Ind. Eng. Chem. Res. 2004, 43, 7697-7701
7697
APPLIED CHEMISTRY Synthesis of Octyl Palmitate in Various Supercritical Fluids Giridhar Madras,* Rajnish Kumar, and Jayant Modak Department of Chemical Engineering, Indian Institute of Science, Bangalore 560012, India
Octyl palmitate was enzymatically synthesized from palmitic acid and octanol. The synthesis of octyl palmitate was investigated in three supercritical fluids, namely, carbon dioxide, methane, and ethane, using three different enzymes. The effects of temperature, initial water content, alcohol concentration, and enzyme loading on the conversion were determined. Among the enzymes, Novozym 435 (immobilized) catalyzed the reaction, resulting in the highest conversion compared to the other lipases, irrespective of the solvent used. Further, Novozym 435 was active over a wider range of temperatures (35-65 °C) compared to the other two enzymes. The lowest esterification (76%) was obtained in supercritical carbon dioxide, while the highest esterification was obtained in supercritical methane (85%). The results thus indicate that for this reaction, among the systems investigated, supercritical methane and Novozym 435 are the best supercritical fluid and enzyme, respectively. Introduction The use of biocatalysts suspended in nonaqueous media has received much attention in the past few years.1 Supercritical fluids have been considered as interesting alternatives to conventional organic solvents.2-5 These fluids offer some advantages over organic solvents, such as solubilization of hydrophobic compounds, ease of recovery of enzyme, and the possibility of performing reactions that are thermodynamically unfavorable in water (e.g., synthesis of esters and amides). The fluids also exhibit unique transport properties and an adjustable solvent power, which may allow an integrated process of reaction and downstream separation of products and unreacted substrates, yielding a final product almost free of solvent. Octyl palmitate has applications in cosmetics, pharmaceuticals, and food and chemical industries.6,7 It is used as a low-temperature plasticizer for poly(vinyl chloride), vinyl choride copolymers, polystyrene, ethyl cellulose, and synthetic rubber and also in making lubricants and water-resistant coatings.8,9 Octyl palmitate is usually produced by chemical catalytic esterification,10 which has several disadvantages, such as many side products and high energy consumption. The enzymatic synthesis of a mixture of 2-ethylhexyl esters of fatty acids by transesterification of rapeseed oil and 2-ethylhexanol, in which monoesters, monoglycerides, diglycerides, and triglycerides were included, was investigated,11 but it was difficult to separate octyl palmitate from the mixture. The kinetics of palmitic acid esterification with isopropyl alcohol catalyzed by immobilized lipase Novozym 435 to synthesize isopropyl palmitate has been investigated.12 Among supercritical fluids, the use of carbon dioxide is attractive because of its low toxicity and cost and * To whom correspondence should be addressed. Tel.: 91-80-2293 2321. Fax: 91-80-360 0683. E-mail: giridhar@ chemeng.iisc.ernet.in.
environmental friendliness, but it is perhaps the worst supercritical fluid to use as a solvent.5 Enzyme activity and stability have been shown to be moderately/ adversely affected upon exposure to supercritical carbon dioxide. The effect of carbon dioxide on the transesterification of vinyl butyrate by benzyl alcohol was studied, and propane was better than carbon dioxide on the catalytic activity of subtilisin,13 which was consistent with the results obtained by other investigators.14 To our knowledge, the synthesis of octyl palmitate in supercritical fluids has not been investigated. Therefore, the objective of this study is to investigate the various parameters that influence the synthesis of octyl palmitate in various supercritical fluids such as supercritical carbon dioxide (SCC), methane (SCM), and ethane (SCE). The reactions were catalyzed by the three different commercial enzymes Novozym 435 (immobilized), Lipolase 100T (immobilized), and Hog Pancreas Lipase (HPL; free enzyme). Materials and Methods Materials. Octan-1-ol and palmitic acid were obtained from SD Fine Chemicals Ltd. (Mumbai, India) and desiccated before use. The immobilized enzymes, Novozym 435 and Lipolase 100T, were gifted by Novo Nordisk (Bagsvaerd, Denmark), while the free enzyme, HPL, was obtained from Fluka Chemie AG (Buchs, Switzerland). All enzymes were stored at -15 °C and desiccated for 1 day at room temperature before use. Carbon dioxide (98%; Vinayaka gases, Bangalore, India) was used after dehydration by passing the gas through a silica gel column, and the purity was 99.5%. Methane (99.5%) and ethane (99.5%) (Bhoruka Gases, Karnataka, India) were used without further purification. All solvents were of high-performance liquid chromatography (HPLC) grade and were distilled and filtered with a 0.45-µm membrane filter before use. Methods. Reactions were performed in a 7-mL stainless steel batch reactor by adding the requisite amount
10.1021/ie049551e CCC: $27.50 © 2004 American Chemical Society Published on Web 10/23/2004
