I n d . Eng. Chem. Res. 1989,28, 1017-1020
so t=
[
(XP hXB(1) F(R + 1) 1 - exp C V xB.(l)XD(l) - X B ( l )
)]
(25)
Literature Cited Diwekar, U. M. Simulation, Design and Optimisation of Multicomponent Batch Distillation Columns. Ph.D Thesis, IIT Bombay, India, 1988. Diwekar, U. M.; Malik, R. K.; Madhavan, K. P. Optimal reflux rate policy determination for multicomponent batch distillation columns. Comput. Chem. Eng. 1987, 11, 629-637.
1017
Happel, J. Process Plant Components. In Chemical Process Economics; Wiley: New York, 1958. Houtman, J. P. W.; Hussain, A. Design calculations for batch distillation column. Chem. Eng. Sci. 1956,5, 178-187. Rippin, D. W. T. Simulation of single and multiproduct batch chemical plants for optimal design and operation. Comput. Chem. Eng. 1983, 7, 137-156. Robinson, E. R.; Goldman, E. R. The Simulation of multicomponent batch distillation processes on a small digital computer. Br. Chem. Eng. 1969, 14, 809-812.
Received for review December 8, 1988 Revised manuscript received April 3, 1989 Accepted April 26, 1989
Supercritical Fluid Isolation of Monocrotaline from Crotalaria spectabilis Using Ion-Exchange Resins Steven T. Schaeffer,+Leon H. Zalkow,t and Amyn S. Teja* School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0100
In our previous studies, we have successfully extracted monocrotaline, a hepatotoxic pyrrolizidine alkaloid of chemotherapeutic interest, from the seeds of Crotalaria spectabilis using supercritical carbon dioxide-ethanol mixtures. However, the presence of highly soluble lipid material in the seeds resulted in rather low monocrotaline purity in the extracts-24% by mass or less. The purity could be improved t o values as high as 49% monocrotaline by mass by using a two-stage process in the temperature-solubility crossover region. However, pressure changes were still required to obtain the product. In this work, we show t h a t monocrotaline purities greater than 95% by mass can be obtained by the combination of cation-exchange resins with supercritical fluid extraction without any pressure changes. This represents a new application of supercritical extraction which is particularly attractive for the isolation of alkaloids, drugs, and other biological materials.
1. Introduction Supercritical fluid extraction has found increasing acceptance as a separation process in the biochemical and pharmaceutical industries principally due to the temperature sensitivity of the compounds of interest and the need for physiologically inert solvents. Its application, however, has been somewhat limited by the inherent nonselectivity of the preferred solvent-carbon dioxide-to the compounds of interest. Moreover, biomolecules often have low solubilities in supercritical fluids, and they occur only in trace amounts in mixtures containing highly soluble lipid materials, making isolation quite difficult. Judicious selection of a cosolvent by matching acid-base sites or polarity (Dobbs et al., 1987) can improve the extraction selectivity. However, further separations are still required to complete the isolation. In a previous study (Schaeffer et al., 1988a), we showed that supercritical carbon dioxide-ethanol mixtures can be used to extract the pyrrolizidine alkaloid, monocrotaline, from the seeds of Crotalaria spectabilis. Monocrotaline is of interest because of its role as an environmental toxin (Mattocks, 1986) and as a precursor in the development of semisynthetic pyrrolizidine alkaloids (Gelbaum et al., 1982) for the treatment of cancer. The conventional preparative separation of monocrotaline from Crotalaria spectabilis is quite difficult and expensive. In our de-
* To whom
correspondence should be addressed. Present address: E. I. d u Pont de Nemours & Co., Inc., P.O. Box 2042, Wilmington, NC 28402. *Also School of Chemistry, Georgia Institute of Technology.
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velopment of a supercritical fluid based extraction alternative, we found that extraction selectivities toward monocrotaline as high as 24% could be achieved prior to the noticeable depletion of the seed components and as high as 40% after this depletion. The balance of the extracts consisted of lipid components normally found in seeds. Monocrotaline purities as high as 49% by mass were achieved by using a two-stage process in the temperature-solubility crossover region (Schaeffer et al., 1988b). While all of the monocrotaline could be removed using this process, a great deal of postprocessing is still required to isolate pure monocrotaline. In this work, we have investigated an alternative designed to improve the monocrotaline extraction purity. The new process is based on supercritical extraction of the seed material followed by adsorption of the monocrotaline on an ion-exchange resin. As such, it represents a novel approach to the isolation of alkaloids from plant materials.
