9536 J. Agric. Food Chem., Vol. 54, No. 25, 2006
Andjelkovic et al.
yield according to the above-described procedure. The 1H NMR spectra
of all conjugated vegetable oils correspond closely to the previously
reported spectra (21).
transesterification and determine the suitability of this method
for the synthesis of CLA.
EXPERIMENTAL PROCEDURES
Kinetic Study. Aliquots were taken at different times during the
conjugation reaction and subjected to the same workup as previously
described. Samples were analyzed by both 1H NMR spectroscopy and
GC-MS to determine the progress of the reaction.
Materials. The vegetable oils used in this study were Planters peanut
(PNT), Mazola corn (COR), Wesson soybean (SOY), low-saturation
soybean (LSS), Hain safflower (SAF), and Superb linseed (LIN) oils.
All of the oils were purchased in local supermarkets, except the Superb
linseed and low-saturation soybean (Select Oil) oils, which were
supplied by Archer Daniels Midland Co. (Decatur, IL) and Zeeland
Food Service, Inc. (Zeeland, MI), respectively. All of the oils were
used without further purification. RhCl3‚3H2O was provided by
Kawaken Fine Chemicals Co. Ltd. (Japan) and used as received. (p-
CH3C6H5)3P and absolute ethanol were purchased from Aldrich
Chemical Co. and used as received. The CLA gas chromatography (GC)
standards methyl cis,cis-9,12-octadecadienoate [(9Z,12Z)-CLA], methyl
cis,trans-9,11-octadecadienoate [(9Z,11E)-CLA], methyl trans,cis-10,-
12-octadecadienoate [(10E,12Z)-CLA], methyl cis,cis-9,11-octadeca-
dienoate [(9Z,11Z)-CLA], methyl trans,trans-9,11-octadecadienoate
[(9E,11E)-CLA], and cis,trans-11,13-octadecadienoic acid (>97%
purity) were purchased from Matreya Inc. (Pleasant Gap, PA). Prior to
GC analysis, cis,trans-11,13-octadecadienoic acid was converted to its
methyl ester by acid-catalyzed esterification in methanol to afford
methyl cis,trans-11,13-octadecadienoate [(11Z,13E)-CLA]. The methyl
esters of palmitoleic (C16:1), oleic (C18:1), and trans-vaccenic (t-C18-
1) acids and all saturated fatty acid standards were obtained from
NuChek Prep, Inc. (Elysian, MN). The chlorobis(cyclooctene)rhodium
dimer, [RhCl(C8H14)2]2, was synthesized according to a previously
published procedure (22). Standard grade silica gel (porosity 60 Å,
particle size 32-63 µm, surface area 500-600 m2/g) was purchased
from Sorbent Technologies (Atlanta, GA) and used as received. SPEX
certiprep Rh standard (RhCl3 in a matrix of 10% HCl) has been used
as a calibration standard for ICP-MS analysis.
CLA Analysis by GC. Approximately 20 mg of triglyceride, 1 mL
of 3 N methanolic HCl (Supelco), and 150 µL of hexane were added
to a test tube, which was tightly capped, and incubated in a water bath
at 65 °C for 50 min. After the solution was cooled to room temperature,
1 mL of hexane and 8 mL of water were added to the test tube, and
the resulting solution was mixed thoroughly and left overnight at room
temperature for phase separation. The hexane solution containing the
CLA methyl esters was separated and quantified by gas chromatogra-
phy. To improve the separation and reduce the separation time, the
following oven temperature program was used: step 1, temperature
ramp from 180 to 200 °C at 5 °C/min; step 2, isothermal hold at 200
°C for 6 min; step 3, temperature ramp from 200 to 220 °C at 10 °C/
min; step 4, temperature ramp from 220 to 230 °C at 5 °C/min; step 5,
isothermal hold at 230 °C for 6 min. Helium gas was used as a carrier
gas, and the column flow rate was 1.0 mL/min. The temperatures of
the inlet and detector were 280 and 320 °C, respectively. The flow
rates of air, hydrogen gas, and makeup gas (He) at the detector were
350, 35, and 38.3 mL/min, respectively. The area of each peak was
integrated by Chemstation software (Hewlett-Packard Co., Wilmington,
DE), and the peak areas were used to calculate the fatty acid
composition.
Sample Preparation and ICP-MS Analysis. The conjugated
vegetable oils were purified by flash chromatography on silica gel using
n-pentane as a solvent. After removal of the ethanol from the crude
reaction mixture under reduced pressure, 150 mL of conjugated oil
was diluted with 300 mL of n-pentane and passed through a glass frit
packed with 60, 90, or 120 g of silica gel to obtain dark red, orange,
or yellow solutions, respectively. The solvents were removed under
reduced pressure, and the remaining solution was used for ICP-MS
analysis to determine the Rh and Sn content. On the basis of the initial
concentrations of Rh and Sn, 1 mg/mL samples were prepared by
mixing a small amount of conjugated oil with 1% HNO3 in 50 mL
volumetric flasks. The flasks were shaken vigorously to facilitate
dissolution of the Rh and Sn salts in water and used for analysis the
next day. A blank sample (1 mg/mL) was prepared by mixing regular
SOY and DI water in a volumetric flask. After the run, the blank signal
was subtracted from all of the subsequent data. The Rh and Sn contents
in the samples were calculated using a calibration curve obtained from
the analysis of RhCl3 and SnCl4 standard solutions. The original
concentration of the standard (1000 ppm) was diluted volumetrically
to 100, 10, and 1 ppb with DI water, and the resulting solutions were
used for the calibration.
