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Continuous subcritical water extraction of medicinal plant essential oil: comparison with conventional techniques
Talanta 51 (2000) 1179 – 1185 www.elsevier.com/locate/talanta
Continuous subcritical water extraction of medicinal plant essential oil: comparison with conventional techniques L. Ga´miz-Gracia, M.D. Luque de Castro * Analytical Chemistry Di6ision, Faculty of Sciences, Uni6ersity of Co´rdoba, E-14004 Co´rdoba, Spain Received 13 October 1999; received in revised form 29 December 1999; accepted 29 December 1999
Abstract A subcritical extractor equipped with a three-way inlet valve and an on/off outlet valve has been used for performing subcritical water extractions (SWE) in a continuous manner for the isolation of the essential oil of fennel, a medicinal plant. The target compounds were removed from the aqueous extract by a single extraction with 5 ml hexane, determined by gas-chromatography-flame ionization (GC-FID) and identified by mass spectrometry (MS). The proposed extraction method has been compared with both hydrodistillation and dichloromethane manual extraction. Better results have been obtained with the proposed method in terms of rapidity, efficiency, cleanliness and possibility of manipulating the composition of the extract. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Subcritical water; Gas chromatography; Essential oil; Medicinal plant
1. Introduction Fennel (Foeniculum 6ulgare) has been used traditionally in medicine for the treatment of several stomach affections (as aerophagia or diarrhea) and obesity. There are also commercial pharmaceuticals with formula based on fennel essential oil. The essential oil from plants has usually been isolated by either steam distillation [1 – 3] or solvent extraction [4 – 6]. These techniques present some shortcomings, namely losses of volatile compounds, low extraction efficiency, long extraction * Corresponding author. Tel.: +34-957-218614; fax: + 34957-218606. E-mail address: [email protected] (M.D. Luque de Castro)
time, degradation of unsaturated compounds and toxic solvent residue, that make mandatory the use of alternative techniques for the extraction of essential oils [7]. Continuous subcritical water extraction is a technique based on the use of water as extractant, at temperatures between 100 and 374°C and pressure high enough to maintain the liquid state. This technique is emerging as a powerful alternative for solid sample extraction [8]. Its use in the field of essences is recent and very promising [9,10] and it appears as a useful alternative to conventional and supercritical CO2 extractions [11]. The aim of this work was to develop a method
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L. Ga´miz-Gracia, M.D. Luque de Castro / Talanta 51 (2000) 1179–1185
for the continuous subcritical water extraction of medicinal essential oils, and compare the results with those obtained by conventional techniques, in order to introduce this advantageous alternative in the pharmaceutical field.
2. Experimental
2.1. Instruments and apparatus The subcritical water extractions were performed using the following assembly: a Shimadzu LC10AD high pressure pump was used to propel the extractant water through the system. The extractor, described elsewhere [7] (a prototype designed and patented by Salvador and Merchan [12]) consisted of a stainless steel cylindrical extraction chamber (150×11 mm id, 14 ml internal volume) and it was closed with screws at both ends in order to permit the circulation of the leaching fluid through it. The screw caps also contained stainless steel filter plates (2 mm in thickness and 1/4 in id) to ensure that the plant material remained in the extraction chamber. This chamber, together with a stainless steel preheater, was located in an oven, designed to work at up to 300°C and its temperature was controlled using a Toho TC-22 temperature controller. A loop made from a 1 m length stainless steel tubing and cooled with water at room temperature, was used to cool the fluid carrying out from the oven to a temperature close to 25°C, thus avoiding losses of volatile components caused by the hot water. A simple laboratory quickfit apparatus which contained a 2000 ml steam generator flask, a distilling flask, a condenser and a receiving vessel was used to perform the hydrodistillation. The analyses of the extracts were all performed by using a Varian Star 3400 gas chromatograph equipped with a SGL-5 fused silica column (25 m × 0.25 mm id, 0.25 mm film thickness) and a flame ionization detector (FID). Finally, a Fison VG Autospec (Micromass Instruments) mass spectrometer was used to identify the compounds in the extracts.
