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Isolation and characterization of wax esters in meadowfoam oil
INDUSTRIALCROPS AND PRODUCTS AN INTERNATIONAL
ELSEVIER
JOURNAL
Industrial Crops and Products 5 (1996) 239-243
Isolation and characterization
of wax esters in meadowfoam oil
Terry A. Isbell *, Kenneth D. Carlson, Thomas P. Abbott, Bliss S. Phillips, Selim M. Erhan, Robert Kleiman New Crops Research, National
Center for Agricultural Utilization Research, Agricultural 1815 N. University Street, Peoria, IL 61604, USA
Research Service, USDA,
Received 20 November 1995; accepted 2 May 1996
Abstract Meadowfoam oil has potential use in the cosmetic industry due to its long chain fatty acids. However. particulates that form when the oil is stored could create problems in this application. Particulate matter found in refined meadowfoam oil samples was isolated by first centrifugation and then crystallization from acetone. The white crystalline solid had a melting point of 77”-78”C, and was characterized as a mixture of wax esters by ‘H nuclear magnetic resonance (NMR), 13C NMR and infrared (IR) analysis. Gas chromatography (GC) analysis of the wax esters indicated a distribution of esters from C4 to C& including odd chain esters. Base hydrolysis of the wax ester and GC analysis of the fatty methyl esters and fatty alcohols indicated a mixture of saturated methyl esters from Cl6 to C32 including small amounts of the odd-chain methyl esters C21-C29 with the predominate methyl ester being C24. The alcohol portion of the wax esters contained saturated chain lengths of CZ0--C3~including odd chains ofC21-C 29. The main alcohol component was saturated Cz4 alkanol. GC-MS (mass spectroscopy) confirmed the GC assignments. A normal phase high performance liquid chromatography (HPLC) technique was developed to determine the amount of wax ester (0.06% to 0.12%) in various types of refined meadowfoam oil. Keywords:
Odd-chain
Meadowfoam fatty alcohols
oil; Limnanthes
alba
seed oil; Saturated
1. Introduction Meadowfoam (limnanthes alba) is a new oil seed crop currently grown in northwestern United States. This winter annual crop is an economical and environmental alternative to the grass seeds being cultivated in the Willamette valley of Oregon. Meadowfoam production has increased steadily since 1984 under the supervision of the Oregon Meadowfoam Growers Association. Meadowfoam seeds yield up to 30% oil. The oil * Corresponding author. Fax: (309) 681-6524.
wax esters;
Fatty alcohols:
Odd-chain
fatty acids;
is composed of long chain fatty acids (Glaser et al., 1994) with 5-eicosenoic acid (64%) as the major fatty acid. The other main components are 5,13docosadienoic acid (19%), 5-docosenoic acid (3%) and 13-docosenoic acid (10%). This unique combination of monoenoic fatty acids makes for a very oxidatively stable oil with an AOM (active oxygen method) value of 200. In comparison, other vegetable oils such as soybean and high oleic sunflower have AOM values of 14 and 90, respectively (Fitch, 1994). Meadowfoam oil is utilized in cosmetic formulations. Due to the many rigorous requirements for cosmetic applications such as low color and high
0926-6690/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SO926-6690(96)000019-2
240
TA. Isbell et al. /Industrial Crops and Products 5 (1996) 239-243
clarity, problems in oil seed development can arise. Recently processed meadowfoam oil developed a precipitate upon storage in a warehouse. Since oil clarity is important for cosmetic applications, the isolation and characterization of this precipitate are necessary. The suspended contaminate was suspected to be a wax, possibly extracted from the seed coat during oil processing. Waxes and nonglycerol esters have been found in other seed oils (Grob et al., 1990; Mariani and Fedeli, 1989; Moulton, 1988) and posed similar crystallization and turbidity problems. In light of these findings, an evaluation of meadowfoam oil for the presence of waxes was initiated. 