Isolation and Purification of Fatty Acid Steryl Esters from Soybean Oil

Sep 10, 2008 - Depending on the source, deodorizer distillates usually have significantly different characteristics, uses, and value. Soybean oil deod...
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Ind. Eng. Chem. Res. 2008, 47, 7013–7018

7013

Isolation and Purification of Fatty Acid Steryl Esters from Soybean Oil Deodorizer Distillate Setiyo Gunawan, Cynthia Fabian, and Yi-Hsu Ju* Department of Chemical Engineering, National Taiwan UniVersity of Science and Technology, 43 Keelung Road, Section 4, Taipei 106-07, Taiwan

Depending on the source, deodorizer distillates usually have significantly different characteristics, uses, and value. Soybean oil deodorizer distillate (SODD) has been suggested as an alternative to marine animals as a natural source of squalene and a good raw material for the production of fatty acid steryl esters (FASEs), tocopherols, free phytosterols and fatty acids. The purpose of this work was to isolate and purify natural FASEs from SODD by a suitable method without degradation of the FASEs. Modified Soxhlet extraction, modified silica gel column chromatography, and binary solvent extraction were employed sequentially in this study to obtain FASEs from SODD. FASEs with a purity of 86.74% and a total recovery of 85.32% could be obtained in the final product. 1. Introduction Fatty acid steryl esters (FASEs) are important raw materials in the production of hormones and vitamins, and they are more effective at lowering serum cholesterol levels than free phytosterols.1 Knowledge of this physiological effect has led to the development of several functional foods, such as salad oil and dressing with added sterol and margarine blended with FASEs. In particular, because FASEs and oils (triacylglycerols, TAGs) completely dissolve each other, much attention is being focused on the addition of FASEs to oil-related foods.2 Soybean oil deodorizer distillate (SODD) has been suggested as an alternative to marine animals as a natural source of squalene3 and a good raw material for the production of FASEs, tocopherols, free phytosterols, and fatty acids.4 Because FASEs are minor components in SODD, it is a challenging task to obtain FASEs with high purity and recovery. Molecular distillation is by far the preferred method for isolating FASEs from SODD. Molecular distillation at 250 °C and 0.02 mmHg has been employed by several investigators2,5,6 for the isolation of two main groups of compounds. FASEs were contained in the residue fraction, which was rich in diacylglycerols (DAGs) and TAGs. Then, the DAGs and TAGs in the residue fraction were selectively hydrolyzed by a lipase,2 resulting in the production of a mixture composed of free fatty acids (FFAs), glycerols, and FASEs. Finally, after two molecular distillations operated in series, FASEs were obtained at 97.3% purity and 87.7% overall recovery. SODD in Taiwan typically contains high levels of FFAs and TAGs, and the efficient removal of these components is crucial for the concentration of FASEs in SODD. FASEs are prone to degradation under high-temperature, alkali, and basic conditions. Converting FFAs and free phytosterols in SODD to FASEs is not desired because FFAs in SODD have a low quality because of the harsh conditions used during soybean oil refining, which results in reactions such as oxidation and cis-trans conversion.7 The purpose of this work was to isolate and purify FASEs from SODD by a suitable method to avoid the degradation of FASEs. A modified Soxhlet extraction was first employed to separate SODD into two fractions based on differences in the polarities of the constituent compounds (see Figure 1). The resulting * To whom correspondence should be addressed. Tel.: 886-227376612. Fax: 886-2-27376644. E-mail: [email protected].

nonpolar lipid fraction (NPLF) is rich in hydrocarbons and FASEs, whereas the polar lipid fraction is rich in FFAs and acylglycerols. The NPLF was then fractionated in a modified silica gel column chromatography to yield an FASE-rich fraction. Then, FASEs with high purity were finally obtained by solvent extraction. 2. Materials and Methods 2.1. Materials. Three samples of SODD with different compositions were donated by TTET Union Corporation (Tainan, Taiwan). Thin-layer chromatography (TLC) aluminum plates (20 cm × 20 cm × 250 µm) were purchased from Merck (Darmstadt, Germany). Silica gel (70-230 mesh) was obtained from Silicycle (Quebec, Canada). Characteristics of the gel according to the manufacturer were as follows: particle size, 60-200 µm; pore size, 60 Å; pH, 7; water content, 6%; and specific surface area, 500 m2/g. Standard cholesteryl stearate; nonacosane; farnesene; cholesta-3,5-diene; squalene; stigmasterol; palmitic acid; stearic acid; oleic acid; linoleic acid; linolenic acid; R-, δ-, and γ-tocopherol; monooleylglycerol; diolein; tristearin; triolein; and tripalmitin were obtained from Sigma Chemicals Company (St. Louis, MO). Standard β-sitosterol (practical grade) was obtained from MP Biomedicals, LLC (Aurora, OH). All solvents and reagents were of either high-

Figure 1. Flowchart showing the separation and purification of FASEs from SODD.