2.2. Reagents Fennel (F. 6ulgare) was collected in the South of Spain (Co´rdoba). A stock standard solution of 5200 mg ml − 1 of nonane (Sigma, St Louis, MO) in HPLC grade hexane (Scharlau, Barcelona, Spain) was prepared. For the liquid–liquid extraction step of the aqueous extracts NaCl, Na2SO4 (Merck, Darmstadt, Germany) and HPLC grade hexane were used as demulsifier, drying agent and extractant, respectively. Bidistilled degassed water purified through a Milli-Q deionizing unit (Millipore) and dichloromethane (Prolabo, France) were used as extractants. All the gases were of 95% purity or higher (Carburos Meta´licos, Barcelona, Spain).
2.3. Sample preparation Fennel was stored at 4°C until analysis. A 4.0-g sample was cut immediately prior to subcritical water, hydrodistillation or organic solvent extraction, in order to avoid losses of volatile components.
2.4. Procedure 2.4.1. Subcritical water extraction (SWE) The cell was filled with fennel cut in small pieces and weighed (4.0-g), and glass wool plugs were inserted in both ends of the cell to prevent the frit from being plugged. After assembling the extraction cell in the oven, this was brought up to the working temperature (150°C) and pressurised with ca. 50 bar of water by opening the inlet needle valve from the pump. The valve was then closed and static extraction was developed for 30 min. The inlet and outlet valves were then opened, water was pumped through the chamber at a flow rate of 2 ml min − 1 and the extract was collected in a vial at room temperature. For kinetic experiments, the vial was replaced at pre-set intervals of 5 min (10 ml each). 2.4.2. Hydrodistillation A 4.0-g portion of fennel was subjected to hydrodistillation in the assembly described above. The steam generator flask was filled with 350 ml
L. Ga´miz-Gracia, M.D. Luque de Castro / Talanta 51 (2000) 1179–1185
of Milli-Q water and heated with a heating mantle. As the water vaporized, the steam passed to the distillation flask containing the fennel. The vapour then passed through the cooled tube where it condensed. The distillate (94 ml) was finally collected into the receiving flask after 4 h distillation.
2.4.3. Liquid– liquid extraction After subcritical and hydrodistillation extractions, a liquid – liquid extraction step was performed, in which volumes of 5 ml of hexane and 65 ml of nonane stock solution (used as extractant and internal standard, respectively) were added to each extract in a separatory funnel, and ca. 0.5 g of NaCl was added to facilitate the breaking of the emulsion. The organic extract was then dried with anhydrous Na2SO4. 2.4.4. Dichloromethane extraction A 4.0-g portion of fennel was placed in a glass screw-top vial and 130 ml of internal standard and 10 ml of dichloromethane were added to the sample. The extraction was performed on a shaker for 24 h [13]. 2.4.5. Chromatographic separation and detection Aliquots of 2 ml of hexane after either SWE or hydrodistillation plus liquid – liquid extraction in Table 1 Optimisation of variables Variable SWE Temperature (°C) Pressure (bar) Flow rate Amount of sample (g)
Range studied
50–200 – 0.5–3.0 2.0–5.0
Gas chromatography Carrier Injection volume (ml) Column Split ratio 1:30/1:6 Carrier flow rate (ml min−1) Temperature gradient (°C min−1) Ramp 1 1–5 Ramp 2 1–5
Optimum value
150 20 2.0 4.0 Helium 2 SGL-5 1:6 1.0
1 5
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both cases, and of 2 ml of dichloromethane from the manual extraction, were injected into an SGL5 fused silica column (25 m×0.25 mm id, 0.25 mm film thickness). The carrier gas (helium) was delivered at a flow rate of 1.0 ml min − 1. The injector and detector temperatures were 250 and 300°C, respectively. The oven temperature was 55°C for 2 min, then increased to 75°C at a rate of 1°C min − 1, kept for 1 min, and finally increased to 250°C at a rate of 5°C min − 1. The split ratio was 1:6.
2.4.6. Gas chromatography-mass spectrometry identification Fractions of the hexane extracts obtained by subcritical fluid extraction and hydrodistillation were collected in order to identify the components by gas chromatography-mass spectrometry. Mass spectra (electron impact) of the compounds were obtained using a Fison VG Autospec (70 eV) by direct injection of 2 ml of the extract into the ionization chamber at a temperature of 250°C.
3. Results and discussion A static-dynamic approach was tested for the isolation of fennel oil and then compared with conventional techniques such as hydrodistillation and organic–solvent extraction. A static extraction period (for enhancing sample–extractant contact thus favoring the attainment of the partition equilibrium) was carried out, followed by a dynamic extraction period in which fresh extractant passed continuously through the extraction chamber, thus displacing the equilibrium to quantitativeness.
