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Phase equilibrium for capsaicin+water+ethanol+supercritical carbon dioxide
Journal of Supercritical Fluids 22 (2002) 87 – 92 www.elsevier.com/locate/supflu
Phase equilibrium for capsaicin+water + ethanol+supercritical carbon dioxide Catarina M.M. Duarte *, Marcelo Crew 1, Teresa Casimiro, Ana Aguiar-Ricardo, Manuel Nunes da Ponte Departamento de Quı´mica, Centro de Quı´mica Fina e Biotecnologia, Faculdade de Cieˆncias e Tecnologia, Uni6ersidade No6a de Lisboa, 2829 -516 Caparica, Lisboa, Portugal Received 21 February 2001; received in revised form 18 July 2001; accepted 17 August 2001
Abstract The possibility of extracting capsaicin with supercritical carbon dioxide from a hydroalcholic mixture containing the alkaloid was investigated. High-pressure phase equilibrium on the quaternary system CO2 + ethanol+water+ capsaicin was measured in order to obtain the separation factor of the natural product from hydro-alcoholic model mixtures. Experiments on phase equilibrium behavior were performed at several pressures (12, 15 and 18 MPa) and temperatures of 40 and 50 °C. The separation factors for three mixtures of different composition— 0.40 water + 0.60 ethanol mass fraction and 0.9 water + 0.10 ethanol mass fraction, containing 0.02% of capsaicin and 0.40 water mass fraction containing 0.04% of capsaicin—are compared. The effect of the water content in the selectivity of the extraction process is discussed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Capsaicin; Alkaloids; Hydroalcoholic mixtures; Vapor– liquid equilibrium; Supercritical fluid extraction
1. Introduction Extraction with supercritical carbon dioxide has found many applications in the processing of natural products [1,2]. Most of these applications involve extraction from solid materials in large high-pressure vessels, in batch or semi-batch mode. Fractionation of liquids in countercurrent * Corresponding author. Tel.: + 351-212948500; fax: + 351-212948385. E-mail address: [email protected] (C.M.M. Duarte). 1 On leave from the Department of Chemistry, University of Nottingham, UK.
extraction columns is a more advantageous process, as it may be performed continuously and using much smaller volumes under pressure. However, in most cases the target substances to be recovered from natural products are trapped in solid material. Fractionation can only be used as a secondary step, after a primary extraction with a liquid solvent. Supercritical carbon dioxide is perceived as a non-toxic solvent. If it is to be used only in a second step of a process, the first extraction should use one of the few other solvents that are also considered suitable for contact with products for human consumption. Hydroalcoholic mixtures
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C.M.M. Duarte et al. / J. of Supercritical Fluids 22 (2002) 87–92
are in this category and they are currently used to extract many different substances from plants. Moreover, phase equilibrium in the ternary system water+ethanol +carbon dioxide has been extensively studied [3]. In order to examine the feasibility of the secondary extraction, the partition coefficient of the substance of interest between high-pressure carbon dioxide and water+ ethanol mixtures must be determined. This work is part of a study designed to provide these data for a series of substances that are commonly extracted from plants, in particular alkaloids. Stahl and collaborators [4] were the first to study the solubility of these substances in supercritical carbon dioxide. Atropine– benzeacetic acid, a-(hydroxylmethyl)-8-methyl-8-azabicyclo [3.2.1] oct-3-yl ester endo (+/ − )-and capsaicin (8-methyl-N-vanillyil-6-nonenamide) are among the most soluble. Atropine was the subject of a previous report [5]. Capsaicin is the ‘hot’ component of paprika and chili peppers and has found numerous applications [6]. It can be extracted from those plants using different solvents, including supercritical carbon dioxide [7] and water+ ethanol mixtures [8]. Zeljko and Steiner [9] reported solubilities of capsaicin in dense CO2 at several experimental conditions. Phase equilibrium in the ternary system water + ethanol+carbon dioxide has been extensively studied [3]. However, the design of the supercritical extraction process to carry out the second stage separation requires a detailed knowledge of the vapor– liquid equilibrium compositions of the quaternary mixtures CO2 +ethanol+water +natural product. The best optimized conditions for the extraction from the solid botanical source with a hydroalcoholic solvent were found to be 0.40 water + 0.60 ethanol mass fraction. After the primary extraction, the composition of the resulting hydroalcoholic mixture can be changed by evaporation. As the solution is evaporated it becomes more viscous. The composition of 0.90 water+ 0.10 ethanol mass fraction corresponds to the limit of acceptable viscosity for the second stage extraction with carbon dioxide in a continuous mode.