2. Materials and methods Meadowfoam oil samples were provided by the Fanning Corp. (Chicago, IL) and the Oregon Meadowfoam Growers Assoc. (Salem, OR). Potassium hydroxide and solvents for crystallization and chromatography were high performance liquid chromatography (HPLC) grade and obtained from Fisher Scientific (Fair Lawn, NJ). Saturated fatty acid methyl ester (FAME) standards were obtained from Alltech Associates, Inc. (Deerfield, IL) HPLC separations were performed on a Spectra Physics 8800 ternary pump system (San Jose, CA) equipped with a Spectra System AS3000 autosampler/injector (Therm0 Separation Products, Fremont, CA) coupled to an evaporative light scattering detector (ELSD) from Varex (Burtonsville, MD). A Dynamax silica column (250 x 4.6 mm i.d., 60 A, 8 pm) purchased from Rainin Instrument Co. (Wobum, MA) was used to separate the wax ester mixtures. Components were eluted from the column with an isocratic hexane : acetone 95 : 5 mixture at a flow rate of 1 ml min-’ . The ELSD drift tube was set at 35°C and the gas flow through the nebulizer was 1.5 standard 1 min-‘, 70 KPa N2. Retention times for eluted peaks: saturated wax ester 2.7 min, jojoba oil 3.0 min and meadowfoam oil 3.5 min. Oil samples containing wax esters to be analyzed by HPLC or gas chromatography (GC) were first warmed to dissolve the precipitated wax esters. GC separations of triglycerides and wax ester components were obtained on a GB-1 2.5 m x 0.32 mm id. column (Foxboro, New Haven, CT) in a
Hewlett Packard 5890 Series II gas chromatograph (Palo Alto, CA) with a flame ionization detector. The following conditions were used for analysis: programmed ramp from 50°C to 350°C at 30°C min-‘, 70 KPa He head pressure, injector at 325°C and detector at 350°C. GC separations for the hydrolysed wax ester products, FAMES and alcohols, were obtained on 2 columns, a polar SP2380 (30 m x 0.25 mm i.d.) and a nonpolar SPB-1 (30 m x 0.25 mm i.d.) both purchased from Supelco (Bellefonte, PA). SP2380 analysis conditions: 172 KPa He head pressure, programmed ramp 150°C to 250°C at 3°C min-’ with a 2 min hold at 25O”C, injector and detector set at 250°C. SPB-1 analysis conditions: 172 KPa He head pressure, programmed ramp 250°C to 350°C at 3°C mini with a 2 min hold at 35O”C, injector and detector set at 250°C. GC-MS (mass spectroscopy) analysis was performed on the hydrolysed wax ester products (FAMES and alcohols) using a Hewlett Packard 5890A GC with a 15 m x 0.25 mm DB-1 column (J & W Scientific, Folsom, CA) and a Hewlett Packard 5970 mass selective detector (Palo Alto, CA). GC conditions: 35 KPa He head pressure, programmed ramp from 185°C to 320°C at 5°C min-‘, injector set at 25O”C, transfer line set at 280°C. MS conditions: mass range 50 to 550 amu, electron multiplier 200 volts relative. ‘H NMR and 13C NMR were performed on a Bruker ARX 400 with a dual 5 mm proton/carbon probe (‘H 400 MHz t3C 100.61 MHz). CDCls served as the solvent in all experiments. ‘H NMR of saturated wax ester: S 4.04 (t, 2H), 2.27 (t, 2H), 1.751.05 (m, 96H) and 0.85 ppm (t, 6H). 13C NMR: S 174.0, 64.4, 34.4, 31.9, 29.7, 29.7, 29.5, 29.5, 29.4, 29.3, 29.1,28.6, 25.9, 25.0,22.7 and 14.1 ppm. Infrared analysis of the wax esters was performed on a Mattson Galaxy 6020 Fourier transform infrared (IT IR) set at 1 cm-’ resolution. Samples were run in KBr cells using CCL as solvent. CC14 also served as the background. IR of wax ester: v 2928.8,2855.5, 1735.5, 1462.5 and 1173.1 cm-‘. The waxes were isolated from two samples, a 1993 oil processed material that was removed from the bottom of a drum and a 1994 processed oil that had a large amount of suspended solids. Both samples were treated by the following isolation tech-
ZA. Isbell et al. /Industrial Crops and Products 5 (1996) 239-243