3.1. Optimisation of 6ariables The variables affecting the subcritical extraction were studied in order to maximise the yield of essential oil in as short a time as possible. With this aim the univariate method was used. The static period was kept constant at a value of 5 min. The optimum values found for the variables are shown in Table 1.
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Fig. 1. Effect of the temperature in the main components of essential oil from fennel.
Fig. 2. Effect of the flow rate in the main components of essential oil from fennel. IS= internal standard.
The temperature of the extraction chamber was the key variable affecting continuous subcritical extraction. The yield increased with temperature up to 150°C. At 175°C, the yield of most of the monoterpene compounds increased, but decreased for the oxygenated compounds, probably due to their degradation (Fig. 1). For temperatures higher than 175°C, a strong burning smell of the extract appeared. A value of 150°C was selected as optimum. The pressure, as demonstrated in previous works [10], had little influence on the yield of essential oil, as long as the extractant water was under liquid state. Thus, a minimum pressure of 20 bar was maintained throughout all extractions. The flow rate was studied in the interval 0.5–3.0 ml min − 1. Increased flow rates enhanced the extraction of most of the monoterpene compounds, while in the case of oxygenated compounds the extraction rate was maximum at 2 ml min − 1, as can
be seen in Fig. 2. A flow rate of 2 ml min − 1 was selected for further experiments. The influence of cutting or grounding the sample was also studied. When the sample was ground, the yield of the essential oil decreased, probably due to losses of volatiles during preparation. Thus, for further experiments, the sample was just cut into small pieces (ca. 1 cm in length). The amount of sample was also studied in the interval 2–5 g (the maximum capacity of the cell), and 4 g was selected as it provided signals high enough. In the manual liquid–liquid extraction, the number of extraction steps was studied by performing a second 5 ml hexane extraction to ensure that no compounds remained in the aqueous phase after the first extraction. Only small peaks of the majority compounds were obtained after injection of the second extract, so a single 5 ml hexane extraction was carried out in subsequent experiments.
L. Ga´miz-Gracia, M.D. Luque de Castro / Talanta 51 (2000) 1179–1185
3.2. Effect of the static extraction time on the o6erall extraction kinetics The influence of the static extraction time on both the extraction kinetics and composition of the oil was studied by performing extractions with subcritical water consisting of a static extraction step of 0, 5, 15 and 30 min followed by 20 min of dynamic extraction (at 2 ml
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min − 1) during which the vial was replaced every 5 min (ca. 10 ml). As can be seen in Fig. 3, increased static extraction times enhance the extraction of most of the compounds. It can also be established that for the monoterpene compounds, 20 ml is enough to complete the extraction, while in the case of the oxygenated ones, at least 40 ml of subcritical water is required.
Fig. 3. Effect of the static extraction time in the main components of essential oil from fennel.
Fig. 4. Chromatograms of the SWE (a), dichloromethane extraction (b) and hydrodistillation (c) of fennel essential oil. Peaks identification: (1) a-pinene; (2) b-pinene; (3) b-myrcene; (4) a-phelandrene; (5) limonene; (6) unknown; (7) camphor; (8) terpinen-4ol; (9) linalyl propanoate; (10) unknown; (11) anethol; (12): copaene.
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Table 2 Comparison of area compound/internal standard ratio for GC of extracts obtained after hydrodistillation, dichloromethane extraction and SWE Compound
Hydrodistillationa
Dichloromethane extractionb
SWEa
a-Pinene b-Pinene b-Myrcene a-phelandrene Limonene Unknown Camphor Terpinen-4-ol Linalyl propanoate Unknown Anethol Copaene
0.265 0.067 0.569 2.397 5.858 25.196 0.697 – 0.154 5.857 25.214 –
0.499 0.140 0.667 2.580 5.576 3.529 – – – 1.272 5.887 –
4.889 0.887 5.469 18.689 51.598 67.732 1.503 0.225 0.781 23.613 68.054 0.234
a b
Extracted in 5 ml of hexane. In 10 ml of dichloromethane.
3.3. Manipulation of the oil composition As a consequence of the relationship between the extraction conditions and qualitative composition of the extract, the latter can be manipulated by changing the conditions of the extraction (temperature, flow rate and static extraction time).
