( 3 ) Biemann, K., Bommer, P., Burlingame, A. L., Mcl'lurray, W. J., Tetrahedron Letters 1963 (28), p. 1969. (4) Bommer, P., McMurray, W., Biemann, K., J . A m . Chem. SOC.86,
1439 (1964). (5) Crable, G. F., Kearns, G. L., Norris, M. S., ANAL. CHEM.32, 13 (1960). (6) Field, F. H., Hastings, S. H., Zbzd., 28, 1248 (1956). (7) Helm, R. V., Latham, D. R., Ferrin,
(12) Pu'ier, A. 0 . ) "Nuclear Masses and
C. R., Ball, J. S., Ibid., 32, 1765 (1960). (8) Jongh, Don C. de, Biemann, K., J . A m . Chem. SOC.8 6 , 67 (1964). (9) Lumpkin, H. E., A~YAL.CHEM.30, 321 11968). (10) Lumpkin, H. E., Aczel, T., Ibid., 36, 181 (1964). (11) Meter, R. A. van, Bailey, C. R., Smith, J. R., Moore, R. T., Allbright, C. S., Jacobson, I. A., Jr., Hylton, V. M., Ball, J. S., Ibzd. 24, 1758 (1952).
Their Determination," H. Hintenberger, ed., pp. 89-102, Pergamon Press, Xew York, 1959.
RECEIVEDfor review August 3, 1964. Accepted September 28, 1964. 12th Annual Conference on LIass Spectrometry, ASTM Committee E-14, Montreal, Canada, June 1964.
Determination of Beta-Olefinic Methyl Groups in Esters of Fatty Acids by Nuclear Magnetic Resonance CURTIS A. GLASS and HERBERT J. DUTTON Northern Regional Research laboratory, ARS, USDA, Peoria, 111.
b An analytical method for determining 15,16-unsaturation in fatty acids by nuclear magnetic resonance spectrometry is described. Utilized in the determination are the low field member of the p-olefinic methyl proton triplet and the central peak of non/3-olefinic methyl proton triplet. The areas of these signals are deiermined by the instrumental integrator and b y paper tracings, and precision of the two methods is compared. Application to the kinetics of hydrogenation is presented.
3.0
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Methyl Oleate
A
in flavor and odor stability of hydrogenated vegetable oils is t,he amount of 15,16unsaturation (p-olefinic) that remains after reduction (2). Det,ermining the amount of this structure is difficult. A4vailable oxidative cleavage methods ( I , 6) are not quantitative because the low-boiling propionic acid fragment is partially lost in solvent evaporation steps. The possibilit,y of using nudear magnetic resonance (XMR) to determine this structural arrangement was first considered by Storey ( 7 ) and by Johnson and Shoolery (4) u-ho noted that the resonance of the terminal methyl protone was shifted slightly downfield by a p-olefinic bond. The latter men concluded that a quant,itative measure of the amount of this grouping was not possible because of overlapping signals. In the present report, various procedures for exploiting this second-order shift, in resonance have been studied, a quantitatire procedure is described, and precision has been determined. N IMPORTANT FACTOR
EXPERIMENTAL
YMR spectra were measured with a I'arian -4-60 spectrometer. Samples were studied as 20% solutions of
r*.
Methyl Linolenate
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NMR spectra of methyl esters of fatty acids
methyl esters in carbon tetrachloride with tetramethylsilane (TMS) and benzene present as internal references. The standard Varian 5-nim. 0.d. tubes and a room-temperature probe were employed throughout. Chemical shifts are given in parts per million a5 7 values (10.00 - p.p.ni. from TMS). The oleate, linoleate, and linolenate were obtained from the Hormel Foundation.