High-pressure phase equilibrium experiments on quaternary mixtures CO2 + ethanol+ water+ capsaicin were performed at several pressures (12, 15 and 18 MPa) and at the temperatures of 40 and 50 °C. The separation factors for three mixtures of different composition— 0.40 water+0.60 ethanol mass fraction and 0.90 water+ 0.10 ethanol mass fraction, containing 0.02% of capsaicin and 0.40 water mass fraction containing 0.04% of capsaicin— are compared. The effect of the water content in the selectivity of the second stage extraction process is discussed.
2. Experimental
2.1. Materials The substances used in this work were: 98% pure capsaicin supplied by Sigma [404-86-4] CAS; 99.8% ethanol from Merck; 99.8% methanol from Merck; 99.998 mol% carbon dioxide from Air Liquide. Doubly distilled water was also used.
2.2. Equilibrium determination The phase equilibrium apparatus is built around a sapphire tube with the following dimensions, height, 15 cm; internal diameter, 1.9 cm; external diameter, 3.2 cm. This type of equilibrium cell was described in detail by Pereira et al. [10] and a similar apparatus was recently described by Gourgouillon and Nunes da Ponte [11]. In this work, modifications were introduced in the cell design to allow for an improved resistance to leakage at low pressures, where the unsupported area type seals used on both ends of the sapphire tube might give problems. Fig. 1 shows the modified version of the equilibrium cell. The Teflon seal is extruded against sapphire by means of two metal pieces tightened one against the other. The cell is initially loaded to approximately half of its height with a mixture of water+ethanol+ capsaicin of known composition, and then purged with a flow of pure CO2, at low pressure. The cell is closed, and the CO2 is introduced up to the desired pressure using a manual pressure genera-
C.M.M. Duarte et al. / J. of Supercritical Fluids 22 (2002) 87–92
tor. The pressure inside the sapphire-cylinder is measured with a pressure transducer (Omega PX931-5KSV calibrated between 0 and 343 MPa (precision, 0.1%; accuracy, 0.15%). The temperature is measured with a platinum-resistance (HART 5616 RTD) in thermal contact with sapphire cylinder and connected to a PID controller (HART 2100). The temperature is assumed to be homogeneous inside the insulated air-bath, heated by means of a 150 W resistance connected to the PID controller and a fan. The typical temperature stability during experiments is 90.01 K. At fixed temperature, a typical equilibration time is 1 h. A magnetic bar activated by a magnet performs stirring inside the cell. Samples are withdrawn from both phases through two high-pressure six-port switching valves, located on the top and the bottom of the cell, into sample loops. The gas in the samples is then expanded into calibrated volumes and the amount of CO2 in each sample is calculated from the measurement of the pressure increase at the working temperature. The sample loops are later flushed with methanol to collect the solute precipitated during the large pressure drop that occurred with the expansion.
2.3. Analytical aspects The samples collected were diluted in 10 ml of methanol. The resulting methanolic solutions of capsaicin were analyzed by Supercritical Fluid Chromatography with APCI-MS detection, using
89
the protocol developed by Davidson and collaborators [12]. The measurements were carried out at the Department of Chemistry of the University of Nottingham, in a Gilson SF3 system, with a 821 regulation valve interfacing into a VG Trio 2000 single quadrupole mass spectrometer (VG Biotech, Altrincham, UK). The column was a standard HPLC column with cyano propyl stationary phase, and the eluent phase a 90% CO2 + 10% methanol mixture. Calibration was obtained via use of standard samples.
3. Results and discussion In this work, two water+ ethanol mixtures of different composition, 0.40 water+ 0.60 ethanol mass fraction and 0.90 water+ 0.10 ethanol mass fraction-were used, in order to study the effect of the water content in the selectivity of the extraction process. Capsaicin was added to these mixtures until a composition of 2× 10 − 4 mass ratio was obtained. For the 0.40 water mixture, an additional mixture with 4× 10 − 4 in capsaicin was also prepared. These concentrations are of the order of magnitude of those obtained in hydroalcoholic extracts from plants. Experiments were carried out at pressures of 12, 15 and 18 MPa, and temperatures of 40 and 50 °C. Samples from the vapor (CO2-rich) phase and liquid (water+ethanol-rich) phase were analyzed for capsaicin content. The compositions of
Fig. 1. Seal detail of the high-pressure equilibrium cell.