nique and yielded the same composition of saturated wax esters. A turbid oil sample (0.36 g) was dissolved in 5 ml of acetone with 1 ml of hexane and the solution heated to boiling in a centrifuge tube. The tube was cooled to room temperature at which point crystals formed. The sample was then centrifuged and the supematant liquid removed. The crystals were redissolved in 5 ml of hot acetone and crystallized at room temperature. The solution was centrifuged and the supematant liquid removed. The 5 ml acetone recrystallization was repeated 3 times to insure complete removal of any residual triglyceride. The crystals were then filtered through a Buchner funnel to give 16.9 mg of saturated wax esters. The combined supematant liquids were concentrated in vacua to give 0.33 g of oil for a total mass recovery of 97%. Isolated saturated wax esters (5.3 mg) were placed in a sealed vial with 1 ml of 0.5 M KOH/methanol. The resulting solution was placed in a 100°C heating block for 1 hour and then cooled to room temperature. One ml of 1.0 M HCUmethanol was added and the vial sealed and placed back in the heating block for 5 min. The reaction mixture was then poured into 2 ml hexane and washed once with 2 ml of H20. The water layer was then extracted once with 1 ml of ethyl acetate and combined with the hexane layer. The organic phase was dried over Na$Od, filtered and concentrated. HPLC confirmed the complete hydrolysis of the wax ester. GC and GC-MS analysis was then performed on this sample. 3. Results and discussion Three samples of meadowfoam oil were compared for turbidity, a 1993 processed oil, a 1994 processed oil and a sludge sample removed from the bottom of a drum that had been stored over winter from a 1993 processed oil. The 1993 oil was free of particulate matter, whereas the 1994 oil had a significant amount of precipitate and the sludge sample appeared to be a partially melted solid at room temperature. However, analyses by GC, HPLC, IR and GC of the oils and their methyl esters indicated that all 3 samples were nearly identical. The lack of information provided by the analytical procedures used indicated that an isolated sample of the particulate material for further evaluation was
241 GE-I (2.5 m x 0.32 mm I d.) column 50°C 10 350°C @ 30°C /ml”
.
‘93
E
Meadowfoam Oil I....,....,....,. 450
500
550
600
J
650
700
750
Time (Seconds]
Fig. 1. GC separation triglycerides.
of meadowfoam
saturated
wax esters and
necessary. Morrison (1982) reported that waxes isolated from sunflower seed oil are nearly insoluble in acetone and recrystallization in this solvent would completely remove the waxes from the oil. In light of Morrison’s findings, an acetone recrystallization on the meadowfoam sludge sample was attempted. The sludge sample was used as the initial sample for particulate isolation since it had the largest quantity of precipitate. Recrystallization from hot acetone of the sludge sample provided crystals with a melting point of 77”-78°C. The melting point of the isolated waxes was similar to the melting point (75”-78°C) of a sunflower seed oil wax obtained by Moulton (1988). GC (Fig. l), HPLC (Fig. 2) and IR analyses of the isolated crystals were considerably different from those of the starting oil samples and the crystals exhibited similar chromatographic behavior to wax esters reported by Grob et al. (1990) and Tulloch (1975). NMR of the crystalline solid indicated the existence of a long chain ester. The definitive protons are a triplet at 4.04 ppm for the -OCH2of the alcohol residue and the LXmethylene triplet at 2.27 ppm for the fatty acid component. In conjunction with the proton data, the 13C spectra confirmed the ester resonance at 174.03 ppm. Similarly, Gunstone (1993) reports a cat-bony1 resonance of 173.96 ppm for the ester of lauryl palmitate. The chemical shift of the -OCHz-R of the meadowfoam wax ester was
T.A. lsbell et al. /Industrial Crops and Products 5 (1996) 239-243
242
Dynamax silica 250 mn x 4.6 mm l.d. Hexone I Acetone 955, I ml / min
Table 1 GC analysis of wax esters and hydrolyzed wax esters Chain length
Wax estera (normalized %)
0.5 0.8 12.0 0.9 21.2 1.8 23.1 2.0 20.3 1.2
16 h!
/
I
2
_
‘93 Sludge
#
‘93 Meadowfoam oil 4
3
5
Time (min.)
