Development of Process Alternatives for Separation and Purification of

Ind. Eng. Chem. Res. 2007, 46, 181-189

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Development of Process Alternatives for Separation and Purification of Isoflavones Benny Harjo,†,‡ Christianto Wibowo,§ Elizabeth J. N. Zhang,| Kathy Q. Luo,| and Ka M. Ng*,† Department of Chemical Engineering and Bioengineering Graduate Program, School of Engineering, The Hong Kong UniVersity of Science and Technology, Clear Water Bay, Hong Kong, and ClearWaterBay Technology, Inc., 20311 Valley BlVd., Suite C, Walnut, California 91789

This article demonstrates the application of an integrative process development approach that combines conceptual design and bench-scale experiments to develop realistic process alternatives for the separation and purification of isoflavones from soybeans. The procedure begins with a generic process structure and the chemistry of the compounds involved. Basic data such as solid-liquid equilibrium phase behavior and chromatograms are then generated. The conceptual design calls for the recovery of the isoflavones as a mixture first using either chromatography or antisolvent crystallization. This step is followed by fractionation of this mixture into pure products, namely, daizein and genistein, using either chromatography or fractional crystallization. Introduction Isoflavones are a group of plant-derived phenolic compounds often known as phytoestrogens because of their estrogenic activity. These compounds can be found in leguminous plants such as peas, beans, and clovers.1 Soybeans are particularly attractive because they are widely available, cheap, and relatively rich in isoflavones (1.2-4.2 mg/g of soybean, depending on the variety, crop year, and growth location).2 There are three major soy isoflavones, known as daidzein (D), genistein (G), and glycitein (GLY), the molecular structures of which are shown in Figure 1. In soybeans, these isoflavones mostly exist as glycosides, i.e., compounds with sugar, in a proportion of approximately 3:6:1 for daidzin, genistin, and glycitin, respectively.3 Recent research has suggested that isoflavone aglycones might be more biologically active than their respective glycosides.4,5 The interest in isoflavones is increasing because of the growing evidence of their health benefits. It has been reported that isoflavones play a role in preventing and treating various cancers, osteoporosis, and cardiovascular diseases and that they exhibit antioxidant activity.6 For example, genistein has been shown to inhibit the growth of breast cancer cells,7 and daidzein has been suggested as a therapeutic agent for the treatment of alcohol abuse.8 At present, isoflavones are mostly marketed as extracts containing a variety of different components. It is desirable that specific isoflavones be recovered in a purified form so as to improve specificity and efficacy. Different procedures for the extraction of isoflavones from various plants have been reported in the literature.9-12 These procedures usually recover a mixture of compounds; require lengthy steps; and do not consider solvent recovery and recycle, * To whom correspondence should be addressed. Fax: +852 23580054. E-mail: [email protected]. † Department of Chemical Engineering, The Hong Kong University of Science and Technology. ‡ Current address: Mitsubishi Chemical Group Science and Technology Research Center, Inc., 1-1 Shiroishi, Kurosaki, Yahatanishi-ku, Fukuoka 806-0004, Japan. § ClearWaterBay Technology, Inc. | Bioengineering Graduate Program, School of Engineering, The Hong Kong University of Science and Technology.