C.M.M. Duarte et al. / J. of Supercritical Fluids 22 (2002) 87–92
90
Table 1 Phase equilibrium compositions of water, ethanol and CO2, on the quaternary system CO2+ethanol+water+capsaicin T (°C)
p (MPa)
Liquid phase
Solubility in CO2 (×10−6 g/g)
xCO2
ywater
yethanol wt % yCO2
0.02% capsaicin in 40% water+60% ethanol 40 12 37.1 44.0 40 15 37.1 44.0 40 18 37.1 43.9 50 12 37.5 44.5 50 15 37.0 43.9 50 18 36.6 43.3
18.9 18.9 19.0 18.0 19.1 20.1
0.8 0.9 1.1 0.7 1.0 1.3
6.3 7.6 8.8 7.0 8.3 9.6
92.9 91.5 90.1 92.3 90.7 89.1
0.04% capsaicin in 40% water+60% ethanol 40 12 37.1 44.0 40 18 37.1 43.9
18.9 19.0
0.8 1.1
6.3 8.8
92.9 90.1
7.6 18.3
0.02% capsaicin in 90% water+10% ethanol 40 12 87.4 7.7 40 15 86.9 7.6 40 18 86.4 7.6 50 12 88.2 7.7 50 15 87.7 7.7 50 18 87.2 7.6
4.9 5.5 6.0 4.1 4.6 5.2
0.2 0.4 0.5 0.2 0.4 0.6
0.8 1.4 1.9 1.6 1.9 2.1
98.9 98.3 97.6 98.1 97.7 97.3
31.5 31.0 24.0 19.8 12.9 14.4
xwater
xethanol wt. %
Gaseous phase
0.51 0.54 0.69 0.16 0.29 0.57
h ?cap
0.51 0.54 0.69 0.16 0.29 0.57 0.4 0.67 83 754 687 59 292 162
Solubility of capsaicin, expressed in terms of mass of capsaicin per mass of carbon dioxide in the gaseous phase, and the separation factors of capsaicin between the gaseous and liquid phases.
both phases in carbon dioxide, water and ethanol were calculated from mass balances, on the basis of the correlation of Duarte et al. [3] of phase equilibrium data of several authors for the CO2 + water + ethanol system. It was assumed that the presence of capsaicin, due to the very small concentrations in both phases, did not significantly affect the equilibrium ratios for the other components. Mass balances of total capsaicin in the cell (gas + liquid phases) showed that, in the case of the initial mixture richer in water, 0.90 water+ 0.10 ethanol mass fraction, some precipitation of solid capsaicin occurred when CO2 was added to the cell. Carbon dioxide acted as anti solvent upon dissolution in the aqueous mixture. Table 1 summarizes the experimental vapor–
liquid equilibrium data. This table shows the equilibrium compositions of water and ethanol in the liquid and gaseous phases, the solubility of capsaicin-expressed in terms of mass of capsaicin per mass of carbon dioxide in the gas phase-and the separation factors of the alkaloid between the gaseous and liquid phases. These later results are expressed as: h=
(wtcapsaicin/wtwater + ethanol)gas (wtcapsaicin/wtwater + ethanol)liquid
In Figs. 2 and 3, the separation factors for the studied mixtures are plotted as a function of pressure for the all studied mixtures. Fig. 2 shows the separation factors obtained for the mixture of 0.40 water+ 0.60 ethanol mass fraction containing 0.02% of capsaicin, at 40 and
C.M.M. Duarte et al. / J. of Supercritical Fluids 22 (2002) 87–92
Fig. 2. Separations factors of capsaicin between the gaseous and liquid phases, as function of pressure. Results obtained for the mixture of 0.40 water + 0.60 ethanol mass fraction containing 0.02 % of capsaicin, at 40 °C (") and 50 °C ( ), and the results for the mixture containing 0.04 % of capsaicin at 40 °C for the 0.4 water ratio mixture().
50 °C, and the results for the mixture containing 0.04% of capsaicin at 40 °C. The three curves are similar and they show the increasing trend of the separation factor with increasing pressure and decreasing temperature. This behavior follows the density variations of carbon dioxide. In Fig. 3, the separation factors, obtained for the mixture of 0.90 water+ 0.10 ethanol mass fraction containing 0.02% of capsaicin, are plotted as a function of pressure at 40 and 50 °C. The separation factor increases with pressure, between 12 and 15 MPa, and shows a slight trend to decrease when pressure increases up to 18 MPa. This behavior suggests that the extraction pressure of the natural product from the hydroalco-
91
holic mixture should be performed close to 15 MPa. At higher pressures the small decrease of the separation factor might be due to the higher solvent power of carbon dioxide with lose of selectivity. The effect of the water content in the separation factor of capsaicin is obviously very large. When the initial hydroalcoholic mixture is richer in water, lower concentrations in capsaicin are allowed in the equilibrium liquid phase due to the less solute–solvent affinity. Therefore, higher ratios of capsaicin in the gaseous phase are obtained. If the experiments are performed with hydroalcoholic mixtures richer in ethanol, lower separation factors are obtained, showing that ethanol prevents the extraction of capsaicin to the gaseous phase. Ethanol acts as co-solvent to both water and carbon dioxide, increasing capsaicin solubility in liquid and gaseous phases. However, this effect is more significant in the aqueous phase, and lower equilibrium concentrations of capsaicin are attained when extracting with carbon dioxide from ethanol richer mixtures. It may be concluded from these results that capsaicin should be easily extracted from waterrich mixtures and that a low content in ethanol is crucial for the success of processing. This means that supercritical fluid extraction of capsaicin should be performed from mixtures containing the maximum quantity of water allowed in the initial hydroalcholic mixture before the alkaloid starts to precipitate with carbon dioxide.