Fig. 2. Normal phase HPLC separation of meadowfoamsaturated wax esters and triglycerides.
64.39 ppm compared to 64.39 ppm reported by Gunstone (1993). The proton and 13C NMR spectra had no signals corresponding to unsaturation, indicating a saturated wax ester. FT IR confirmed the ester moiety with a carbonyl signal at 1736.5 cm-‘. Hydrolysis and transesterification of the saturated wax esters by KOH/methanol followed by HCl/ methanol provided the corresponding methyl esters and alcohols. GC analysis on both polar and nonpolar stationary phases provided ECL (equivalent chain length) values for all the saturated esters and alcohols. These results are reported in Table 1. GC-MS of the esters and alcohols confirmed GC peak assignments. The distribution of acids and alcohols is very similar to wax esters found in sludge samples of sunflower seed oil (Kleiman et al., 1969), including the odd-chain length moieties. An HPLC technique was developed for determining the amount of saturated wax esters in meadowfoam oil by developing a standard curve for the wax ester. Table 2 reports the results of the amount of wax ester in the oils. A striking observation we made is the effect of small amounts of wax on the appearance of the oil. The sludge sample contains the largest amount of wax (5%) and appears to be semisolid even at these low concentrations. Centrifugation of the 1994 oil reduced the wax ester content to nearly half (0.07% vs. 0.12%), and the supernatant oil had about the same wax concentration as the 1993 oil. Neither the 1993 oil nor the centrifuged 1994 oil was turbid at room temperature.
18 20 21 22 23 24 25 26 27 28 29 30 32 44 45 46 41 48 49 50 51 52 53 54 55 56
Hvdrolvzed _ wax ester b FAME Alcohol (normalized %) (normalized %)
11.1 0.7 3.5 0.9
1.4 0.4 19.2 1.8 52.1 2.1 15.6 0.8 3.9 0.8 2.1
8.5 1.8
19.7 3.3 23.0 3.1 19.8 2.4
11.1 1.3 4.3 0.5 1.3
a Wax esters were analyzed on a GB-1 (2.5 m x 0.32 mm i.d) column. b Methyl esters and alcohols were analyzed on a SPB-1 (30 m x 0.25 mm i.d.) and SP2380 (30 m x 0.25 mm i.d.) columns. Table 2 Wax esters in meadowfoam oil samples, by HPLC analysis a Sample
Saturated wax ester (%)
1993 Processed oil 1994 Processed oil Centrifuged 1994 oil 1993 Sludge
0.06 0.12 0.07 5.01
a HPLC separations were made on a Dynamax silica column (25 cm x 4.6 mm i.d.) eluting with hexane/acetone 95 : 5 at 1 ml min-’ .
4. Conclusions Refined meadowfoam oil contains small quantities of long chain saturated wax esters, which can
T.A. Isbell et al./Industrial
Crops and Products 5 (1996) 239-243
be effectively removed by recrystallization from acetone. Furthermore, HPLC can be used as a rapid and effective tool to determine the amount of wax ester present in an oil sample. Concentrations of wax ester greater than 0.1% can cause considerable turbidity in the oil. Consequently, future processing of meadowfoam oil will require a winterization step to meet specialized industrial applications. Acknowledgements The Fanning Corporation provided refined meadowfoam oil and sludge samples. Support for GC-MS was provided by Ronald D. Plattner. David Weisleder performed the NMR experiments. References Fitch, B., 1994. Modified Inform, 5: 1198-1210.
oil may be key to sunflower’s
future.