3.4. Precision of the SWE/chromatographic method The precision (expressed as RSD) of both the chromatographic step and the overall process (extraction and detection) was studied separately. The precision of the chromatographic step was calculated by injecting one of the organic extracts from SWE of 4.0 g of fennel seven times. The average RSD for the eight major components was 3.26%. For studying the precision of the overall process, 5 portions of 4.0 g of fennel were subjected to SWE under the same conditions and the hexane layer obtained after liquid – liquid extraction was injected in the GC. The resulting RSD for the eight major components was 6.77%.
3.5. Comparison of methods The SWE method (30 min of static extraction
followed by 20 min of dynamic extraction at 2 ml min − 1) has been compared with hydrodistillation and manual dichloromethane extraction as conventional alternatives in terms of rapidity, efficiency, cleanliness and possibility of manipulating the composition of the extract.
3.5.1. Rapidity The SWE is clearly quicker than its conventional counterparts. The extraction takes 50 min, whilst 4 and 24 h were required by hydrodistillation and manual dichloromethane extraction, respectively. 3.5.2. Efficiency The most efficient method in terms of quantitative composition of the extract seems to be SWE, as can be seen in the chromatograms of the extracts obtained by the three methods for the same amount of sample, and the values of the area ratios for the main component and the internal standard (Fig. 4 and Table 2, respectively). It must be taken into account that in the case of SWE and hydrodistillation the final extracts was obtained in 5 ml of hexane, and in the case of manual dichloromethane extraction, the final volume was 10 ml.
L. Ga´miz-Gracia, M.D. Luque de Castro / Talanta 51 (2000) 1179–1185
3.5.3. Cleanliness SWE is a very clean method, which avoids both the use of large amounts of organic solvents and residue generation. 3.5.4. Possibility of manipulating the composition of the extract The SWE allows the manipulation of the composition of the extract by changing extraction parameters such as temperature, flow rate and/or static extraction time. This is unfeasible with the conventional methods.
4. Conclusion The continuous SWE of essential oil in medicinal plants (fennel) has been studied. The method offers important advantages over traditional alternatives, namely: shorter extraction times (50 min against 4 h for hydrodistillation and 24 for manual dichloromethane extraction); cost (energy cost is fairly higher for performing hydrodistillation than that required for reaching subcritical conditions); cleaner features (as no a great volume of organic solvent is involved); and the possibility of manipulating the composition of the oil by changing the parameters of the extraction (temperature, flow rate and static extraction time). The method contributes to the automation of pharmaceutical industry.
.
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Acknowledgements The Spanish Comisio´n Interministerial de Ciencia y Tecnologı´a (CICyT) is thanked for financial support (Project no. PB96-1265). The Department of Organic Chemistry (Faculty of Sciences, University of Co´rdoba) is thanked for realising the GC-MS identifications. References [1] J.C. Chalchat, R.P. Garry, A. Michet, Flavour Fragr. J. 6 (1991) 189. [2] M.D. Guille´n, N. Cabo, J. Murillo, J. Sci. Food Agric. 70 (1996) 359. [3] H.L. De-Pooter, E.A. Aboutabl, A.O. El-Shabrawy, Flavour Fragr. J. 10 (1995) 63. [4] R.K. Verma, G.C. Uniyal, M.M. Gupta, Indian J. Pharm. Sci. 52 (1990) 276. [5] M. Taverna, A.E. Baillet, D. Baylock, J. Ass. Off. Anal. Chem. 73 (1990) 206. [6] S.K. Volkov, E.I. Grodnitskaya, J. Chromatogr. 769 (1997) 275. [7] M.D. Luque de Castro, M.M. Jime´nez Carmona, V. Ferna´ndez Pe´rez, Trends Anal. Chem. 18 (1999) 708. [8] M.D. Luque de Castro, M.M. Jime´nez Carmona, Trends Anal. Chem. 17 (1998) 441. [9] A. Basile, M.M. Jime´nez Carmona, A. Clifford, J. Agric. Food Chem. 46 (1998) 5205. [10] M.M. Jime´nez Carmona, J.L. Ubera, M.D. Luque de Castro, J. Chromatogr. 855 (1999) 625. [11] S.H.R. Al-Saidi, A. Clifford, A. Basile, Fresenius J. Anal. Chem. (1999) submitted for publication. [12] USA Patent no. 5, 400, 642, 1995. [13] J.M. Bowman, M.S. Braxton, M.A. Churchill, J.D. Hellie, S.J. Starrett, G.J. Ellis, S.D. Ensley, S.J. Maness, C.D. Meyer, J.R. Seller, Y. Hua, R.S. Woosley, D.J. Butcher, Microchem. J. 56 (1997) 10.
