ΔG(CH2) in Biphasic Systems of Water and Bis(trifluoromethylsulfonyl

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ΔG(CH2) in Biphasic Systems of Water and Bis(trifluoromethylsulfonyl)Imide-Based Ionic Liquids Filipa M. Maia, Oscar Rodríguez, and Eugénia A. Macedo* LSRE−Laboratory of Separation and Reaction Engineering−Associate Laboratory LSRE/LCM, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal ABSTRACT: Partition coefficients for a series of four dinitrophenylated (DNP) amino acids were experimentally determined in four biphasic systems, composed of hydrophobic ionic liquids (ILs) and water. The ILs were 1-alkyl-3methylimidazolium bis(trifluoromethyl-sulfonyl)imide, [Cnmim][NTf2] (n = 2, 4, 6, and 8). DNP-amino acids distribute preferentially to the water-rich (top) phase. The experimental partition coefficients decrease as the size of the alkyl side chain of both the DNP-amino acid and the IL increase. The experimental data allowed the calculation of the free energy of transfer of a methylene group (CH2) between the two equilibrium phases, ΔG(CH2). This thermodynamic function is regarded to be a good measure of the relative hydrophobicity of the phases. This property was found to be independent of the size of the alkyl side chain in the ILs. This fact indicates that in this family of ILs the anion contributes much more significantly to the hydrophobicity of the compound than the alkyl chain in the cation.

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INTRODUCTION The separation and recovery of biomolecules of interest are key steps for a successful biotechnological process. The purification of the desired molecule and the elimination of any biproducts or other components are of the utmost importance in order to obtain an added value product. One of the separation techniques most frequently used for this purpose is liquid− liquid extraction. This separation process is widely used in industry due to several advantages over other processes, such as its great selectivity and low cost, the use of simple equipment, the easiness of scale-up, the ability to operate in a continuous mode, and the achievement of high product recoveries.1−5 Traditionally, liquid−liquid extraction processes depend heavily on the use of organic solvents immiscible with water, allowing the coexistence of two liquid phases. However, most of these solvents are potentially harmful to biomolecules, to the environment, and to people working with them, as they are toxic, flammable, and volatile.6 For this reason, the search for alternative greener solvents (for example, ionic liquids or supercritical fluids) has been growing rapidly. Ionic liquids (ILs) have been presented as highly beneficial alternative solvents for liquid−liquid extraction.7 They present several characteristics which make them suitable for this kind of process, like low vapor pressure and flammability, high chemical and thermal stability, high selectivity and solvation capability, wide temperature range for the liquid phase, and easiness of recovery.8−12 Additionally, they have been labeled as “designer solvents” because it is possible to tune some physical-chemical properties (melting point, viscosity, density, or hydrophobicity) just by introducing changes in the structures of their constituent ions. Such changes might just be setting the size of an alkyl side chain or introducing different functional groups © XXXX American Chemical Society

in the structure of an ion. ILs can therefore be tailored to a specific process, like for example the extraction of a specific target molecule. Regarding the applicability of ILs in biotechnological processes, several studies have proved that many ILs are biocompatible, ensuring that the stability of proteins and enzymes is maintained (and sometimes even improved) in the presence of ILs.13−19 For example, several studies have shown that different ILs have a stabilizing effect on α-chymotrypsin.16,20,21 Weaver et al.22 also showed that the thermal stability of lysozyme and interleukin-2 increases with the concentration of IL choline dihydrogen phosphate. Tamura et al.23 also demonstrated that cytochrome c maintains its thermal stability and redox activity in some polar ILs. Very recently, Ventura et al.24 were able to significantly increase the activity of commercial enzyme Candida antarctica lipase B using IL 1decyl-3-methylimidazolium chloride, [C10mim][Cl]. They also showed that the enzyme activation energy was not affected by the presence of the IL, which suggests that the IL does not induce any structural changes in the enzyme. Instead, they claim that the increase in the activity is explained by the formation of microemulsions caused by self-aggregation of the IL. Qian et al.25 were able to improve the biocompatibility of laccase-based biocathodes by applying a surface overcoating of IL 1-butyl-3-methylimidazolium hexafluorophosphate, [C4mim][PF6], with good bioelectrocatalytic activity toward O2. In a recent work, Bian et al.26 immobilized the enzyme papain in an IL-decorated mesoporous SBA-15, using IL Received: October 31, 2012 Accepted: April 23, 2013

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dinitrophenyl)-L-alanine ≥ 97 %, N-(2,4-dinitrophenyl)-DL-nleucine ≥ 97 %). In Table 1, the description of all ILs and DNP-amino acids is presented. 1-Propanol was supplied by Merck (99.8 %, LC).