10.1021/ie800346x CCC: $40.75  2008 American Chemical Society Published on Web 09/10/2008

7014 Ind. Eng. Chem. Res., Vol. 47, No. 18, 2008

performance liquid chromatography (HPLC) grade or analytical reagent grade and were obtained from commercial sources. 2.2. Extraction of the NPLF from SODD. Foreign material was removed from SODD at 50 °C by using 7-µm Advantec filter paper (Toyo Roshi Kaisha Ltd., Tokyo, Japan). Silica gel was kept in a furnace at 150 °C for 1 h to remove its water content. A Soxhlet extractor, equipped with a temperatureinsulating jacket and connected to a condenser system, was used as described by Gunawan et al.3 SODD (20 g) was dissolved in hexane (150 mL), and silica gel (60 g) was added to this solution. The mixture was magnetically stirred at 300 rpm and room temperature for 1 h. Afterward, SODD was coated onto silica gel by removing the hexane at 60 °C and 160 mmHg. Then, SODD-loaded silica gel was packed into an extraction thimble, and the top surface of the thimble was covered with cotton to prevent spillage. A thermocouple was inserted into the thimble, and the thimble was placed inside the Soxhlet extractor. The less-polar lipids were extracted with hexane (350 mL), which was heated. The hexane vapor traveled up the distillation arm and flooded into the chamber housing the thimble. The condenser ensured that any hexane vapor condensed and dripped back down into the chamber housing the thimble. The chamber containing the thimble slowly filled with hexane at the controlled temperature (-6 °C). A refrigerated circulating bath was used with ethyl alcohol as the refrigerant, which flowed through the jacket of the extraction chamber. When the chamber was almost full, it was automatically emptied by a siphon sidearm, which returned the hexane to the distillation flask. After 11 h, the desired compounds were concentrated in the distillation flask. The solution in the distillation flask was then filtered, and the resulting hexane extractive is referred to as the NPLF. The NPLF of SODD from sample C was used for the next study. 2.3. Elution of FASEs from the NPLF. A modified version of the classical silica gel column chromatograph (300 mm × 4 mm i.d. glass tube) equipped with a jacket, a valve to control the flow rate of eluent, and a condenser system was used as described by Gunawan and Ju.8 Ethyl alcohol was used as the refrigerant and was circulated in the jacket to control the column (packing region) temperature. Silica gel (30 g) was kept in a furnace at 150 °C for 1 h to remove its water content. Next, a slurry of silica gel in hexane was poured into the column, which had previously been half-filled with hexane. The exit of the column was plugged with 1 g of cotton to retain the silica gel, and a thermocouple was inserted above the cotton. The hexane was allowed to drain slightly during packing. The top surface of the silica gel was covered with 1 g of cotton, and a thermocouple was placed above the cotton. The hexane level was lowered until it was ca. 100 mL above the cotton at the top. The NPLF (3 g) was added to the column at room temperature (23 ( 1 °C). The column was eluted with a mobile phase (50 mL of hexane) that was put into a 500-mL roundbottom flask at the start of the run and was heated. The mobilephase vapor traveled up the distillation arm and flooded into the column housing the silica gel and the NPLF. The condenser ensured that any mobile-phase vapor condensed and dripped back down into the column. The column was slowly eluted with the mobile phase at controlled flow rate and temperature. The first fraction, which contained most hydrocarbons, was obtained using hexane as the mobile phase at a flow rate of 4.0 ( 0.18 mL/min after 4.5 h. The next fraction, which was pure squalene, was obtained at a hexane flow rate of 33.33 ( 2.23 mL/min after an additional 2.5 h. The third fraction, which contained most FASEs in the NPLF, was obtained after eluting the column

