for the detection of aromatic compounds on paper chromatograms. Because chromatography causes spots to enlarge by diffusion, quantities of 50 to 100 Y may be required for good detection on a chromatogram. LITERATURE CITED
(1) Branch, G. E. IC., Calvin, M., “Theory
of Organic Chemistry,” pp. 70 E., 118-19, Prentice-Hall, S e w York, 1941.
( 5 ) I b i d . , 30, 542 (1958).
(6) Orchin, )I.,
Reggel, L., Woolfolk, E. O., J . -4m.Chern. SOC.69, 1225 (1947). (7) Orchin, bl., Woolfolk! E. O., Zbid.,
(2) Gillam, *4., Stern, E. S., “Introduction to Electronic Absorption Spectrescopy in Organic Chemistry,” pp. 127-8, Edward Arnold, London, 1954. (3) Laskowski, D. Grabar, D. G., >fCcTOne, w.c.,h A L . CHEM. 2 5 , 1400 (1953). (4) Laskowski, D. E., McCrone, W. C., Ibid., 26, 1497 (1954).
68, 1727 (1946).
RECEIVED for review January 18, 1955. Accepted September 2, lyj8, a o r k supported by Research Grant E-l081(c), from the Sational Institutes of Health, U.S. Public Health Service.
Analysis of Long-Chain Fatty Acids by Gas-Liquid Chromatography Micromethod for Preparation of Methyl Esters WILHELM STOFFEL, FLORENCE CHU, and EDWARD H. AHRENS, Jr. Rockefeller Institute, 66th Street and York Avenue, New York 2 1 , N. Y. An essential prerequisite for the analysis of lipide mixtures of biological origin by gas-liquid chromatography (GLC) is the quantitative formation and isolation of the constituent methyl esters. A micromethod is described involving interesterification with methanol and hydrochloric acid. By sublimation the methyl esters are isolated from the reaction products in a pure form ready for gas-liquid chromatography. The methylation and sublimation can be done with ease on a large number of samples. This method eliminates the use of alkali and diazomethane, which may lead to isomerization or pyrazoline formation.
A
method for forination of methyl esters of long-chain fatty acids necessarily precedes analysis by gas-liquid chromatography. The requirements of an optimal micromethod are: quantitative yield, absence of change in double-bond structure of highly unsaturated acids, and technical convenience. Saponification of fatty acids esterified as glycerides, phosphatides, and sterol esters can be followed by methylation with diazomethane. However, this procedure suffers some disadvantages-yields may be poor because of the formation of addition products of diazomethane a t ethylene bonds (pyrazolines) (4); structural changes in double bonds may occur during saponification ( 1 ) ; and the isolation of soaps from nonsaponifiable contaminants is rarely completely satisfactory. Formation of fatty acid methyl esters by interesterification, on the RELIABLE
other hand, has proved in this laboratory t o be technically simpler, milder, and more quantitative than diazomethanolysis. Yields of methyl esters from glycerides, phosphatides, and cholesterol esters are nearly quantitative, alterations in double bond structures are avoided, and the technical aspects are relatively simple. Completeness of interesterification d e w n d s an acid medium and the ahsence of water. In an acid medium,
the electron-donating Lapacity of the is greater oxygen in water, H-Q-H, than that of the oxygen in methanol, CH8-g--H, and, therefore, methylation demands strictly anhydrous conditions. In the absence of water,
H
R,
Hon-eJ-er, if water is present,
R
H
As Reaction 2 has preference over Reaction 1, methyl ester formation by interesterification is hindered in the presence of water. I n the method described below, any nonsaponifiable contaminants which may be present are eliminated by sublimation of the methyl esters. This feature assumes considerable importance when methyl esters are formed from cholesterol esters; the mixture of methyl esters applied to the gas-liquid column can be evaluated quantitatively only if free from cholesterol. Similarly, the triglycerides isolated by silicic acid chromatography from mixtures of naturally occurring lipides (2) are frequently contaminated by free cholesterol; the methyl esters formed by interesterification are conveniently freed of cholesterol by sublimation. The procedure has proved satisfactory for interesterification of safflower oil glycerides, soybean phosphatides, and cholesterol palmitate, as well as for glycerides, sterol esters, and phosphatides isolated from human serum by silicic acid chromatography ( 2 ) . Absence of changes in polyenoic acids was verified by ultraviolet and infrared spectrophotometry and by degradation studies ( 5 ) .
IO’ METHOD
Reagents.
Dry hydrochloric acid
(5%) in superdry methanol ( 6 ) . VOL. 31, NO. 2, FEBRUARY 1959
307
Sodium sulfatesodium bicarbonate mixture, reagent grade, anhydrous, 4 to 1 mixture by weight. Petroleum ether, 30"to 60" C., JIerck, reagent grade, redistilled. Benzene, Merck, reagent grade, dried over sodium and distilled. Apparatus. Microinteresterification assembly, 19/38 (S), consisting of round-bottomed test tubes, Liebig condensers, cold fingers, and six-place manifold, with nitrogrn inlet. (Available in toto from Metro Industries, 11-38 31st Ave., Long Island City 6, S.Y., catalog No. ME-517. or piecemeal.) AIcLeod gage, High vacuum pump, cold-trapped. Interesterification. The esters or acids to be methylated (1 t o 10 mg.) are dissolved in 4 ml. of 5% hydrochloric acid in superdry methanol and 0.5 ml. of dry benzene in a 15-ml. microsublimation tube t o which a condenser with a calcium chloride moisture trap is connected. The mixture is refluxed in a silicone bath a t 80' t o 100" C. for 2 hours, with frequent shaking a t the start to dissolve the lipide mixture. After cooling t o room temperature, two volumes of water are added, and the methyl esters are extracted three times with 3 ml. of petroleum ether. The pooled extracts are simultaneously neutralized and dried over sodium sulfatesodium hicarbonate mixture for 1 hour. The esters are then quantitatively transferred with petroleum ether to a second niicrosublimation tube and the solvent is evaporated to dryness a t reduced pressure in a 40' C. n-ater bath.
