barbituric acid ( 5 ) . For microgram quantities, stannous chloride in 2N hydrochloric acid (10) was used, with a wave length of 470 mp, as recommended by Maynes and McBryde ( 9 ) . Total volumes and path lengths were varied, depending upon the size of sample. This method could be used efficiently for samples containing as little as 2 y of rhodium in 25 ml. with a 5-cm. path length, if the sample mas sufficiently acidic during the heating period. Determination of Iridium. For milligram amounts, precipitation by 2-mercaptobenzothiazole ( I ) was used. The microgram samples were analyzed colorimetrically by stannous chloride dissolved in concentrated hydrobromic acid ( 2 ) . Prior to analysis, and folloxing the digestion to remove organic matter, the samples were boiled with 5 ml. of hydrobromic acid until the volume was reduced to 1 ml. During boiling the beaker was covered tightly with a cover glass, so that fumes were allowed to escape only a t the lip of the beaker. This boiling seemed necessary, as it was difficult to reconvert iridium from the nitro form. In the analysis, a heating period of 90 seconds was adopted. Because the reproduction of the solution composition was
uncertain, the standards and samples were analyzed simultaneously. I n all cases, the reference blank and standards were carried through the entire procedure. For the standards, the eluent from the column operations was salted with known amounts of the elements under consideration. Where rhodium or iridium was determined independently, these standards agreed with those determined on solutions taken directly from the stock bottles. When rhodium and iridium were mixed together in the procedure and a separation was performed, the standards appeared to be slightly lower than those determined directly, although 100% recovery was obtained for the separation on standard solutions directly. The reason for these low results has not been determined, but they may be due to any one of several concealed factors. The results of the analyses are recorded in Tables I and 11.
aid in the form of a research scholarship which enabled them to carry out this research. LITERATURE CITED
( 1 ) Barefoot, R. R . , McDonnell, W.J., Beamish, F. E., ANAL. CHERI. 23, 514 (1951). ( 2 ) Berman, S. S., illCBr!.de, TI-. A. E., Analyst 81, 566 (1956). ( 3 ) Berman, S. S., RicBryde, W.A . E., Can. J . Chem. 36, 835-52 (1958). ( 4 ) Coburn, H. G., Beamish, F. E., Lewis, C. L., ASAL. CHEN. 28, 1297 (1956). ( 5 ) Currah, J. E., &IcBrq.de, JT. A . E., Cruickshank, A. J., Beamish, F. E., ISD. EKG.CHEM.,ASAL. ED. 18, 120 f1946). (6, Gilchris,t, R., J . Researcn S a t l . Bur. Stan,?a-ds 30,89 (1943). Analyst 1 7 1 K ember, ?rT. F., Wells, R. -4.> 80, T35 (1955).
( 8 ) >I [ac,Uevin, W. AI., McKay, E. S., ANAL.CHI211. 29, 1220 (1957). ( 9 ) Maynet4 , A. D., McBryde, \I7.A. E., .4na/yst 79, 230 (1954). (10) Sandell, E. B., “Colorimetric Determination of Traces of Metals,” 2nd ed., DD. 523-5, Interscience, Xew York,
ibo.
ACKNOWLEDGMENT
The authors are grateful to the Ontario Research Foundation for financial
( 1 1 ) Westland, A. D . , Beamish, F. E., Mikrochtm. Acta 10, 1474 (1956). RECEITEDfor review October 14, 1957. hccepted Ma!. 9. 1958.
Cholesteryl Esters of Long-chain Fatty Acids Infrared Spectra and Separation by Paper Chromatography J. A. LABARRERE, J. R. CHIPAULT, and W. 0.LUNDBERG The Hormel Institute, University of Minnesota, Austin, Minn. Pure cholesteryl laurate, myristate, palmitate, stearate, oleate, linoleate, and linolenate were prepared and used to develop analytical procedures for the separation and identification of these compounds. Melting points, specific rotations, and infrared and near-infrared spectra were obtained. Infrared analysis can be used for identification of only a few milligrams of pure, unknown samples. Model mixtures containing both saturated and unsaturated esters were separated by ascending reversed-phase paper chromatography using two solvent systems (acetone-ethanol-formic acidwater, and chloroform-methanol-formic acid-water) consecutively on the same paper strip. The amount of each compound required for this purpose is approximately 10 to 20 y.