[Simim][Cl]. They proved that papain immobilized on this material showed a higher specific activity than that on SBA-15, which they claim to be a consequence of the electrostatic attraction between the cation of the IL and the negatively charged papain. Rehmann et al.27 have measured the activity of laccase in the presence of 63 ILs. They used water-miscible and water immiscible ILs, which included imidazolium, pyridinium, ammonium, and phosphonium cations. They determined that in general, water-immiscible ILs allowed the highest enzyme activities, especially the ones with the biś (trifluoromethylsulfonyl)imide anion. Dominguez et al.28 also showed that IL 1-butyl-3-methylimidazolium chloride, [C4mim][Cl] has a stabilizing effect on laccase, as they observed little activity decay within three weeks. Some theoretical work has also been conducted regarding the extraction of amino acids using (IL + water) systems. For example, Seduraman at al.29 studied the extraction of tryptophan in these kinds of systems, using ILs [C4mim][PF6], [C8mim][PF6], and [C8mim][BF4] with molecular dynamics simulations. They were able to confirm the importance of the hydrophobicity/hydrophilicity of the ions in the extraction process. All of these studies, among others, come to show that there are several biocompatible ILs which may be suitable for the liquid−liquid extraction of biomolecules. The application of ILs on these fields has been studied for several families of molecules, like proteins,30,31 amino acids,4,32−35 antibiotics,2,36−39 hormones,40 DNA,41 among others. In this work, the partition coefficients of four dinitrophenylated (DNP) amino acids in four biphasic (IL + water) systems were experimentally determined. The ILs used for the biphasic systems were 1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)-imide, [Cnmim][NTf2], where the alkyl group was ethyl, butyl, hexyl, and octyl. These ILs were chosen because they are liquid at 296.15 K, they have low viscosity when compared to other ILs (which makes them easier to work with), and they are highly hydrophobic, so they form systems with two liquid phases when in contact with water. The solutes used were N-(2,4-dinitrophenyl)glycine (DNP-Gly), N-(2,4-dinitrophenyl)-L-alanine (DNP-Ala), N(2,4-dinitrophenyl)-DL-n-valine (DNP-Val), and N-(2,4-dinitrophenyl)-DL-n-leucine (DNP-Leu). These DNP amino acids were selected because they only differ on the size of the alkyl side chain of the molecule, that is, in the number of methylene (CH2) groups. Therefore, it is possible to determine the free energy of transfer of a methylene group between the equilibrium phases through the experimental partition coefficients. This information is useful because it allows the quantification of the relative hydrophobicity of the equilibrium phases, which may lead to a better understanding of the interactions of ILs with other components.

Table 1. Description of Chemicals product

short name

1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide 1-methyl-3-octylimidazolium bis(trifluoromethylsulfonyl)imide N-(2,4-dinitrophenyl)glycine N-(2,4-dinitrophenyl)-L-alanine

