Ionic Liquids as Green Solvents - American Chemical Society

Chapter 20

Enzymatic Catalysis in Ionic Liquids and Supercritical Carbon Dioxide 1

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Pedro Lozano , Teresa De Diego , Daniel Carrié , Michel Vaultier , and José L. Iborra Downloaded by COLUMBIA UNIV on July 19, 2012 | http://pubs.acs.org Publication Date: August 26, 2003 | doi: 10.1021/bk-2003-0856.ch020

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Departamento de Bioquímica y Biología Molecular Β e Inmunología, Facultad de Química, Universidad de Murcia, P.O. Box 4021, E-30100 Murcia, Spain Université de Rennes, Institut de Chimie, U M R CNRS 6510, Campus de Beaulieu. Av. Général Leclerc, 35042, Rennes, France 2

The suitability of ionic liquids (ILs) and supercritical carbon dioxide (scCO ) as reaction media for enzyme catalyzed synthesis was studied. Ionic liquids exhibited an over-stabilizing effect (up 2,500-times) on α-chymotrypsin and Candida antarctica lipase Β during thermal deactivation. All the assayed ILs were adequate media for lipase-catalyzed transesterification. A continuous biphasic reactor combining free lipase-ILs (catalytic phase) and scCO (extractive phase) was used for butyl butyrate synthesis and the kinetic resolution ofrac-1-phenylethanol.The synthetic activity and operational stability of this green bioreactor was dependent on the supercritical conditions, revealing high catalytic efficiency even at extreme temperatures (120 and 150 °C). 2

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Introduction One active area of current research in biotechnology is biocatalysis in nonconventional media, involving all reaction systems with a reduced water content, e.g. organic solvents (1). However, in many cases, the use of organic solvents in biocatalytic reactions is seriously limited by their denaturative action on proteins, and it is necessary to use enzyme stabilization strategies at the same time (e.g. immobilization, chemical modification, presence of polyols, etc) (2,3). Furthermore, organic solvents are usually volatile liquids that evaporate into the atmosphere with detrimental effects to the environment and human health.

© 2003 American Chemical Society In Ionic Liquids as Green Solvents; Rogers, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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240 Enzymatic reactions based on neoteric solvent, e.g. ionic liquids (ILs) and supercritical fluids, appear to be promising alternative media for developing integral green chemical processes, because of the physical and chemical characteristics that these solvents possess. ILs are organic salts composed of cations (e.g. alkylimidazolium, alkylammonium, etc.) and anions (e.g. BF , PF , Tf N, etc.) that are liquids at room temperature, with negligible vapour pressure and excellent chemical and thermal stability. Additionally, their solvent properties can be finely tuned by changing either the anion or the alkyl substituents in the cation (4). Recently, the use of ionic liquids as reaction media has been extended to biocatalytic processes, and excellent results have been obtained for many different synthetic reactions catalyzed by lipases and proteases (5-9) Supercritical fluids, i.e. fluids at temperatures and pressures slightly above the critical points (e.g. 31 C and 7.38 MPa for C0 ), exhibit unique combined properties, such as liquid-like density, gas-like diffusivity and viscosities. Their solvent characteristics can be reliably controlled by changing the pressure or the temperature, and they have been successfully used for several large-scale extraction industrial processes (e.g. caffeine-free coffee) (10). Supercritical C 0 has been widely tested as a reaction medium for biocatalysis, but it has been shown to have a direct adverse effect on enzyme activity, including decrease in pH of the enzyme microenvironment (11), covalent modification of free amino groups on enzymes to form carbamates (12), and deactivation by pressurization/depressurization clycles (13). In this work, the effect of different Dus on the stability of two enzymes (Candida antarctica lipase B, CALB, and α-chymotrypsin) is shown, and a continuous green bioreactor, based on the use of CALB-ILs and scC0 , is studied. Two different reactions (butyl butyrate synthesis and the kinetic resolution of rac-l-phenylethanol) are used as reaction models to analyze the catalytic efficiency of this system, including under extreme conditions (e.g. at 120 and 150 °C). 4