as most of them are not intended for large-scale production. This article addresses the development of conceptual process alternatives for the separation and purification of soy isoflavones that are suitable for manufacturing processes. The separation objective is to obtain isoflavones daidzein (D) and genistein (G), each as a pure product. Preliminary Conceptual Design Using a previously developed systematic procedure for synthesizing phytochemical manufacturing processes,13 process alternatives for the manufacture of soy isoflavones can be generated. Starting with the chemistry of the compounds involved and a generic process structure, heuristics and guidelines are used to select feasible separation methods. The overall process consists of four parts (Figure 2). The first part involves extraction of isoflavones from soybean and separation of insoluble materials from the extract. Because isoflavones are mostly present as glycosides in nature (only 2-5% are present as aglycones14), a hydrolysis step is needed to convert the glycosides in the extract to aglycones. The third step is the removal of the remaining glycosides, released sugars, and other impurities present in the hydrolyzed extract. Finally, a fractionation step is needed to obtain isoflavones D and G as separate products. Because isoflavone glycosides are polyphenols with several hydroxyl groups and a sugar moiety, they are expected to be soluble in water. Consequently, polar solvents such as alcohols and their aqueous mixtures are normally used as extraction solvents, which leads to co-extraction of other water-soluble compounds. Hydrolysis results in the formation of isoflavone aglycones, which are practically insoluble in water.15 The solubility difference between the aglycones and other compounds in the hydrolyzed extract can be exploited for impurity removal. Because G contains an additional hydroxyl group, isoflavones D and G are expected to have different solubilities. Therefore, chromatography and crystallization are good choices for the fractionation step. In the chromatography-based process, the mixture of isoflavones is separated into D-rich and G-rich fractions. The pure isoflavones can then be crystallized from these fractions to give the final products. In the crystallization-

10.1021/ie061027f CCC: $37.00 © 2007 American Chemical Society Published on Web 12/06/2006

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Ind. Eng. Chem. Res., Vol. 46, No. 1, 2007

Figure 1. Molecular structures of major soy isoflavones.

Figure 2. Outline of the manufacturing process of soy isoflavones.

based process, fractional crystallization can be employed to obtain isoflavones D and G in pure form without prior chromatographic separation. Bench Tests A series of bench-scale experiments were performed to provide adequate data for conceptual design. These include extraction, hydrolysis, and impurity removal both by chromatography and by antisolvent crystallization. Soybean flour (Sigma-Aldrich, St. Louis, MO) was used as the starting raw material. Isoflavones daidzein (D), genistein (G), and glycitein (GLY) with purity greater than 99% (LC Labs, Inc., Woburn, MA) were used to prepare standard mixtures for quantitative analysis.

HPLC analysis was performed on an Agilent 1100 Series chromatograph (including a pumping system, degasser, autosampler, and UV-DAD detector) using a reverse-phase C18 column (Phenomenex, Prodigy 5 µm, 250 × 4.6 mm). The column temperature was maintained at 40°C using a thermostat. UV spectra were recorded and isoflavone peak areas were quantified at a wavelength of 255 nm. The mobile phase used was acidified water with 0.1% acetic acid and methanol. The flow rate was 1.0 mL/min, and the gradient in the mobile phase was linear from 0 min (30% methanol) to 45 min (50% methanol), whereas from 45 to 50 min, methanol was kept constant at 50%. The sample injection volume was 10 µL. All solutions were filtered through 0.45-µm membranes (Millipore, Billerica, MA) before analysis to prevent any solid from entering the HPLC column. The methanol (Mallinckrodt, Hazelwood, MO) used was HPLC grade. Acetic acid was obtained from Fisher Scientific, Leicestershire, U.K. Water was supplied by a Milli-Q water purifier system (Millipore). These commercially available chemicals were used without any further purification. Extraction and Hydrolysis Both soybean extraction and hydrolysis have been investigated by several researchers. For example, Zheng et al.16 reported that the preferred extraction temperature was between 50 and 60 °C, that the ratio of plant material to solvent mixture (grams to milliliters) in the extraction process varied from 1:1 to 1:3, and that the hydrolysis could be accomplished by subjecting the soy extract to acid hydrolysis in a hydrochloric acid/water mixture. Murphy et al.17 compared various organic solvents for isoflavone extraction and found that methanol and a mixture of acetone and acetonitrile were effective extraction solvents for soybean flour. Our bench-scale experiments were performed in a similar manner without any attempt to optimize the operating conditions.

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Figure 3. Extraction and hydrolysis results: (a) Chromatogram of the soy extract before hydrolysis, (b) chromatogram of the soy extract after hydrolysis.

