reagent Co., Ltd.) and methylurea (>98%, Adamas Reagent Co., Ltd.) were all used without further purication. The inorganic salt K2HPO4 was purchased from Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin (BSA) was purchased from Sinopharm Chemical Reagent Co., Ltd., and ovalbumin (OVA) and trypsin (Try) were purchased from Shanghai Source Biological Technology Co., Ltd. 2.2. Apparatus UV-vis spectra were recorded using a UV2450 spectrophotometer (SHIMADZU, Japan). FTIR spectra were recorded on a Spectrum One FTIR spectrometer (Perkin Elmer, U.S.). The CD spectra were measured with a MOS-500 Circular Dichroism Spectrometer (Bio-Logic, France). The DLS measurements were carried out using a Zetasizer Nano-ZS90 (Malvern Instruments, U.K.). Aer being dried, the samples were imaged using a JEOL JEM-3010 transmission electron microscope. 2.3. Synthesis and characterization of DESs In this work, four kinds of deep eutectic solvent were synthesized by stirring two eutectic mixtures at 80 C until a homogeneous, colorless liquid was formed. The investigated deep eutectic solvents (as shown in Table 2) were based on quaternary ammonium salts (ChCl, TMACl and TPABr) and hydrogenbond donors (HBDs) (urea and methylurea) with a certain molar ratio (1 : 2) of quaternary ammonium salt to HBD. As an
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Scheme 1
DES-based ATPS for the extraction of proteins.
example, the synthetic route of ChCl–urea DES is shown in Fig. 1. The structures of all of the synthetic DESs were conrmed by FT-IR, which is shown in ESI Fig. S1.† 2.4. Extraction and determination of proteins in both phases An appropriate mass (1.6 g) of the DES was put into a 10 mL centrifuge tube. Then, 2.0 mL K2HPO4 aqueous solution with a
Table 2
concentration of 0.60 g mL1 was added and 10 mg protein was mixed in. Aerwards, the mixture was shaken vigorously in a shaker for 10 min to ensure the transfer of the proteins into the DES-rich top phase. Aer extraction, the concentrations of the proteins in the top phases were determined by measuring the absorbance at 278 nm for BSA, OVA and Try using a UV2450 UVvis spectrophotometer. The data were measured three times to guarantee accuracy, and each standard curve was made at least
Structures of the quaternary ammonium salts and hydrogen-bond donors in the four investigated deep eutectic solvents Mole ratio of salt to HBD
Abbreviation
Choline chloride–urea
1:2
DES(a)
Tetramethylammonium chloride–urea
1:2
DES(b)
Tetrapropylammonium bromide–urera
1:2
DES(c)
Choline chloride– methylurea
1:2
DES(d)
DES
Quaternary ammonium salts
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Hydrogen-bond donors
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Fig. 1
Paper
Synthetic route of ChCl–urea deep eutectic solvent.
three times to reduce articial errors and guarantee good correlation coefficients. The standard curves were y ¼ 0.00291 + 0.61535x, R2 ¼ 0.99984 (BSA), y ¼ 0.01607 + 0.89953x, R2 ¼ 0.99971 (OVA), y ¼ 0.00154 + 0.13881x, R2 ¼ 0.99993 (Try). The linearity for analyzing BSA, OVA and Try was in the concentration range of 0.05–1.00 mg mL1, 0.10–1.00 mg mL1 and 0.05– 1.00 mg mL1 with the correlation coefficient 0.99984, 0.99971 and 0.99993, respectively. The extraction efficiency (E) was calculated using the following equation:29 E¼
Ct Vt Ct Vt þ Cb Vb
where Ct and Cb are concentrations of the proteins in the DESrich top phase and phosphate-rich bottom phase, respectively. Vt and Vb stand for the volume of the top phase and bottom phase, respectively. Vt and Vb were calculated by dividing the number of centimeters of top phase and bottom phase of 1 mL volume.
3.