Continuous subcritical water extraction of medicinal plant essential oil: comparison with conventional techniques L. Ga´miz-Gracia, M.D. Luque de Castro * Analytical Chemistry Di6ision, Faculty of Sciences, Uni6ersity of Co´rdoba, E-14004 Co´rdoba, Spain Received 13 October 1999; received in revised form 29 December 1999; accepted 29 December 1999
Abstract A subcritical extractor equipped with a three-way inlet valve and an on/off outlet valve has been used for performing subcritical water extractions (SWE) in a continuous manner for the isolation of the essential oil of fennel, a medicinal plant. The target compounds were removed from the aqueous extract by a single extraction with 5 ml hexane, determined by gas-chromatography-flame ionization (GC-FID) and identified by mass spectrometry (MS). The proposed extraction method has been compared with both hydrodistillation and dichloromethane manual extraction. Better results have been obtained with the proposed method in terms of rapidity, efficiency, cleanliness and possibility of manipulating the composition of the extract. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Subcritical water; Gas chromatography; Essential oil; Medicinal plant
1. Introduction Fennel (Foeniculum 6ulgare) has been used traditionally in medicine for the treatment of several stomach affections (as aerophagia or diarrhea) and obesity. There are also commercial pharmaceuticals with formula based on fennel essential oil. The essential oil from plants has usually been isolated by either steam distillation [1 – 3] or solvent extraction [4 – 6]. These techniques present some shortcomings, namely losses of volatile compounds, low extraction efficiency, long extraction * Corresponding author. Tel.: +34-957-218614; fax: + 34957-218606. E-mail address: [email protected] (M.D. Luque de Castro)
time, degradation of unsaturated compounds and toxic solvent residue, that make mandatory the use of alternative techniques for the extraction of essential oils [7]. Continuous subcritical water extraction is a technique based on the use of water as extractant, at temperatures between 100 and 374°C and pressure high enough to maintain the liquid state. This technique is emerging as a powerful alternative for solid sample extraction [8]. Its use in the field of essences is recent and very promising [9,10] and it appears as a useful alternative to conventional and supercritical CO2 extractions [11]. The aim of this work was to develop a method
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L. Ga´miz-Gracia, M.D. Luque de Castro / Talanta 51 (2000) 1179–1185
for the continuous subcritical water extraction of medicinal essential oils, and compare the results with those obtained by conventional techniques, in order to introduce this advantageous alternative in the pharmaceutical field.
2. Experimental
2.1. Instruments and apparatus The subcritical water extractions were performed using the following assembly: a Shimadzu LC10AD high pressure pump was used to propel the extractant water through the system. The extractor, described elsewhere [7] (a prototype designed and patented by Salvador and Merchan [12]) consisted of a stainless steel cylindrical extraction chamber (150×11 mm id, 14 ml internal volume) and it was closed with screws at both ends in order to permit the circulation of the leaching fluid through it. The screw caps also contained stainless steel filter plates (2 mm in thickness and 1/4 in id) to ensure that the plant material remained in the extraction chamber. This chamber, together with a stainless steel preheater, was located in an oven, designed to work at up to 300°C and its temperature was controlled using a Toho TC-22 temperature controller. A loop made from a 1 m length stainless steel tubing and cooled with water at room temperature, was used to cool the fluid carrying out from the oven to a temperature close to 25°C, thus avoiding losses of volatile components caused by the hot water. A simple laboratory quickfit apparatus which contained a 2000 ml steam generator flask, a distilling flask, a condenser and a receiving vessel was used to perform the hydrodistillation. The analyses of the extracts were all performed by using a Varian Star 3400 gas chromatograph equipped with a SGL-5 fused silica column (25 m × 0.25 mm id, 0.25 mm film thickness) and a flame ionization detector (FID). Finally, a Fison VG Autospec (Micromass Instruments) mass spectrometer was used to identify the compounds in the extracts.
2.2. Reagents Fennel (F. 6ulgare) was collected in the South of Spain (Co´rdoba). A stock standard solution of 5200 mg ml − 1 of nonane (Sigma, St Louis, MO) in HPLC grade hexane (Scharlau, Barcelona, Spain) was prepared. For the liquid–liquid extraction step of the aqueous extracts NaCl, Na2SO4 (Merck, Darmstadt, Germany) and HPLC grade hexane were used as demulsifier, drying agent and extractant, respectively. Bidistilled degassed water purified through a Milli-Q deionizing unit (Millipore) and dichloromethane (Prolabo, France) were used as extractants. All the gases were of 95% purity or higher (Carburos Meta´licos, Barcelona, Spain).