with a solvent mixture (hexane/ethyl acetate ) 99:1, v/v) as the mobile phase at 33.33 ( 2.23 mL/min for 3 h. The remaining compounds that were still absorbed on the silica gel were eluted from the column with ethyl acetate at 33.33 ( 2.23 mL/min for 1 h. 2.4. Distillation of the NPLF. The NPLF was subjected to vacuum distillation for 10 min, under the following conditions: NPLF ) 1 g, pressure ) 5 mTorr, temperature ) 220 ( 1 °C. All FASEs remained in the residue, which was rich in acylglycerols. Most hydrocarbons and FFAs were isolated in the distillate. 2.5. Purification of FASEs by Binary Solvent Extraction. Impurities (aldehydes, ketones, squalene, tocopherols, FFAs, and acylglycerols) in the third fraction obtained from modified silica gel column chromatography of the NPLF were extracted using a binary solvent at room temperature. Sample (0.5 g) was thoroughly mixed with 40 mL of a mixture of water and acetone for 15 min using a Vortex-Genie 2 mixer (Scientific Industries, New York). The mixture was then centrifuged (7000 × g) at room temperature for 15 min by using Avanti J-25 (Beckman Coulter, Fullerton, CA). The supernatant, which contained most of the impurities, was discarded. FASEs were collected in the precipitate. This process was repeated several times until most impurities were removed, as confirmed by the high-temperature gas chromatography (HT-GC) and TLC analyses. The final precipitate was collected and the residual solvent was removed to obtain high-purity FASEs. 2.6. Distillation of the Third Fraction. The third fraction obtained from modified silica gel column chromatography of the NPLF was introduced to a simple vacuum distillation, under the following operation conditions: NPLF ) 0.5 g, pressure ) 5 mTorr, temperature ) 220 ( 1 °C, and time ) 10 min. FASEs were contained in the residue, which was also rich in acylglycerols. All FFAs were isolated in the distillate. 2.7. Determination of FFA Content. FFA contents as oleic acid were determined by the American Oil Chemists’ Society (AOCS) official method using phenolphthalein as an indicator.9 Sample was dissolved in ethyl alcohol at 60 °C and FFAs contained in the sample were neutralized with sodium hydroxide solution. The sample mass and the volume of sodium hydroxide used were used to calculate the contents of FFAs. 2.8. Analysis by TLC and HT-GC. Compounds in each fraction were identified by TLC and HT-GC using authentic standards as described by Gunawan et al.3 TLC plates were developed in hexane. After the plates had been dried in air, spots on each plate were visualized by exposing the chromatogram to iodine vapor. Spots for FASEs and steroidal hydrocarbons were detected by spraying with a fresh solution of 50 mg of ferric chloride in a mixture of 90 mL of water, 5 mL of acetic acid, and 5 mL of sulfuric acid. After the plates had been heated at 100 °C for 3-5 min, FASEs and steroidal hydrocarbons were indicated by a red-violet color.10 The contents of hydrocarbons, FASEs, free phytosterols, tocopherols, and acylglycerols in each fraction were determined by HT-GC as described by Gunawan et al.3 The chromatographic analysis was performed on a TLC plate and a Shimadzu GC-17A (Kyoto, Japan) gas chromatograph equipped with a flame ionization detector. Separations were carried out on a DB5HT (5%-phenyl)methylpolysiloxane nonpolar column (15 m × 0.32 mm i.d.; Agilent Technologies, Palo Alto, CA). The temperatures of the injector and the detector were both set at 370 °C. The temperature of the column was started at 80 °C, increased to 365 °C at 15 °C/min, and maintained at 365 °C for 8 min. The split ratio was 1:50 using nitrogen as the carrier

Ind. Eng. Chem. Res., Vol. 47, No. 18, 2008 7015 Table 3. Compositions of the NPLFs Obtained from SODD with Different Initial FASE Contentsa–c

Table 1. External Standard Calibration Curve Parameters of Components in SODDa A ) wC

w

R2

cholesteryl stearate squalene R-tocopherols stigmasterol monooleylglycerol diolein tristerarin

48.772 123.99 98.812 144.29 1974.5 185.31 115.56

0.9989 0.9999 0.9969 0.9979 0.9979 0.9989 0.9971

compounds FASEs tocopherols free phytosterols FFAs

a

A, area; C, analyte concentration (mg/kg, ppm); w, calibration factor; R2, squared correlation coefficient.

otherse

Table 2. Compositions of SODD Obtained with Different Initial FASE Contents (wt %)a compounds

sample A

sample Bb

sample C

FASEs tocopherols free phytosterols FFAs acylglycerols othersc

1.80 ( 0.28 7.73 ( 0.95 6.20 ( 0.50 46.46 ( 0.63 17.80 ( 0.65 19.98 ( 1.74

3.91 ( 0.39 6.40 ( 0.85 5.36 ( 0.19 45.38 ( 2.13 23.30 ( 2.20 17.06 ( 0.16

4.12 ( 0.23 14.89 ( 0.22 11.25 ( 0.14 41.63 ( 0.50 10.37 ( 0.12 17.74 ( 0.70

a

Averages of three independent measurements. b Reference 3. c Hydrocarbons, aldehydes, ketones, pesticides, herbicides, and breakdown products of tocopherols and free phytosterols.

