Anal. Chem. 1980, 52, 253-254
253
Characterization of Olefinic Double Bonds in Linear Unsaturated Fatty Acids Using Fluorine Magnetic Resonance Spectrometry Michelle V. Buchanan and James W. Taylor" Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706
F'' NMR can be employed generally on stereospecific hexafluoroacetone derivatives of linear unsaturated fatly acid esters to yield the position and geometry of the original carboncarbon double bond. Complications arise in derivative formation when the double bond is in the number two position, and NMR alone is not sufficient for positional characterization when the double bond is near the center of an acid with a chain length greater than 16 carbons.
Table I. Chemical Shifts of HFA Derivatized Methyl Esters of Unsaturated Fatty Acids vs. HFB cis acids palmitoleic (cis-9-hexadecenoic) petroselenic (cis-6-octadecenoic) oleic (cis-9-octadecenoic) erucic (cis-13-docosenoic)
EXPERIMENTAL The samples of elaidic, petroselenic, palmitoleic, and erucic acids were obtained from Sigma Chemical Co., St. Louis, Mo. Oleic acid was supplied by Matheson, Coleman, and Bell, East Rutherford, N.J. and trans-2-hexadecenoic acid was purchased from ICN Pharmaceuticals, Plainview, N.Y. All samples were used as received and were converted into methyl esters to prevent possible reaction of the acid group with the HFA (4-8). The acids were esterified by stirring for 30 min in a solution containing chloroform, methanolic hydrochloric acid, and cupric acetate (9). The reaction mixture was then extracted into hexane, dried, and the excess hexane removed with a rotary evaporator. The unsaturated methyl ester was stereospecifically converted into a trans-bromohydrin, which was then condensed with HFA to form a cyclic ketal. A detailed account of the experimental process for the preparation of the HFA derivatives may be found in a previous paper (10). The proton-decoupled '9 NMR spectra were obtained at 94.176 MHz using a Varian XL-100/15 NMR spectrometer with the Varian FT Accessory and Varian 620-L computer. The spectra were recorded a t an ambient probe temperature of 28 "C, and with a deuterium lock. The samples were run with 50 p L of the compound and 10 pL of hexafluorobenzene (HFR) dissolved in 300 pL of acetone-&. The chemical shifts of the compounds were referenced to the sharp singlet of HFR (136 Hz downfield from a HFB standard sealed in a capillary). Values of Aum were estimated from the distance (in hertz) between two peaks in the A3B3spectral pattern (3). These values were confirmed by simulated spectra obtained on the IJniversity 0003-2700/80/0352-0253$01 0010
7918.53 791 1.91 7917.93 7916.50
trans acid elaidic (trans-9-octadecenoic)
The position and geometry of a carbon-carbon double bond have been shown to affect the activity of biological molecules ( 1 , 2 )and also t o cause a difference in the chemical reactivity of isomeric olefinic compounds. T h e physical properties of isomeric olefinic compounds, however, do not differ greatly, and this often makes the position and geometry of a double bond difficult to determine. In a n earlier paper ( 3 ) ,we described a method t o accomplish this characterization whereby alkenes were first stereospecifically derivatized using hexafluoroacetone (HFA) t o form a cyclic ketal. T h e "F NMR spectra of the derivatized olefins yielded information on both the geometry and position of the double bond in the original olefin from the chemical shift and AuAB values, respectively. I n this study, the 19F NMR characterization of olefinic double bonds is extended to the methyl esters of unsaturated long chain (CIG-C2,) fatty acids. T h e advantages as well as the limitations of this analytical application are discussed.
Hz
7 839.06
of Wisconsin Chemistry Department's Harrisl'i computer using Bothner-By and Castellano's LAOCNY self-iterative program ( 1 2 ) . The simulated spectra were plotted on a Bruker WH 270 NMR spectrometer (BNC 12 computer) with a revised version of the ITRCAL six-spin self-iterative program 13).