7698
Ind. Eng. Chem. Res., Vol. 43, No. 24, 2004
of reactants and enzyme in the reactor. The reactor, loaded with reactants and enzyme, was pressurized with respective gases and then immersed in a water bath maintained at the desired temperature of (0.5 °C. The initial pressure was 68, 130, and 46 bar at 298 K for carbon dioxide, methane, and ethane, respectively. This corresponds to densities of 0.7, 0.1, and 0.33 g/mL or 16, 6, and 11 mol/L for carbon dioxide, methane, and ethane, respectively. The pressurized reactor was then immersed in a water bath maintained at the desired temperature ((0.5 °C). Depending on the temperature investigated, the pressure of the reactor increased to 75 -100 bar for carbon dioxide. Because the volume of the reactor is constant, an increase in the temperature of the system increases the pressure of the system but the density of the supercritical fluid is constant. The reactor was equipped with a pressure gauge to ensure that the system operated at the same pressure throughout the reaction. The pressure in the reactor at all reaction conditions was measured with an accuracy of (1 bar. Thus, all of the reactions were carried out at constant density for various temperatures. After the reaction time, the reactions were terminated by depressurizing the reactor, and then 2 cm3 of acetonitrile was added to the system to remove the product and enzyme. The amounts of substrates for the reactions were chosen so that the concentrations of the reactants were low enough such that they dissolved completely in supercritical fluids but high enough to ensure detection without significant error. Because the solubilities of the substrates in supercritical fluids are not available in the literature, a method was developed to ensure that the substrates taken were completely soluble in the supercritical fluid. In this technique, a two-reactor system was designed to investigate the effect of solubility/ mixing in the reaction. The enzyme was added into one reactor, and acid and alcohol were added into the other reactor. Thus, there was no physical contact between the reactants and the enzyme. The concentrations of the reactants and the enzyme in the two-reactor setup were taken to be identical with the concentrations in the single reactor. The reactors were then individually pressurized. After pressurization, they were connected through a small steel tube, and the connecting valves were opened and incubated at the required temperatures. In this setup, the substrates and the enzyme are not in physical contact with each other in the tworeactor setup. Therefore, the substrates and the enzyme can come in contact with each other only through the solvent (supercritical) phase. The conversion was identical with that obtained in the single-reactor system, confirming that the system is completely mixed and solubility does not play a role in determining the conversion of the substrates. The operation of pressurizing and depressurizing the reactor takes less than 5 min. The enzyme was settled by centrifugation at 4000 rpm for 2 min. The influence of water on the reaction kinetics was investigated by adding water with the reactant before pressurizing the system. The effect of stirring on the conversion to octyl palmitate was studied in various supercritical fluids in the presence of Novozym 435. Stirring was achieved by lateral movement of the reactors at different oscillations. There was no effect of stirring on conversion, which is consistent with our previous study.15 Multiple experiments indicated that the error in the determination of the percent esterification was approximately 2%. However, because
Figure 1. Effect of temperature on the esterification reaction in different media with 15 mg of palmitic acid and optimum octanol concentration for 6 h with 5 mg of (a) HPL, (b) Lipolase, and (c) Novozym 435 in various supercritical fluids. Legend: b, SCC; 2, SCM; 1, SCE.
a design of the experiments was not performed to evaluate the reaction conditions, the word optimum used in the manuscript simply refers to the value that yields the maximum conversion at the given reaction conditions. Analysis. The reaction samples were analyzed by a HPLC system consisting of a pump (Waters 501, Wayland, MA), a reverse-phase column (0.39 cm × 25 cm, µBondapak C18), and an injector (Rheodyne 7010, with an injection loop of 250 mm3) equipped with an UV detector (GBC Scientific Equipment LC 1205, Dandenong, Victoria, Australia) at 212 nm. The samples were analyzed by an acetonitrile/water (90:10, v/v) solution at a flow rate of 1 mL/min. For construction of the calibration curve, the ester was chemically synthesized by Fischer’s method, was purified by washing the reaction mixture with water and a saturated NaHCO3
Ind. Eng. Chem. Res., Vol. 43, No. 24, 2004 7699
Figure 2. Effect of enzyme concentration on the esterification reaction in different media with 15 mg of palmitic acid and optimum octanol concentration at the optimum temperature for 6 h with (a) HPL, (b) Lipolase, and (c) Novozym 435 in various supercritical fluids. See Figure 1 for legend.
