Infrared Analysis of Methyl Stearates Containing Deuterium W. K. Rohwedder, C. R. Scholfield, H e n r y Rakoff,’ J a n i n a Nowakowska, a n d H. J. Dutton Northern Regional Research Laboratory, Peoria, Ill.
61604
Infrared spectra of methyl stearates specifically labeled with deuterium at or near the 9-10 carbon atoms were measured to provide a basis for quantitative determination of deuterium content and to determine the relative amounts of CHD and CD2 on unknown stearates. Methyl stearates containing deuterium as follows: 9- or lO-d,; erythro3,lO-dz; threo9,10-d,; 6,7-d2; 9,9,10- or 9,10,10-d3; 9,10,11,12-d4; 9,10, 12,13-d4; 9,9,10,10-dc; and 9,10,12,13,15,16-d6 were prepared for making band assignments. Methyl stearate prepared by the catalytic deuteration of unsaturated methyl esters had two shoulders on the 2148 cm-l C-D peak, whereas the same esters reduced with hydrazine hydrate-ds had only a single peak without shoulders. The peak at 2148 cm-l results from the C-D stretching vibration of the CHD group while the shoulders come from symmetric and asymmetric stretching of C-D in the CD2 group. Integrated linear absorbance gave good quantitative correlations with deuterium content determined mass spectrometrically. The infrared spectrum is given of methyl esters of a mixture of perdeutero fatty acids extracted from the algae Scenedesmus obliques grown in 99.7% DzO.
ATTEMPTSTO ESTABLISH a n infrared spectrophotometric method for deuterium content of methyl stearate based o n peak absorptivities produced two different calibration curves, one for stearates from deuteriohydrazine-reduced esters; the other for stearates from catalytically deuterated esters. Moreover, the catalytically deuterated esters gave two shoulders one on either side of the central band for the C-D stretching frequency, as compared to the single peak structure of the deuteriohydrazine-reduced ester. Although infrared spectra of methyl acetate ( I ) , ethyl acetate (2), and methyl laurate (3) with deuterium in terminal groups have been compared with hydrogen compounds, little work has been done o n the infrared spectra of esters containing deuterium in the middle of the chain. Heterogeneous catalytic deuteration of unsaturated fatty esters causes extensive exchange of deuterium for carbonbonded hydrogen ( 4 , 5). The work reported here relates the configuration of deuterium, CHD, or CDz, on the carbon chain to infrared spectra and sets up the basis for a n analytical procedure to determine quantitatively the average deuterium content of saturated esters. EXPERIMENTAL
The compounds examined are given in Table I. The methyl stearate-9 or 10-dl was prepared by treating methyl oleate with disiamylborane (bis-3-methyl-2-butyIborane)in purified diglyme followed by refluxing with deuterioacetic Present address, Chemistry Department, Parsons College, Fairfield, Iowa 52556 (1) B. Nolin and R. N. Jones, Can. J . Chem., 34, 1382 (1956). (2) B. N o h and R. N. Jones, Zbid., 34, 1392 (1956). (3) R. N. Jones, Ibid., 40, 301 (1962). (4) N. Dinh-Nguyen and R. Ryhage, Acta Chem. Scand., 13, 1032 (1959). (5) W. K. Rohwedder, E. D. Bitner, H. M. Peters, and H. J. Dutton, J. Am. Oil Chemists’ SOC.,41, 33 (1964). 820 *
ANALYTICAL CHEMISTRY
No. 1 2 3 4 5
6 7 8
9 10 11 12 13 14 15 16 17
Table I. Deuterium Contents and Absorptivities of Deuterio Stearates Integrated Absorpabsorptivity/ tivity/ Average average average D atoms D atoms D atoms Per Per Per Methyl stearate molecule molecule molecule 9 or 0.50 0.0459 9.94 erythro-9,10-dz 1.68 0.0499 11.6 0.0494 threo-9,10-dz 1.66 11.5 erythro-6,7-d2 1.73 11.8 0.0509 9,9,10 or 9,10,10-d3 2.58 ... 11.6 3.38 9,10,11,12-d4 0.0493 13.4 3.48 9,10,12,13-d4 11.7 0.0483 9,9,10,10-d4 3.46 12.7 9,10,12,13,15,16-ds 5.15 11.1 0.0468 0.391 Catalytic, 20 %a 0.0578 14.4 0.994 0.0473 Catalytic, 40 13.1 1.50 Catalytic, 60 % 13.0 0.0426 Catalytic, 80 2.13 0.0386 12.0 2.01 Catalytic, 100% 0.0398 12.7 11.9 4.34 0.0338 Catalytic, 100% 6.21 0.0313 Catalytic, 100% 11.6 Perdeuterio algae, methyl (CHI) ester mixture 32.57 ...