Acknowledgements
Fig. 3. Separation factor of capsaicin between the gaseous and liquid phases, as function of pressure, obtained for the mixture of 0.90 water +0.10 ethanol mass fraction containing 0.02 % of capsaicin, at 40 °C (") and 50 °C ( ).
The authors are grateful for financial support from the European Commission through Contract ERBFAIRCT962003. Marcelo Crew thanks for financial support from the European Commission through SOCRATES/ERASMUS 98/99. The authors thanks Keith Helliwell and Derek Petri from WR&S (UK), George Davidson from University of Nottingham (UK), Bernard Marty from Microlithe (France) and Erol Drevici from Genex (Germany) for helpful discussions.
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References [1] G. Brunner, Processing of natural materials by supercritical gases, in: M. Perrut, P. Subra (Eds.), Proceedings of the 5th Meeting on Supercritical Fluids. Materials and Natural Products Processing, vol. 2, 1998, p. 413. [2] M. Mukhopadhyay, Natural Extracts Using Supercritical Carbon Dioxide, CRC Press, Boca Raton, FL, 2000 (ISBN: 0849308194). [3] C.M.M. Duarte, A. Aguiar Ricardo, T. Casimiro, N. Ribeiro, M. Nunes da Ponte, Correlation of vapor – liquid equilibrium for carbon dioxide + ethanol+ water at temperatures from 35 °C to 70 °C, Sep. Sci. Technol. 35 (14) (2000) 2187. [4] E. Stahl, W. Schilz, E. Schu¨ tz, E. Willing, A quick method for the microanalytical evaluation of the dissolving power of supercritical gases, Angew. Chem. Int. Ed. Engl. 17 (1978) 731. [5] C.M.M. Duarte, A. Aguiar-Ricardo, M. Nunes da Ponte, K. Dost, G. Davidson, Processing of atropine using supercritical CO2: a preliminary study, in: M. Poliakoff, M.W. George, S.M. Howdle (Eds.), Proceedings of the 6th Meeting on Supercritical Fluids. Chemistry and Materials, 1999, p. 59.
[6] M. Majeed, V. Badmaev, L. Prakash, S. Natarajan, S. Gopinathan, Capsaicin —The Anti-arthritic Phytochemical, Nutriscience Publishers, Piscataway, NJ, 1997 (ISBN: 0-9647856-2-5). [7] U. Nguyen, M. Anstee, D.A. Evans, Extraction and fractionation of spices using supercritical carbon dioxide, in: M. Perrut, P. Subra (Eds.), Proceedings of the 5th Meeting on Supercritical Fluids. Materials and Natural Products Processing, vol. 2, 1998, p. 523. [8] C.M.M. Duarte, M. Crew, A. Aguiar-Ricardo, M. Nunes da Ponte, Processing of capsaicin using supercritical CO2, in: M. Perrut, E. Reverchon (Eds.), Proceedings of the 7th Meeting on Supercritical Fluids. Particle Design-Materials and Natural Products Processing, vol. 2, 2000, p. 681. [9] K. Zeljko, R. Steiner, Solubility of capsaicin in dense CO2, J. Supercrit. Fluids 5 (1992) 251. [10] P.J. Pereira, M. Gonc¸ alves, B. Coto, E. Gomes de Azevedo, M. Nunes da Ponte, Phase equilibria of CO2 + a-tocopherol at temperatures from 292 K to 333 K and pressures up to 26 MPa, Fluid Phase Equilibria 91 (1993) 133. [11] D. Gourgouillon, M. Nunes da Ponte, High pressure phase equilibria for poly(ethyleneglycol)s + CO2: experimental results and modelling, Phys. Chem. Chem. Phys. 1 (1999) 5369. [12] G. Davidson, personal communication, 1999.