Microsublimation. After the microsublimation tube is fitted to the cold finger, a vacuum of 0.2 i: 0.15 mm. of mercury is produced. The tube is then lowered into a silicone bath at 60" 2" C. for 60 minutes. The assembly is disconnected after cooling, and the sublimed methyl esters are rinsed off with petroleum ether into a glass-stoppered tube. After evaporation of solvent, the preparation is now ready for application to the gas-liquid chromatography column.
*
The sublimation technique is described for use with l to 5 mg. of methyl esters. If greater quantities are sublimed, an increased sublimation time may be required. Use of a manifold permits several sublimations to be carried out simultaneously.
-
phorus content of the aliquot selected for interesterification remained in the water washes and yields of methyl esters were quantitative, indicating completeness of methylation. Benzene is incorporated in the interesterification mixture in order to assure complete solution of the lipides. This is essential in the case of the cholesterol esters which are not completely soluble in methanol. Unless the lipides are completely dissolved, interesterification is incomplete. When these precautions are observed during the interesterification of cholesterol esters, the recoveries of methyl esters on the cold finger and of free cholesterol in the residue are quantitative. LITERATURE CITED
COMMENTS
l l i Chahine. M. H.. Cousins. E. R.. Feuge, R.'O., J . Am. Oil Chehists' Soc: 35, 396 (1958). ( 2 ) Hirsch, J., Ahrens, E. H., Jr., J . Biol. Chem. 233, 311 (1958). (3) Koehler, A. E., Hill, E., Zbid., 179, 1 (1949). (4) Jluller, E., "Seuere Anschauungen der Organischen Chemie," p. 450, Springer-Verlag, Berlin, 1957. ( 5 ) Stoffel, K., Ahrens, E. H., Jr., J. Am. Chem. Soc., in press. (6) Vogel, A, 'LPracticalOrganic Chemistry," 3rd ed., pp. 169, 179, Longmans, Green, S e w York, 1956. \
I n pilot experiments with soybean phosphatides, safflower oil, and cholesterol palmitate, the recovery of methyl esters was better than 95% of theory. When free cholesterol was added to the methyl esters prior to sublimation, it remained quantitatively as residue in the microsublimation tube, and the methyl esters were LiebermannBurchard negative. However, sublimation a t 85" C. and 0.2 mm. of mercury permits traces of cholesterol to collect on the cold finger, more a t higher temperatures. I n the case of soybean phosphatides, the entire phos-
I
RECEIVEDfor review June 23, 1958. Accepted October 20, 1958. Sup orted in part by the U. S. Public Health &vice (H-2539).
Determination of Quinquevalent, Trivalent, and Organic Phosphorus in the Atmosphere and in Aqueous Solutions ROY MAY Occupational Health Branch, Division of Health and Safety, Tennessee Valley Authority, Wilson Dam, Ala.
A simple and rapid method for the microdetermination of quinquevalent, trivalent, and organic phosphorus in the atmosphere and in aqueous solutions is based on selective oxidation of the organic phosphorus, which is readily converted into the inorganic phosphate with hot, alkaline ammonium persulfate solutions, but is resistant to attack from this oxidizing agent in hydrochloric acid solutions. The trivalent phosphorus, being more easily oxidized than the organic phosphorus, i s converted into orthophosphate under either condition. The amount of the three types of phosphorus compounds in a sample can b e calculated from the differences in the quinquevalent phosphorus found in three separate
308 *
ANALYTICAL CHEMISTRY
aliquots: (1 ) without oxidizing and following, (2) hydrochloric acid, and (3) alkaline persulfate oxidation.
I
x A STUDY of environmental contamination in a plant manufacturing intermediate compounds for the production of nerve gas (organophosphonates), a need arose for a method to determine low concentrations of orthophosphate, and trivalent and organic phosphorus in aqueous solutions. Such a mixture of phosphorus compounds resulted from the hydrolysis of phosphorus oxychloride, phosphorus trichloride, and organic phosphorus compounds during atmospheric sampling in the plant. Because of the highly toxic characteristics
associated with organic phosphorus compounds, it was most important to determine the atmospheric concentration of the organic phosphorus constituent. A method was required which would determine quantitatively the organic phosphorus in the presence of the inorganic phosphorus compounds. The two generally used methods for the assaying of micro quantities of phosphorus are the colorimetric molybdenum blue (5) and the phosphovanadomolybdate (4), or some modifications. They both require that the phosphorus be in the orthophosphate state. The trivalent phosphorus may be converted to the phosphate by a number of wet oxidizing agents, such as perchloric acid, nitric acid and bromine, and per-