Extraction took place in a batch vessel with stirring, using 40 g of soybean flour and 120 mL of methanol as the extraction solvent. The temperature was maintained at around 60 °C using a water bath. After 12 h, the extract was separated from the insoluble materials by means of vacuum filtration using Whatman No.1 filter paper. The soy extract was then anaylzed using HPLC. As evident from the chromatogram (Figure 3a), most of the isoflavones extracted from the soybean are in glycoside form, with sizable daidzin and genistin peaks at around 10 and 15 min, respectively. Hydrolysis was performed by adding 5 mL of 37% hydrochloric acid (Fisher Scientific) to approximately 55 mL of the extract mixture. The hydrolysis temperature was maintained at around 70 °C. After approximately 6 h, the mixture was filtered through 0.45-µm membranes and subjected to HPLC analysis. The chromatogram of the hydrolyzed mixture (Figure 3b) suggests that the hydrolysis conditions were sufficient to convert most of isoflavone glycosides to their aglycones, as the daidzin and genistin peaks are significantly smaller and the D and G peaks (at 33 and 43 min, respectively) are larger in size. Judging from the area ratio of the daidzin and genistin peaks before and after hydrolysis, the conversion of the glycosides to aglycones is estimated to be 93.5%. The chromatogram also features a

significant new peak at about 6.5 min, which probably corresponds to the released sugar. Development of Impurity Removal Processes Impurity Removal by a Chromatography-Based Process. The chromatogram of the hydrolyzed extract (Figure 3b) also reveals that undesired compounds as well as the remaining isoflavone glycosides have much shorter retention times than the aglycones. This opens the possibility of using chromatography to remove the impurities from the hydrolyzed extract. To verify this hypothesis, chromatography experiments were performed using a preparative-scale reverse-phase C18 column (Waters, XTerra 7 µm, 300 × 19 mm, Milford, MA). An isocratic elution operation, instead of gradient elution, was employed because it is more suitable for a large-scale process. The flow rate was 8 mL/min, the feed injection volume was 0.5 mL, and the eluent was methanol/water (50/50, v/v). The UV detection wavelength of 225 nm for isoflavones was the same as that used for analytical HPLC. Because the soy extract might contain macromolecular impurities such as soluble proteins, ultrafiltration was performed before chromatography. The ultrafiltration unit was of centrifuge type (Centricon Plus20, Millipore) with a molecular cutoff of 5000 Da.

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Figure 4. Impurity removal based on the chromatographic process: (a) Chromatogram from the preparative column, (b) proposed process flowsheet.

The chromatogram is shown in Figure 4a. It can be observed that, although the peaks are wider than the HPLC peaks, a relatively clear-cut separation between the impurities and the isoflavones was achieved in the preparative column. This provides a good indication that a reasonable separation can be achieved in a commercial plant even if the peaks overlap somewhat because of the poorer separation performance obtained at larger scales. In practice, a simulated moving bed (SMB) chromatographic process is often preferable for large-scale production because of its higher productivity and yield and lower solvent consumption compared to conventional liquid chromatography.18 Therefore, the proposed chromatography-based process flowsheet for impurity removal (Figure 4b) assumes the use of an SMB unit. The unit can be designed to produce an extract stream that contains only the isoflavones as a mixture and a raffinate stream containing the impurities. Most of the eluent in the extract and raffinate streams can be recovered in the evaporators (S1 and S2) and recycled. The amount of evaporation can be controlled so as to produce a concentrated solution or a precipitate. It might be necessary to completely remove the solvent from the extract stream and recover the isoflavone mixture in solid form. This

mixture can be redissolved in another solvent for further separation. Makeup eluent is necessary to compensate for any loss of eluent to the purge stream. The performance of the proposed process can be analyzed through modeling and simulation using the chromatogram in Figure 4a as a starting point. Such design and optimization issues have been discussed in detail by others19-21 and are not included here. It is worth noting that the extract after hydrolysis was found to be dark yellowish. Carbon adsorption was performed by adding 15 mg of active carbon into 5 mL of sample mixture, which was then stirred for approximately 1 h. Afterward, the carbon was separated using a 0.2-µm filter (Millipore), and the solution was subjected to HPLC analysis. The color of the mixture became brighter after carbon adsorption, but the chromatogram did not show any difference compared to that of the mixture before adsorption. A possible explanation is that color-forming substances were present in the mixture but did not appear at the HPLC detection wavelength. To ensure that these color-forming substances are removed, carbon adsorption is included in the process. Impurity Removal by Antisolvent Crystallization. The HPLC results (Figure 3a,b) confirm the hypothesis that the