Phase equilibrium for capsaicin+water + ethanol+supercritical carbon dioxide Catarina M.M. Duarte *, Marcelo Crew 1, Teresa Casimiro, Ana Aguiar-Ricardo, Manuel Nunes da Ponte Departamento de Quı´mica, Centro de Quı´mica Fina e Biotecnologia, Faculdade de Cieˆncias e Tecnologia, Uni6ersidade No6a de Lisboa, 2829 -516 Caparica, Lisboa, Portugal Received 21 February 2001; received in revised form 18 July 2001; accepted 17 August 2001
Abstract The possibility of extracting capsaicin with supercritical carbon dioxide from a hydroalcholic mixture containing the alkaloid was investigated. High-pressure phase equilibrium on the quaternary system CO2 + ethanol+water+ capsaicin was measured in order to obtain the separation factor of the natural product from hydro-alcoholic model mixtures. Experiments on phase equilibrium behavior were performed at several pressures (12, 15 and 18 MPa) and temperatures of 40 and 50 °C. The separation factors for three mixtures of different composition— 0.40 water + 0.60 ethanol mass fraction and 0.9 water + 0.10 ethanol mass fraction, containing 0.02% of capsaicin and 0.40 water mass fraction containing 0.04% of capsaicin—are compared. The effect of the water content in the selectivity of the extraction process is discussed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Capsaicin; Alkaloids; Hydroalcoholic mixtures; Vapor– liquid equilibrium; Supercritical fluid extraction
1. Introduction Extraction with supercritical carbon dioxide has found many applications in the processing of natural products [1,2]. Most of these applications involve extraction from solid materials in large high-pressure vessels, in batch or semi-batch mode. Fractionation of liquids in countercurrent * Corresponding author. Tel.: + 351-212948500; fax: + 351-212948385. E-mail address: [email protected] (C.M.M. Duarte). 1 On leave from the Department of Chemistry, University of Nottingham, UK.
extraction columns is a more advantageous process, as it may be performed continuously and using much smaller volumes under pressure. However, in most cases the target substances to be recovered from natural products are trapped in solid material. Fractionation can only be used as a secondary step, after a primary extraction with a liquid solvent. Supercritical carbon dioxide is perceived as a non-toxic solvent. If it is to be used only in a second step of a process, the first extraction should use one of the few other solvents that are also considered suitable for contact with products for human consumption. Hydroalcoholic mixtures
0896-8446/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 6 - 8 4 4 6 ( 0 1 ) 0 0 1 1 4 - 0
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C.M.M. Duarte et al. / J. of Supercritical Fluids 22 (2002) 87–92
are in this category and they are currently used to extract many different substances from plants. Moreover, phase equilibrium in the ternary system water+ethanol +carbon dioxide has been extensively studied [3]. In order to examine the feasibility of the secondary extraction, the partition coefficient of the substance of interest between high-pressure carbon dioxide and water+ ethanol mixtures must be determined. This work is part of a study designed to provide these data for a series of substances that are commonly extracted from plants, in particular alkaloids. Stahl and collaborators [4] were the first to study the solubility of these substances in supercritical carbon dioxide. Atropine– benzeacetic acid, a-(hydroxylmethyl)-8-methyl-8-azabicyclo [3.2.1] oct-3-yl ester endo (+/ − )-and capsaicin (8-methyl-N-vanillyil-6-nonenamide) are among the most soluble. Atropine was the subject of a previous report [5]. Capsaicin is the ‘hot’ component of paprika and chili peppers and has found numerous applications [6]. It can be extracted from those plants using different solvents, including supercritical carbon dioxide [7] and water+ ethanol mixtures [8]. Zeljko and Steiner [9] reported solubilities of capsaicin in dense CO2 at several experimental conditions. Phase equilibrium in the ternary system water + ethanol+carbon dioxide has been extensively studied [3]. However, the design of the supercritical extraction process to carry out the second stage separation requires a detailed knowledge of the vapor– liquid equilibrium compositions of the quaternary mixtures CO2 +ethanol+water +natural product. The best optimized conditions for the extraction from the solid botanical source with a hydroalcoholic solvent were found to be 0.40 water + 0.60 ethanol mass fraction. After the primary extraction, the composition of the resulting hydroalcoholic mixture can be changed by evaporation. As the solution is evaporated it becomes more viscous. The composition of 0.90 water+ 0.10 ethanol mass fraction corresponds to the limit of acceptable viscosity for the second stage extraction with carbon dioxide in a continuous mode.