Per cent concentration of deuterium in hydrogen used to catalytically saturate the esters.
acid (6, 7). Some reduction of the ester, as well as reduction of the double bond, occurred t o give four products: methyl stearate, stearyl acetate, methyl oleate, and what was probably oleyl acetate. The methyl stearate and stearyl acetate were separated from the methyl oleate and oleyl acetate o n a reversed phase liquid chromatographic column by the method of Hirsch (8) with a rubber stationary phase and 88% acetone as the moving phase. The methyl stearate was separated from the stearyl acetate by saponification, extraction of the stearyl alcohol with ethyl ether, and reesterification of the stearic acid. Compounds 2 through 9 in Table I were prepared by reduction of the unsaturated acid with hydrazine hydrate-& (9, IO). The appropriate acid was dissolved in purified dioxane (11) and hydrazine hydrate-do was added while the solution was stirred on a n oil bath a t ca. 50” C . A stream 2 was passed through the reaction mixture. Deuterioof dry 0 hydrazine was added as needed, and reduction was continued until gas-liquid chromatographic analysis showed 85-90 stearate. The stearate esters were separated from the un(6) H. C. Brown, “Hydroboration,” W. A. Benjamin, Inc., New York, 1962. (7) H. C. Brown and K. Murray,J. Am. Chem. Soc., 81,4108 (1959). (8) J. Hirsch, Colloq. Intern. Centre Natl. Rech. Sci. (Paris), 99, 11 (1960). (9) F. Aylward and C. V. N. Rao, J. Appl. Chem., 6, 248 (1956). (10) C . R. Scholfield, E. P. Jones, Janina Nowakowska, E. Selke, and H. J. Dutton, J . Am. Oil Chemists’ Soc., 38, 208 (1961). (11) L. %. Fieser, “Experiments in Organic Chemistry,” D. C. Eeath and Co., Boston, 1941, p. 369.
2.5
4
100
Wavelength, microns 6 7
5
8
9 10
12
15
20
i
I60=
Capillary Film
.
E 20-
d /
5. Methyl stearate.9,9,10 or 9,10,10
IA' \
-
Frequency, cm-1
-Methyl Stearate
C H ~ O ~ ~ C H Z ~ ~ C H ~
0 Methyl Stearate.9,9,10,10.1r
__---Methyl Stearate.6,7& CH~~C[CH~J~CHOCHOICH,~~~CHI
0
_-_ Methyl Stearate.9,10,12,13.dc
C H ~ O ~ J C H Z I J C ~ ~ C ~ ~ I C H Z ~ ~ ~ ~CH306[ ~ CH2)$HDCHOCH2CHOCHO[CH2]4CH3 0 0
Figure 1. Infrared spectra of methyl stearate and methyl deuteriostearates saturated esters on a rubber column (8). Compounds 2, 3 and 4 were prepared E:om oleic, elaidic, and petroselinic acids respectively. Compound 5 was made from oleic acid-9 or -lO-dl, in turn prepared from methyl stearolate, disiamylborane, and acetic acid-d,, CH3COOD. Compounds 6, 7, 8, and 9 came from trans-9,trans-11-octadecadienoic, linoleic, stearolic, and linolenic acids, respectively. Compounds 10, 11, 12, and 13 were produced from methyl oleate by catalytic reduction in a glass manometric apparatus a t 40" C and atmosphuic pressure with a 5 % palladiumon-carbon catalyst and a gas mixture of hydrogen and 20, 40, 60, and 80% deuterium, respectively. Compounds 14, 15, and 16 were prepared siniilarly but with 99.7 % deuterium gas to reduce methyl oleate, methyl linoleate, and methyl linolenate, respectively. The free acids were made from methyl oleate, petroselinate, elaidate, linoleate, and linolenate-all from The Hormel InstitLte. Stearolic acid was obtained by bromination-dehydrobromination of methyl oleate (12). The rrans-9,-rruns-ll-octiidecadienoicacid was prepared by the method of Schneider, Gast, and Teeter (13). The hydrazine hydrate-&, 98 atom D, was purchased from Merck, Sharp & Dohme of Canada, Ltd.; the deuterium gas, from Liquid Carbonic Corp. A mixture of methyl esters of perdeuterio long-chain fatty acids, Compound 17, was extracted from lyophilized cells of green algae Scenedesmks obliques (Merck, Sharp & Dohme). The cells were grown ir. 99.7% deuterium oxide and