RESULTS AND DISCUSSION A series of five long chain unsaturated fatty acids, given in Table I, were derivatized with HFA. A sixth acid, trans2-hexadecenoiq was not successfully derivatized. It was suggested by Dalton, Dutta, and Jones (12),that the reaction of an olefin with N-bromosuccinitnide in dimethyl sulfoxide and water proceeds through the formation of a bromonium ion and that high electron density a t the double bond is necessary for this reaction. Owing to the presence of the carboxyl group on the double bond, this requirement is not met with truns-2-hexadecenoic acid, and thus the bromohydrin precursor for the HFA derivatization reaction does not appear t o be formed. The results of the I9F NMR analysis, given in 'Fable I, show t h a t the geometry of the original double bond may be determined by the chemical shift of the derivatives. These chemical shift values are approximately the same as in the analysis of linear alkenes (3). T h e cis isomers of the acids appear at approximately 7916 Hz (84.06 ppm) from HFR and the trans isomers at 7839 Hz (83.24 ppm). This indicates that the I9F NMR analysis for double bond geometry in olefinic compounds is unaffected by the presence of either the carboxyl group or the carbon chain length. In the analysis of alkenes using the HFA derivatization technique, both substituents on the dioxolane ring were aliphatic hydrocarbon chains. However, in the derivatives of the long chain unsaturated acids, one mbstituent is aliphatic, but the other is aliphatic with a terminal methyl ester group, that is -(CH2),COOCH,, where n = 1 or greater. As was seen in the study of the HFA derivatized alkenes (31, the overall symmetry of the derivatized molecule is reflected by the "F spectral parameters. The effect of the presence of the ester group is especially evident in the AuAn values of the HFA derivatives, as shown in Table 11. For t>xample,the Aum value for the eladic ester (trans-9-octadecenoic acid) is slightly larger than zero. Although the double bond is in the middle of the molecule (at the C9 position of the 18-carbon acid), the presence of the ester group reduces the overall :;ymmetry of c' 1980 American Chemical Society
254
ANALYTICAL CHEMISTRY, VOL. 52, NO. 2, FEBRUARY 1980
T a b l e 11. E s t i m a t e d V a l u e s of A U A B for HFA M e t h y l Esters of U n s a t u r a t e d Fatty A c i d s
-
m
Derivatized AVAB ,
cis esters
Hz
' 1
0
II
palmitoleic
( R , = C,H,,
R, = C,H,,COCH,)
7.3
0
+
II
oleic
( R , = C,H,,
R, = C,H,,COCH,) 0
:
II
erucic
( R , = C,H,,
R, = C,,H,,COCH,)
7.2
0
II
petroselenic ( R , = C,,H,,
R, = C,H,COCH,)
0
II
R, = C,H,,COCH,)
1
2
3
4
5
6
7
8
9
1
0
1
1
1
2
BOND POSITION Figure 1. Plot of AuABvs. bond position for five HFA derivatized cis alkenes (+) and four derivatized cis unsaturated fatty acid methyl esters
0
( R , = C,H,,
0
7.0
trans esters
elaidic
b
A
7.1
>O
the molecule. This makes the two trifluoromethyl groups of the HFA derivative experience nonequivalent environments. The 19FNMR spectrum yields, therefore, an A3B3spectrum. In this case the AUABvalue is only slightly larger than zero. This can be compared to the true singlet (A6 spectrum) with a Aum value of zero, which was found with trans-5-decene (3). Although the Aum values of the derivatized esters change with double-bond position, as seen in Table 11, the Aum values are consistent with chain length. This can be seen by comparing the oleic ester (from an 18-carbon acid) and the erucic ester (from a 22-carbon acid). Each of these acids has an 8-carbon hydrocarbon chain as the shorter of the two substituents on the dioxolane ring, and both yield 19F NMR spectra with AuAB values of approximately 7.2 Hz. However, in contrast to the alkene results where the Aum values followed the general pattern of being determined by the shorter of the two alkyl chains, the unsaturated esters, with a carboxyl group as one of the dioxolane ring substituents, do not always follow this trend. For example, HFA derivatized petroselenic ester (from an 18-carbon acid) has an alkyl group with 11 carbons as one substituent and a 4-carbon alkyl chain with a terminal methyl ester group (-(CH2)4COOCH3)as the other substituent on the dioxolane ring. The small Aum value for