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Downloaded by COLUMBIA UNIV on July 19, 2012 | http://pubs.acs.org Publication Date: August 26, 2003 | doi: 10.1021/bk-2003-0856.ch020

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Materials and Methods Materials Soluble CALB (Novozym 525, EC 3.1.1.3, from Novo Nordik S.A.) was washed by ultrafiltration to eliminate all the low molecular weight additives, obtaining an enzyme solution of 5.75 mg/mL, as determined by Lowry's method. α-Chymotrypsin (EC 3.4.21.1) type II from bovine pancreas, substrates, solvents and other chemicals were purchased from Sigma-Aldrich-Fluka Chemical Co, and were of the highest purity available.

In Ionic Liquids as Green Solvents; Rogers, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

241 Synthesis of ionic liquids l-Ethyl-3-methylimidazoliurn tetrafluoroborate [Emim][BF ] and 1-butyl-3methylimidazolium tetrafluoroborate, [Bmim][BF ] were prepared according to Suarez et al (14). l-Ethyl-3-methylimidazolium triflimide [Emim][Tf N] and 1buthyl-3-methylimidazolium triflimide, [Emim]fTf N], were synthesized following the procedure described by Bonhôte et al (15). l-Butyl-3methylimidazolium hexafluorphosphate, [Bmim][PF ] was prepared according to Huddleston et al (16). 4

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Stability of α-chymotrypsin in liquid media Twenty microliters of 0.5% w/v α-chymotrypsin were added to different screw-capped vials of 1 mL total volume containing 980 μL of water, 3.2 M sorbitol in water, [Bmim][PF ], or 0.1M N-acetyl-L-tyrosine ethyl ester (ATEE) in [Bmim][PF ]. The resulting solutions were homogenized by shaking, and then incubated at 50 °C. At regular intervals, aliquots of 50 were extracted and the residual esterase activity was measured, as described previously (3). 6

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Stability of CALB in liquid media Ten microliters of 0.3% w/v CALB solution in water were mixed with 400 μΐ of 1-butanol, or ILs ([EmimHBF ], [EmimHTf N], or [Bmim]IPF ]) containing (or not) 30 μΐ. vinyl butyrate in different screw-capped vials, and the resulting solutions were incubated at 50°C. Then, at selected incubation time, 90 μΐ of substrate solution (1.68 M vinyl butyrate in 1-butanol) were added to each vial, and the synthetic activity was followed and analyzed by GC (see next sections). 4

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Butyl butyrate synthesis in ILs Thirty microliters (236 μπιοί) of vinyl butyrate and 110 μL (1.21 mmol) of 1butanol were added to different screw-capped vials of 1 mL total capacity containing 300 μΐ of ILs ([Emim][BF ], [Emim][Tf N], [Bmim][PF ], or [Bmim][Tf N]), or hexane, or 1-butanol. The reaction was started by adding 10 μL of 0.3% w/v CALB solution in water, and run at 50 °C in an oil bath, shaking for 1 h. At regular time intervals, 20 μΐ aliquots were taken and suspended in 1 mL of hexane. The biphasic mixture was strongly shaken for 3 min to extract all substrates and product into the hexane phase. For hexane and 1-butanol reaction media, aliquots were dissolved in acetone: HC1 (99:1 v/v) to stop the reaction. Samples were analyzed by GC. 4

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242 Enzymatic reactions in ILs-scC0

