Figure 1 indicates that an RSC value of 0.7-0.8 fits the selenium values very well. Consequently the mass spectrographic data corrected by this factor should yield absolute values of selenium in GaP accurate to better than =k50 %. The data of Figure 1 strongly suggest that the RSC values of zinc, silicon, germanium, tin, sulfur, and tellurium fall between 1.5 and 0.5, the lower values being favored where the data is scattered. An RSC of about 1.3 is indicated for copper in the GaAs crystal. This should also be a particularly well characterized sample since the copper was diffused into the GaAs and the problem of inclusions is absent. CONCLUSION
The results of this comparison of mass spectrographic data with spectrophotometric analyses indicate that, for impurities zinc, silicon, germanium, tin, sulfur, selenium, and tellurium in GaP and for copper in GaAs, the spark source mass spectrograph is capable of yielding concentrations accurate to within a factor of 2 or 3 by assuming a Relative Sensitivity Coefficient of unity. More specifically, this comparison indicates an RSC of 0.7 to 0.8 for selenium and probably the lower end of the range 1.5 to 0.5 for the other elements. An RSC of 1.3 for copper in GaAs was obtained.
Since these impurities vary widely in their properties (volatility, position in periodic table, mass etc.) it is a reasonable assumption that many other impurity elements in GaP could be measured in the same way with the same accuracy. Furthermore the selenium results suggest that equally homogeneous samples doped with other elements at levels high enough to be amenable to analysis by spectrophotometric or other standard techniques might be preparable. If so, a comparison of mass spectrographic data with that of these standard techniques could yield reliable RSC data. Then, mass spectrographic analyses which could be made on low concentration samples not generally amenable to analysis by other techniques could yield more accurate data than heretofore possible. ACKNOWLEDGMENT
We are indebted to M. Kowalchik and L. Derick for the growth of the GaP crystals and to K. B. Wolfstirn for the GaAs crystal. RECEIVED for review June 28, 1966. Accepted December 22, 1966.
Separation of Muscle Lipids into Classes by Nonchromatographic Techniques Irwin Hornstein,l Patrick F. Crowe, and Jules B. Ruck U.S. Department of Agriculture, Agricultural Research Sercice, Beltsoille, Md. Muscle lipids can be separated into phospholipids, free fatty acids, triglycerides, and cholesterol in a test tube procedure that eliminates time consuming chromatographic steps. Phospholipids are separated from the total lipid extract by adsorption on activated silicic acid. The free fatty acids in the supernatant a r e in turn adsorbed on an anion exchange resin. The solution, free of phospholipids and of free fatty acids, is saponified with alcoholic potassium hydroxide. After acidification, a hexane extract is obtained containing the glyceride fatty acids which are produced on sponification, and cholesterol. The fatty acids are adsorbed on an anion exchange resin and total cholesterol remains in solution. Fatty acids are converted to methyl esters directly on the resin. Phospholipids adsorbed on silicic acid are transmethylated to produce the methyl esters of the phospholipid fatty acids. The methyl esters a r e analyzed by gas chromatography.
separation by TLC can lead to oxidative losses of highly unsaturated fatty acids and recovery of the phospholipids may be incomplete (1, 2). Phospholipids have been removed from lipid extracts by adsorption on activated silicic acid (3, 4) and free fatty acids have been removed from lipid extracts by adsorption on anion exchange resins (5). The phospholipid adsorption is, however, not clean (6) and because the free fatty acids are not adsorbed quantitatively, an internal standard must be used. These separations have been improved and the following procedure has been developed for the rapid separation of lipids into the FFA, TG, and PL without the use of chromatographic procedures and for the subsequent conversion of the fatty acids in each class to methyl esters.
THISLABORATORY determines the composition of intramuscular lipids from different muscles in animals of varying ages. We separate total lipids into phospholipids (PL), triglycerides (TG), and free fatty acids (FFA) and then determine the fatty acid composition of each of the classes. These studies require many lipid analyses; lipids usually are separated into classes by thin layer chromatography (TLC) or column chromatography. These separations are time consuming; columns and plates must be prepared and the development time is appreciable. In addition, if proper care isn't exercised,
Reagents. Silicic acid 100-mesh powder, analytical reagent (Mallinckrodt Chemical Works, St. Louis, Mo.) is activated just prior to use by heating for ' 1 2 hour at 120" C.