[C2mim] [NTf2] [C4mim] [NTf2] [C6mim] [NTf2] [C8mim] [NTf2] DNP-Gly DNP-Ala

N-(2,4-dinitrophenyl)-DL-n-valine N-(2,4-dinitrophenyl)-DL-n-leucine

DNP-Val DNP-Leu

supplier

purity

Iolitec

99 %

Iolitec

99 %

Iolitec

99 %

Iolitec

99 %

Sigma Research Organics Sigma Research Organics

≥ 99 % ≥ 97 % ≥ 97 % ≥ 97 %

All products were used as received, without any additional purification. Deionized water was used in all experiments. Sample mass was determined by weighing using an Adam Equipment balance model AAA250L, precise to within ± 0.2 mg. Methods. The determination of the partition coefficients of the dinitrophenylated (DNP) amino acids at 296.15 K was carried out for all (IL + water) systems. Stock solutions of DNP amino acids in water with a concentration of 0.2 wt % were prepared. Know volumes of IL and water were added to 2 mL eppendorf tubes to prepare the biphasic systems. Volumes were measured with an automatic pipet (Eppendorf Multipette Xstream). The ratio of volumes chosen for each component depended on the solute partition in the corresponding systems. Six replicates of each system were prepared and volumes of 0 μL (blank) to 100 μL of each DNP amino acid stock solution were added to each tube. The corresponding volumes of water (100 μL to 0 μL) were also added so that the compositions of IL and water were maintained in all replicates. Samples were vigorously mixed using a vortex mixer during 5 min to reach equilibrium after which phase separation was enhanced by centrifugation at 104 rpm for 15 min. Samples were collected from the top (water-rich) and bottom (IL-rich) phases and diluted conveniently in water and 1-propanol, respectively. The samples were then analyzed by UV/vis spectroscopy (Thermo Electron Corp., model UV1) at a wavelength of 362 nm. Absorbance was lower than 2.0 in all cases. For each DNP amino acid in each solvent, suitable calibration lines were determined for adequate concentration ranges, with each equilibrium phase present in the same amount as in the diluted samples. All calibration lines were linear. Partition coefficients were obtained from the slope of the straight line found when concentrations of the top phase are plotted against concentrations of the bottom phase (all concentrations in mg/ mL). By increasing the concentration of solute in different replicates it becomes possible to determine the thermodynamic partition coefficients, which are concentration independent. This allows confirmation that there are no aggregation effects.

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MATERIALS AND METHODS Materials. Ionic liquids 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C2mim][NTf2], 1-butyl-3methylimidazolium bis(trifluoromethylsulfonyl)imide [C 4 mim][NTf 2 ], 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C6mim][NTf2] and 1-methyl3-octylimidazolium bis(trifluoromethyl-sulfonyl)imide [C8mim][NTf2], with purities ≥ 99 %, were bought from Iolitec. Dinitrophenylated (DNP) amino acids were obtained from Sigma (N-(2,4-dinitrophenyl)glycine ≥ 99 %, N-(2,4-dinitrophenyl)-DL-n-valine ≥ 97 %), and Research Organics (N-(2,4B

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Table 2. Experimental Partition Coefficients of DNP-Amino Acids in Systems (Ionic Liquid + Water) at 293.15 K and Atmospheric Pressure

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Solute\IL

[C2mim][NTf2]

[C4mim][NTf2]

DNP-Gly DNP-Ala DNP-Val DNP-Leu

145.4 ± 1.4 51.2 ± 1.5 15.42 ± 0.50 5.921 ± 0.086

55.94 ± 0.84 24.50 ± 0.78 6.90 ± 0.28 2.60 ± 0.11

RESULTS AND DISCUSSION The theory applied to determine the free energy of transfer for methylene groups between equilibrium phases has been explained elsewhere.33,34,42−44 When a solute, i, is transferred between two phases, α and β, the free energy change associated with the process, ΔG, is obtained from the difference of chemical potentials of the solute, μi, in each phase: ΔG = μi β − μiα

(1)

(2)

where R is the universal gas constant, T is the temperature, and Ci is the concentration of component i in each phase (α and β). At equilibrium, ΔG = 0 and the ratio of concentrations that appears on the right-hand side of eq 2 becomes the partition coefficient of solute, Ki:

Ki =

Ciβ Ciα

(3)

By convention, units for concentration are in molarity. Therefore, ΔG* can be expressed as a function of the partition coefficient: ΔG* = −RT ln K i

(4)

Deeper details on such derivations are shown in the literature.45,46 If we now apply the group contribution concept, the free energy of transfer can be regarded as the contribution of two terms, obtained by dividing the molecules into an alkyl part (by summation of n methylene groups) and a nonalkyl part. This allows the formulation of the logarithm of partition coefficients as follows:43,47,48

ln K = C + En(CH 2)

[C8mim][NTf2]