243
Glaser, L., Ahmed, I. and Parker, H., 1994. Meadowfoam oil and polyols expand vegetable oil markets. Ind. Uses Agric. Mater., 4: 11-13. Grob, K., Lanfranchi, M. and Marian, C., 1990. Evaluation of olive oils through the fatty alcohols, the sterols and their esters by coupled LC-GC. J. Am. Oil Chem. Sot.. 67: 626-634. Gunstone, ED., 1993. High resolution 13C NMR spectra of long chain acids, methyl esters,glycerol esters, wax esters, nitriles. amides, alcohols and acetates. Chem. Phys. Lipids, 66: 189193. KJeimaftttliR., Earle, F.R. and Wolff, LA.. 1969. Wax esters from sunflower oii tank settlings. J. Am. Oil Chem. Sot.. 46: 505. Mariani, C. and Fedeii, E;, 1989. Minor components of vegetable oils: nonglyceridic esters. Ital. Sostanze Grasse, 66: 397-401. Morrison, W.H., 1982. Rapid determination of wax in sunflower seed oil. J. Am. Oil Chem. Sot., 59: 284-285. Moulton, K.J., 1988. Turbidimetric measurement of wax in sunflower oil. J. Am. Oil Chem. Sot., 65: 367-368. Tulloch, AI?, 1975. Chromatographic analysis of natural waxes. J. Chromatogr. Sci., 13: 403-407.
ELSEVIER
JOURNAL
Industrial Crops and Products 5 (1996) 239-243
Isolation and characterization
of wax esters in meadowfoam oil
Terry A. Isbell *, Kenneth D. Carlson, Thomas P. Abbott, Bliss S. Phillips, Selim M. Erhan, Robert Kleiman New Crops Research, National
Center for Agricultural Utilization Research, Agricultural 1815 N. University Street, Peoria, IL 61604, USA
Research Service, USDA,
Received 20 November 1995; accepted 2 May 1996
Abstract Meadowfoam oil has potential use in the cosmetic industry due to its long chain fatty acids. However. particulates that form when the oil is stored could create problems in this application. Particulate matter found in refined meadowfoam oil samples was isolated by first centrifugation and then crystallization from acetone. The white crystalline solid had a melting point of 77”-78”C, and was characterized as a mixture of wax esters by ‘H nuclear magnetic resonance (NMR), 13C NMR and infrared (IR) analysis. Gas chromatography (GC) analysis of the wax esters indicated a distribution of esters from C4 to C& including odd chain esters. Base hydrolysis of the wax ester and GC analysis of the fatty methyl esters and fatty alcohols indicated a mixture of saturated methyl esters from Cl6 to C32 including small amounts of the odd-chain methyl esters C21-C29 with the predominate methyl ester being C24. The alcohol portion of the wax esters contained saturated chain lengths of CZ0--C3~including odd chains ofC21-C 29. The main alcohol component was saturated Cz4 alkanol. GC-MS (mass spectroscopy) confirmed the GC assignments. A normal phase high performance liquid chromatography (HPLC) technique was developed to determine the amount of wax ester (0.06% to 0.12%) in various types of refined meadowfoam oil. Keywords:
Odd-chain
Meadowfoam fatty alcohols
oil; Limnanthes
alba
seed oil; Saturated
1. Introduction Meadowfoam (limnanthes alba) is a new oil seed crop currently grown in northwestern United States. This winter annual crop is an economical and environmental alternative to the grass seeds being cultivated in the Willamette valley of Oregon. Meadowfoam production has increased steadily since 1984 under the supervision of the Oregon Meadowfoam Growers Association. Meadowfoam seeds yield up to 30% oil. The oil * Corresponding author. Fax: (309) 681-6524.