isoflavone aglycones (which are always eluted last) are less soluble in water. This is because a gradient elution from 70% to 50% water was used in HPLC, which implies that watersoluble compounds were eluted first when the eluent was rich in water. Therefore, antisolvent crystallization is a promising alternative for separating isoflavone aglycones from the hydrolyzed extract. Naturally, water is a suitable choice for the antisolvent. Antisolvent crystallization was performed by adding a known amount of water, varied from 15 to 150 mL, into 15 mL of hydrolyzed extract mixture. The mixture was stirred for 1 h at room temperature. After crystallization, the mixture was filtered through a 0.2-µm membrane to separate the solids from the mother liquor. The amount of solid cake collected on the filter was approximately 1.2-5.7 mg, depending on the amount of water added. The cake was washed with about 10 mL of water and then allowed to dry inside the fumehood. For analysis, the solids were dissolved in 3.5 mL of methanol and sonicated for 15 min to ensure complete dissolution before being injected into the HPLC column. Figure 5a shows the chromatogram of the recovered solids from the antisolvent crystallization experiment using an antisolvent to feed (S/F) ratio of 4. It is evident that most of the impurities in the hydrolyzed extract were removed by the antisolvent crystallization, as indicated by the absence of shortretention-time peaks. These results demonstrate that antisolvent crystallization can be an attractive alternative to chromatographic separation, which is generally more expensive. Figure 5b shows the effect of the S/F ratio on the yields of the three key soy isoflavones. The yield in mass percentage is defined as the amount of isoflavone obtained in the crystallized solids divided by the initial amount of the same isoflavone in the hydrolyzed extract. In general, the higher the S/F ratio, the higher the yields of isoflavones D and G because of their decreasing solubilities in the solvent mixture. As expected, the undesired isoflavone GLY also crystallized out, and its yield followed a trend similar to that of the other two isoflavones. Figure 5c shows the concentration of isoflavone GLY in the recovered solids (solvent-free basis) as a function of S/F ratio. The results in Figure 5b and c suggest that, above S/F ) 4, there was no significant increase in the yield of D and G and the content of GLY in the recovered solids remained almost the same. Therefore, in the interest of maximizing the yield while keeping the process flow rates as low as possible, an S/F ratio of 4 should be chosen. The flowsheet for this process alternative is depicted in Figure 5d. As in the chromatography-based alternative, carbon adsorption process is included in this step to ensure that the feed mixture is free of color-forming substances. The isoflavones are obtained as a solid product, which might need to be redissolved for further processing. The solvent should be selected on the basis of the downstream fractionation process. Because a significant amount of D remains dissolved after the antisolvent crystallization, a portion of the mother liquor can be mixed with the fresh feed (hydrolyzed extract), thus creating a recycle scheme. Development of Fractionation Process Alternatives As mentioned earlier, chromatography and crystallization are likely to be good choices for fractionating the mixture of isoflavones obtained in the impurity removal step, so as to obtain isoflavones D and G as separate, pure products. The development of these two process alternatives is discussed in the next two sections.