RESULTS AND DISCUSSION
Figure 1 presents the S M R spectra of methyl oleate and niethyl linolenate The sharl) signal at 2.65 7 is a second reference, benzene. The folloiving assignments for methyl oleate are as made previouily ( 3 ): the signal a1woximating a tripkt at 4.63 7 , Olefinic protons, a sharp signal at 6 . 3 i 7 , niethoxy VOL. 36, NO. 13, DECEMBER 1964
2401
t
90
80
where X is the fraction of 15,16unsaturated esters and 1 6 0 and Is, are the integrated intensities a t 66 C.P.S. and 54 c.p.s. This relation was tested by plotting the ratio of the area a t 66 c.p.s. to the area a t 54 c.p.s. against the ratio of linolenate to linoleate in weighed standard mixtures. The areas were determined by weighing tracing paper cutouts of the signals on an analytical balance, and from the integral curve recorded on the chart. When the areas were determined by weighed paper tracings, the standard deviation was 0.126 (equivalent to about 15% near 0% linolenate and about 457- near 75% linolenate).
lo (P*
Figure 2. Superimposed NMR spectra of methyl regions of methyl oleate and methyl linolenate (1 0 X )
protons of the methyl ester group; the broad region at 7.92 T , protons of methylenes alpha to olefinic bonds or alpha to carbonyl groups; the strong signal at 8.70 T , protons of insulated methylenes. The protons of the terminal methyl group give a signal a t 9.10 T , which is split into a triplet by the adjacent methylene (apparent coupling constant of 4.8 c.p.s.). At the right, 10.00 T , is the T l l S signal. The linolenate spectrum has these same signals but differs in that the olefinic proton signal is three times as large as in the oleate, a new signal occurs at 7.20 T , a-diolefinic methylene protons, and the methyl signal is shifted. The terminal @-olefinicmethyl protms iiroduce a signal at, 9.02 T , which is also split into a tril)let by the adjacent methylene but with a wider splitting (aliiiarent couliling constant of 7.2 C.l,.".).
The suiierpsition of these tril)lets is shown in Figure 2. This is a 10-times a, and the lack of register is real and represents a chemical shift of 0.08 11.p.m. -IFa result, the lowfield member of the linolenate triplet is well isdated. The presenre or absence of this -ignal is obvious by visual inqiection ( 7 ) and even this qualit,ative inforination is frequently useful. Quantitative estimation of the polefinic methyl grouping is not readily apparent because the o ~ e r l a p from strong nearby signals is frequently much greater than the desired signals of the methyl protons. Johnson and Shoolery ( 4 ) used the other signals nientioned here and, with very fine instrument at ion, oht ained excellent precision Cor the determination of unsaturatiun and molecular weight. The signal for the @-olefinic methyl protons was not used in their work since the overlappiny signals prevented highly accurate analysis. 2402
ANALYTICAL CHEMISTRY
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Figure 3. NMR spectrum of methyl region of 50:50 methyl linolenate-methyl linoleate mixture showing regions used in analytical method for 15,16-unsaturation
(10x1 The basis for the somewhat empirical method described here is apparent in Figure 3, which presents the YhIR ylectrum of the methyl region of 50: 50 methyl linolenate-methyl linoleate. Shaded at 66 c.p.s. is the low-field member of the triplet of methyl protons influenced by P-unsaturation. Shaded at 54 c.11.a. is the central I)eak of the triplet of methyl protons not influenced by p-unsaturation. Backgrounds approximated in this manner are admittedly subject to some judgment of the operator, but, as will be seen, are apparently reproducible. .4s the 66-c.11.s. signal is proportional to the 15,16-unsaturation, so is the 54-c.p.s. signal to the non-15-unsaturated material. One expects, therefore, that the ratio of the 6 6 - c . 1 ~signal area to the 54-c.p.s. signal area will be linearly related to the ratio of the l5,16-unsaturated material to the remainder of the material present:
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.:l.ObO
0.5-
Figure 4. Plot of area fraction at 66 C.P.S. taken by instrument integrator (upper) and b y weighed paper tracings (lower)
Areas determined from the instrument integrator resulted in somewhat better precision. The standard deviation was 0.043 (equivalent to about 5y0 near 0% linoleriate and about 1.5% near 75% linolenatej. The latter method would be useful over this range, but the necessity of applying analyses of concentrations based on ratios is clumsy. Also, the variable precision (the ratio approaching infinity a t 100% linolenatej is undesirable. To overcome these difficulties, the ratio of the area a t 66 C.P.A.to the sum of the areas a t 66 and 54 c.p.s. was plotted against the fraction of linolenate in the standard mixtures. The data obtained from the instrument integrator on eight mixtures of methyl linoleate and methyl linolenate are plotted in Figure 4. On the X-axis is plotted the fraction of linolenate in weighed standard mixtures. On the Y-axis is plotted the ratio of the area at
Table I. Comparison of @-OlefinicMethyl Protons and Olefinic Protons Found by NMR in a Series of Samples from Hydrogenation of Methyl Linolenate with Nickel Catalyst
Uptake of Hp, mole8
Olefinic protons, NMR
Per cent of original
@-Olefinic methyl protons, NR4R
0 0.5 1.0 1.5 2.0
5.81 4.53 3.65 2.85 2.11
97 76 61 48 35
3.00 2.28 1,38 0.81 0.48
Per cent of original 100 76 46 27 16
Another application is in the analysis
form for concurrent analysis by gas liquid chromatography (GLC). However, other types of material can be analyzed because the chemical shift and coupling are unaffected by distant parts of the molecule. I n Figure 6 is the spectrum of a mixture of linseed and safflower oil triglycerides in which 34,770 15,16-unsaturation was detect,ed by XMR and 40.1y0 octadecatrienoate by GLC.