11. Experimental Section The apparatus used in our investigation has been discussed in detail in a previous paper (Schaeffer et al., 1988a) and is shown schematically in Figure 1. Pressurized carbon dioxide (B) was filtered, liquified in an ice bath (C), and fed into one head of an Eldex dualhead metering pump (E). The cosolvent from a graduated cylinder (D) was filtered and fed into the other head of the metering pump. The solvent and cosolvent were pumped to the system pressure before entering the constant-temperature bath (F). The mixture was homogenized in a Kenics static mixer (I) and fed into the equilibrium cell (K) (a Jergeson level gauge). The equilibrium 0 1989 American Chemical Society
1018 Ind. Eng. Chem. Res., Vol. 28, No. 7, 1989
Figure 1. Apparatus used in this study.
cell was packed with alternating layers of crushed seeds and glass beads, which facilitated the attainment of equilibrium between the solid and fluid phases. The equilibrium mixture exited the cell and passed through a tubular vessel (N) (71/2cm3, 64x1. x 5/16-in.i.d.) containing 3 g of DOWEX 50W-8X cation-exchange resin in the H+ form (5.1 mequiv of adsorption/dry g) layered with glass beads. The fluid mixture then passed through a high-pressure sampling valve (0)and a Mettler-Parr DMA-512 fluid densiometer (Q) and was depressurized in a heated micrometering valve (U). The solute and a portion of the cosolvent were retained in a collection vessel (V) immersed in an ice bath. The gas mixture then passed through two rotameters (W) (for flow visualization), a gas sampling valve (X), and a wet test meter (Z) (for flow totalization). The gas composition was determined in a Perkin-Elmer 900 gas chromatograph (Y)with a flame ionization detector and a 2% OV-101 on 6-ft X 'I8-in. Chemasorb column. The GC was used principally to determine the amount of cosolvent in the depressurized gas, and no seed volatiles were detected. A valve (S) located at the bath exit was used to flush any components that had deposited in the exterior lines or metering valve due to pressure or temperature fluctuations using solvent from vessel R. The system pressure was maintained constant by a Grove Industries back-pressure regulator (J)controlled to f0.03 MPa by high-pressure nitrogen (A). The nitrogen pressure gauge (P2) and the system pressure gauge (Pl) were Heise gauges calibrated with a Budenburg deadweight gauge. The precision of the pressure measurements was f0.03 MPa. The bath temperature was controlled to f O . l K with a Haake 1.6-kW temperature controller (G). The bath (Tl) and cell (T2) type K thermocouples were calibrated to *O. 1 K total accuracy with a NBS-certified platinum resistance thermometer and were measured with a Fluke digital multimeter (H). The DMA-512 fluid densiometer was calibrated using carbon dioxide and high-pressure nitrogen a t the experimental temperature and pressure. The estimated accuracy of the densities was f O . l kmol/m3. The amounts of cosolvent and solute deposited in the collection vessel were determined gravimetrically in a Mettler balance accurate to f O . l mg. Trace impurities present in the cation-exchange resin were removed by flushing with supercritical carbon dioxide and carbon dioxide-ethanol mixtures prior to the actual experiments. The cation-exchange resin was activated to the H+form by passing 80 mL of 1 N HCl in vessel L through the bed. Residual C1- and H+were removed by flushing with distilled water. After the supercritical mixture had passed through the resin bed, any entrapped compounds were removed by flushing the resin with 80 mL of 95 wt % ethanol-5 wt % water. The adsorbed components were released from the resin by flushing sequen-