2.3. Sample preparation Fennel was stored at 4°C until analysis. A 4.0-g sample was cut immediately prior to subcritical water, hydrodistillation or organic solvent extraction, in order to avoid losses of volatile components.
2.4. Procedure 2.4.1. Subcritical water extraction (SWE) The cell was filled with fennel cut in small pieces and weighed (4.0-g), and glass wool plugs were inserted in both ends of the cell to prevent the frit from being plugged. After assembling the extraction cell in the oven, this was brought up to the working temperature (150°C) and pressurised with ca. 50 bar of water by opening the inlet needle valve from the pump. The valve was then closed and static extraction was developed for 30 min. The inlet and outlet valves were then opened, water was pumped through the chamber at a flow rate of 2 ml min − 1 and the extract was collected in a vial at room temperature. For kinetic experiments, the vial was replaced at pre-set intervals of 5 min (10 ml each). 2.4.2. Hydrodistillation A 4.0-g portion of fennel was subjected to hydrodistillation in the assembly described above. The steam generator flask was filled with 350 ml
L. Ga´miz-Gracia, M.D. Luque de Castro / Talanta 51 (2000) 1179–1185
of Milli-Q water and heated with a heating mantle. As the water vaporized, the steam passed to the distillation flask containing the fennel. The vapour then passed through the cooled tube where it condensed. The distillate (94 ml) was finally collected into the receiving flask after 4 h distillation.
2.4.3. Liquid– liquid extraction After subcritical and hydrodistillation extractions, a liquid – liquid extraction step was performed, in which volumes of 5 ml of hexane and 65 ml of nonane stock solution (used as extractant and internal standard, respectively) were added to each extract in a separatory funnel, and ca. 0.5 g of NaCl was added to facilitate the breaking of the emulsion. The organic extract was then dried with anhydrous Na2SO4. 2.4.4. Dichloromethane extraction A 4.0-g portion of fennel was placed in a glass screw-top vial and 130 ml of internal standard and 10 ml of dichloromethane were added to the sample. The extraction was performed on a shaker for 24 h [13]. 2.4.5. Chromatographic separation and detection Aliquots of 2 ml of hexane after either SWE or hydrodistillation plus liquid – liquid extraction in Table 1 Optimisation of variables Variable SWE Temperature (°C) Pressure (bar) Flow rate Amount of sample (g)
Range studied
50–200 – 0.5–3.0 2.0–5.0
Gas chromatography Carrier Injection volume (ml) Column Split ratio 1:30/1:6 Carrier flow rate (ml min−1) Temperature gradient (°C min−1) Ramp 1 1–5 Ramp 2 1–5
Optimum value
150 20 2.0 4.0 Helium 2 SGL-5 1:6 1.0
1 5
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both cases, and of 2 ml of dichloromethane from the manual extraction, were injected into an SGL5 fused silica column (25 m×0.25 mm id, 0.25 mm film thickness). The carrier gas (helium) was delivered at a flow rate of 1.0 ml min − 1. The injector and detector temperatures were 250 and 300°C, respectively. The oven temperature was 55°C for 2 min, then increased to 75°C at a rate of 1°C min − 1, kept for 1 min, and finally increased to 250°C at a rate of 5°C min − 1. The split ratio was 1:6.
2.4.6. Gas chromatography-mass spectrometry identification Fractions of the hexane extracts obtained by subcritical fluid extraction and hydrodistillation were collected in order to identify the components by gas chromatography-mass spectrometry. Mass spectra (electron impact) of the compounds were obtained using a Fison VG Autospec (70 eV) by direct injection of 2 ml of the extract into the ionization chamber at a temperature of 250°C.
3. Results and discussion A static-dynamic approach was tested for the isolation of fennel oil and then compared with conventional techniques such as hydrodistillation and organic–solvent extraction. A static extraction period (for enhancing sample–extractant contact thus favoring the attainment of the partition equilibrium) was carried out, followed by a dynamic extraction period in which fresh extractant passed continuously through the extraction chamber, thus displacing the equilibrium to quantitativeness.