solution to remove acid, and was further purified by silica gel chromatography. Results and Discussion The synthesis of compounds in supercritical fluids is influenced by a wide variety of parameters such as temperature, density, pressure, enzyme activity, enzyme specificity, and water and substrate concentrations. Influence of Temperature. The effect of temperature on the esterification reaction was first investigated. All of the reactions were investigated by conducting the reactions for 6 h at the concentration of octanol that yielded the maximum conversion of palmitic acid to octyl palmitate, which was determined by conducting the
Figure 3. Kinetics of the esterification reaction in different media with 15 mg of palmitic acid and optimum octanol concentration at the optimum temperature with 5 mg of (a) HPL, (b) Lipolase, and (c) Novozym 435 in various supercritical fluids. See Figure 1 for legend.
reactions at different octanol concentrations, as discussed below. The reactions were conducted with 15 mg of palmitic acid (8.3 mM) and 5 mg of enzymes in a 7-cm3 stainless steel batch reactor. In SCC and SCM, 100 mm3 of octanol (90.1 mM) was used, while in SCE, 60 mm3 of octanol (54 mM) was used for the reactions because these are the amounts of octanol that produce the maximum conversion of palmitic acid to octyl palmitate, as explained below in the section that discusses the influence of the molar ratio of acid to alcohol on the reaction. The influence of temperature on esterification is shown in Figure 1a-c for the synthesis in HPL, Lipolase 100T, and Novozym 435, respectively. The temperature at which the maximum conversion was achieved was around 55 °C for all of the
7700
Ind. Eng. Chem. Res., Vol. 43, No. 24, 2004
Figure 4. Effect of octanol concentration on the esterification reaction in different media with 15 mg of palmitic acid at the optimum temperature for 6 h with 5 mg of (a) HPL, (b) Lipolase, and (c) Novozym 435 in various supercritical fluids. See Figure 1 for legend.
Figure 5. Effect of water addition concentration on the esterification reaction in different media with 15 mg of palmitic acid and optimum octanol concentration at the optimum temperature for 6 h with 5 mg of (a) HPL, (b) Lipolase, and (c) Novozym 435 in various supercritical fluids. See Figure 1 for legend.
enzymes in all of the cases, except in the case of HPLcatalyzed reaction in SCM, where it was 45 °C. Enzyme Loading. The effect of enzyme loading was investigated to determine the minimum amount of enzyme to get the maximum conversion in different supercritical fluids. The effect of enzyme loading was studied by adding various amounts of enzyme to reaction mixtures with the same reactant concentrations as those described previously and was carried out for 6 h. Parts a-c of Figure 2 show the effect of enzyme loading on the conversions. The minimum amount of enzyme ranges from 3 to 5 mg, depending upon the enzyme and solvent used. A further increase in the amount of enzyme did not increase the conversion appreciably. Kinetics. The kinetics of the reaction was studied by incubating the reaction system for different times. As
shown in Figure 3a-c, the conversion did not appreciably increase after 3 h for Novozym 435 catalyzed reactions and after 6 h with the other two enzymes in SCC and SCM. However, for reactions in SCE, there was no appreciable increase in the conversion after 2 h for all of the enzymes. Influence of Acid/Alcohol Molar Ratio. The effect of acid/alcohol molar ratio on the conversion was investigated by adding various amounts of alcohol to 15 mg of palmitic acid (8.3 mM). Parts a-c of Figure 4 show the influence of octanol addition in the conversion of the reactions in different media and with different enzymes. The amounts of alcohol that yielded the maximum conversion were 100, 100, and 60 mm3 in SCM, SCC, and SCE, respectively. At higher octanol concentration, the conversion decreases. This indicates that the de-
Ind. Eng. Chem. Res., Vol. 43, No. 24, 2004 7701
activation of enzyme occurs when octanol present in the reaction system exceeds a certain level. Esterification reactions were conducted in SCE by varying the substrate concentration in Novozym 435 (results not shown). Results indicate that both octanol and palmitic acid deactivate the enzyme. A total of 60 mm3 of octanol and 15 mg of palmitic acid were found to give maximum conversion in SCE. The effect of substrate concentration on the esterification of isopropyl palmitate was investigated, and both palmitic acid and isopropyl alcohol deactivate the reaction when present in excess,12 which is consistent with this study. Influence of Water. Water plays a vital role in noncovalent interactions that allow the enzyme to retain its native conformation. In the complete absence of water, enzymes are not able to maintain an active conformation, thus hindering its ability to act as a catalyst.16,17 However, an excess of water can hinder the synthesis of esters because of the occurrence of hydrolysis. Thus, the amount of water needed is specific to each solvent/substrate/enzyme system. To determine the effect of water on the reaction, all of the reactants and enzymes were put in a desiccator for 12 h to remove water. Before reaction, different amounts of water were added in the system along with other reactants. Parts a-c of Figure 5 show the effect of water addition and initial water content that yields the maximum conversion for the reactions ranging from 1 to 5 mm3 (7.939.7 mM). At higher water content, the conversion decreases sharply. This indicates that the water content that would yield the maximum conversion is dependent on the solvent/substrate/enzyme system. Conclusions The synthesis of octyl palmitate was investigated in SCC, SCM, and SCE using three different enzymes. The effects of temperature, initial water content, alcohol concentration, and enzyme loading on the conversion were determined. Irrespective of the supercritical fluid, the highest conversion was obtained when Novozym 435 (immobilized) was used to catalyze the reaction. Further, the conversion was highest in SCM. There could be several reasons for the increased conversion to octyl palmitate in SCM. It is possible that the solvation of the reactants is high in SCM. However, among the supercritical fluids used, the density of methane was the lowest. This indicates that the conformation of the enzyme may be more favorable in SCM compared to its conformation in other systems. It is, of course, possible that other factors such as the solvent dielectric constant and mass transfer could play a key role in determining why Novozym 435/SCM is the best for conducting this reaction.