tially with 80 mL of 1 N NH,OH and 80 mL of distilled water from vessel M. The seeds of Crotalaria spectabilis (approximately 5 mm in diameter) were prepared by milling to 1 mm and sieving to 850 ym. Approximately 19 g of the 850-pm fraction containing the inner seed material was placed in the equilibrium cell. The extracts were analyzed for monocrotaline content by using a lH NMR technique developed by Molyneaux et al. (1979) using a Gemini 300-MHz NMR spectrometer. The extracts were dissolved in CDC1, using p-dinitrobenzene as an internal standard. This technique yielded the mass of monocrotaline with an estimated accuracy of &7%. As little as 0.5 mass 7'0 monocrotaline could be detected by using the NMR technique. Source and Purity of Materials. Coleman instrument-grade carbon dioxide (>99%) was obtained from Matheson Gas Company. Pure ethanol (>99.9%) was obtained via reactive distillation of Fisher HPLC-grade ethanol (-99%) with magnesium metal catalyzed with iodine. The seeds of Crotalaria spectabilis were harvested in Nov 1984 in Clarke County, GA. Analysis of the seeds revealed that they contained 1.9% monocrotaline and 2.5% hexane-extractable material by mass. 111. Results The supercritical fluid extracts of Crotalaria spectabilis contain monocrotaline, a basic alkaloid, and lipid material. In the separation of the alkaloid caffeine from coffee, Zosel (1981) recommended extraction with carbon dioxide-water mixtures followed by either adsorption of the caffeine onto activated carbon or absorption of the caffeine into liquid water. In our case, activated carbon would be a nonselective adsorbent, as it would collect both the monocrotaline and the lipid material. Also, regeneration of the activated carbon involves either burning off the adsorbate or disposal of the activated carbon-both of which are economically and environmentallyundesirable. Absorption of monocrotaline into liquid water is possible; however, the solubility of monocrotaline in liquid water is quite low (Schaeffer, 1988). Also, the ethanol cosolvent would also be absorbed, and the resulting decrease in the ethanol concentration in the fluid phase would cause the lipids to precipitate. Moreover, the low solubility of monocrotaline (Schaeffer, 1988) in carbon dioxide-water mixtures makes the use of water a poor cosolvent. Gelbaum et al. (1982) used a cation-exchange resin to isolate monocrotaline from an ethanol solution. The same cation-exchange resin was therefore adopted as a potentially highly selective second-stage adsorbent in our work. In theory, a cation-exchange resin should adsorb all basic compounds from a fluid phase. Since monocrotaline is a basic alkaloid and there are no other alkaloids or basic materials in the seeds of Crotalaria spectabilis, this adsorbent should therefore remove only the monocrotaline from the fluid phase. This was found to be the case in our work, as discussed below. Carbon Dioxide-Ethanol-Crotalaria specta bilis : Cation-ExchangeResin Adsorption System. Supercritical mixtures of carbon dioxide and ethanol were used to extract the soluble components of the crushed seeds of Crotalaria spectabilis. The resulting mixture (at the system temperature and pressure) was then passed through a bed of cation-exchange resin. The effect of temperature and pressure on the ability of the resin to separate monocrotaline was investigated, and the results were found to be independent of the adsorption conditions (Table I). As an example, at 308.15 K and 10.34-MPa pressure, 93.6 g of the carbon dioxide-ethanol mixture passed sequentially
Ind. Eng. Chem. Res., Vol. 28, No. 7 , 1989 1019 Table I. Results of Cation-Exchange Resin Adsorption experiments 4 2 3 conditions 1 EtOH EtOH EtOH EtOH cosolvent av concn, mol 70 4.0 5.3 3.8 4.3 temp, K 308.15 308.15 328.15 328.15 pressure, MPa 10.34 22.15 10.34 22.15 mass solvent, g 93.6 89.3 93.0 60.6 unadsorbed fraction total mass, mg 154.5 455.0 20.3 306.9 0 monocrotaline mass, mg 0 0 0 entrapped fraction total mass, mg 30.4 39.9 2.4 29.8 monocrotaline mass, mg 0 0 0 0 adsorbed fraction 3.0 12.7 total mass, mg 9.3 8.9 monocrotaline mass, mg 9.0 8.9+ 3.0 12.3 100 97 monocrotaline purity, % 97 100+ monocrotaline yieldo 88 100+ 62 100
i! 5
HZO
Monocrotaline
0.7 328.15 22.15 67.6 171.3
0 20.7 0
3.3 3.1 94 92
aMonocrotaline yield represents the ratio of the mass of monocrotaline adsorbed on the exchange resin to the theoretical amount of monocrotaline in the fluid phase under these conditions (data from Schaeffer et al. (1988a) for ethanol cosolvent and from Schaeffer (1988) for water cosolvent).