gas with a linear velocity of 30 cm/s at 80 °C. A 20-mg sample was dissolved in 1 mL of ethyl acetate, and a 1-µL sample of this solution was taken and injected into the HT-GC instrument. External standard calibration curves were obtained using 0.2-20 mg of pure standards. Cholesteryl stearate was selected for the determination of the FASE calibration factor and was used for all FASEs. The calibration factors of squalene, stigmasterol, R-tocopherol, monooleylglycerol, diolein, and tristearin were used to quantify squalene, free phytosterols, tocopherols, monoacylglycerols (MAGs), DAGs, and TAGs, respectively. The parameters obtained for the external standard calibration curves are listed in Table 1. 2.9. Statistical Analysis. The reliability of the results was checked by statistical analyses. The standard deviation (SD) of the measures was calculated from the difference between the value obtained for an individual experiment, x, and the mean value of three independent experiments, jx, using the formula SD )

[∑ (x - x) ]/(n - 1) _

2

acylglycerols

(1)

where n represents the total number of experiments. The statistical significance of the effects of each parameter was systematically checked by the p-value method.11 3. Results and Discussion 3.1. Characterization. Compositions of the three samples of SODD obtained from the deodorization step during chemical refining of soybean oil are listed in Table 2. From Table 2, it is apparent that the SODD used in this study has lower FFA contents (40-50%) than the SODD obtained from physical refining (>70%).12 The FFA and TAG contents in sample C were significantly lower (p < 0.05), whereas the tocopherol and free phytosterol contents were significantly higher (p < 0.05) than those in samples A and B. The FASE content was significantly lower (p < 0.05) in sample A than in samples B and C. 3.2. Extraction of the NPLF from SODD. It has been shown that a modified Soxhlet extraction, which is much easier to operate than molecular distillation, can concentrate hydro-

sample A

sample Bd

sample C

6.11 ( 0.91 (92.85 ( 2.98) 2.15 ( 1.03 (7.50 ( 3.18) 0.35 ( 0.15 (1.51 ( 0.56) 37.03 ( 5.14 (21.74 ( 2.13) 1.26 ( 0.53 (3.79 ( 1.04) 46.53 ( 3.43 (70.69 ( 5.84)

12.19 ( 0.30 (94.32 ( 3.01) 2.39 ( 0.51 (9.83 ( 2.18) 0.41 ( 0.14 (2.09 ( 0.65) 35.05 ( 2.77 (20.41 ( 1.46) 3.49 ( 1.80 (5.89 ( 3.08) 40.19 ( 0.06 (75.91 ( 1.78)

14.05 ( 1.57 (92.47 ( 3.40) 3.98 ( 0.97 (6.67 ( 1.41) 0.29 ( 0.20 (0.64 ( 0.40) 31.71 ( 1.13 (19.18 ( 1.29) 2.95 ( 0.55 (6.05 ( 1.66) 40.88 ( 2.57 (65.67 ( 5.95)

a Averages of three independent experiments. b Values reported as contents (wt %), with recoveries (%) in parentheses. c Recovery ) {[(NPLF mass, g) × (content of the compound in the NPLF, %)]/ [(SODD mass, g) × (content of the compound in SODD, %)]} × 100%. d Reference 3. e Hydrocarbons, aldehydes, ketones, pesticides, herbicides, and breakdown products of tocopherols and free phytosterols.