Results and discussion
3.1. Phase behavior of DES-based ATPS In a system consisting of K2HPO4 aqueous solution (0.70 g mL1, 2.0 mL), the effect of the mass of the DES on the formation of an ATPS was studied at room temperature. The results are shown in Fig. 2a. A further increase in the mass of the DES guarantees an increase in the volume of the DES-rich top phase, and a decrease in the volume of the bottom phase. Moreover, the volume ratio of top phase to bottom phase linearly increases with a slope of 1.11. In order to investigate the effect of K2HPO4 on the formation of the two-phase system and its capability for phase separation, various concentrations of K2HPO4 (2.0 mL) were added at room temperature to the system with a certain amount of ChCl–urea DES (1.4 g). The results are shown in Fig. 2b. The experiments indicated that further increases in the concentration of K2HPO4 ensure a slight increase in the volume of the bottom phase, and a decrease in the volume of the top phase. Moreover, the volume ratio of top phase to bottom phase linearly decreases with a slope of 1.96. 3.2. Selection of DES for the extraction of proteins Four kinds of DES have been investigated for the extraction of three proteins (BSA, OVA and Try). The extraction efficiencies are shown in ESI Fig. S2.† It is obvious that different DESs have different abilities to extract various proteins. According to Fig. S2,† BSA was chosen as the protein for this research, and
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ChCl–urea DES was the best extraction solvent. We can also improve the extraction efficiency of OVA and Try by changing the type of DES, mass of DES, concentration of K2HPO4, temperature, separation time, pH value or the other conditions. However, in this study, we aimed for BSA partitioning. Maybe in future work, we can also apply this method to other proteins. 3.3. Single factor experiments 3.3.1. Effect of the mass of DES. Using BAS as an example, the effect of the mass of ChCl–urea DES on the protein distribution in the two phases was studied, and the results are illustrated in Fig. 3a. A ChCl–urea/K2HPO4 (0.7 g mL1, 2.0 mL) ATPS was used. 10 mg BSA was added. As shown in Fig. 3a, 85.88–99.91% of BSA was transferred into the DES-rich top phase. When the mass of the DES varied from 0.8 g to 1.4 g, the extraction efficiency increased rapidly. The reason is that, with a higher concentration of DES, the number of formed DES micelles gradually increased, so that more BSA could be aggregated by the DES. However, when the DES mass was increased to 1.6 g, the extraction efficiency did not obviously increase any more. So the phenomenon indicated that 1.4 g was the optimum mass of DES for the ATPS, which was adopted in subsequent work. 3.3.2. Effect of the concentration of K2HPO4. As an example, Fig. 3b shows the effect of the amount of K2HPO4 on BSA distribution. ChCl–urea DES (1.4 g)/K2HPO4 (2.0 mL) ATPS was used and 10 mg BSA was added. As indicated in this gure, the extraction efficiency changes with various concentrations of K2HPO4 over the range of 0.5–1.0 g mL1. It is clear that the extraction efficiency increases with an increasing concentration of K2HPO4 solution below 0.6 g mL1, but it decreases when the concentration of K2HPO4 is higher than 0.6 g mL1. The protein structure is dependent on the hydrophobic–hydrophilic balance. K2HPO4 is a strong salting-out salt and large amounts of K2HPO4 in a protein solution can increase hydrophobic interactions, leading to the reduction of the solubility of protein in water. The reason for the decrease in extraction efficiency when the K2HPO4 concentration was higher than 0.6 g mL1 might be because the molecular structure of the protein is also controlled by the water molecules at the protein surface. When the bottom phase is highly hydrophilic, hydrogen bonding interactions between the water molecules at the protein surface and the amino acid residue drive the protein to transfer into the DES-rich top phase. Therefore, it is likely that the hydrophobic interactions, salting-out effect and hydrogen bonding interactions act as the important driving force for the protein to transfer into the DES-rich top phase. Therefore, the optimum
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(a) Effect of the amount of the DES on the formation of an ATPS at 25 C. 2 mL K2HPO4 solution (0.70 g mL1) was added into the ATPS. (b) Effect of the amount of K2HPO4 on the formation of an ATPS at 25 C. 1.4 g DES was added into the ATPS. Fig. 2
salt concentration was determined as 0.6 g mL1 and was used in the following experiments. 3.3.3. Effect of the separation time. Separation time is an obvious inuencing factor on the extraction efficiency. The extraction of BSA was carried out over a time range of 2–30 min, and the time dependence of the extraction efficiency is illustrated in Fig. 3c. ChCl–urea (1.4 g)/K2HPO4 (0.6 g mL1, 2.0 mL) ATPS was adopted and 10 mg BSA was added. It is shown that within 8 minutes, the amount of BSA transferred into the DESbased top phase grows linearly with an increase in separation time. When the separation time was increased to 8 min, almost
99.49% of BSA was extracted into the top phase, and the extraction efficiency was no longer increased with increasing separation time. Therefore, the DES-based ATPS in this work has a high-efficiency and saves time. 3.3.4. Effect of the temperature. As an example, the extraction efficiency of BSA was studied over a temperature range of 10–50 C, and the results are presented in Fig. 3d. ChCl–urea (1.4 g)/K2HPO4 (0.6 g mL1, 2.0 mL) ATPS was used and 10 mg BSA was added. It is obvious that at temperatures below 40 C, the BSA that was transferred into the DES-based top phase remained virtually unchanged, keeping a high
Fig. 3 Effect of mass of DES (a), concentration of K2HPO4 (b), separation time (c), and temperature (d) on the extraction efficiency in the DES/ K2HPO4 ATPS.