through the extraction vessel and the adsorption column (Table I). The average concentration of ethanol was 4 mol '70.The amount of solute collected a t the system outlet was 154.5 mg and contained no monocrotaline. At the end of the run, the adsorption column was depressurized, and its contents were flushed with 95 w t '70ethanol-5 w t % water. The mass of solute was 30.4 mg and contained no monocrotaline. The presence of the solute in the adsorption column (but not adsorbed) was principally due to the tendency of the resin to entrap compounds in its porous structure. Also, a portion of this fraction resulted from solute precipitation on depressurization. The fact that no monocrotaline was detected in either fraction could be attributed to the low flow rates through the adsorption column, which allow all the monocrotaline to be adsorbed. Since the significance of the mass-transfer rate on solubility was carefully eliminated in the extraction vessel, the effect of mass transfer on adsorption in the adsorption column was negligible. A t some point, the adsorption capacity of the resin could be approached, although this was not the case in our studies. The adsorption column was then flushed with 1 N NH,OH, and the adsorbed species were released from the resin. The mass of the solute collected in this manner was 9.3 mg and was found to contain 9.0 mg of monocrotaline. The overall and monocrotaline solubilities, 2.07 X and 1.0 x mass fractions, respectively, are within the experimental accuracy of the equilibrium solubilities measured in our previous work: i3'70 overall solubility and i7 % monocrotaline solubility (Schaeffer et al., 1988a). A t all conditions, the material adsorbed onto the cation-exchange resin was a white crystalline-structured solid. NMR analysis of this material revealed that it contained monocrotaline at concentrations greater than 95 mass % . While this represents the quantitative limit of the NMR analysis technique, the adsorbate spectrum were consistent with the spectrum of pure monocrotaline (Figure 2). Lipid components, had they been present in the adsorbed fraction, would clearly have been evident in the low chemical shift range of the NMR spectra. The material that was not adsorbed was analyzed by NMR and was found to contain lipid material and no monocrotaline. Thus, the cation-exchange resin completely removes and isolates monocrotaline from the fluid phase. Carbon Dioxide-Water-Crotalaria spectabilis : Cation-Exchange Resin Adsorption System. Water has been demonstrated to be quite effective in the removal
a
7
6
5
4
3
2
1
Chemical Shift (ppm)
Cation Exchange Resin Adsorbate
a
7
6
5
4
3
2
1
Chemical Shift (ppm)
Figure 2. NMR spectra of monocrotaline and cation-exchange resin adsorbate.
Solvent t Co-Solvent
Recycle
-__
v
Constant Temperature Bath
Figure 3. Schematic diagram of potential industrial process for supercritical fluid isolation of basic alkaloids from complex substrates.
of the alkaloids caffeine and nicotine from natural products. Therefore, the carbon dioxide-water-Crotalaria spectabilis system was also investigated. The objective here was to determine if the use of cation-exchange resins could be extended to the carbon dioxide-water supercritical phase as well as to determine if the adsorption effectiveness is dependent on the cosolvent. Once again, the adsorbate was found to be 94% monocrotaline, and all of the monocrotaline was removed from the fluid phase. Also, no lipid material was detected in the adsorbate (Table I). Based on these results, it is expected that the supercritical extraction/ion-exchange process can be employed in the extraction of other basic alkaloids, e.g., nicotine and caffeine, from plant materials.
IV. Discussion The use of cation-exchange resin as an adsorbent for the isolation of basic compounds represents a significant development in the field of supercritical fluid extraction. Industrial processes based on this concept could be quite efficient and inexpensive since there is no need to reduce the pressure to remove the solute. The economic penalty of recompressing the solvent could be reduced by simply using a recirculator (Figure 3). Since pure monocrotaline is obtained in the combined supercritical extraction/ionexchange process, further processing would not be re-
Ind. Eng. Chem. Res. 1989, 28, 1020-1024
1020
quired. It is possible that cosolvents other than water and ethanol may be used provided they do not deactivate the exchange resin. There are two possible drawbacks to the use of exchange resins. Firstly, there may be a tendency for the resin to deactivate in the presence of the solvent power of supercritical fluids and cosolvents. However, this was not observed in the present work. Secondly, the resin could entrap other compounds from the fluid. This latter problem can be overcome by periodically flushing the resin bed with a neutral solvent or by loosely packing the resin. A logical extension of this work is in the application of anion-exchange resins to the removal of acidic components, such as fatty acids, rosin acids, and various acidic drugs, from supercritical fluids. V. Conclusions