carbons and FASEs into the NPLF. A modified Soxhlet extraction was used to separate crude rice bran oil13 and soybean oil deodorizer distillate3 into two fractions based on differences in the polarities of the constituent compounds. Table 3 shows the compositions of the NPLFs obtained from SODD with different initial FASEs contents. The component recoveries in each step of the separation were calculated with the equation recovery of individual step ) {[(weight of the product, g) × (content of the compounds in the product, %)]/ [(weight of feed, g) × (content of the component in feed, %)]} × 100% (2) The total recovery was calculated by multiplying the recoveries of the individual steps. Whereas the FASE contents in the NPLFs increased with increasing initial FASE content in the SODD, the recoveries of FASEs were not significantly different (p > 0.05) among the NPLFs obtained in this study. It can be seen that, for the three samples studied, this method is capable of eliminating tocopherols, free phytosterols, FFAs, and acylglcerols from SODD by87.99-95.68%,97.26-99.76%,76.13-82.11%,and91.03-97.25%, respectively. 3.3. Elution of FASEs from the NPLF. The NPLF of sample C was a yellowish liquid at room temperature. Aliphatic, steroidal, sesquiterpene, and triterpene (squalene) hydrocarbons represented about 40-50% of the NPLF. Because of this high content, efficient removal of these hydrocarbons and FFAs is crucial for high-purity FASEs to be obtained. Repeated modified Soxhlet extraction, solvent crystallization (hexane, acetone, acetic acid, methanol, and isopropanol), and solvent extraction using a mixture of solvents (water/acetone and water/methanol) did not result in a successful separation (data not shown). By subjecting the NPLF to distillation at 220 °C and 5 mTorr for 10 min, it was possible to concentrate most of the FFAs and aliphatic, steroidal, and sesquiterpene hydrocarbons in the distillate, whereas most of the free phytosterols and all of the FASEs and acylglycerols remained in the residue fraction, as shown in Table 4. The disadvantages of distillation are that it results in the degradation of FASEs and a decrease of the squalene recovery. It was found that the total amounts of FFAs and free phytosterols increased as a result of FASE degradation. To check this FASE degradation, standard cholesteryl stearate (white solid at 25 °C) was heated at 220 °C and 5 mTorr for 10 min. The results showed that cholesteryl stearate was degraded

7016 Ind. Eng. Chem. Res., Vol. 47, No. 18, 2008 Table 4. Comparison of the Fractionations of the NPLFs Obtained by Modified Silica Gel Column Chromatography and Distillation (wt %)a-c modified silica gel column chromatography compound FASEs tocopherols free phytosterols FFAs acylglycerols squalene others

distillation

first fraction

second fraction

third fraction

residue

NDd (0) ND (0) ND (0) ND (0) ND (0) 0.53 ( 0.50 (4.82 ( 1.26) 99.47 ( 0.50e (97.45 ( 2.95)

ND (0) ND (0) ND (0) ND (0) ND (0) 98.23 ( 0.12 (92.36 ( 3.89) 1.77 ( 0.12e (0.19 ( 0.02)

79.99 ( 4.48 (97.38 ( 3.86) 1.45 ( 0.44 (12.14 ( 4.62) ND (0) 2.96 ( 0.51 (2.11 ( 0.41) 5.46 ( 1.66 (24.13 ( 7.38) 1.00 ( 0.53 (2.68 ( 1.58) 6.13 ( 5.58f (1.93 ( 1.81)

68.71 ( 3.58 (96.76 ( 1.13) 3.62 ( 1.39 (33.75 ( 1.03) 0.71 ( 0.22 (99.28 ( 3.72) 1.12 ( 0.05 (0.73 ( 0.13) 14.05 ( 2.36 (99.81 ( 0.27) 9.03 ( 0.36 (23.38 ( 1.68) 2.76 ( 1.18g (3.31 ( 1.34)

a Averages of three independent experiments. b Values reported as contents (wt %), with recoveries (%) in parentheses. c Recovery ) {[(fraction mass, g) × (content of the compound in the fraction, %)]/[(NPLF mass, g) × (content of the compound in the NPLF, %)]} × 100%. d Not detected. e Aliphatic, steroidal, and sesquiterpene hydrocarbons. f Aldehydes and ketones. g Mainly steroidal hydrocarbons and breakdown products of tocopherols and free phytosterols.

to stearic acid and cholesterol as evidenced by HT-GC analysis, and it was brownish solid at 25 °C. Results obtained by applying a modified silica gel column chromatography on the NPLF are also reported in Table 4. Details on the operation of modified silica gel column can be found elsewhere.8 It can be seen that modified silica gel column chromatography is capable of concentrating squalene in the second fraction (purity >98%, 92.36% recovery) and FASEs in the third fraction (79.99% purity, 97.38% recovery). In the third fraction, the FASE contents are significantly higher than (p < 0.05), whereas the recoveries are not significantly different (p > 0.05) from those obtained by distillation. It is clear from Table 4 that modified silica gel column chromatography is capable of isolating squalene and FASEs from the NPLF with high purity and recovery. 3.4. Purification of FASEs from the Third Fraction of the NPLF. The third fraction of the NPLF from sample C is a yellowish solid at room temperature. Analysis of this fraction showed the presence of a small amount of squalene but the absence of aliphatic, steroidal, and sesquiterpene hydrocarbons as evidenced by HT-GC and TLC analyses using pure hexane as the mobile phase (data not shown). Acetone is used mainly as a solvent and intermediate in chemical production. It also has food uses as an extraction solvent for fats and oils and as a precipitating agent in sugar and starch purification. The current interest in acetone comes from the fact that acetone was removed from the list of toxic chemicals. No evidence is available to suggest that acetone is carcinogenic in humans or animals.14 Acetone has been used to measure the phospholipid contents in vegetable oils by the nephelometric method15 and to enrich phospholipid contents in commercial soybean lecithin.16 In another study, acetone crystallization followed by filtration was used to determine wax ester contents in vegetable oils.17,18 This method is more accurate when applied to oils rich in crystallizable wax esters, such as crude sunflower oil or rice bran oil.19 In this study, impurities in the third fraction obtained from modified silica gel column chromatography of NPLFs were extracted at room temperature using a water/acetone mixture as the solvent. It was found that water-to-acetone volume ratios of more than 30:70 did not result in good separations (data not shown). When pure acetone was employed as the solvent, the recoveries of FASEs in the precipitate were quite low, as can