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extraction efficiency. When the temperature was kept at 50 C or higher, BSA was denaturated because of heat treatment. Therefore, the temperature was controlled to not exceed 40 C to ensure that the protein remained unchanged in the separation process. The extraction efficiency maintained a slow growth when the temperature was increased slowly. A possible reason for this phenomenon is that the hydrophobic interactions are enhanced with increasing temperature, but when the temperature is too high the hydrogen bonding interactions between the water molecules at the protein surface and the amino acid residue are destroyed, the formation of hydrogen bonds are less inuenced by hydrophobic interactions so the hydrophobic effect is weakened. It is worth mentioning that incremental increases in temperature facilitated protein extraction but this was not obvious, so room temperature makes the separation process more convenient.
Table 4. Apparatus precision was investigated by measuring the top phase solution ve times, in parallel, under the same conditions. The RSD of extraction efficiency is 0.55% which indicates that the precision of the UV-vis spectrometer is excellent (taking into account ve copies of the same sample measured under the same conditions). The value of RSD is 0.83%, indicating that the detection method has good repeatability. The stability experiment was performed by taking a sample detected continuously over ve days under the same conditions. The results show the RSD of extraction efficiency is 0.98%, proving that the sample is recoverable within ve days.
Table 4 The results of the precision, repeatability and stability experiments
Precision measurement results (n ¼ 5)
3.4. Orthogonal experiment The optimized conditions were determined through an orthogonal experiment using the mass of DES (factor A), concentration of K2HPO4 (factor B), temperature (factor C) and separation time (factor D). The results of the orthogonal experiment on the four factors and three levels are shown in Table 3. K1, K2 and K3 are the average extraction efficiencies of each factor in each of the levels. R is the difference value between the highest and the lowest values of K. The bigger the difference of R, the more inuence the factor has on the extraction efficiency. Table 3 shows that the order of importance of each factor is ADBC and the optimized conditions are A3B1C2D3, when the average extraction efficiency reaches up to 99.94%, 99.72%, 100.05% and 100.05% (each measured three times). 3.5. Methodological study
Repeats
1
2
3
4
5
Absorbance Extraction efficiency (%) RSD (%)
0.326 99.78 0.55
0.324 99.17
0.327 100.08
0.323 98.86
0.327 100.08
Repeatability measurement results (n ¼ 5) Sample number
1
2
3
4
5
Absorbance Extraction efficiency (%) RSD (%)
0.326 99.78 0.83
0.321 98.26
0.324 99.17
0.319 97.65
0.322 98.56
Stability measurement results (n ¼ 5) Day number
1
2
3
4
5
Absorbance Extraction efficiency (%) RSD (%)
0.326 99.78 0.98
0.319 97.65
0.322 98.56
0.327 100.08
0.324 99.17
The method used in this study was validated by precision, repeatability and stability experiments. The results are shown in Table 3
Results of orthogonal experiment L9 (34)
Experiment
A DES (g)
B K2HPO4 (g mL1)
C T ( C)
D t (min)
E (%)
1 2 3 4 5 6 7 8 9 K1
1(1.2) 1 1 2(1.4) 2 2 3(1.6) 3 3 94.07
1(0.6) 2(0.7) 3(0.8) 1 2 3 1 2 3 96.68
1(20) 2(25) 3(30) 2 3 1 3 1 2 95.35
1(4) 2(8) 3(10) 3 1 2 2 3 1 93.69
93.34 95.08 93.80 97.05 90.95 93.38 99.64 99.32 96.77 Major and minor order: A > D > B > C optimized conditions: A3B1C2D3
K2 K3 R Optimal level
93.79 98.58 4.78 A3
95.12 94.65 2.03 B1
96.30 94.80 1.50 C2
96.03 96.72 3.04 D3
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3.6. Extraction mechanism
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3.6.1. UV-vis spectroscopy. In order to examine the protein conformation before and aer extraction, UV-vis spectra were investigated for BSA. ESI Fig. S3† shows the UV-vis spectra of BSA in pure water and in the DES-rich top phase aer
extraction. It is clear that the maximum absorption peak of BSA in the DES-rich top phase still existed at 278 nm, and the conformations of BSA before and aer extraction were similar, suggesting that there are no chemical bonds between BSA molecules and DESs.