A novel supercritical fluid technique has been developed to isolate monocrotaline from a complex mixture. This two-stage process employs a cation-exchange resin in series with a supercritical extraction step and has been shown to yield almost pure monocrotaline. A further advantage of the exchange resin is that no pressure changes are employed in the supercritical extraction process. A major economic disadvantage of supercritical fluid extraction is therefore eliminated. Similar processes can also be de-
signed for other plant or biological materials. Literature Cited Dobbs, J . M.; Wong, J. M.; Lahiere, R. J.; Johnston, K. P. Modification of Supercritical Fluid Phase Behavior using Polar CoSolvents. Ind. Eng. Chem. Res. 1987, 26, 56-62. Gelbaum, L. T.; Gordon, M. M.; Miles, M.; Zalkow, L. H. Semisynthetic Pyrrolizidine Alkaloid Antitumor Agents. J . Org. Chem. 1982, 47, 2501-2504. Mattocks, A. R. Chemistry and Toxicology of Pyrrolizidine Alkaloids; Academic Press: Orlando, FL, 1986. Molyneaux, R. J.; Johnson, A. E.; Roitman, J . N.; Benson, M. E. Chemistry of Toxic Range Plants. Determination of Pyrrolizidine Alkaloid Content and Composition in Senecio Species by Nuclear Magnetic Resonance Spectroscopy. J . Agric. Food Chem. 1979, 27(3), 494-499. Schaeffer, S. T. Extraction and Isolation of Monocrotaline from Crotalaria spectabilis using Supercritical Fluids. Ph.D. Thesis, Georgia Institute of Technology, Atlanta, 1988. Schaeffer, S. T.; Zalkow, L. H.; Teja, A. S.Extraction of Monocrotaline from Crotalaria spectabilis using Supercritical Carbon Dioxide and Carbon Dioxide-Ethanol Mixtures. Biotechnol. Bioeng. 1988a, in press. Schaeffer. S. T.: Zalkow. L. H.: Teia. A. S. Suoercritical Extraction of Monocrotaline from Croia1a;iu spectabilis in the Cross-Over Region. AIChE J . 1988b, 34, 1740-1’742. Zosel, K. Process for the Decaffeination of Coffee. US Patent 4,260,639, 1981. Received for reuieu! November 8, 1988 Accepted April 4, 1989
Styrene/Ethylbenzene Separation Using Facilitated Transport through Perfluorosulfonate Ionomer Membranes Carl A. Koval,* Terry Spontarelli, and Richard D. Noblet Department of Chemistry a n d Biochemistry, Campus Box 215, University of Colorado, Boulder, Colorado 80309
Steady-state fluxes of styrene and ethylbenzene through perfluorosulfonate ionomer membranes exchanged with sodium and silver ions are reported. Due to the reversible formation of complexes with silver, the fluxes of styrene and ethylbenzene are enhanced when Na+ is replaced with Ag+. Since styrene forms a more stable complex, its flux is enhanced significantly more than that of ethylbenzene. This effect is investigated for different membrane thicknesses, membrane solvents, and permeate feed concentrations. Facilitation factors (Ag+ membrane flux/Na+ membrane flux) range from 50 to 590 for styrene and from 9 to 84 for ethylbenzene. Separation factors (styrene permeability/ethylbenzene permeability) are about 2 for Na+-exchanged membranes and range from 8 to 36 for Ag+-exchanged membranes. The data are analyzed qualitatively utilizing factorial analysis and quantitatively by comparison to the predictions of a mathematical model. Facilitated transport through liquid membranes, which relies on the reversible formation of a carrier permeate complex, is a potentially selective and efficient separation technique. The selectivity of a facilitated transport membrane (FTM) separation is mainly dependent on the selectivity of the carrier-permeate complexation reaction and the relative solubilities of the permeates in the membrane phase. The properties of the FTM support material can have large effects on the rate, selectivity, and stability of the separation. Membranes derived from ion-exchange materials have recently received considerable attention with respect to their structural, physical, and chemical properties (Elliott and Redepenning, 1984; Quezado et al., 1984; Szentirmay et al., 1986; Herrera and Yeager, 1987). The use of ionomer materials as a FTM support material *Address correspondence to this author. Also associated with the Department of Chemical Engineering, Campus Box 424.
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has been reported for the separation of gaseous mixtures such as C02/CH4 and ethylene/ethane (Way et al., 1987; LeBlanc et al., 1980). Ionomer membranes containing solvents display greater physical strength than liquid membranes immobilized on macroporous supports. They are more stable in the sense that the ionic carrier cannot be lost from the membrane if exchangeable ions are not present in the contacting phases. Also, since carrier solubility is dictated by ion-exchange site density and not by physical solubility limits, higher carrier loadings are possible. Fluxes of liquid-phase olefins such as 1-hexene and 1,5-hexadiene can be enhanced by a factor of over 400 when Na+ ions are replaced by olefin-complexing Ag+ ions in water-swollen Nafion membranes (Koval and Spontarelli, 1988). The reversible complexation of olefins by aqueous Ag+ is a well-known reaction (Beverwijk et al., 1970). The stability of the Ag+-olefin complex is dependent on electronic factors and on steric hindrance around the olefin 0 1989 American Chemical Society