Chromatography-Based Fractionation Process. The preparative chromatography column data can also be used to develop a chromatography-based process alternative for obtaining pure isoflavones D and G. Such an alternative is particularly desirable if chromatography has already been chosen for impurity removal, because the isoflavone-rich stream from the impurity removal step can be fed directly to this process stage without having to evaporate too much eluent. Again, with largescale production in mind, an SMB process is assumed. From the chromatogram in Figure 4a, D is the least adsorbed component among the three key isoflavones, and G is the most adsorbed. Therefore, the extract stream will be rich in isoflavone G, whereas the raffinate stream will be rich in D. The undesired isoflavone GLY, whose peak is between those of D and G, will be distributed in both streams. Figure 6 shows the proposed flowsheet for this process alternative. Distillation units (S1 and S2) are used to concentrate the extract and raffinate streams, as well as to recover the eluent. The bottom products are sent to crystallizers C1 and C2, which produce pure D and pure G, respectively, in solid form. Because the mother liquor streams from the crystallizers still contain isoflavones, they are recycled to the feed stream. Partial purging is necessary to provide an outlet for GLY, thereby avoiding accumulation. Detailed design of such a chromatography/ crystallization hybrid process has been discussed elsewhere.22,23 Crystallization-Based Fractionation Process. A process alternative based only on fractional crystallization24 can be developed. The mixture of isoflavones from the impurity removal step can be dissolved in an appropriate solvent. Then the desired products D and G can be crystallized as pure products under different crystallization conditions as dictated by the solid-liquid equilibrium (SLE) phase behavior of the system. In general, such a fractionation can be achieved when the SLE behavior features a shift in the location of the doublesaturation point (the composition at which both D and G are saturated) at two different temperatures.25 In practice, the choice of these two temperatures is influenced by various factors such as solvent boiling point, utility, thermal decomposition, side reactions, and so on. Obviously, the SLE behavior depends strongly on the choice of solvent, which is more difficult to control when the feed already contains a particular solvent. For this reason, this alternative is more suitable for use in combination with the antisolvent-crystallization-based impurity removal process, for which the isoflavones are obtained as a solid (solvent-free) mixture. As a starting point, the SLE phase behavior of a ternary system containing D, G, and solvent is considered. The composition of the double-saturation point was measured at two judiciously selected temperatures. Known excess amounts of pure isoflavones D (white powder) and G (yellow powder) were mixed with the solvent. The mixture was kept in a temperaturecontrolled sonication bath at the desired temperature for 2 h, so that some of both isoflavones dissolved into the solvent and equilibrium was achieved between the liquid and the remaining solids. After that, a small amount of mother liquor was sampled from the mixture using a syringe and analyzed, after dilution, using HPLC. The sampling was done through a 0.2-µm syringe filter (Advantec, Dublin, CA) to ensure that there was no solid in the sample. To verify that the mother liquor composition corresponded to the double-saturation point, a visual check was conducted on the undissolved solids to ensure that both white and yellow-colored solids were still present. An additional run with a longer sonication time (3 h) was also performed using the same initial composition. Identical results were obtained,

Figure 5. Impurity removal based on the antisolvent crystallization process: (a) Chromatogram of the precipitated solid at S/F ) 4, (b) yield of key isoflavones versus S/F ratio, (c) composition of the isoflavone GLY in the recovered solids versus S/F ratio, (d) proposed process flowsheet.

186 Ind. Eng. Chem. Res., Vol. 46, No. 1, 2007


Figure 6. Proposed process flowsheet for fractionation of isoflavones D and G based on the chromatographic process. Table 1. Double-Saturation Points of the Ternary Systems of D, G, and Solvent

solvent methanol ethanol ethyl acetate acetone

temperature (° C)

mass fraction of D at the double-saturation point (solvent-free basis), xD

15 60 15 60 15 60 15 60

0.212 0.240 0.327 0.279 0.088 0.114 0.254 0.239

∆xD 0.028 0.048 0.026 0.015

Table 2. Solubility Data for the Ternary Systems of D, G, and Ethanol temperature (°C)

mass fraction of D in solution (10-3)

mass fraction of G in solution (10-3)