then extracted with ethyl ether and methanol. After the lipid extract was saponified with KOH, unsaponifiable matter was removed by ether extraction before recovery of fatty acids. Methyl esters were formed by treatment with methanol and BFB. The approximate composition of the mixture was methyl palmitate 16%, palmitoleate 11 %, oleate 34%, linoleate 1 7 z , and sma:ler amounts of other esters. The identifications are tentative because all gas-liquid chromatographic peaks seemed to have slightly shorter retention time than the corresponding undeuterated esters. Infrared spectra of' thtse fatty acids were measured on a Perkin-Elmer 621 spectrophotometer with potassium bromide cells. The integrated absorbance spectra were measured in carbon tetrachloride solution, 20 to 65 m g h l depending on deuterium content, in 1-rnm cells. For these spectra, minor absorptions of unaeuteriited esters in the 240(t2000 cm-1 region were compensated by an equivalent solution of methyl stearate in the reference. beam. Deuterium contents were (12) K. S . Tenny, S. C . Gupta, R. F. Nystrom, and F. A. Kumrnerow, J . Am. Oil Chemi.its' SOC.,40, 172 (1963). (13) W. J. Schneider, L. E. Gast, and H. M. Teeter, Ibid., 41, 605
(1964).
22'00 ' 21b0 frequency. cm-I
Figure 2. sorptions
'
2 DO
Carbon-deuterium stretching frequency ab-
Numbered compounds correspond to those in Table I Zalculated from the parent peaks of the mass spectra measured on a Nuclide 12-9OG mass spectrometer with a 150' C glass Iinlet, 250' C source temperature and 70-volt electron energy. I
RESULTS AND DISCUSSION
The complete spectrum of nondeuterated methyl stearate is given in Figure 1 with the spectra of the deuterated compounds superimposed wherever there is a significant difference. The hreo and erythro forms of methyl stearate-9,10-d2gave identi:a1 spectra. Only the region from 2400 to 2000 cm-l was studied extensively in this paper. The nondeuterated methyl stearate spectrum has almost no ibsorption between 2400 to 2000 cm-l where C-D stretching Frequencies occur. The curves that characterize all the samples in this region are shown in Figure 2. The compounds known t o contain only one deuterium atom per :arbon atom-i.e., No, 1-4, 6, 7, 9 of Table I, have curves similar to curve 7 of methyl stearate-9,10,12,13-d4 in Figure 2. The methyl stearate-9,10,11 ,12-d4 spectrum (not shown) differs in that it is noticeably broader than the other curves i t 2148 cm-1. The spectra of compounds 15 and 16 in Table [ prepared with 100% deuterium are qualitatively similar t o :urve 14 in Figure 2. The curves of the samples catalytically ieuterated with 20, 40, 60, and 80% deuterium are shown in Figure 2. The exchange which takes place during catalytic deuteration rapidly dilutes the gas phase deuterium with hydrogen, and the amount of deuterium absorbed by the sample somewhat depends on catalyst activity and stirring Iconditions. Variation in either is probably the reason why :ompound 13 prepared with 80z deuterium contains more deuterium than compound 14 prepared with 100% deuterium. The curves for methyl stearate-9,9,10 or -9,10,10-d3, sample 5, and methyl stearate-9,9,10,10-d4, sample 8, which contain two atoms of deuterium on one carbon atom, are also shown in Figure 2. Table I1 is compiled by use of the approximation YC-D = vc--a/l.36 (14). Allowing for inaccuracies in the approximation, it appears that the absorptions a t 2191 and 2102 cm-* are asymmetric and symmetric C-D stretching vibrations of (14) Koji Nakanishi, "Infrared Absorption Spectroscopy," Holden-
Day, San Francisco, 1962. VOL. 39, NO. 7, JUNE 1 9 6 7
821
3
25 100
Wavelength, microns 5 6 1
4
9 10
8
12
15
0.25-
20
0.20
s 80I
Y 60c
-
-
5 40-
) . .