Table 5. Comparison of Results Obtained by Solvent Extraction and Distillation Operated on the Third Fraction of the NPLF (wt %)a-c water/acetone (v/v) compounds FASEs others

d

0:100

10:90

distillation 20:80

residue

98.64 ( 1.07 94.95 ( 0.64 88.81 ( 1.63 88.59 ( 1.38 (25.04 ( 1.68) (58.99 ( 3.98) (68.85 ( 4.09) (85.56 ( 4.00) 1.36 ( 1.07 5.05 ( 0.64 9.96 ( 1.19 10.41 ( 1.38 (2.86 ( 1.67) (26.92 ( 1.91) (65.91 ( 5.20) (97.82 ( 2.04)

a Averages of three independent experiments. b Values reported as contents (wt %), with recoveries (%) in parentheses. c Recovery ) {[(fraction mass, g) × (content of the compound in the fraction, %)]/ [(third fraction of the NPLF mass, g) × (content of the compound in the third fraction of the NPLF, %)]} × 100%. d Mainly acylgycerols.

be seen from Table 5. Apparently, most FASEs still dissolved in the acetone phase. As the percentage of water in the water/ acetone mixture increased, the solubility of water-insoluble compounds such as FASEs and acylglycerols decreased. As a result, the FASE recoveries were significantly higher (p < 0.05) whereas the FASE contents were significantly lower (p < 0.05) as the percentage of water was increased. The precipitation fractions obtained using water-to-acetone volume ratios of 10: 90 and 20:80 were yellowish and semisolid at room temperature. A white solid at room temperature was obtained when pure acetone was the solvent. In the precipitate obtained for a water/acetone ratio of 20: 80, the FASE content was not significantly different (p > 0.05), whereas the recovery was significantly lower (p < 0.05), compared to those obtained from distillation (residue). However, the residue fraction obtained from distillation was a brownish solid at room temperature as a result of the degradation of FASEs. Therefore, solvent extraction with a mixture of water and acetone as the solvent is preferred because of the absence of thermal degradation and also because of the higher-purity FASEs obtained. The method proposed in this work provides better separation of FASEs with higher purity and recovery when applied to lipid fractions with high initial FASEs contents and low contents of acylglycerols and hydrocarbons. The energy cost for solvent recovery should be economically feasible because acetone does not form an azeotrope with water.20 HT-GC chromatograms of FASE fractions collected from separation steps used in this study are shown in Figure 2. Figure 2A is the chromatogram of sample C with an initial FASE content of 4.12%. The NPLF consists mostly of aliphatic, steroidal, and sesquiterpene hydrocarbons as can be seen from

Ind. Eng. Chem. Res., Vol. 47, No. 18, 2008 7017

Figure 2. HT-GC chromatogram profiles of (A) sample C, (B) NPLF, (C) the third fraction from modified silica gel column chromatography, (D) the precipitation fraction from solvent extraction (water/acetone ) 20:80, v/v), and (E) the supernatant fraction from solvent extraction. Table 6. Enrichment of FASEs Using a Sequence of Separation Steps with SODD Sample C as the Starting Materiala separation process modified Soxhlet extraction modified silica gel column chromatography binary solvent extractione

Figure 3. Flowchart showing the water/acetone extraction sequence on the third fraction of the NPLF. Table 7. Precipitation Fractions Obtained by Combination Solvent Extraction on the Third Fraction of the NPLF (wt %)a-c compounds