T
present interest in the relationship between cholesterol metabolism and atherosclerosis, and in the physiological role of cholesteryl esters HE
1466
ANALYTICAL CHEMISTRY
of long-chain fatty acids, has resulted in numerous attempts to separate and identify these compounds in model mixtures and in lipides obtained from animal tissues. Methods for characterizing the individual esters, however, generally are based upon analysis of the mixed fatty acids obtained by saponification. Direct analysis by separation of the esters is preferable in all cases, and becomes a necessity when xorking with esters isotopically labeled in the cholesterol moiety. I n previous experiments on direct identification of mixtures of cholesteryl esters by chromatographic separation (4,11, 19, 20, 22, 26), difficulties 11 ere met with cholesteryl esters of longchain fatty acids, and also with esters of saturated and unsaturated fatty acids when present together. Michalec (20) and Zimmerman (26) have obtained nearly identical R , values for the cholesteryl esters of oleic acid and long-chain saturated fatty acids. Paper chromatography of cholesteryl linoleate has been mentioned recently (18),
but the linolenate ester apparently has not been examined previously by this technique. The infrared spectra of cholesterol and of the cholesteryl esters of a fen short-chain fatty acids hare appeared in the literature (5, 14, 23), and Holman and Edmondson ( I S ) have obtained the near-infrared spectrum of cholesterol. Our knowledge of the infrared spectra of cholesteryl esters of long-chain fatty acids, however, has been limited to the partial spectrum of cholesteryl laurate, in carbon disulfide, published by Freeman et al. (6, 7 ) , and to these authors’ observations that the fatty acid composition did not appreciably alter the solution spectra of cholesteryl esters. I n the present study, the cholesteryl esters of lauric, myristic, palmitic, stearic, oleic, linoleic, and linolenic acids have been prepared ; infrared spectrometry and paper chroma tography were used for identifying and separating these compounds.
EXPERIMENTAL
Starting Materials. T h e saturated acids were prepared at T h e Hormel Institute by distillation a n d crystallization, a n d were free of unsaturated impurities (Wijs iodine value, 0 ) . The melting points were: lauric, 44" C.; myristic, 53.5to 54' C. ; palmitic 63.5 C. : and stearic, 69" C. The unsaturated acids were obtained from The Hormel Foundation, and had the following iodine values: oleic, 89.80 (theory 89.87); linoleic, 180.10 (theory 181.00); linolenic, 273.0 (theory 273.57). Oleic and linoleic acids were the natural isomers and m r e free of trans unsaturation. Linolenic acid had been prepared by a bromination-debromination procedure and contained trans double bonds corresponding to a n estimated maximum content of less than 15% of a ci.. cis, trans isomer. Cholesterol (U.S.P. from Sutritional Biochemical Corp.) had been prepared from the srsinal cord of cattle and w r i fied via the dibromide; melting Iyoint, 148.5" C.; [cu]2,0, -39.5'. Preparation of Cholesteryl Esters. Thc esters were prepared by the method of Swell and Treadnell (%), except t h a t oxalyl chloride (Eastman) was used instead of thionyl chloride as t h e chlorinating agent. The use of thionyl chloride with unsaturated fatty acids is impractical (1, 95). and, furthermore, oxalyl chloride is removed more easily from the reaction mixtures. The esterifications were performed under pure nitrogen, and the unsaturated esters n-ere stored under vacuum a t -20" c. Melting Points and Optical Rotations. Report,ed melting points are uncorrected. and were obtained wit'li a Fisher-Johns apparatus. Optical rotations were measured on 5% chloroform solutions with a Bellingham and Stanley polarimeter equipped with a sodium vapor lamp. Spectrophotometric Studies. Infrared spectra were obtained with a Perkin-Elmer 3lodel 21 double-beam infrared spectrophotometer equipped d h sodium chloride optics. Solution spectra n-ere measured in a 0.1-mm. cell, using 10% solutions in carbon disulfide (2.0 to 4.2. 