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Sixty-five microliters of 0.9% w/v CALB solution in water was mixed with 2 mL of ILs ([Emim][Tf N] or [Bmim][Tf NJ) in a test-tube, and then 3 g of dry Celite were added to absorb the enzyme-EL solution. The final mixture was placed in the cartridge of an ISCO 220SX high pressure extraction apparatus of 10 mL total capacity. Reactions (butyl butyrate synthesis or the kinetic resolution of rac- 1-phenylethanol) were carried out by continuous pumping of a substrate solution (0.38 M vinyl butyrate and 0.76 M 1-butanol in hexane for the former, or 50 mM vinyl propionate and lOOmMrac-1-phenylethanol in hexane for the latter) at 0.1 mL/min, and mixed with the scC0 flow of the system at different pressures and temperatures (Figure 1). The reactor was continuously operated for 4h. 2

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Figure /. Experimental set-up of the bioreactor with lLs-scC02» Substrates and products were fully soluble in scC0 , and the reaction mixtures were recovered by continuous depressurising through a calibrated heated restrictor (1 mL/min, 70 °C) for 30 minute steps. Samples were analysed by GC. In all cases, the synthetic activity of each enzyme-DL system was continuously tested by operation/storage cycles. During the storage steps (20 h/d), the cartridge was maintained under dry conditions at room temperature. 2


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GC analysis Analyses were performed with a Shimadzu GC-17A instrument equipped with a FID detector. Samples from the butyl butyrate synthetic reactions were analyzed by a Nukol™ column (15 m χ 0.53 mm, Supelco), using propyl acetate as internal standard and the following conditions: carrier gas (nitrogen) at 8 kPa (20 mL/min total flow); temperature program: 45 °C, 4 min, 8 °C/min, 133 °C, split ratio, 5:1; detector, 220 °C. One unit of activity was defined as the amount of enzyme that produces 1 μπιοί of butyl butyrate per min. Samples from the kinetic resolution ofrac-1-phenylethanolwere analyzed by a Beta DEX-120 column (30 m χ 0.25 mm χ 0.25 μπι, Supelco), using butyl butyrate as internal standard and the following conditions: carrier gas (He) at 1 MPa (205 mL/min total flow); temperature programme: 60°C, 10°C/min, 130°C; split ratio, 100:1; detector, 300°C. One unit of activity was defined as the amount of enzyme that produces 1 μπιοί of (R)-l-phenylethyl propionate per min.

Results and Discussion Stability and activity of α-chymotrypsin and CALB in ILs In all cases, both α-chymotrypsin and CALB thermal stabilities were followed by a first-order deactivation kinetic, allowing determination of half-life of the enzymes at 50 °C. The influence of the different media on enzyme stability could be comparatively analyzed by a parameter called "protective effect", defined as the ratio of enzyme half-life time in the presence of ILs (or solvent) to the half-life time without any ILs (using water for α-chymotrypsin and 1-butanol for CALB) (see Figure 2). In the case of α-chymotrypsin, IBmim][PF ] has a clearly stabilizing effect (140-times), although lower than that of classical polyhydric additives used for enzyme stabilization, such as sorbitol (700-times) (see Figure 2 I). The protective effect might be explained by changes in the microenvironment of the enzyme produced by the presence of either IL or polyol. Under thermal stress, a greater degree of protein unfolding takes place, resulting in a disruption of all interactions responsible for the maintenance of the tertiary structure (e.g. hydrogen, van der Walls, etc). Sorbitol increased the organization of water molecules around the enzyme by means of hydrogen bonds, maintaining the solvophobic interactions inside the protein core, which resulted in the clear enhancement of enzyme stability (3). For small molecules (e.g. water, methanol, etc) dissolved in ILs, the local solute structure around ILs is determined by competition between the solute-IL interactions and the interaction between the solvent ions (17). However, in the case of proteins, ILs should be considered as a 6


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fvSSl Without substrateΒ δ 3 With substrate

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Figure 2. Protective effect of different media on the α-chymotrypsin (I) and CALB (II) stability at 50°C. A water; B, [Bmim][PF ]; C, 3.2M Sorbitol; D, 100 mM ATEE in [Bmiml[PF ]; A', 1-butanol; B\ [Emim][BF ]; C\ [Emim][NTf ]; D\ [Bmim][PF ]. t