= 200! 4000
0
3500
3000
2500
r 2000 1400 1000 400 1800 1600 Frquency, cm -1
1200
800
600
Figure 3. Infrared spectrum of methyl esters of long-chain perdeuterio acids Average Atoms o f Deuterium per Molecule the CD? groups. There can be no symmetric and asymmetric splitting with the C H D group because the C-H and C-D fundamental frequencies are different ; consequently the compounds with only one deuterium atom per carbon atom would be expected to show only a single C-D stretching frequency corresponding t o the R3CH group stretching frequency. The band shifts seen in the trideuterio compound compared to the tetradeuterio compound are probably caused by a n overlap with the center band. The catalytically deuterated compounds have a spectrum which shows that they are a mixture of CD2 and C H D groups as one would expect considering the amount of exchange known to take place during heterogeneous catalytic deuteration. The curves of Figure 2 show that the size of the shoulders becomes smaller as deuterium content is reduced, again as one would expect because the lower the deuterium content the less the chance of two deuterium atoms on one carbon atom. This change in the shape of the absorption band explains why the “absorptivity/average D atoms per molecule” values in column 3, Table I, change for compounds 10 through 16 because this value is measured a t only one frequency, 2148 ern-'. The reason for the reduction in the absorptivity values for compound 16 compared t o 15 and 15 compared to 14 is probably that it is due to exchanged deuterium around the 9-10 position overlapping exchanged deuterium around the 12-1 3 and 15-16 positions forming more CD2groups. The complete infrared spectrum of the methyl (CH3) esters from the fat of perdeuterio algae is shown in Figure 3. The peaks in the 2800-3000 cm-l region are due to the C H 3 group used to esterify the free acids. The peaks at 2199 and 2097 cm-’ correspond to 2191 and 2102 cm-l assigned to the asymmetric and symmetric stretching frequencies in Table 11. Because there is little hydrogen on the acid chain, there is no absorption corresponding t o 2148 cm-l and all the groups must be gem dideuterio groups, CD,. The absorptivities a t 2148 cm-l for almost all the compounds are plotted against the average atoms of deuterium per molecule in Figure 4. The values fall along two curves, one for the catalytically deuterated samples, the other for the hydrazine-deuterated samples. The formation of two curves
Table 11. Stretching Frequencies C-H (Ref. 14) C-D R2CH2asym. RaCH &CH* sym.
2925 2890
2151 2125
i650
2096
_ I
322
(Calcd) C-D (Measd)
4 N A L V X 4 1 CHEMISTRY
2191 2148 2102 ~
-.--
.
Figure 4. Carbon-deuterium absorptivity a t 2148 cm-1 cs. average atoms of deuterium per molecule Numbers refer to componnds in Table I
Average Atoms o f Deuterium per Molecule Figure 5. Carbon-deuterium integrated absorptivity from 2000 to 2400 cm-’ cs. average atoms of deuterium Numbers refer to compounds in Table I
is what one would expect as the deuterium atoms in gem dideuterio groups CD2 d o not absorb a t 2148 cm-l. The catalytically deuterated samples containing smaller amounts of deuterium, compounds 10 and 11, fall nearer the hydrazine line than the catalytic line because it is unlikely that these samples have more than one atom of deuterium per molecule and thus they cannot form gem dideuterio groups. The integrated absorptivities from 2000 to 2400 cm-l of all the compounds, except the algae sample, are plotted in Figure 5 against the average atoms of deuterium per molecule. Because all points fall along one straight line, the absorption due to each C-D bond must be independent of its relationship to C-D and C-H bonds. While the shape of infrared curves changes owing to variation in configuration of the deuterium, integrated absorptivity is sufficiently proportional ?o the number of C-D bonds so that in compounds of this ?orm It can be used as a quantitative measure of deuterium sontent. The shape o f the curves gives a qualitative indica-
tion of the composition as to C H D and C D 2 groups. This indication adds greatly to our knowledge of the composition of these deuterated compounds. ACKNOWLEDGMENT
We thank E. D. Bitner for preparing the methyl stearate9,9,10 or -9,10,10-d3 a i d methyl stearate-9,9,10,10-d4, and Wilma Schneider for providing the trans-9,trans-1 l-octadecadienoic acid.
RECEIVED for review January 3, 1967. Accepted March 9, 1967. Presented a t Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., February 1966. The Northern Regional Research Laboratory is headquarters for the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. Mention of trade or company names is for identification only and does not imply endorsement by the Department.