G ratio total recovery (%) 3.41 19.42 21.56

92.47b 90.05c 62.00d

Initial FASE content of 4.12%. b Total recovery ) {[(NPLF mass, g) × (content of FASEs in the NPLF, %)]/[(SODD mass, g) × (content of FASEs in SODD, %)]} × 100%. c Total recovery ) {[(third fraction of the NPLF mass, g) × (content of FASEs in the third fraction of the NPLF, %)]/[(SODD mass, g) × (content of FASEs in SODD, %)]} × 100%. d Total recovery ) {[(precipitate mass, g) × (content of FASEs in precipitate, %)]/[(SODD mass, g) × (content of FASEs in SODD, %)]} × 100%. e Water/acetone (20:80, v/v). a

Figure 2B. These hydrocarbons represent about 40-50% of the NPLF. The third fraction collected from modified silica gel column chromatography is mostly FASEs (Figure 2C). The precipitate fraction, which was obtained after extracting the previous fraction with a mixed solvent (water/acetone ) 20: 80, v/v), contains high-purity FASEs (Figure 2D). The supernatant is mostly FFAs (Figure 2E). To clarify the effectiveness of separation steps used in this study on the isolation and purification of FASEs, the ratio G is introduced (Table 6) as G ) FASE content in the fraction/FASE content in the SODD (3) It can be seen that the FASE enrichment (G ratio) of SODD samples A-C in modified Soxhlet extraction was 3.39, 3.12, and 3.41, respectively. A 21.56-fold increase in FASE content could be achieved in the precipitate fraction after the application of modified Soxhlet extraction, modified silica gel column chromatography, and solvent extraction (water/acetone ) 20: 80, v/v) to sample C.

FASEs othersd amount (%)e

precipitate 1

precipitate 2

precipitate 3

96.55 ( 0.62 (23.20 ( 0.21) 3.45 ( 0.62 (2.91 ( 1.35) 23.24 ( 2.06

86.82 ( 0.20 (56.16 ( 1.73) 11.17 ( 1.27 (26.22 ( 2.30) 62.17 ( 1.66

70.78 ( 1.41 (15.32 ( 5.97) 28.40 ( 0.35 (10.38 ( 4.72) 14.60 ( 1.53

a Averages of three independent experiments. b Values reported as contents (wt %), with recoveries (%) in parentheses. c Recovery ) {[(fraction mass, g) × (content of the compound in the fraction, %)]/ [(third fraction of the NPLF mass, g) × (content of the compound in the third fraction of the NPLF, %)]} × 100%. d Mainly acylgycerols. e Amount ) [(precipitate mass, g)/(third fraction of the NPLF mass, g)] × 100%.

Because the optimal FASE purity attainable after water/ acetone extraction (water/acetone ) 20:80, v/v) was about 88.81% with 68.85% recovery, the combination of water/acetone extractions is proposed to obtain higher purity and recovery as shown in Figure 3. After subjecting the third fraction of the NPLF to solvent extraction, three FASE fractions could be obtained in different purities, i.e. 96.55% (23.20% recovery), 86.82% (56.16% recovery), and 70.78% (15.32% recovery), by using mixed solvents with water-to-acetone volume ratios of 0:100, 10:90, and 20:80, respectively, as can be seen in Table 7. 4. Conclusions By combining a modified Soxhlet extraction, a modified silica gel column chromatography, and water/acetone extractions, the FASE fraction could be obtained with a high purity (86.74%) and a high total recovery (85.32%) from SODD with an initial FASE content of 4.12%. According to the results, this separation process can yield the FASE fraction from SODD without

7018 Ind. Eng. Chem. Res., Vol. 47, No. 18, 2008

degradation of the FASEs. The advantage of the process is that, starting with SODD, high-purity squalene and FASEs can be obtained. In addition, the polar fraction (PLF in Figure 1) contains most of the tocopherols and free physterols and can be processed further to obtain pure tocopherols and free phytosterols. Nomenclature A ) peak area C ) analyte concentration (mg/kg, ppm) DAGs ) diacylglycerols FASEs ) fatty acid steryl esters FFAs ) free fatty acids G ) FASE enrichment ratio HPLC ) high-performance liquid chromatography HT-GC ) high-temperature gas chromatography MAGs ) monoacylglycerols n ) total number of experiments NPLF ) nonpolar lipid fraction p ) probability SD ) standard deviation of the measures SODD ) soybean oil deodorizer distillate TAGs ) triacylglycerols TLC ) thin-layer chromatography w ) analyte calibration factor x ) parameter value for an individual experiment jx ) mean parameter value for three independent experiments Acknowledgment This work was supported by a grant (NSC94-2214-E011004) from the National Science Council of Taiwan. Literature Cited (1) Moreau, R. A.; Whitaker, B. D.; Hicks, K. B. Phytosterols, phytostanols, and their conjugates in foods: Structural diversity, quantitative analysis, and health-promoting uses. Prog. Lipid Res. 2002, 41, 457. (2) Hirota, Y.; Nagao, T.; Watanabe, Y.; Suenaga, M.; Nakai, S.; Kitano, M.; Sugihara, A.; Shimada, Y. Purification of steryl esters from soybean oil deodorizer distillate. J. Am. Oil Chem. Soc. 2003, 80, 341. (3) Gunawan, S.; Kasim, N. S.; Ju, Y. H. Separation and purification of squalene from soybean oil deodorizer distillate. Sep. Purif. Technol. 2008, 60, 128. (4) Winters, R. L. In Proceedings: World Conference on Emerging Technologies in the Fats and Oils Industry; Baldwin, A. R., Ed.; American Oil Chemists’ Society: Champaign, IL, 1986; p 186.