3.1. Optimisation of 6ariables The variables affecting the subcritical extraction were studied in order to maximise the yield of essential oil in as short a time as possible. With this aim the univariate method was used. The static period was kept constant at a value of 5 min. The optimum values found for the variables are shown in Table 1.
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Fig. 1. Effect of the temperature in the main components of essential oil from fennel.
Fig. 2. Effect of the flow rate in the main components of essential oil from fennel. IS= internal standard.
The temperature of the extraction chamber was the key variable affecting continuous subcritical extraction. The yield increased with temperature up to 150°C. At 175°C, the yield of most of the monoterpene compounds increased, but decreased for the oxygenated compounds, probably due to their degradation (Fig. 1). For temperatures higher than 175°C, a strong burning smell of the extract appeared. A value of 150°C was selected as optimum. The pressure, as demonstrated in previous works [10], had little influence on the yield of essential oil, as long as the extractant water was under liquid state. Thus, a minimum pressure of 20 bar was maintained throughout all extractions. The flow rate was studied in the interval 0.5–3.0 ml min − 1. Increased flow rates enhanced the extraction of most of the monoterpene compounds, while in the case of oxygenated compounds the extraction rate was maximum at 2 ml min − 1, as can
be seen in Fig. 2. A flow rate of 2 ml min − 1 was selected for further experiments. The influence of cutting or grounding the sample was also studied. When the sample was ground, the yield of the essential oil decreased, probably due to losses of volatiles during preparation. Thus, for further experiments, the sample was just cut into small pieces (ca. 1 cm in length). The amount of sample was also studied in the interval 2–5 g (the maximum capacity of the cell), and 4 g was selected as it provided signals high enough. In the manual liquid–liquid extraction, the number of extraction steps was studied by performing a second 5 ml hexane extraction to ensure that no compounds remained in the aqueous phase after the first extraction. Only small peaks of the majority compounds were obtained after injection of the second extract, so a single 5 ml hexane extraction was carried out in subsequent experiments.
L. Ga´miz-Gracia, M.D. Luque de Castro / Talanta 51 (2000) 1179–1185
3.2. Effect of the static extraction time on the o6erall extraction kinetics The influence of the static extraction time on both the extraction kinetics and composition of the oil was studied by performing extractions with subcritical water consisting of a static extraction step of 0, 5, 15 and 30 min followed by 20 min of dynamic extraction (at 2 ml
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min − 1) during which the vial was replaced every 5 min (ca. 10 ml). As can be seen in Fig. 3, increased static extraction times enhance the extraction of most of the compounds. It can also be established that for the monoterpene compounds, 20 ml is enough to complete the extraction, while in the case of the oxygenated ones, at least 40 ml of subcritical water is required.
Fig. 3. Effect of the static extraction time in the main components of essential oil from fennel.
Fig. 4. Chromatograms of the SWE (a), dichloromethane extraction (b) and hydrodistillation (c) of fennel essential oil. Peaks identification: (1) a-pinene; (2) b-pinene; (3) b-myrcene; (4) a-phelandrene; (5) limonene; (6) unknown; (7) camphor; (8) terpinen-4ol; (9) linalyl propanoate; (10) unknown; (11) anethol; (12): copaene.
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Table 2 Comparison of area compound/internal standard ratio for GC of extracts obtained after hydrodistillation, dichloromethane extraction and SWE Compound
Hydrodistillationa
Dichloromethane extractionb
SWEa
a-Pinene b-Pinene b-Myrcene a-phelandrene Limonene Unknown Camphor Terpinen-4-ol Linalyl propanoate Unknown Anethol Copaene
0.265 0.067 0.569 2.397 5.858 25.196 0.697 – 0.154 5.857 25.214 –
0.499 0.140 0.667 2.580 5.576 3.529 – – – 1.272 5.887 –
4.889 0.887 5.469 18.689 51.598 67.732 1.503 0.225 0.781 23.613 68.054 0.234
a b
Extracted in 5 ml of hexane. In 10 ml of dichloromethane.
3.3. Manipulation of the oil composition As a consequence of the relationship between the extraction conditions and qualitative composition of the extract, the latter can be manipulated by changing the conditions of the extraction (temperature, flow rate and static extraction time).
3.4. Precision of the SWE/chromatographic method The precision (expressed as RSD) of both the chromatographic step and the overall process (extraction and detection) was studied separately. The precision of the chromatographic step was calculated by injecting one of the organic extracts from SWE of 4.0 g of fennel seven times. The average RSD for the eight major components was 3.26%. For studying the precision of the overall process, 5 portions of 4.0 g of fennel were subjected to SWE under the same conditions and the hexane layer obtained after liquid – liquid extraction was injected in the GC. The resulting RSD for the eight major components was 6.77%.