of products from the hydrogenation of samples of methyl linolenate. To be internally consistent, it is desirable to compare 15,16-unsaturation to olefinic protons found by XMR. The method for obtaining an integral reference is based on the olefinic protons. Areas corresponding to olefinic protons and to the total protons of the material are determined by the instrument infegrator. The ratio, R, of these areas is then the ratio of olefinic protons to the total protons. One can assume that the particular sample in question was obtained from methyl linolenate by hydrogenation and write:
R=- 6 - X 32
+X
Where X is a number of protons between 0 and 6, and solving for X :
- 32R x = 6___ l + R
+
From 32 X protons and the total proton area, the area per proton was calculated. The number of olefinic protons obtained in this manner was related to known compositions. The standard deviation calculated from 17 points was 5.2%. iipplication of this analytical
..
CPS
Figure 5 .
NMR spectrum of methyl region of methyl linoleate preparation (10x1
66 c.p.s. to the sum of the areas at 66 and 54 C.P.S. The standard deviation was 4.87, The lower plot in Figure 4 shows the same data but with the areas obtained by weighing tracing paper cutouts. The fit is better, having a standard deviation of 3.00jo0. From the goodness of fit and the ease of obtaining the areas, this method is preferred. Figure 5 shows the XMR spectrum of the methyl proton region of a preparation of supposedly pure methyl linoleate. The small amount of 15,16unsaturated material is apparently an impurity. With the standard deviation given above, the absolute value is not significant, but the detection of this small signal is important as a qualitative measure of purity. Samples examined have usually been methyl esters, since this is the best
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Figure 6.
NMR spectrum of methyl region of mixture of linseed and safflower oil
(lox)
VOL. 3 6 , NO. 13, DECEMBER 1964
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technique to the study of selective hydiogenation is illustrated in Table I, which shows sampling a t intervals during reduction of methyl linolenate with nickel catalyst poisoned with nickel sulfide. From these samplings it is apparent that the three p-olefinic methyl protons are decreasing more rapidly than the six olefinic protons and that either the attack on the 15,16-double bond is somewhat selective or bond migration is occurring. Future plans call for the continued study, through NMR analysis, of the products of partial hydrogenation of vegetable oils, acids, and esters, as well
as effect of catalysts and conditions on the structure of these products. This application is the subject of a subsequent communication ( 5 ) . The ultimate goal will be to minimize or aroid 15,16-unsaturation in the final products and thus to enhance flavor and odor stability in edible oils. LITERATURE CITED
(1) Criegee, R., Record Chem. Progr., Kresqe-Hooker Sci. Lib. 18, 111 (1957). ( 2 ) Ditton, H. J., Lancaster, C. R., Evans, C. D., Cowan, J. C., J . Am. Oil Chemists' SOC.28, 115 (1951). ( 3 ) Hopkins, C. Y., Bernstein, H. J., Can. J . Chem. 37, 775 (1959).