wax esters;
Fatty alcohols:
Odd-chain
fatty acids;
is composed of long chain fatty acids (Glaser et al., 1994) with 5-eicosenoic acid (64%) as the major fatty acid. The other main components are 5,13docosadienoic acid (19%), 5-docosenoic acid (3%) and 13-docosenoic acid (10%). This unique combination of monoenoic fatty acids makes for a very oxidatively stable oil with an AOM (active oxygen method) value of 200. In comparison, other vegetable oils such as soybean and high oleic sunflower have AOM values of 14 and 90, respectively (Fitch, 1994). Meadowfoam oil is utilized in cosmetic formulations. Due to the many rigorous requirements for cosmetic applications such as low color and high
0926-6690/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SO926-6690(96)000019-2
240
TA. Isbell et al. /Industrial Crops and Products 5 (1996) 239-243
clarity, problems in oil seed development can arise. Recently processed meadowfoam oil developed a precipitate upon storage in a warehouse. Since oil clarity is important for cosmetic applications, the isolation and characterization of this precipitate are necessary. The suspended contaminate was suspected to be a wax, possibly extracted from the seed coat during oil processing. Waxes and nonglycerol esters have been found in other seed oils (Grob et al., 1990; Mariani and Fedeli, 1989; Moulton, 1988) and posed similar crystallization and turbidity problems. In light of these findings, an evaluation of meadowfoam oil for the presence of waxes was initiated. 2. Materials and methods Meadowfoam oil samples were provided by the Fanning Corp. (Chicago, IL) and the Oregon Meadowfoam Growers Assoc. (Salem, OR). Potassium hydroxide and solvents for crystallization and chromatography were high performance liquid chromatography (HPLC) grade and obtained from Fisher Scientific (Fair Lawn, NJ). Saturated fatty acid methyl ester (FAME) standards were obtained from Alltech Associates, Inc. (Deerfield, IL) HPLC separations were performed on a Spectra Physics 8800 ternary pump system (San Jose, CA) equipped with a Spectra System AS3000 autosampler/injector (Therm0 Separation Products, Fremont, CA) coupled to an evaporative light scattering detector (ELSD) from Varex (Burtonsville, MD). A Dynamax silica column (250 x 4.6 mm i.d., 60 A, 8 pm) purchased from Rainin Instrument Co. (Wobum, MA) was used to separate the wax ester mixtures. Components were eluted from the column with an isocratic hexane : acetone 95 : 5 mixture at a flow rate of 1 ml min-’ . The ELSD drift tube was set at 35°C and the gas flow through the nebulizer was 1.5 standard 1 min-‘, 70 KPa N2. Retention times for eluted peaks: saturated wax ester 2.7 min, jojoba oil 3.0 min and meadowfoam oil 3.5 min. Oil samples containing wax esters to be analyzed by HPLC or gas chromatography (GC) were first warmed to dissolve the precipitated wax esters. GC separations of triglycerides and wax ester components were obtained on a GB-1 2.5 m x 0.32 mm id. column (Foxboro, New Haven, CT) in a
Hewlett Packard 5890 Series II gas chromatograph (Palo Alto, CA) with a flame ionization detector. The following conditions were used for analysis: programmed ramp from 50°C to 350°C at 30°C min-‘, 70 KPa He head pressure, injector at 325°C and detector at 350°C. GC separations for the hydrolysed wax ester products, FAMES and alcohols, were obtained on 2 columns, a polar SP2380 (30 m x 0.25 mm i.d.) and a nonpolar SPB-1 (30 m x 0.25 mm i.d.) both purchased from Supelco (Bellefonte, PA). SP2380 analysis conditions: 172 KPa He head pressure, programmed ramp 150°C to 250°C at 3°C min-’ with a 2 min hold at 25O”C, injector and detector set at 250°C. SPB-1 analysis conditions: 172 KPa He head pressure, programmed ramp 250°C to 350°C at 3°C mini with a 2 min hold at 35O”C, injector and detector set at 250°C. GC-MS (mass spectroscopy) analysis was performed on the hydrolysed wax ester products (FAMES and alcohols) using a Hewlett Packard 5890A GC with a 15 m x 0.25 mm DB-1 column (J & W Scientific, Folsom, CA) and a Hewlett Packard 5970 mass selective detector (Palo Alto, CA). GC conditions: 35 KPa He head pressure, programmed ramp from 185°C to 320°C at 5°C min-‘, injector set at 25O”C, transfer line set at 280°C. MS conditions: mass range 50 to 550 amu, electron multiplier 200 volts relative. ‘H NMR and 13C NMR were performed on a Bruker ARX 400 with a dual 5 mm proton/carbon probe (‘H 400 MHz t3C 100.61 MHz). CDCls served as the solvent in all experiments. ‘H NMR of saturated wax ester: S 4.04 (t, 2H), 2.27 (t, 2H), 1.751.05 (m, 96H) and 0.85 ppm (t, 6H). 13C NMR: S 174.0, 64.4, 34.4, 31.9, 29.7, 29.7, 29.5, 29.5, 29.4, 29.3, 29.1,28.6, 25.9, 25.0,22.7 and 14.1 ppm. Infrared analysis of the wax esters was performed on a Mattson Galaxy 6020 Fourier transform infrared (IT IR) set at 1 cm-’ resolution. Samples were run in KBr cells using CCL as solvent. CC14 also served as the background. IR of wax ester: v 2928.8,2855.5, 1735.5, 1462.5 and 1173.1 cm-‘. The waxes were isolated from two samples, a 1993 oil processed material that was removed from the bottom of a drum and a 1994 processed oil that had a large amount of suspended solids. Both samples were treated by the following isolation tech-