Fig. 4
The size distribution of the DES aggregates in aqueous solution at different DES concentrations. (a) 0.20; (b) 0.30; (c) 0.40; (d) 0.50 g mL1.
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3.6.2. The microscopic structure of the DES-rich top phase. Examination of the microscopic structure of the DES-rich top phase furthers our understanding of the separation process. As shown in Fig. 4, the DES solution is not microscopically homogeneous, but polydisperse. The average sizes are 574, 619.2, 687.5 and 968.6 nm, when the concentrations are 0.20, 0.30, 0.40, 0.50 g mL1, respectively. The size of the DES aggregates increases with an increasing concentration of DES. Then, TEM images of the DES (0.5 g mL1), BSA (5.0 mg mL1) and a mixture (0.5 g mL1 DES + 5.0 mg mL1 BSA) were taken as shown in Fig. 5. Fig. 5a shows the conformation of DES aggregates, Fig. 5b shows the appearance of BSA aggregates, and Fig. 5c showed the distribution of the mixture aggregates (scale bars 0.2 mm, 0.2 mm and 0.5 mm, respectively). From Fig. 5c, it can be seen that the DES aggregates encircle the BSA aggregate, which is the main driving force in the uptake of protein by the DES-based ATPS.
4. Conclusions This is the rst report for the extraction of proteins with a deep eutectic solvent-based ATPS. The greatest benet of the proposed method is that the adapted extraction solvent is green and environmentally friendly. In comparison with ionic liquids, deep eutectic solvents display more superiority as they are nontoxic, biodegradable, and have an atom utilization rate of 100% in the synthesis process. By adjusting the types of quaternary ammonium salts and HBDs, different kinds of DESs can be obtained. In addition, since the synthetic materials of DESs are abundant and inexpensive, DESs are expected to be applied successfully in large-scale industrial production. As a result, the present method uses innovative extraction media (DESs) that can replace traditional ILs, and demonstrates higher extraction efficiencies in DES-based ATPSs than in ILATPSs. Besides the single factor experiments, the orthogonal experiment was performed to acquire the optimum conditions for the extraction of proteins with this DES-based ATPS. These data fully illustrated the choices of factors used in this system. It is worth mentioning that the aggregation and surrounding phenomenon plays an important role in the extraction process. Along with the other driving forces involved in the partitioning of the protein between the DES-rich phase and phosphate phase, the aggregation and surrounding phenomenon could be applied to a variety of different samples and exhibit potential value. But much to our regret, it is hard to back-extract the targeted protein free from the DES in this work. We chose a low concentration of SDS aqueous solution as a stripping reagent but we failed because DES is hydrophilic and so we were unable to separate the DES phase from the aqueous solution. Maybe it will be the direction of our future research. Fig. 5 TEM images of the aggregates for DES (0.5 g mL1), BSA (5.0 mg mL1) and DES (0.5 g mL1) + BSA (5.0 mg mL1). (a) DES; (b) BSA; (c) DES + BSA.
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Acknowledgements The authors greatly appreciate the nancial support of the National Natural Science Foundation of China (no. 21175040;
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no. 21375035; no. J1210040) and the Foundation for Innovative Research Groups of NSFC (Grant 21221003).