± ± ± ±

15.7 ± 1.0 8.25 ± 0.56 2.24 ± 0.13 0.735 ± 0.042

29.78 15.63 3.308 1.244

0.78 0.39 0.079 0.046

hydrocarbons are good examples of hydrophobic chemicals, ΔG(CH2) can be regarded as a measurement of the relative hydrophobicity between phases α and β. In this work, the series of homologous solutes was a set of four dinitrophenylated (DNP) amino acids. The experimental partition coefficients of the DNP-amino acids in the four systems studied are presented in Table 2. Partition coefficients for all DNP-amino acids in all systems are higher than 1, except for DNP-leucine in the system with IL [C8mim][NTf2]. Because partition coefficients are defined as the ratio between concentrations on the top and bottom phases, and most of the IL remains in the bottom phase (ILrich phase), this means that the solutes distribute preferentially to the water-rich phase (top). The partition coefficients for each system vary as follows: Kgly > Kala > Kval > Kleu. Therefore, the longer the side chain in the amino acid, the higher its affinity toward the IL-rich phase. Regarding the errors associated with the experimental partition coefficients, presented in Table 2, the average relative error was 3.6 %. Regarding the four different systems composed of ILs and water, it is observed that for each DNP-amino acid, partition coefficients decrease as the length of the side chain in the ILs increases. Therefore, the higher partition coefficients are obtained with IL [C2mim][NTf2], and the lower are obtained with IL [C8mim][NTf2]. Figure 1 presents the relationships between the logarithm of the partition coefficients and the equivalent number of methylene groups (CH2) in the four DNP-amino acids. The number of equivalent methylene groups is slightly different than the real number of methylene groups (0, 1, 3, and 4 for DNP-glycine, alanine, valine, and leucine, respectively). The reason for this has been previously explained.43 The relation-

The contribution to ΔG that results directly from the transfer of solute i between the two phases is calculated as ⎛Cβ ⎞ ΔG* = ΔG − RT ln⎜⎜ iα ⎟⎟ ⎝ Ci ⎠

[C6mim][NTf2]

(5)

where n(CH2) is the number of methylene groups in the alkyl chain, parameter C represents the contribution of the nonalkyl moiety to the partition coefficient, and parameter E represents the contribution of one methylene group. If a series of solutes is now considered that differ only in the number of methylene groups, it is possible to represent the logarithm of partition coefficients against the number of equivalent methylene groups in the molecule, and thus a straight line should be obtained. The slope of such line is parameter E and the y-intercept is parameter C. From eqs 4 and 5, ΔG(CH2) can be calculated from parameter E as ΔG(CH2) = −RTE

(6)

Figure 1. Logarithm of experimental partition coefficients (Ki) as a function of the equivalent number of methylene groups in the alkyl chain of the DNP amino acids.

ΔG(CH2) represents a measurement of the difference in the affinities of the equilibrium phases for a CH2 group. Since C

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Table 3. Parameters C and E (eq 5), Linear Regression Coefficient and Free Energy of Transfer of a Methylene Group, ΔG(CH2) C

ionic liquid [C2mim][NTf2] [C4mim][NTf2] [C6mim][NTf2] [C8mim][NTf2]

5.05 4.17 3.62 2.98

± ± ± ±

r2

E 0.02 0.11 0.22 0.20

−0.871 −0.844 −0.893 −0.843

± ± ± ±

0.009 0.044 0.090 0.083

0.999 0.995 0.980 0.981

ΔG(CH2)/kcal·mol−1 0.513 0.497 0.525 0.496

± ± ± ±

0.005 0.026 0.053 0.049

order with the increase in the size of the alkyl side chain of the ILs. We believe this can be explained by the fact that in this series of ILs, the anion provides the most significant contribution to the hydrophobicity of the compound. Thus, the changes in the size of the alkyl chains do not contribute significantly to the increase or decrease of the hydrophobicity of the IL. The errors associated with ΔG(CH2) are rather large for ILs [C6mim][BF4] and [C8mim][BF4] because of the deviations from linearity mentioned above. If this is taken into consideration, the value of ΔG(CH2) can be considered constant for this series of ILs. This type of experiments has been previously conducted for other groups of ILs. Our group has determined the free energy of transfer of a methylene group for systems containing [BF4]−based ILs ([C6mim][BF4], [C8mim][BF4], and [C10mim][BF4])33 and for other [NTf2]−-based ILs ([C3mpip][NTf2], [C3mpyrr][NTf2], and [C4mpyrr][NTf2]).34 The values obtained in those two works are presented in Figure 2, for ease of comparison. The values obtained in this work for ΔG(CH2) are in the same range as the values previously obtained for [NTf2]−-based ILs, which varied between 0.512 and 0.585 kcal/mol. On the other hand, the values obtained for [BF4]−-based ILs are mostly lower than the values obtained in this work (between 0.391 and 0.498 kcal/mol). This was expected, since [NTf2]− is a rather more hydrophobic anion than [BF4]−. It is important to note that these results for ΔG(CH2) for [NTf2]−-based ILs are similar to those obtained for polymer-salt ATPS (0.2−0.7 kcal/mol)49−51 rather than polymer−polymer ATPS (0.05−0.2 kcal/mol).48,52