ZA. Isbell et al. /Industrial Crops and Products 5 (1996) 239-243
nique and yielded the same composition of saturated wax esters. A turbid oil sample (0.36 g) was dissolved in 5 ml of acetone with 1 ml of hexane and the solution heated to boiling in a centrifuge tube. The tube was cooled to room temperature at which point crystals formed. The sample was then centrifuged and the supematant liquid removed. The crystals were redissolved in 5 ml of hot acetone and crystallized at room temperature. The solution was centrifuged and the supematant liquid removed. The 5 ml acetone recrystallization was repeated 3 times to insure complete removal of any residual triglyceride. The crystals were then filtered through a Buchner funnel to give 16.9 mg of saturated wax esters. The combined supematant liquids were concentrated in vacua to give 0.33 g of oil for a total mass recovery of 97%. Isolated saturated wax esters (5.3 mg) were placed in a sealed vial with 1 ml of 0.5 M KOH/methanol. The resulting solution was placed in a 100°C heating block for 1 hour and then cooled to room temperature. One ml of 1.0 M HCUmethanol was added and the vial sealed and placed back in the heating block for 5 min. The reaction mixture was then poured into 2 ml hexane and washed once with 2 ml of H20. The water layer was then extracted once with 1 ml of ethyl acetate and combined with the hexane layer. The organic phase was dried over Na$Od, filtered and concentrated. HPLC confirmed the complete hydrolysis of the wax ester. GC and GC-MS analysis was then performed on this sample. 3. Results and discussion Three samples of meadowfoam oil were compared for turbidity, a 1993 processed oil, a 1994 processed oil and a sludge sample removed from the bottom of a drum that had been stored over winter from a 1993 processed oil. The 1993 oil was free of particulate matter, whereas the 1994 oil had a significant amount of precipitate and the sludge sample appeared to be a partially melted solid at room temperature. However, analyses by GC, HPLC, IR and GC of the oils and their methyl esters indicated that all 3 samples were nearly identical. The lack of information provided by the analytical procedures used indicated that an isolated sample of the particulate material for further evaluation was
241 GE-I (2.5 m x 0.32 mm I d.) column 50°C 10 350°C @ 30°C /ml”
.
‘93
E
Meadowfoam Oil I....,....,....,. 450
500
550
600
J
650
700
750
Time (Seconds]
Fig. 1. GC separation triglycerides.
of meadowfoam
saturated
wax esters and
necessary. Morrison (1982) reported that waxes isolated from sunflower seed oil are nearly insoluble in acetone and recrystallization in this solvent would completely remove the waxes from the oil. In light of Morrison’s findings, an acetone recrystallization on the meadowfoam sludge sample was attempted. The sludge sample was used as the initial sample for particulate isolation since it had the largest quantity of precipitate. Recrystallization from hot acetone of the sludge sample provided crystals with a melting point of 77”-78°C. The melting point of the isolated waxes was similar to the melting point (75”-78°C) of a sunflower seed oil wax obtained by Moulton (1988). GC (Fig. l), HPLC (Fig. 2) and IR analyses of the isolated crystals were considerably different from those of the starting oil samples and the crystals exhibited similar chromatographic behavior to wax esters reported by Grob et al. (1990) and Tulloch (1975). NMR of the crystalline solid indicated the existence of a long chain ester. The definitive protons are a triplet at 4.04 ppm for the -OCH2of the alcohol residue and the LXmethylene triplet at 2.27 ppm for the fatty acid component. In conjunction with the proton data, the 13C spectra confirmed the ester resonance at 174.03 ppm. Similarly, Gunstone (1993) reports a cat-bony1 resonance of 173.96 ppm for the ester of lauryl palmitate. The chemical shift of the -OCHz-R of the meadowfoam wax ester was
T.A. lsbell et al. /Industrial Crops and Products 5 (1996) 239-243
242
Dynamax silica 250 mn x 4.6 mm l.d. Hexone I Acetone 955, I ml / min
Table 1 GC analysis of wax esters and hydrolyzed wax esters Chain length
Wax estera (normalized %)
0.5 0.8 12.0 0.9 21.2 1.8 23.1 2.0 20.3 1.2
16 h!