identity of remaining solid

15

1.83 2.40 2.16 3.74 2.31 1.51 0 3.09 3.04 3.62 4.87 6.41 2.61 1.49 0

0 0.99 1.60 7.70 7.40 7.89 8.09 0 1.21 2.22 12.0 11.2 10.6 11.0 16.6

D D D D, G G G G D D D D D, G G G G

60

indicating that 2 h of sonication was sufficient to achieve equilibrium for this system. The double-saturation points were measured at 15 and 60 °C for four common organic solvents in pharmaceutical processing, namely, methanol, ethanol, ethyl acetate, and acetone. Table 1 summarizes the results, reported as solvent-free compositions. It is evident from the results that, in all four ternary systems, the location of the double-saturation point at the two temperatures shifted slightly, with the D-G-ethanol ternary system showing the largest movement. Because a larger shift in the double-saturation point composition generally translates to a higher per-pass crystallization yield, ethanol was selected as the crystallization solvent to be studied further. More experimental data were then collected to construct the SLE phase diagram of the ternary system of D, G, and ethanol. Mixtures of pure D and pure G in various proportions were prepared, and a sufficient amount of ethanol was added to completely dissolve one of the two isoflavones. After sonication of the mixture at constant temperature for 2 h, a small amount

Figure 7. Fractionation of isoflavones D and G based on the crystallization process: (a) SLE phase diagram of D + G + EtOH at 15 and 60 °C showing the process points, (b) proposed process flowsheet based on temperature swing, (c) purge versus GLY concentrations in the crystallizers.

of mother liquor was sampled and analyzed using HPLC. The identity of the remaining solid was visually determined from its color. The data are summarized in Table 2 and plotted on an SLE phase diagram (Figure 7a) to outline the phase boundaries at 15 °C (blue curve) and at 60 °C (red curve). The phase boundaries at a given temperature define four regions on the diagram, in which a single phase exists or multiple phases exist in equilibrium. For clarity, these regions are marked only for 15 °C in Figure 7a. Region L is the unsaturated liquid region, where no crystal can be obtained. Regions D(s) + L and G(s) + L are two-phase regions where pure D and pure G,

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Table 3. Material Balance Calculation Results for Different Purge Ratios, with S/F ) 4 and a Basis of 100 kg/h Mixed Isoflavones Feed (Solvent-Free) flow rate (kg/h) purge ratio (% of stream 5)

stream 1

stream 2

stream 3

stream 4

stream 5

10 20 30 40 50

34 918 27 813 22 749 19 206 15 476

27 287 21 735 17 778 15 010 13 016

17 869 14 227 11 635 9 823 8 517

15 221 12 117 9 909 8 365 7 253

38 687 34 641 32 355 31 844 30 752

respectively, can be crystallized out. The blue-dotted lines define the D(s) + G(s) + L region, where both D and G crystallize out. With the SLE phase diagram in hand, the crystallization-based separation process can be synthesized. The process path is depicted on the phase diagram in Figure 7a, and the corresponding flowsheet is shown in Figure 7b. Note that all compositions are shown on a GLY-free basis, even though GLY is actually present in the feed. The feed composition (32.0% D, 62.5% G, and 5.5% GLY, by mass) is taken from the results of the antisolvent crystallization test with an S/F value of 4. This feed stream (not shown in Figure 7a) is mixed with ethanol or a recycle stream (point 5) such that the resulting mixture (point 1) is inside the D(s) + L region at 60 °C. Therefore, isoflavone D crystallizes out upon cooling to 60 °C inside the first crystallizer (C1), leaving a mother liquor (point 2) with a composition close to the double-saturation point at 60 °C. To obtain isoflavone G, some ethanol is added to this mother liquor to produce a mixture inside the G(s) + L region at 15 °C (point 3). Cooling to 15 °C inside the second crystallizer (C2) causes G to crystallize out, and the mother liquor composition is given by point 4. Some ethanol is evaporated from this stream to produce recycle stream 5, which is partially purged to avoid accumulation of GLY and any other impurities in the process. Note that makeup ethanol is necessary to compensate for the unavoidable loss of ethanol to the purge stream. Because of the recycle stream, the concentration of impurities in the process will be higher than that in the feed. The presence of too much impurity in the crystallizer feed might affect product purity through cocrystallization or inclusion. Crystallization tests with different amounts of impurities in the feed would be needed to determine the maximum allowable concentration of various impurities. Obviously, the impurity concentration in the crystallizer feed streams at steady state depends on the purge ratio (fraction of stream 5 being purged). Figure 7c shows the relationship between the GLY concentration in the process, the purge ratio, and the overall recovery of the desired isoflavones, based on material balance calculations. The GLY concentration shown in the figure is for the feed stream of C2 in mass percentage on a solvent-free basis. (The GLY concentration in the feed stream of C1 is slightly lower.) If, for example, the maximum allowable GLY concentration in both crystallizers is 10%, then a purge ratio of about 16% is required, corresponding to a 50% overall recovery of the desired isoflavones. The flow rates of various streams in the process for different purge ratios are summarized in Table 3. From such material balance calculations, a preliminary estimate of the process economics can be obtained. Note that, because the production capacity is typically low and isoflavones are high-value fine chemicals, the process might still be economically feasible despite the relatively low throughput as indicated by the high recycle flow rate.