(4) Johnson, L. F., Shnolery, J. X., ANAL. CHEM.34, 1136 (1962). ( 5 ) Johnston, A. E., Glass, C. A,, Dutton, H. J., Abstracts of Papers 22, Am. Oil Chemists' SOC., Minneapolis, llinn., October 1963. (6) Jones, E. P., Stolp, J. A., J . Am. Oil Chemists' SOC.35, 71 (1958). (7) Storey, W. H., Jr., Zbid., 37, 676 (1960). RECEIVEDfor review May 4, 1964. Acce ted September 30, 1964 14th Pittsgurgh Conference on Analytical Chemistry and Applied Spectroscopy, March 4-8, 1963. Mention of firm names or trade products does not imply that they are endorsed or recommended by the Dept. of Agriculture over other firms or similar products not mentioned.
Internal Standard X- Ray Spectrographic Procedure for the Determination of Calcium, Barium, Zinc, and Lead in Hydrocarbons W. E. BURKE, 1. S. HINDS, G. E. DEODATO, E. D. SAGER, Jr., and R. E. BORUP Cities Service Research and Development Co., Drawer #2, Cranbury, N. J.
b A procedure for determination of calcium, barium, zinc, and lead concentrations in lubricating oils and lead content of gasolines is described. An internal standard holder is used to suspend a pellet of an appropriate reference element within a standard Philips sample cup containing the sample to b e analyzed. The intensities of the reference element and the sample element are used to calculate the intensity the sample element would have in the calibration curve mediums. This technique adequately compensates for interelemental effects and achieves good agreement with standard chemical procedures. Procedural modification may also enable lower atomic number determinations. The calculations and principles involved should b e applicable to absorptiometry procedures.
X
is a useful tool for qualitative analyses. Accurate quantitative analyses, however, have been complicated by absorption and enhancement effects of all elements (including the element sought) in the samlile being analyzed. Prominent among the various procedures used to compensate for these matrix absorption effects are direct comparison with a standard, dilution techniques, and internal standard procedures. Direct coinparison with a standard has been most successful when used for -RAY SPECTROGRAPHY
2404
ANALYTICAL CHEMISTRY
quality control where the composition of the standard is very similar to that of the sample being analyzed ( I , 8, 9). Analyses of unknown and dissimilar matrices, however, often give highly erroneous results. Dilution techniques (7, 12), although widely used, decrease both the seneitivity and the accuracy of the analyses. Internal standard procedures include the use of background lines as references (5) and the physical addition of a suitably chosen element to the sample being analyzed (2, 3 ) . The use of background lines has not been ton successful. Physical addition of a suitably chosen element has been most successful when the added element's absorption edge and emission line hare been very close to that of the element sought. Most internal standard procedures, however, have not completely compensated for matrix absorption effects. I n addition, physically adding an internal standard entails additional time for sample preparation. Jones (6) utilized a reference element in the form of an iron rod which was attached to the cover of a standard Philips sample holder. He ratioed the manganese intensity of the sample to that of the iron rod suspended in the sample and thereby successfully determined the manganese content of gasolines. This unique internal standard procedure thus avoided the sample preparation usually involved in internal standard methods. Gunn ( 4 ) suyiended disks of molybdenum, nickel, iron, manganese, titanium, and potassium bromide in white oil
samples and thereby measured the absorption effect of these elements in varying white oil depths. The procedure outlined below also utilizes a suspended reference element within the sample to be analyzed. The reference element is protected from sample contamination (when necessary) by a Mylar window. Matrix absorption effects are corrected with a Bouguer absorption law calculation. EXPERIMENTAL
Apparatus. The instrument used is a Philips Electronics inverted sample, three-position x-ray spectrograph. Instrumental parameters are tungsten target a t 50 kv., 45 m a ; flow proportional counter with P-10 gas; helium atmosphere; and lithium fluoride crystal. The reference element holder (shown in Figure 1) has a micrometer-type adjustment and is suspended within a standard Philips sample cup. As shown, the pellet can be protected by a l/4-mil Mylar cover. The height of the reference element within the sample cup is varied to obtain a desired sample thickness and is fixed by means of the lock plug. Reagents. NBS calcium 2-ethyl hexantate, NBS barium cyclohexane butyrate, XES zinc cyclohexane butyrate, NBS lead cyclohexane butyrate. Procedure. Calibration standards of calcium, bsrium, zinc, and lead in white oil and lead in iso-octane are prepared as instructed by the detailed directions given with each NBS standard sample.