/
I
2
_
‘93 Sludge
#
‘93 Meadowfoam oil 4
3
5
Time (min.)
Fig. 2. Normal phase HPLC separation of meadowfoamsaturated wax esters and triglycerides.
64.39 ppm compared to 64.39 ppm reported by Gunstone (1993). The proton and 13C NMR spectra had no signals corresponding to unsaturation, indicating a saturated wax ester. FT IR confirmed the ester moiety with a carbonyl signal at 1736.5 cm-‘. Hydrolysis and transesterification of the saturated wax esters by KOH/methanol followed by HCl/ methanol provided the corresponding methyl esters and alcohols. GC analysis on both polar and nonpolar stationary phases provided ECL (equivalent chain length) values for all the saturated esters and alcohols. These results are reported in Table 1. GC-MS of the esters and alcohols confirmed GC peak assignments. The distribution of acids and alcohols is very similar to wax esters found in sludge samples of sunflower seed oil (Kleiman et al., 1969), including the odd-chain length moieties. An HPLC technique was developed for determining the amount of saturated wax esters in meadowfoam oil by developing a standard curve for the wax ester. Table 2 reports the results of the amount of wax ester in the oils. A striking observation we made is the effect of small amounts of wax on the appearance of the oil. The sludge sample contains the largest amount of wax (5%) and appears to be semisolid even at these low concentrations. Centrifugation of the 1994 oil reduced the wax ester content to nearly half (0.07% vs. 0.12%), and the supernatant oil had about the same wax concentration as the 1993 oil. Neither the 1993 oil nor the centrifuged 1994 oil was turbid at room temperature.
18 20 21 22 23 24 25 26 27 28 29 30 32 44 45 46 41 48 49 50 51 52 53 54 55 56
Hvdrolvzed _ wax ester b FAME Alcohol (normalized %) (normalized %)
11.1 0.7 3.5 0.9
1.4 0.4 19.2 1.8 52.1 2.1 15.6 0.8 3.9 0.8 2.1
8.5 1.8
19.7 3.3 23.0 3.1 19.8 2.4
11.1 1.3 4.3 0.5 1.3
a Wax esters were analyzed on a GB-1 (2.5 m x 0.32 mm i.d) column. b Methyl esters and alcohols were analyzed on a SPB-1 (30 m x 0.25 mm i.d.) and SP2380 (30 m x 0.25 mm i.d.) columns. Table 2 Wax esters in meadowfoam oil samples, by HPLC analysis a Sample
Saturated wax ester (%)
1993 Processed oil 1994 Processed oil Centrifuged 1994 oil 1993 Sludge
0.06 0.12 0.07 5.01
a HPLC separations were made on a Dynamax silica column (25 cm x 4.6 mm i.d.) eluting with hexane/acetone 95 : 5 at 1 ml min-’ .
4. Conclusions Refined meadowfoam oil contains small quantities of long chain saturated wax esters, which can
T.A. Isbell et al./Industrial
Crops and Products 5 (1996) 239-243
be effectively removed by recrystallization from acetone. Furthermore, HPLC can be used as a rapid and effective tool to determine the amount of wax ester present in an oil sample. Concentrations of wax ester greater than 0.1% can cause considerable turbidity in the oil. Consequently, future processing of meadowfoam oil will require a winterization step to meet specialized industrial applications. Acknowledgements The Fanning Corporation provided refined meadowfoam oil and sludge samples. Support for GC-MS was provided by Ronald D. Plattner. David Weisleder performed the NMR experiments. References Fitch, B., 1994. Modified Inform, 5: 1198-1210.
oil may be key to sunflower’s
future.
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