High-pressure phase equilibrium experiments on quaternary mixtures CO2 + ethanol+ water+ capsaicin were performed at several pressures (12, 15 and 18 MPa) and at the temperatures of 40 and 50 °C. The separation factors for three mixtures of different composition— 0.40 water+0.60 ethanol mass fraction and 0.90 water+ 0.10 ethanol mass fraction, containing 0.02% of capsaicin and 0.40 water mass fraction containing 0.04% of capsaicin— are compared. The effect of the water content in the selectivity of the second stage extraction process is discussed.
2. Experimental
2.1. Materials The substances used in this work were: 98% pure capsaicin supplied by Sigma [404-86-4] CAS; 99.8% ethanol from Merck; 99.8% methanol from Merck; 99.998 mol% carbon dioxide from Air Liquide. Doubly distilled water was also used.
2.2. Equilibrium determination The phase equilibrium apparatus is built around a sapphire tube with the following dimensions, height, 15 cm; internal diameter, 1.9 cm; external diameter, 3.2 cm. This type of equilibrium cell was described in detail by Pereira et al. [10] and a similar apparatus was recently described by Gourgouillon and Nunes da Ponte [11]. In this work, modifications were introduced in the cell design to allow for an improved resistance to leakage at low pressures, where the unsupported area type seals used on both ends of the sapphire tube might give problems. Fig. 1 shows the modified version of the equilibrium cell. The Teflon seal is extruded against sapphire by means of two metal pieces tightened one against the other. The cell is initially loaded to approximately half of its height with a mixture of water+ethanol+ capsaicin of known composition, and then purged with a flow of pure CO2, at low pressure. The cell is closed, and the CO2 is introduced up to the desired pressure using a manual pressure genera-
C.M.M. Duarte et al. / J. of Supercritical Fluids 22 (2002) 87–92
tor. The pressure inside the sapphire-cylinder is measured with a pressure transducer (Omega PX931-5KSV calibrated between 0 and 343 MPa (precision, 0.1%; accuracy, 0.15%). The temperature is measured with a platinum-resistance (HART 5616 RTD) in thermal contact with sapphire cylinder and connected to a PID controller (HART 2100). The temperature is assumed to be homogeneous inside the insulated air-bath, heated by means of a 150 W resistance connected to the PID controller and a fan. The typical temperature stability during experiments is 90.01 K. At fixed temperature, a typical equilibration time is 1 h. A magnetic bar activated by a magnet performs stirring inside the cell. Samples are withdrawn from both phases through two high-pressure six-port switching valves, located on the top and the bottom of the cell, into sample loops. The gas in the samples is then expanded into calibrated volumes and the amount of CO2 in each sample is calculated from the measurement of the pressure increase at the working temperature. The sample loops are later flushed with methanol to collect the solute precipitated during the large pressure drop that occurred with the expansion.
2.3. Analytical aspects The samples collected were diluted in 10 ml of methanol. The resulting methanolic solutions of capsaicin were analyzed by Supercritical Fluid Chromatography with APCI-MS detection, using
89
the protocol developed by Davidson and collaborators [12]. The measurements were carried out at the Department of Chemistry of the University of Nottingham, in a Gilson SF3 system, with a 821 regulation valve interfacing into a VG Trio 2000 single quadrupole mass spectrometer (VG Biotech, Altrincham, UK). The column was a standard HPLC column with cyano propyl stationary phase, and the eluent phase a 90% CO2 + 10% methanol mixture. Calibration was obtained via use of standard samples.
3. Results and discussion In this work, two water+ ethanol mixtures of different composition, 0.40 water+ 0.60 ethanol mass fraction and 0.90 water+ 0.10 ethanol mass fraction-were used, in order to study the effect of the water content in the selectivity of the extraction process. Capsaicin was added to these mixtures until a composition of 2× 10 − 4 mass ratio was obtained. For the 0.40 water mixture, an additional mixture with 4× 10 − 4 in capsaicin was also prepared. These concentrations are of the order of magnitude of those obtained in hydroalcoholic extracts from plants. Experiments were carried out at pressures of 12, 15 and 18 MPa, and temperatures of 40 and 50 °C. Samples from the vapor (CO2-rich) phase and liquid (water+ethanol-rich) phase were analyzed for capsaicin content. The compositions of
Fig. 1. Seal detail of the high-pressure equilibrium cell.