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liquid immobilization support, rather than as a solvent, because multipoint enzyme-IL (e.g. ionic, hydrogen, van der Walls, etc) interactions may occur. In this way, ILs resulted as an external and flexible supramolecular net able to maintain the active protein conformation under denaturing conditions (7). In presence of the substrate, the symmetrical charge distribution produced in the active centre of α-chymotrypsin produced ensures internal rigidification of the protein structure, which results in over-stabilization (up to 2500-times). These phenomena were also observed for CALB (Figure 2 II), where the three ILs assayed exhibited a poor protective effect against enzyme deactivation, whereas in the presence of substrate, all the ILs demonstrated an extremely good protective effect, which correlated with their polarity as follows: [Emim][BF] < [Emim] [Tf N] < [Bmim][PF] (8). 4

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CALB-catalyzed butyrate synthesis in ILs The ability of CALB to catalyze butyl butyrate synthesis from butyl vinyl ester and 1-butanol in these ILs was also studied at 50 °C and 2% v/v water content. Since synthetic activity is a kinetically controlled process, the efficiency of the enzyme activity can be expressed by the synthetic activity and the selectivity parameter (ratio between the synthetic and hydrolytic activities). Figure 3 shows the synthetic activity exhibited by the enzyme in different ILs


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Figure 3. Activity of CALB for butyl butyrate synthesis in different reaction media. A, hexane; B, 1-butanol; C, [Emim][BF ]; A [Emim][NTf ]; E, [Bmim][PF ]. 4

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([Emim][BF ], [Emim][Tf N] and [Bmim][PF ]) and organic solvents (hexane and 1-butanol). As can be seen, all the ILs acted as suitable reaction media for the enzymatic synthesis, which involves an appropriate solubilization of substrates and products, and an active enzyme conformation. Thus, for all ILs, the synthetic activity was (up to 2-times) higher than that observed in the organic solvents assayed. Also, for all cases, the selectivity parameter was higher than 94% due to the low water content of these reaction media. 4

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CALB-catalyzed butyl butyrate synthesis in ILs-scC0

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A continuous biphasic enzymatic reactor was designed, taking into account the excellent properties of ILs to maintain enzymes in an active conformation, as well as the ability of scC0 to transport hydrophobic substrates and products (see Figure 1) (18). As has been described, scC0 is highly soluble in certain ILs (up to 0.6 mol fraction), while ILs are insoluble in scC0 (19). Thus, a new concept for a continuous phase-separable biocatalytic reactor is proposed, whereby a homogeneous free enzyme solution is "dissolved" into the IL phase (catalytic phase), and substrates and/or products reside largely in the supercritical phase (extractive phase). Two kinetically-controlled synthetic processes catalyzed by CALB were considered as reaction models to analyze the efficiency of this proposed enzymatic reactor, as follows: a simple transesterification reaction towards a primary alcohol (1-butanol), and the kinetic resolution of a sec-alcohol (rac-1 -phenylethanol). 2

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246 Figure 4 shows the evolution of the activity and half-life time of CALB[Bmim][Tf NJ system for the butyl butyrate synthesis as a function of the scC0 density, determined by the experimental conditions (40°C, 15.0 MPa; 50 °C 12.5 MPa; and 100 °C, 15.0 MPa, respectively). As can be seen, lipase was able to catalyze the transesterification reaction in all the assayed supercritical conditions, being the activity increasing as scC0 density decreased. This may be related with an improvement in the mass-transport efficiency of the supercritical fluid for exposing substrates to the enzyme microenvironment, thus enhancing the synthetic activity. The negative effect of C 0 on the enzyme action was also observed because the synthetic activity was 10-times lower than that observed in ILs without scC0 (see Fig. 3). On the other hand, the enzyme stability was reduced by a decrease in scC0 density, probably due to the associate increase in temperature (from 40 to 100 °C). Two additional observations were made, firstly that butyl butyrate was not synthesized in the absence of enzyme, and secondly, no enzyme activity was detected at the exit of the reactor, which means that the enzyme molecules "dissolved" into the IL must be considered as immobilized. Once again, the selectivity parameter in all cases was higher than 96%, which remained constant throughout all the operation periods due to the low water content (2% v/v in the IL phase). 2