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References 1 G. T. Wei, Z. S. Yang and C. J. Chen, Anal. Chim. Acta, 2003, 488, 183–188. 2 M. Matsumoto, K. Mochiduki, K. Fukunishi and K. Kondo, Sep. Purif. Technol., 2004, 40, 97–103. 3 S. Shah and M. N. Gupta, Bioorg. Med. Chem. Lett., 2007, 17, 921–928. 4 C. E. Song and E. J. Roh, Chem. Commun., 2000, 837–842. 5 F. Favre, H. Olivier-Bourbigou, D. Commereuc and L. Saussine, Chem. Commun., 2001, 1360–1363. 6 D. R. MacFarlane, M. Forsyth, P. C. Howlett, J. M. Pringle, J. Sun, G. Annat, W. Neil and E. I. Izgorodina, Acc. Chem. Res., 2007, 40, 1165–1173. 7 D. J. Couling, R. J. Bernot, K. M. Docherty, et al., Green Chem., 2006, 8, 82–90. 8 A. S. Wells and V. T. Coombe, Org. Process Res. Dev., 2006, 10, 794–798. 9 A. P. Abbott, G. Capper and D. L. Davies, Chem. Commun., 2003, 70–71. 10 A. P. Abbott, D. Boothby, G. Capper, D. L. Davies and R. K. Rasheed, J. Am. Chem. Soc., 2004, 126, 9142–9147. 11 A. P. Abbott and K. J. McKenzie, Phys. Chem. Chem. Phys., 2006, 8, 4265–4279. 12 K. Haerens, E. Matthijs and K. Binnemansc, Green Chem., 2009, 11, 1357–1365. 13 L. Wei and Y. Fan, Chemistry, 2011, 74, 333–338. 14 A. P. Abbott, D. Boothby, G. Capper, D. L. Davies and R. K. Rasheed, J. Am. Chem. Soc., 2004, 126, 9142–9147.
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Analyst
15 A. M. Azevedo, P. A. J. Rosa and I. F. Ferreira, Sep. Purif. Technol., 2009, 65, 31–33. 16 J. S. Becker, O. R. T. Thomas and M. Franzreb, Sep. Purif. Technol., 2009, 65, 46–49. 17 L. Li, F. Liu and X. X. Kong, Anal. Chim. Acta, 2002, 452, 321– 327. 18 D. H. Chen, X. W. Chen, Y. Shu and J. H. Wang, Talanta, 2008, 75, 1270–1278. 19 Z. Du, Y.-L. Yu and J.-H. Wang, Chem.–Eur. J., 2007, 13, 2130– 2137. 20 Z. Li, X. Liu, Y. Pei, J. Wang and M. He, Green Chem., 2012, 14, 2941–2950. 21 Y. Pei, J. Wanga, K. Wua, X. Xuana and X. Lu, Sep. Purif. Technol., 2009, 64, 288–295. 22 P. YuanChao, L. ZhiYong, L. Li, W. JianJi and W. HuiYong, Sci. China: Chem., 2010, 53, 1554–1560. 23 C. K. Su and B. H. Chiang, Process Biochem., 2006, 41, 257– 261. 24 G. A. Gomes, A. M. Azevedo, M. R. Aires-Barros and D. M. F. Prazeres, Sep. Purif. Technol., 2009, 65, 22–27. 25 J. H. Wang, D. H. Cheng, X. W. Chen, Z. Du and Z. L. Fang, Anal. Chem., 2007, 79, 620–624. 26 A. M. Azevedo, A. G. Gomes, P. A. J. Rosa, I. F. Ferreira, A. M. M. M. O. Pisco and M. R. Aires-Barros, Sep. Purif. Technol., 2009, 65, 14–18. 27 B. Mokhtarani, R. Karimzadeh, M. Amini and S. D. Manesh, Biochem. Eng. J., 2008, 38, 241–248. 28 M. M. Bora, S. Borthakur, P. C. Rao and N. N. Dutta, Sep. Purif. Technol., 2005, 45, 153–156. 29 S. Saravanan, J. R. Rao, T. Murugesan, B. U. Nair and T. Ramasami, Chem. Eng. Sci., 2007, 62, 969–978.
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