Acknowledgment The authors thank the Department of Biotechnology, India, and the Ministry for Human Resources Development for financial assistance. Literature Cited (1) Tramper, J.; Vermue, M. H.; Beeftink, H. H.; Stockar, U. Biocatalysis in non-conventional media. Prog. Biotechnol. 1992, 8, 291. (2) Nakamura, K. Biochemical reactions in supercritical fluids. Trends Biotechnol. 1990, 8, 288. (3) Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M.; Wilke, C. R. Enzyme catalysis in a supercritical fluid. Biotechnol. Lett. 1985, 7, 325. (4) Kamat, S.; Beckman, E. J.; Russell, A. J. Enzyme activity in supercritical fluids. Crit. Rev. Biotechnol. 1995, 15, 41. (5) Mesiano, A. J.; Beckman, E. J.; Russell, A. J. Supercritical Biocatalysis. Chem. Rev. 1999, 99, 623. (6) Breton, L.; Liviero, C. Cosmetic Compositions Containing Cinnamic Acid or Its Derivatives for Assisting Skin Peeling. FR2778560 A1, 1999. (7) Kuwahara, H.; Minematsu, H.; Kawaguchi, T. Adhesive Agents for Medical Use. JP 11104229 A2, 1999. (8) Johnson, R. W.; Fulizi, F. Industrial Fatty Acids and Its Function; China Light Industry Press: Beijing, China, 1992. (9) Opdyke, D. L. J. Monographs on fragrance raw materials. Food Cosmet. Toxicol. 1981, 19, 237. (10) Endo, T. Lubricants for High-Speed Synthetic Fiber Spinning and Lubricant Additives. JP 10008377 A2, 1998 (11) Linko, Y. Y.; Lamsa, M.; Huhtala, A.; Linko, P. Lipase catalyzed transesterification of rapeseed oil and 2-ethyl-1-hexanol. J. Am. Oil Chem. Soc. 1994, 71, 1411. (12) Garcia, T.; Sanchez, N.; Martinez, M.; Aracil, J. Enzymatic synthesis of fatty esters: Part I. kinetic approach. Enzyme Microb. Technol. 1999, 25, 584. (13) Carvalho, B. D. I.; Sampaio, C. D. T.; Barreiros, S. Solvent effects on the catalytic activity of subtilisin suspended in compressed gases. Biotechnol. Bioeng. 1996, 49, 399. (14) Kamat, S.; Critchley, G.; Beckman, E. J.; Russell, A. synthesis of acrylates in organic solvents and supercritical fluids: III. Does carbon dioxide covalently modify enzymes? J. Biotechnol. Bioeng. 1995, 46, 610. (15) Kumar, R.; Madras, G.; Modak, J. M. Enzymatic synthesis of ethyl palmitate in supercritical carbon dioxide. Ind. Eng. Chem. Res. 2004, 43, 1568. (16) Srivastava, S.; Modak, J. M.; Madras, G. Enzymatic synthesis of flavors in supercritical carbon dioxide. Ind. Eng. Chem. Res. 2002, 41, 1940. (17) De, B. K.; Bhattacharyya, D. K.; Bhandu, C. Enzymatic synthesis of fatty alcohol esters by alcoholysis. J. Am. Oil Chem. Soc. 1999, 76, 45.
Received for review May 24, 2004 Revised manuscript received September 2, 2004 Accepted September 11, 2004 IE049551E