C.M.M. Duarte et al. / J. of Supercritical Fluids 22 (2002) 87–92
90
Table 1 Phase equilibrium compositions of water, ethanol and CO2, on the quaternary system CO2+ethanol+water+capsaicin T (°C)
p (MPa)
Liquid phase
Solubility in CO2 (×10−6 g/g)
xCO2
ywater
yethanol wt % yCO2
0.02% capsaicin in 40% water+60% ethanol 40 12 37.1 44.0 40 15 37.1 44.0 40 18 37.1 43.9 50 12 37.5 44.5 50 15 37.0 43.9 50 18 36.6 43.3
18.9 18.9 19.0 18.0 19.1 20.1
0.8 0.9 1.1 0.7 1.0 1.3
6.3 7.6 8.8 7.0 8.3 9.6
92.9 91.5 90.1 92.3 90.7 89.1
0.04% capsaicin in 40% water+60% ethanol 40 12 37.1 44.0 40 18 37.1 43.9
18.9 19.0
0.8 1.1
6.3 8.8
92.9 90.1
7.6 18.3
0.02% capsaicin in 90% water+10% ethanol 40 12 87.4 7.7 40 15 86.9 7.6 40 18 86.4 7.6 50 12 88.2 7.7 50 15 87.7 7.7 50 18 87.2 7.6
4.9 5.5 6.0 4.1 4.6 5.2
0.2 0.4 0.5 0.2 0.4 0.6
0.8 1.4 1.9 1.6 1.9 2.1
98.9 98.3 97.6 98.1 97.7 97.3
31.5 31.0 24.0 19.8 12.9 14.4
xwater
xethanol wt. %
Gaseous phase
0.51 0.54 0.69 0.16 0.29 0.57
h ?cap
0.51 0.54 0.69 0.16 0.29 0.57 0.4 0.67 83 754 687 59 292 162
Solubility of capsaicin, expressed in terms of mass of capsaicin per mass of carbon dioxide in the gaseous phase, and the separation factors of capsaicin between the gaseous and liquid phases.
both phases in carbon dioxide, water and ethanol were calculated from mass balances, on the basis of the correlation of Duarte et al. [3] of phase equilibrium data of several authors for the CO2 + water + ethanol system. It was assumed that the presence of capsaicin, due to the very small concentrations in both phases, did not significantly affect the equilibrium ratios for the other components. Mass balances of total capsaicin in the cell (gas + liquid phases) showed that, in the case of the initial mixture richer in water, 0.90 water+ 0.10 ethanol mass fraction, some precipitation of solid capsaicin occurred when CO2 was added to the cell. Carbon dioxide acted as anti solvent upon dissolution in the aqueous mixture. Table 1 summarizes the experimental vapor–
liquid equilibrium data. This table shows the equilibrium compositions of water and ethanol in the liquid and gaseous phases, the solubility of capsaicin-expressed in terms of mass of capsaicin per mass of carbon dioxide in the gas phase-and the separation factors of the alkaloid between the gaseous and liquid phases. These later results are expressed as: h=
(wtcapsaicin/wtwater + ethanol)gas (wtcapsaicin/wtwater + ethanol)liquid
In Figs. 2 and 3, the separation factors for the studied mixtures are plotted as a function of pressure for the all studied mixtures. Fig. 2 shows the separation factors obtained for the mixture of 0.40 water+ 0.60 ethanol mass fraction containing 0.02% of capsaicin, at 40 and
C.M.M. Duarte et al. / J. of Supercritical Fluids 22 (2002) 87–92
Fig. 2. Separations factors of capsaicin between the gaseous and liquid phases, as function of pressure. Results obtained for the mixture of 0.40 water + 0.60 ethanol mass fraction containing 0.02 % of capsaicin, at 40 °C (") and 50 °C ( ), and the results for the mixture containing 0.04 % of capsaicin at 40 °C for the 0.4 water ratio mixture().
50 °C, and the results for the mixture containing 0.04% of capsaicin at 40 °C. The three curves are similar and they show the increasing trend of the separation factor with increasing pressure and decreasing temperature. This behavior follows the density variations of carbon dioxide. In Fig. 3, the separation factors, obtained for the mixture of 0.90 water+ 0.10 ethanol mass fraction containing 0.02% of capsaicin, are plotted as a function of pressure at 40 and 50 °C. The separation factor increases with pressure, between 12 and 15 MPa, and shows a slight trend to decrease when pressure increases up to 18 MPa. This behavior suggests that the extraction pressure of the natural product from the hydroalco-
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holic mixture should be performed close to 15 MPa. At higher pressures the small decrease of the separation factor might be due to the higher solvent power of carbon dioxide with lose of selectivity. The effect of the water content in the separation factor of capsaicin is obviously very large. When the initial hydroalcoholic mixture is richer in water, lower concentrations in capsaicin are allowed in the equilibrium liquid phase due to the less solute–solvent affinity. Therefore, higher ratios of capsaicin in the gaseous phase are obtained. If the experiments are performed with hydroalcoholic mixtures richer in ethanol, lower separation factors are obtained, showing that ethanol prevents the extraction of capsaicin to the gaseous phase. Ethanol acts as co-solvent to both water and carbon dioxide, increasing capsaicin solubility in liquid and gaseous phases. However, this effect is more significant in the aqueous phase, and lower equilibrium concentrations of capsaicin are attained when extracting with carbon dioxide from ethanol richer mixtures. It may be concluded from these results that capsaicin should be easily extracted from waterrich mixtures and that a low content in ethanol is crucial for the success of processing. This means that supercritical fluid extraction of capsaicin should be performed from mixtures containing the maximum quantity of water allowed in the initial hydroalcholic mixture before the alkaloid starts to precipitate with carbon dioxide.