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Figure 4. Effect of scC0 density on the activity and half-life time of CALB-[Bmim}lTfjN} for butyl butyrate synthesis. 2


247 CALB-catalyzed kinetic resolution of rac-1-phenylethanol in ELs-scC0

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Another enzymatic property, enantioselectivity, was also tested for this continuous lipase-EL-scC0 reactor using two different ILs, [Emim][Tf Nl and [Bmim][Tf N], in four supercritical conditions (50 °C, 15 MPa; 100°C 15 MPa; 120°C, 10 MPa; and 150 °C, 10 MPa). In all cases, similar catalytic behaviour was observed. As an example, Figure 5 depicts the activity, selectivity and enantioselectivity profiles during operation cycles, shown by the CALB[Emim][Tf N]-catalyzed kinetic resolution ofrac-1-phenylethanolin scC0 at 120 °C and 10 MPa. As can be seen, for all the assayed cycles the enantioselectivity of the enzyme was maintained at the maximum level (ee> 99.9%), because the (S)-lphenylethyl propionate isomer was not detected at all, and the (R)-l-phenylethyl propionate isomer was always obtained at a rate of conversion higher than 35 %. However, the initial synthetic activity and selectivity of the enzyme for this synthetic reaction were lower than in the case of butyl butyrate synthesis because of the drop in nucleophilic power of the hydroxyl group from 1-butanol (primary position) to rac-1-phenylethanol (secondary position). Furthermore, two interesting phenomena were observed in Figure 5. Firstly, the enzyme was active and stable (t = 13 cycles) at this extreme temperature (120 °C), demonstrating the important protective effect of this IL against enzyme deactivation by heat and/or C0 . Secondly, the selectivity profile showed a hyperbolic increase (from 35% to 98%) with an increasing number of operation cycles, which could be related with the non-optimized initial water content in the IL phase (2% v/v). 2

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Figure 5. Activity, selectivity and enantioselectivity profiles of CALB[Emim][Tf2N] system for continuous kinetic resolution of rac1-phenylethanol at 120 °C and 10 MPa.



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Figure 6. Effect of temperature on the synthetic activity and half-life time of different CALB-IL (·, [Emim][Tf N]; A, [Bmim][Tf N]) systems for continuous kinetic resolution of rac-1-phenylethanol in scC0 at 15.0 and 10.0 MPa, respectively. 2

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249 The decrease in nucleophilicity of the sec-alcohol, together with an excess in water content, may have involved a decrease in the selectivity of the catalytic action of the enzyme compared with the butyl butyrate synthesis. In this case, the continuous "drying" of the IL-phase by the flow of substrate-scC0 , as well as the consumption of free water molecules by the hydrolytic activity during operation cycles, would produce an enhancement in selectivity. These results clearly pointed to the need to optimize the initial water content in the IL-enzyme system, as well as its control during continuous operation processes. Figure 6 shows the evolution of synthetic activity and half-life time of both CALB-IL ([Emim][Tf N] and [Bmim][Tf N]) systems, for the kinetic resolution of rac-1-phenylethanol, as a function of the temperature of scC0 at two pressures (10.0 and 15.0 MPa). As can be seen, the biocatalytic system was active in all the assayed conditions, exhibiting a decrease in both catalytic parameters as temperature increased. The initial activity profile was practically independent of the nature of the assayed IL. However, it is necessary to point to the significant protective effect these media had on the enzyme activity even in extreme thermal conditions, [Emim][Tf N] providing the best stability. In this way, a mesophilic biological macromolecule designed to work at room temperature can maintain its catalytic activity for several daily cycles (t = 2 cycles) of continuous operation at 150 °C. 2