(5) Shimada, Y.; Nakai, S.; Suenaga, M.; Sugihara, A.; Kitano, M.; Tominaga, Y. Facile purification of tocopherols from soybean oil deodorizer distillate in high yield using lipase. J. Am. Oil Chem. Soc. 2000, 77, 1009. (6) Watanabe, Y.; Hirota, Y.; Nagao, T.; Kitano, M.; Shimada, Y. Purification of tocopherols and phytosterols by two-step in situ enzymatic reaction. J. Am. Oil Chem. Soc. 2004, 81, 339. (7) Mendes, M. F.; Pessoa, F. L. P.; Uller, A. M. C. An economic evaluation based on experimental study of the vitamin E concentration present in deodorizer distillate of soybean oil using supercritical CO2. J. Supercrit. Fluids 2002, 23, 257. (8) Gunawan, S.; Ju, Y. H. Design and operation of a modified silica gel column chromatography. J. Chin. Inst. Chem. Eng., manuscript submitted. (9) Official Methods and Recommended Practices of the American Oil Chemists’ Society, 5th ed.; American Oil Chemists’ Society: Champaign, IL, 1997; Method Ca 5a-40. (10) Fried, B. Lipids. In Handbook of Thin-layer Chromatography; Sherma, J., Fried, B., Eds.; Marcel Dekker: New York, 1996; p 704. (11) Montgomery, D. C. Design and Analysis of Experiments, 6th ed.; John Wiley and Sons: New York, 2005; p 96. (12) Verleyen, T.; Verhe, R.; Garcia, L.; Dewettinck, K.; Huyghebaert, A.; Greyt, W. D. Gas chromatographic characterization of vegetable oil deodorization distillate. J. Chromatogr. A 2001, 921, 277. (13) Gunawan, S.; Vali, S. R.; Ju, Y. H. Purification and identification of rice bran oil fatty acid steryl and wax esters. J. Am. Oil Chem. Soc. 2006, 83, 449. (14) Integrated Risk Information System (IRIS); U.S. Environmental Protection Agency: Washington, DC. Available at http://www.epa.gov/ncea/ iris (accessed Jan 2008). (15) Official Methods and Recommended Practices of the American Oil Chemists’ Society, 5th ed.; American Oil Chemists’ Society: Champaign, IL, 1997; Method Ca 19-86. (16) De, B. K.; Bhattacharyya, D. K. Physical refining of rice bran oil in relation to degumming and dewaxing. J. Am. Oil Chem. Soc. 1998, 75, 1683. (17) Rajam, L.; Kumar, D. R. S.; Sundaresan, A.; Arumughan, C. A novel process for physically refining rice bran oil through simultaneous degumming and dewaxing. J. Am. Oil Chem. Soc. 2005, 82, 213. (18) Vandana, V.; Karuna, M. S. L.; Vijayalakshmi, P.; Prasad, R. B. N. A simple method to enrich phospholipid content in commercial soybean lecithin. J. Am. Oil. Chem. Soc. 2001, 78, 555. (19) Vali, S. R.; Ju, Y. H.; Kaimal, T. N. B.; Chern, Y. T. A process for the preparation of food grade rice bran wax and the determination of its composition. J. Am. Oil. Chem. Soc. 2005, 82, 57. (20) Kuk, M. S.; Tetlow, R.; Dowd, M. K. Cottonseed extraction with mixture of acetone and hexane. J. Am. Oil. Chem. Soc. 2005, 82, 609.

ReceiVed for reView March 2, 2008 ReVised manuscript receiVed May 29, 2008 Accepted June 17, 2008 IE800346X