Concluding Remarks One of the major challenges in process development is the conceptualization of a realistic process that is viable for commercial manufacturing. This is particularly true for naturalproduct processes, for which basic information such as thermodynamic and physical properties of the key components involved are seldom available. In such a situation, conceptual design must proceed alongside bench-scale experiments, which provide the missing information in a practical and systematic manner. This exercise results in feasible process alternatives that can be evaluated via material balance calculations. Using such an approach, process alternatives based on the use of chromatography and crystallization for separating and purifying soy isoflavones have been developed. These unit operations are used both for removing impurities and for obtaining the desired isoflavones as separate pure products. For process development, cost estimation and overall process optimization remain to be done. For product development, quantification of the quality of the isoflavone products is needed. For example, biological assays for measuring estrogenic and antioxidant activities should be developed to determine the activities of the purified daidzein and genistein. Such efforts are currently underway. Acknowledgment We thank Mr. Alex Chan and Mr. Stephen Chan for their help with the experiments. Financial support from the Research Grants Council (Grant HKUST602704) is also gratefully acknowledged. Notation D ) daidzein G ) genistein GLY ) glycitein L ) liquid phase xi ) mass fraction of component i Literature Cited (1) Wu, Q. L.; Wang, M. F.; Simon, J. E. Analytical Methods to Determine Phytoestrogenic Compounds. J. Chromatogr. B 2004, 812, 325. (2) Wang, H. J.; Murphy, P. A. Isoflavone Composition of American and Japanese Soybeans in Iowa: Effects of Variety, Crop Year, and Location. J. Agric. Food Chem. 1994, 42, 1674. (3) Wang, H. J.; Murphy, P. A. Mass Balance Study of Isoflavones during Soybean Processing. J. Agric. Food Chem. 1996, 44, 2377. (4) Izumi, T.; Piskula, M. K.; Osawa, S.; Obata, A.; Tobe, K.; Saito, M.; Kataoka, S.; Kubota, Y.; Kikuchi, M. Soy Isoflavone Aglycones are Absorbed Faster and in Higher Amounts than Their Glucosides in Humans. J. Nutr. 2000, 130, 1695. (5) Setchell, K. D. R.; Brown, N. M.; Zimmer-Nechemias, L.; Brashear, W. T.; Wolfe, B. E.; Kirschner, A. S.; Heubi, J. E. Evidence for Lack of Absorption of Soy Isoflavone Glycosides in Humans, Supporting the Crucial Role of Intestinal Metabolism for Bioavailability. Am. J. Clin. Nutr. 2002, 76, 447. (6) Setchell, K. D. R. Phytoestrogens: The Biochemistry, Physiology, and Implications for Human Health of Soy Isoflavones. Am. J. Clin. Nutr. 1998, 68, 1333S. (7) Messina, M. J. Legumes and Soybeans: Overview of Their Nutritional Profiles and Health Effects. Am. J. Clin. Nutr. 1999, 70, 439S. (8) Leung, A. Y.; Foster, S. Encyclopedia of Common Natural Ingredients Used in Food, Drugs and Cosmetics, 2nd ed.; Wiley: New York, 1996. (9) Farmakalidis, E.; Murphy, P. A. Isolation of 6′′-O-acetylgenistin and 6′′-O-acetyldaidzin from Toasted Defatted Soyflakes. J. Agric. Food Chem. 1985, 33, 385. (10) Gugger, E.; Grabiel, R. D. Process for Production of Isoflavone Fractions from Soy. U.S. Patent 6,033,714, 2000.