A New Convenient Method for the Rapid Quantitative Determination of Unsaturation via Hydrogenation Charles A. Brown, Shanti C. Sethi, and Herbert C. Brown R. B. Wetherill Laboratcry, Purdue Unicersity, Lufujette, Ind. 47907 A new procedure for the in situ preparation of highly active hydrogenation catalysts has been combined with the automatic valve for the generation of hydrogen from sodium borohydride to provide a new, convenient technique for the determination of unsaturation through quantitative hydrogenation. Results are reported for hydrogencationof several compounds, including different vegetable oils and the difficultyreduced Ag,lO-~ctalin.The method is rapid, simple, quantitative, and offers major advantages for the determination of unsa1:uration in many types of compounds as compared with other known methods.
FOR M A N Y YEARS the standard method for determining the degree of unsaturation (often expressed as the iodine value, i.e., centigrams of iodine absorbed per gram of compound) has relied on the reaction of iodine chloride with the olefin. This procedure leaves much to be desired. The reagent, IC1, is difficult to store and to keep as a standardized solution. The addition of the reagent to the olefin must be carried out in the dark under rigidly prescribed conditions. Moreover, even under the best conditions the results for conjugated olefins are consi 3erably lower than the correct values, especially troublesome with certain fats and oils. Finally, there is always a danglx that some substitution will occur during the addition reaction. Attempts have been made to circumvent these difficulties by using catalytic hydrogenation to determine the degree of unsaturation. Such hydrogenation methods, as are currently available, suflfei from several handicaps. First, it is necessary to ensure thai the catalyst is totaliy saturated with hydrogen before andlys8is, or high results will be obtained. Second, it is often recommended that an elaborate purification train be used to ensure that the hydrogen gas is free of reducible substances and catalyst poisons. Third, it is necessary to have carefully ttierrnostated gas burets to measure the hydrogen uptake at a constant temperature. This requires ai-* air-conditioned iabine: anu associated equipment. Finall!:, the procedure IS often time-consuming. For example, thc method o f Pack e! al. ( I ) for hydrogenating fats and oiis a t 60" C required 2 hours. An improved procedure for the (1) F. C. Pack, R W. Planck, and F. G . Doliear, J . Am. Oil Chemiisrs' SOC., 29, 227 (1952;.
determination of unsaturation in petroleum fractions has recently been described (2). The hydrogenation method here described is rapid, simple, quantitative, and offers major advantages for the determination of unsaturation in many types of compounds as compared with the IC1 method and other hydrogenation methods. The present method is based on the new, highly active hydrogenation catalysts readily prepared in situ by the reaction of sodium borohydride with appropriate metal salts (3, 4) and the generation of hydrogen of high purity by treatment of standard solutions of sodium borohydride with appropriate aqueous acid solutions (5). These developments avoid many of the errors in the earlier procedures arising from the presence of poisons in the laboratory or plant atmosphere and the presence of minor amounts of reducible impurities in the hydrogen source. The generation of hydrogen can be made simple and automatic by the use of a simple pressure-actuated mercury valve which permits the borohydride solution to be added to the acid only when the internal pressure of the system drops below atmospheric (6, 7). This system has proved to be both versatile and highly useful for laboratory scale hydrogenations (7). Slightly modified, it has proved to be equally valuable for the determination of unsaturation. In the recommended procedure, a standardized borohydride solution is used to measure the hydrogen uptake. Because the buret contains only a liquid (this solution), it need not be thermostated. The only requirements for accuracy are that the hydrogenator vessel be thermostated and that the pressures at the beginning and end of the reaction be the same. The former is assured by a small constant-temperature bath, the latter by our analytical procedure. This method has been used to hydrogenate from 0.2 to 2.0 mmoles of compound (2) M. Sedlak, ANAL.CHEM., 38, 1503 (1966). ( 3 ) H.C. Brown and C. A . Brown. J . Am. Chem. SOC..84. 1494. 2827 (1962; (4) I-I. C. Brown and C. A. Brown. Terrahedron. SUDD!. 8. Part I. 149 (1966). (5) H . C. Brown and C. A . Brown, J . Am. Chem. SOC.,84, 1493 (1962). (6) C. A. Brown and H. C. Brown, Zhid., 84,2829 (I%-, (7) C. A. Brown, and H. C. Brown, "', Org. Ciieni., 31, 3Y89 (1966, _
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