Characterization. All 1H NMR spectroscopic analyses of the
conjugated vegetable oils and other compounds were recorded in CDCl3
using a Varian Unity spectrometer at 300 MHz. The methyl esters of
CLA were separated and quantified using an HP6890 series gas
chromatograph (Hewlett-Packard Co., Wilmington, DE) equipped with
an autosampler and flame ionization detector. A SUPELCOWAX-10
capillary column (30 m × 0.25 mm × 0.25 µm film thickness, Supelco,
Bellefonte, PA) was used for separation. ICP-MS analysis was
performed on a Hewlett-Packard 4500 series ICP-MS spectrometer.
The experimental conditions, such as the forward power (1200 W),
carrier gas flow (1.2 L/min), sample flow rate (250 µL/min), and
sampling depth (7.8 mm), were set for optimal sensitivity.
General Conjugation Procedure. To 10 g (34 mmol) of methyl
linoleate in 5 mL of absolute EtOH were added 25 mg (0.034 mmol,
0.1 mol %) of [RhCl(C8H14)2]2, 41.4 mg (0.136 mmol, 0.4 mol %) of
(p-CH3C6H5)3P, and 62 mg (0.272 mmol, 0.8 mol %) of SnCl2‚2H2O.
The reaction flask was evacuated and refilled with Ar three times and
the solution stirred in an oil bath at 60 °C for 24 h. After removal of
the ethanol under vacuum, the remaining mixture was dissolved in
n-pentane and purified by flash chromatography on silica gel. The
product obtained in almost quantitative yield was 87.1% conjugated
(Figure 1b). 1H NMR spectral data of conjugated methyl linoleate
(CDCl3): δ 0.88 (t, 3 H, CH3CH2), 1.18-1.43 (m, 14 H, CH2CH2CH2-
CH3 and OCCH2CH2(CH2)4), 1.53-1.68 (m, 2 H, OCCH2CH2), 2.00-
2.20 (m, 4 H, allylic), 2.23-2.32 (t, 2 H, OCCH2), 2.70-2.80 (t, signal
for residual bisallylic protons), 3.65 (s, 3 H, OCH3), 5.22-6.33 (m, 4
H, CHdCHsHdCH).
Conjugation of Methyl Linolenate. The methyl ester of linolenic
acid with 92% conjugation was obtained in quantitative yield according
to the above-described procedure. 1H NMR spectral data of conjugated
methyl linolenate (CDCl3): δ 0.81-1.05 (m, 3 H, CH3CH2), 1.20-
1.50 (m, 12 H, CH2CH2CH3 and OCCH2CH2(CH2)4), 1.53-1.68 (m, 2
H, OCCH2CH2), 1.90-2.23 (m, 6 H, 4 H, allylic, and 2 H, OCCH2
overlapped), 2.70-2.80 (t, signal for residual bisallylic protons), 3.65
(s, 3 H, OCH3), 5.20-5.50 (m, 2 H, vinylic), 5.55-5.73 (m, 1 H,
vinylic), 5.80-6.21 (m, 2 H, vinylic), 6.23-6.54 (m, 1 H, vinylic).
Conjugation of the Vegetable Oils. Conjugated PNT, COR, SOY,
SAF, and LIN oils with >95% conjugation were obtained in >99%
RESULTS AND DISCUSSION
Calculation of the Percent Conjugation. The conjugation
(C, %) for methyl linoleate, methyl linolenate, and all vegetable
oils was determined by 1H NMR spectroscopic analysis. Figure
1
1 shows the H NMR spectra of the methyl esters of regular
and conjugated linoleic acids along with their representative
structures and peak assignments. The structure on the top right
represents (9Z,11E)-CLA, which is only one of several possible
structural isomers of the methyl ester of CLA. The signal at
3.65 ppm (A) in both spectra corresponds to protons in the
methoxy group of the methyl ester. The vinylic hydrogens (F)
of the nonconjugated ester (Figure 1a) are typically detected
at 5.2-5.4 ppm, while the methylene protons positioned between
the two CdC bonds, also known as the bisallylic protons (G),
are observed at 2.7-2.8 ppm. The signal for the bisallylic
protons indicates that the CdC bonds in the methyl ester are
nonconjugated. Conversely, the vinylic hydrogens (F) of the
conjugated ester (Figure 1b) are detected in the 5.2-6.4 ppm