3.5. Comparison of methods The SWE method (30 min of static extraction
followed by 20 min of dynamic extraction at 2 ml min − 1) has been compared with hydrodistillation and manual dichloromethane extraction as conventional alternatives in terms of rapidity, efficiency, cleanliness and possibility of manipulating the composition of the extract.
3.5.1. Rapidity The SWE is clearly quicker than its conventional counterparts. The extraction takes 50 min, whilst 4 and 24 h were required by hydrodistillation and manual dichloromethane extraction, respectively. 3.5.2. Efficiency The most efficient method in terms of quantitative composition of the extract seems to be SWE, as can be seen in the chromatograms of the extracts obtained by the three methods for the same amount of sample, and the values of the area ratios for the main component and the internal standard (Fig. 4 and Table 2, respectively). It must be taken into account that in the case of SWE and hydrodistillation the final extracts was obtained in 5 ml of hexane, and in the case of manual dichloromethane extraction, the final volume was 10 ml.
L. Ga´miz-Gracia, M.D. Luque de Castro / Talanta 51 (2000) 1179–1185
3.5.3. Cleanliness SWE is a very clean method, which avoids both the use of large amounts of organic solvents and residue generation. 3.5.4. Possibility of manipulating the composition of the extract The SWE allows the manipulation of the composition of the extract by changing extraction parameters such as temperature, flow rate and/or static extraction time. This is unfeasible with the conventional methods.
4. Conclusion The continuous SWE of essential oil in medicinal plants (fennel) has been studied. The method offers important advantages over traditional alternatives, namely: shorter extraction times (50 min against 4 h for hydrodistillation and 24 for manual dichloromethane extraction); cost (energy cost is fairly higher for performing hydrodistillation than that required for reaching subcritical conditions); cleaner features (as no a great volume of organic solvent is involved); and the possibility of manipulating the composition of the oil by changing the parameters of the extraction (temperature, flow rate and static extraction time). The method contributes to the automation of pharmaceutical industry.
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Acknowledgements The Spanish Comisio´n Interministerial de Ciencia y Tecnologı´a (CICyT) is thanked for financial support (Project no. PB96-1265). The Department of Organic Chemistry (Faculty of Sciences, University of Co´rdoba) is thanked for realising the GC-MS identifications. References [1] J.C. Chalchat, R.P. Garry, A. Michet, Flavour Fragr. J. 6 (1991) 189. [2] M.D. Guille´n, N. Cabo, J. Murillo, J. Sci. Food Agric. 70 (1996) 359. [3] H.L. De-Pooter, E.A. Aboutabl, A.O. El-Shabrawy, Flavour Fragr. J. 10 (1995) 63. [4] R.K. Verma, G.C. Uniyal, M.M. Gupta, Indian J. Pharm. Sci. 52 (1990) 276. [5] M. Taverna, A.E. Baillet, D. Baylock, J. Ass. Off. Anal. Chem. 73 (1990) 206. [6] S.K. Volkov, E.I. Grodnitskaya, J. Chromatogr. 769 (1997) 275. [7] M.D. Luque de Castro, M.M. Jime´nez Carmona, V. Ferna´ndez Pe´rez, Trends Anal. Chem. 18 (1999) 708. [8] M.D. Luque de Castro, M.M. Jime´nez Carmona, Trends Anal. Chem. 17 (1998) 441. [9] A. Basile, M.M. Jime´nez Carmona, A. Clifford, J. Agric. Food Chem. 46 (1998) 5205. [10] M.M. Jime´nez Carmona, J.L. Ubera, M.D. Luque de Castro, J. Chromatogr. 855 (1999) 625. [11] S.H.R. Al-Saidi, A. Clifford, A. Basile, Fresenius J. Anal. Chem. (1999) submitted for publication. [12] USA Patent no. 5, 400, 642, 1995. [13] J.M. Bowman, M.S. Braxton, M.A. Churchill, J.D. Hellie, S.J. Starrett, G.J. Ellis, S.D. Ensley, S.J. Maness, C.D. Meyer, J.R. Seller, Y. Hua, R.S. Woosley, D.J. Butcher, Microchem. J. 56 (1997) 10.