Ind. Eng. Chem. Res., Vol. 46, No. 1, 2007 189 (11) Walter, E. D. Genistin (an Isoflavone Glucoside) and its Aglucone, Genistein, from Soybeans. J. Am. Chem. Soc. 1941, 63, 3273. (12) Bombardelli, E.; Gabetta, B. Soya Extract, Process for its Preparation and Pharmaceutical Composition. U.S. Patent 6,607,757, 2003. (13) Harjo, B.; Wibowo, C.; Ng, K. M. Development of Natural Product Manufacturing Processes: Phytochemicals. Chem. Eng. Res. Des. 2004, 82 (A8), 1010. (14) Coward, L.; Barnes, N. C.; Setchell, K. D. R.; Barnes, S. Genistein and Daidzein and Their β-Glycosides Conjugates: Anti-tumor Isoflavones in Soybean Foods from American and Asian Diets. J. Agric. Food Chem. 1993, 41, 1961. (15) Budavari, S.; O’Neil, M. J.; Smith, A.; Heckelman, P. E. Eds. The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 11th ed.; Merck: Rahway, NJ, 1989. (16) Zheng, B. L.; Yegge, J. A.; Bailey, D. T.; Sullivan, J. L. Process for the Isolation and Purification of Isoflavones. U.S. Patent 5,679,806, 1997. (17) Murphy, P. A.; Barua, K.; Hauck, C. C. Solvent Extraction Selection in the Determination of Isoflavones in Soy Foods. J. Chromatogr. B 2002, 777, 129. (18) Antia, F. D. A Simple Approach to Design and Control of Simulated Moving Bed Chromatographs. In Scale-up and Optimization in PreparatiVe Chromatography: Principles and Biopharmaceutical Applications; Rathore, A. S., Velayudhan, A., Eds.; Marcel Dekker: New York, 2003; p 173. (19) Ruthven, D. M.; Ching, C. B. Counter-current and Simulated Counter-current Adsorption Separation Processes. Chem. Eng. Sci. 1989, 44, 1011.

(20) Mazzotti, M.; Storti, G.; Morbidelli, M. Optimal Operation of Simulated Moving Bed Units for Nonlinear Chromatographic Separations. J. Chromatogr. A 1997, 769, 3. (21) Biressi, G.; Ludemann-Hombourger, O.; Mazzotti, M.; Nicoud, R. M.; Morbidelli, M. Design and Optimization of a Simulated Moving Bed Unit: Role of Deviations from Equilibrium Theory. J. Chromatogr. A 2000, 876, 3. (22) Lorenz, H.; Sheehan, P.; Seidel-Morgenstern, A. Coupling of Simulated Moving Bed Chromatography and Fractional Crystallization for Efficient Enantioseparation. J. Chromatogr. A 2001, 908, 201. (23) Fung, K. Y.; Ng, K. M.; Wibowo, C. Synthesis of Chromatography-Crystallization Hybrid Separation Processes. Ind. Eng. Chem. Res. 2005, 44, 910. (24) Dye, S. R.; Ng, K. M. Fractional Crystallization: Design Alternatives and Tradeoffs. AIChE J. 1995, 41, 2427. (25) Wibowo, C.; Ng, K. M. Unified Approach for Synthesizing Crystallization-Based Separation Processes. AIChE J. 2000, 46, 1400.

ReceiVed for reView August 4, 2006 ReVised manuscript receiVed October 17, 2006 Accepted October 25, 2006 IE061027F

Development of Process Alternatives for Separation and Purification of

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