5.0 to 6.1, and 7.2 to 15.0 microns), and in tetrachloroethylene (4.2 to 5.0 and 6.1 to 7.2 microns). Solid state spectra were obtained directly n-ith solid films and by the potassium 1x0mide disk method. Year-infrared curves, from 0.9 t o 3.0 microns, w r e obtained with 5 to 8y0 solutions in carbon tetrachloride, ivith a Beckman DK-1 Fpectrophotometer, using a 1-mi. cell. Paper Chromatography. The method used was a modification of procedures developed a t The Horniel Institute (9:16. ?dl. Mixtures of sat,urated esters only, or of unsaturated esters only. \\-ere separated effectively by dewloping the chromatograms in a solvent mixt,ure consisting of chloroform (40 m1.j. methanol (60 ml.), 98Yc formic acid (1 ml.). and water ( 5 nil.) (chloroform solvent). Unsaturated e?ters could be separated also by wing a
Table I. Melting or Transition Point and Specific Rotation Melting or Transition Specific Rotation, [01]*2 Cholestery1 Ploint, C. Found Literature Ester Found Literature -27 8 -27 5 a Laurate 90.8-91.5 91.8-92.0a -25 4 -26 5a Myristate 70,0-70.5 73.0-80.0° -24 9 -25 4a Palmitate 75.0-i5.5 75 -80.5' -23 9 -25 l a Stearate 80.0-80.5 71 -79.5" -24 -4 -21 6a Oleate 46.3-47 0 455 -24 7 -23 9c 39.0-40.3 42.0-42 5' Linoleate -24 3 -24 3 c Linolenate 32 5-33.5 49 - 7 4 ~ a
(23). (8).
(20).
mixture of acetone (50 ml.), ethanol (50 ml.), 98% formic acid (15 ml.) - 1 s . a CHEM. 29, 329 (1957). (18) Michalec, C., Intern. Conf. on Biochem. Problems of Lipids, 4th Conf., Essential Fattl- .icids. ()siord. July 15-18, 1957. , (19) Michalec, C,, .\-at ii I' 11'isse nsch often 42, 509 (1955); Biochimi. et Biophys. -4cta 19, 187 (19561. (20) Michalec, C., Jirgl. I-.,Podziniek, J., Erperienfia 13, 342 19.57: . 1
(21) Page, I. M., Rudy. T., Bi'ochenz. 2. 220, 304 (1930). ('22) Quaife, M. L., Geyer, R. P., Bolliger, H. R., Division of Biological Chemist,ry, 130th Meeting. hCS, Atlantic City, X. J., Septenilier 1956. ("3) Rosenkrantz, H . . hlilhorat, A . T., Farber, AI., J . Bioi. C'hern. 195, 509 (1952). (24) Schlenk, H.! Geilernian: J. L., Tillotson, J. -4.:Ilangold, H. K., J . Am. Oil ChejJci.st.s' SOC.34, 377 (1957). ( 2 5 ) Swell] L , Treadwell. C R , J . Bzol. rhem. 212, 141 (1955, ( 2 6 ) Zimmerman, G.. Bidae G., Pharvrazie 1 1 , 715 (1956,.
RECEIVED for revier December 21, 1957. Acrepted April 17, 1958. Supported in part by a grant-in-aid from Oscar AIayer & Co. and by The Hormel Foundation. Hormel Institute Publication KO.165.
Determination of Water in Liquid Bromine-Correction In the infrared quantitative anal-
(2) Bladon, P., Fabian, J. ?*I.,Henbest,
p i s data [R. B. Duvall and L. R.
tion Spectra of Steroids," pp. 41-3, Interscience, New York, 1953. !6) Freeman, 5 . K., Lindgren, F. T., S g . T. C.: Kichols, A , V., J . B i d . Cheni. 203, 293 (1953). 1~7)Zbid., 227, 449 (1957).
Kiley, -%SAL. CHEX 30, 549 (195S)l the range presented should read 0-100 p.p.ni., not 0-100 %, and the accuracy 1' + 0 3 p.p.m., not i 0 . 5 %. The final note in the method should read: ,.Relative absorbance is given as the slope of the Beer's law concentration curve used expressed in terms of absorbance per 100 p.p.ni. of constituent."
H. B., Koch, H. P., Wood, G. W., J . C'hem. SOC.1951, 2402. , 3 1 Chapman, D., Ibid., 1956, 55, 2522. (4) Dieckert, J. W., Reiser, R., J . Am. Oil Chemists' SOC.33, 123 (1956). (5) Dobriner, K., Katzenellenbogen, E. R.! Jones, R. X.) "Infrared Absorp-