Acknowledgements
Fig. 3. Separation factor of capsaicin between the gaseous and liquid phases, as function of pressure, obtained for the mixture of 0.90 water +0.10 ethanol mass fraction containing 0.02 % of capsaicin, at 40 °C (") and 50 °C ( ).
The authors are grateful for financial support from the European Commission through Contract ERBFAIRCT962003. Marcelo Crew thanks for financial support from the European Commission through SOCRATES/ERASMUS 98/99. The authors thanks Keith Helliwell and Derek Petri from WR&S (UK), George Davidson from University of Nottingham (UK), Bernard Marty from Microlithe (France) and Erol Drevici from Genex (Germany) for helpful discussions.
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References [1] G. Brunner, Processing of natural materials by supercritical gases, in: M. Perrut, P. Subra (Eds.), Proceedings of the 5th Meeting on Supercritical Fluids. Materials and Natural Products Processing, vol. 2, 1998, p. 413. [2] M. Mukhopadhyay, Natural Extracts Using Supercritical Carbon Dioxide, CRC Press, Boca Raton, FL, 2000 (ISBN: 0849308194). [3] C.M.M. Duarte, A. Aguiar Ricardo, T. Casimiro, N. Ribeiro, M. Nunes da Ponte, Correlation of vapor – liquid equilibrium for carbon dioxide + ethanol+ water at temperatures from 35 °C to 70 °C, Sep. Sci. Technol. 35 (14) (2000) 2187. [4] E. Stahl, W. Schilz, E. Schu¨ tz, E. Willing, A quick method for the microanalytical evaluation of the dissolving power of supercritical gases, Angew. Chem. Int. Ed. Engl. 17 (1978) 731. [5] C.M.M. Duarte, A. Aguiar-Ricardo, M. Nunes da Ponte, K. Dost, G. Davidson, Processing of atropine using supercritical CO2: a preliminary study, in: M. Poliakoff, M.W. George, S.M. Howdle (Eds.), Proceedings of the 6th Meeting on Supercritical Fluids. Chemistry and Materials, 1999, p. 59.
[6] M. Majeed, V. Badmaev, L. Prakash, S. Natarajan, S. Gopinathan, Capsaicin —The Anti-arthritic Phytochemical, Nutriscience Publishers, Piscataway, NJ, 1997 (ISBN: 0-9647856-2-5). [7] U. Nguyen, M. Anstee, D.A. Evans, Extraction and fractionation of spices using supercritical carbon dioxide, in: M. Perrut, P. Subra (Eds.), Proceedings of the 5th Meeting on Supercritical Fluids. Materials and Natural Products Processing, vol. 2, 1998, p. 523. [8] C.M.M. Duarte, M. Crew, A. Aguiar-Ricardo, M. Nunes da Ponte, Processing of capsaicin using supercritical CO2, in: M. Perrut, E. Reverchon (Eds.), Proceedings of the 7th Meeting on Supercritical Fluids. Particle Design-Materials and Natural Products Processing, vol. 2, 2000, p. 681. [9] K. Zeljko, R. Steiner, Solubility of capsaicin in dense CO2, J. Supercrit. Fluids 5 (1992) 251. [10] P.J. Pereira, M. Gonc¸ alves, B. Coto, E. Gomes de Azevedo, M. Nunes da Ponte, Phase equilibria of CO2 + a-tocopherol at temperatures from 292 K to 333 K and pressures up to 26 MPa, Fluid Phase Equilibria 91 (1993) 133. [11] D. Gourgouillon, M. Nunes da Ponte, High pressure phase equilibria for poly(ethyleneglycol)s + CO2: experimental results and modelling, Phys. Chem. Chem. Phys. 1 (1999) 5369. [12] G. Davidson, personal communication, 1999.