ships presented in Figure 1 were expected to be linear, since they are described by eq 5. Very good linearity was obtained for ILs [C2mim][NTf2] and [C4mim][NTf2], with r2 > 0.99. Slightly worse results were obtained for [C6mim][NTf2] and [C8mim][NTf2], with r2 ≈ 0.98 (Table 3). Parameters C and E from eq 5 were obtained from the linear regressions presented in Figure 1. They equal the value of the y-intercept and the slope, respectively (eq 5). These values are also presented in Table 3, together with the calculated ΔG(CH2) obtained from eq 6. Considering that the IL-rich phase in these systems is the bottom phase, and taking into consideration that the partition coefficients were defined as the ratio between the concentrations on the top and bottom phases, then ΔG(CH2) refers to the transfer of methylene groups from the IL-rich phase to the water-rich phase. The calculated values for ΔG(CH2) are positive for all systems, indicating that such transfer is not spontaneous. Besides, it also points out that IL-rich phases are more hydrophobic than aqueous phases, as expected. The parameter C in eq 5 is observed to decrease as the alkyl chain in the cation of the IL increases. This indicates that the contribution of the nonalkyl part of the solutes to the partition coefficient decreases as the size of the cation side chain increases. This means that as the alkyl chain of the IL increases, interactions of the solute polar moiety with the solvent become less significant in respect to the interactions of its nonpolar moiety (alkyl chain of the DNP-amino acids). Figure 2 shows the representation of ΔG(CH2) against the number of CH2 groups present in the alkyl chain of the IL, n(CH2)IL. No significant differences are observed in the free energy of transfer of a methylene group. Therefore, the relative hydrophobicity of the IL-rich phase does not follow a specific

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CONCLUSIONS The partition coefficients of a series of four DNP-amino acids were experimentally determined in four biphasic systems composed of IL and water, using ILs [Cnmim][NTf2] (with n = 2, 4, 6, and 8). It was observed that the solutes distribute preferentially to the water-rich phase of the biphasic systems. The experimental partition coefficients decrease as the size of the alkyl side chain in the amino acid increases. They also decrease as the size of the alkyl side chain in the ILs increases. From the experimental partition coefficients, it was possible to calculate the free energy of transfer of a methylene group between equilibrium phases, ΔG(CH2) for each system. It was found that this property remains basically constant for the series of ILs, which means that the size of the alkyl side chain in the IL does not contribute significantly to the hydrophobicity of the compound. This is attributed to a larger contribution from the very hydrophobic anion.

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Figure 2. Free energy of transfer of a methylene group, ΔG(CH2), as a function of the number of CH2 groups in the alkyl side chain of the ILs: ○, [Cnmim][NTf2]; Δ, [Cnmpip][NTf2];34 □, [Cnmpyrr][NTf2];34 × , [Cnmim][BF4].33.

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Corresponding Author

*Tel.: + 351 22508 1653. Fax: + 351 22508 1674. E-mail: [email protected]. D

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Funding

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This work is supported by project PEst-C/EQB/LA0020/2011, financed by FEDER through COMPETE−Programa Operacional Factores de Competitividade and by FCT− Fundaçaõ para a Ciência e a Tecnologia. F.M.M. and O.R. acknowledge financial support of a Ph.D. Grant of FCT (SFRH/BD/44087/ 2008) and Programme Ciência 2007 (FCT), respectively. Notes

The authors declare no competing financial interest.

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