this acid (7.0 Hz) does not seem to fit with that of a 5-carbon chain (double bond a t the c6 position). This may be seen in Figure 1,where Aum is plotted as a function of bond position using the AYAB values obtained from the cis linear olefins (3) and cis unsaturated esters. The values of AUAB are seen to first decrease as a function of bond position and then level off to a constant value of about 7 Hz. The AuAB value for petroselenic ester fits better when plotted as a double bond in the 1 2 position (Clz) than a t the c6 position, where the value of Aum should be about 7.6 Hz. From this, it appears that the presence of the methyl ester group on the short alkyl chain of petroselenic ester effectively makes the 11-carbon alkyl chain determine the AuAB value. Figure 1may also be used to estimate the limit of usefulness of the Aum values in characterizing the double-bond position. For example, the difference in hum for double bonds a t C7 and c8 is about 0.1 Hz. This value approaches the limit of the Aum estimation technique ( 3 ) . However, if the difference in Aum is less than 0.1 Hz, as it is in the case of all double-bond positions greater than (28, the double-bond positions cannot easily be distinguished. Therefore, the upper limit of usefulness appears to be a t about the c8 position. This means that a 16-carbon alkene may be analyzed for all double-bond positions, but in an 18-carbon alkene, for example, the dou-
(4 ble-bond position from C3 to C7 could be determined whereas a double bond toward the center of the molecule, a t c8 or C9, would be difficult to distinguish. On the other hand, the double-bond position of an unsaturated acid could be determined only in those compounds where R1 = C8 or less (Table 11). This means that other analytical techniques would need to be employed in addition to 19FNMR to completely characterize a long chain olefinic compound with respect to both double-bond position and geometry. As was demonstrated by McCloskey and co-workers (13, 14),the mass spectrometric analysis of cyclic ketal derivatives of fatty acid esters and alkenes can be utilized to provide positional information on olefinic bonds. Furthermore, the analysis of cyclic HFA ketal derivatized olefinic compounds by mass spectrometry has been shown to be a very definitive method of determining the original double bond position (10, 15). Therefore, the techniques of mass spectrometry and 19F NMR could be jointly used to identify both the double-bond position and geometry using the hexafluoroacetone derivatives of olefinic compounds with more than 16 carbons.
ACKNOWLEDGMENT We thank David Hillenbrand for his aid with the NMR spectra, and the staff of the Instrumentation Center, particularly Paul Bender.
LITERATURE CITED (1) Jacobson, M.; Beroza, M.; Bull, D. L.; Bullock, H. R.; Chamberlain, W. F.; McGovern, T. P.; Redfern, R. E.; Sarmiento, R.; Schwarz, M.; Sonnet, P. E.; Wakabayashi, N.; Waters, R. M.; Wright, J. E. In "Insect Juvenile Hormones", Menn, J. J.. Beroza, M. Eds.; Academic Press: New York, 1972; p 263. (2) Shorey, H. H. "Animal Communication by Pheremones", Academic Press: New York, 1976; p 86. (3) Buchanan, M. V.; Hillenbrand, D. F.; Taylor J. W. Anal. Chem. 1977, 49,2146. (4) Leader, G. R. Anal. Chem. 1970, 42, 16. (5) Leader, G. R. Appl. Spectrosc. Rev. 1976, 11, 287. (6) Ho, F. F.-L. Anal. Chem. 1973, 45, 603. (7) Ho, F. F.-L. Anal. Chem. 1974, 46, 496. (8) Ho, F. F.-L.; Kohler, R. R. Anal. Chem. 1974, 46, 1302. (9) Hoshi, M.; Williams, M.; Kishimoto, Y. J. Lipid Res. 1973, 14, 599. (10) Johnson, B. M.; Taylor, J. W. Anal. Chem. 1972, 44, 1438. (1 1) Bothner-By, A. A.; Castellano, S. M. I n "Computer Programs for Chemistry", Vol. 1, Detar, D. F., Ed.; W. A. Benbmln: Reading, Mass., 1968; pp 10-53. (12) DaRon, D. R.; Dutta, V. P.; Jones, D. C. J . Am. Chem. SOC.1968, 90, 5498. (13) McCloskey, J. A,; McClelland, M. J. J. Am. Chem. SOC. 1985, 87, 5090. (14) Wolff, R. E.; Wolff, G.; McCloskey, J. A. Tetrahedm 1966, 22, 3093. (15) Johnson, B. M.; Taylor, J. W. Org. Mass Spectrom. 1973, 7, 259.
RECEIVED for review June 4,1979. Accepted October 30,1979. This research has been supported by the National Science Foundation under grants MPS-74-23569 and MPS-75-21059.