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Conclusions ILs have been shown to act as excellent agents for stabilizing achymotrypsin and CALB. For both enzymes, the presence of substrate greatly improved their half-life time, resulting in an over-stabilizing effect (up to 2500times). Also, ILs appeared to be adequate media for carrying out synthetic enzymatic processes under in low water conditions. A continuous biphasic reactor based on CALB-EL systems and scC0 have been tested for two different synthetic reactions with excellent results. The system exhibited high activity, enantioselectivity and stability in continuous operation, even in very extreme conditions (e.g. 120 and 150°C). These results clearly show how clean synthetic chemical processes, running as a "black box" directly providing pure products, can be designed easily. In this context, the door for the green chemical industry of the near future is open. 2

Acknowledgements This work was partially supported by the CICYT grants BIO99-0492-C02-01 and PPQ2002-03549. We like to thanks Ms. C. Saez for technical assistance, and Mr. R. Martinez from Novo Espafia, S.A. the gift of Novozym 525.


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References (1) Hailing, P. Enzyme Microb. Technol. 1994, 16, 178-206. (2) Dordick, J. S.Biotechnol.Prog., 1992, 8, 259-267. (3) Lozano, P.; Combes, D.; Iborra, J.L. J. Biotechnol., 1994, 35, 9-18. (4) Carmichael, A.J.; Seddon, K.R. J. Phys. Org. Chem., 2000, 13, 591-595. (5) Erbeldinger, M.; Mesiano, A.J.; Russel, A.J. Biotechnol. Prog. 2000, 16, 1129-1131. (6) Madeira Lau, R; van Rantwijk, F.; Seddon, K.R.; Sheldon, R.A. Org. Lett., 2000, 2, 4189-4191. (7) Lozano, P.; De Diego, T.; Guegan, J.P.; Vaultier, M.; Iborra, J.L. Biotechnol. Bioeng., 2001, 75, 563-569. (8) Lozano, P.; De Diego, T.; Carrié, D.; Vaultier, M.; Iborra, J.L. Biotechnol. Lett., 2001, 23, 1529-1533. (9) Itoh, T.; Akasaki, E.; Kudo, K.; Shirakami, S. Chem. Lett., 2001, 262-263. (10) Jarzebski, A.B.; Malinowski, J.J. Process Biochem., 1995, 30, 343-352. (11) Nakamura, K. Trends Biotechnol., 1990, 8, 288-292. (12) Kamat, S.; Critchley, G.; Beckman, E.J.; Russel, A.J. Biotechnol. Bioeng., 1995, 46, 610-620. (13) Lozano, P.; Avellaneda, Α.; Pascual, R.; Iborra, J.L.Biotechnol.Lett., 1996, 18, 1345-1350. (14) Suarez, P.A.Z.; Dullius, J.E.L.; Einloft, S.; De Souza, R.F.; Dupont, J. Polyhedron, 1996, 15, 1217,-1219. (15) Bonhôte, P.; Dias, A.P.; Papageorgiu, N.; Kalyanasundaram, K.; Grätzel, M. Inorg. Chem., 1996, 35, 1168-1178. (16) Huddleston, J.G.; Willauer, H.D.; Swatloski, R.P.; Visser, A.E.; Rogers, R.D. Chem. Commun. 1998, 1765-1766. (17) Hanke, C.G.; Atamas, N.A.; Lynden-Bell, R.M. Green Chem., 2002, 4, 107111. (18) Lozano, P.; De Diego, T.; Carrié, D.; Vaultier, M.; Iborra, J.L. Chem. Commun., 2002, 692-693. (19) Blanchard, L.A.; Hancu, D.; Beckman, E.J.; Brennecke, J.F. Nature, 1999, 399, 28-29.


Ionic Liquids as Green Solvents - American Chemical Society

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