Present address, USDA, ARS, Human Nutrition Research Division, Food Quality and Use Laboratory. Room 316, Center Bldg., Agricultural Research Center, Beltsville, Md. 20705 352
ANALYTICAL CHEMISTRY
EXPERIMENTAL
(1) N. Pelick, T. L. Wilson, M. E. Miller, and F. M. Angeloni, J. A m . Oil. Chemists' SOC.,42, 393 (1965). ( 2 ) 0. S. Privett, M. L. Blank, D. W. Codding, and E. C. Nickell, Zbid., 42, 381 (1965). (3) R. B. R. Choudhury, and L. K. Arnold, Zbid., 37,87 (1960). (4) L. A. Carlson, J. Atherosclerosis Res., 3, 334 (1963). (5) I. Hornstein, A. Alford, L. E. Elliot, and P. F. Crowe, ANAL. CHEM., 32, 540 (1960). (6) M. Kuchmak, and L. R. Dugan, Jr., J. Am. Oil Chemists' Soc., 40,734 (1963).
The anion exchange resin Dowex 1-X8 200-400 mesh (Dow Chemical Co., Midland, Mich.) is used in the basic form (OH-) and is purified according to the method of Hornstein et al. (5). Methanol-hydrochloric acid, anhydrous 5-10 acid, is prepared by bubbling iydrochloric acid gas through anhydrous methanol. Sodium methoxide, 0.3N solution in methanol, is prepared by slowly adding appmximately 0.9 gram of sodium pieces to 100 ml of methanol. Normality is adjusted by titrating an aliquot and adding the required amount of methanol. The hexane, chloroform and methanol are "distilled in glass" solvents obtained from Burdick and Jackson Laboratories, Muskegon, Mich. Procedure. Total lipid is extracted from ground muscle tissue with a mixed ch oroform-methanol solvent according to the procedure of Bliigh and Dyer (7). The total lipids are isolated in chloroform. The solution is concentrated to 10 ml and normally contains 1 to 2 grams of TG, 0.34.7 gram of PL and 20-200 mi: of FFA plus varying amounts of cholesterol, cholesterol esters, and other minor constituents. Separation of Phospholipids. One milliliter (approximately 0.5 gram) of silicic acid is measured into a graduated 15-ml glass-stoppered centrifuge tube. T o activate the silicic acid the unstoppered tube is8heated for liZ hour at 120" C, then removed from the oven, stoppered, and allowed to come to room temperature. 'Two milliliters of chloroform-hexanediethyl ether (2:l :1) and 0.2 ml of the liquid extract are added in that order to the tube containing the silicic acid. The tube is restoppered, the contents are mixed on a Vortex mixer for 1 minute, allowed to stand for 2 minutes, centrifuged at 1500 rpm for 2 minutes in a clinical-type-centrifuge and the supernatant is decanted into another 15-ml centrifuge tube. The silicic acid which contains the adsorbed phospholipids is washed three times with successive 2-ml portions of the chlorl3form-hexane-diethyl ether solvent. For each wash, the mxture is mixed, centrifuged, and decanted as described above. Further treatment of the adsorbed phospholipids is described under Transmethylation of Phospholipids. Separation of FFA. The volume of the combined washes and supernatant is approximately 8 ml. The supernatant plus washes contains the FFA, the TG, and cholesterol. To this solution is addelj 1 ml of methanol and 2 grams of the purified anion exchange resin Dowex 1-X8. The contents in the glass-stoppered centrifuge tube are mixed on the Vortex for 1 minute and the resin is allowed to settle. The supernatant is decanted into a 25-ml volumetric flask. The anion exchange r:sin is washed five times as above. Each 2-ml wash is in turn decanted into the volumetric flask. The resin retains the FFA. Saponification of Triglycerides. The supernatant from the above step (containing the triglycerides) is made up to 25 ml, and a 5-ml aliquot is pipetted into a graduated centrifuge tube. The solvents are evaporated under a stream of nitrogen with gentle heat supplied by a water bath or hot air blower. Four milliliters of approximately 0.5N KOH in 95% ethanol are added. The air is displaced with nitrogen, the centrifuge tube is closed with a glass stopper, and the mixture is heated on a water bath at 70" C for 30 minutes. The tube is cooled to room temperature, 4 ml of water are added, and the solution is acidified to Methyl Red with 2N HCl. Two milliliters of hexane are added, the mixture is vigorously shaken, and the phases are allowed to separate. The upper hexane layer containing the fatty acids is transferred by means of a 5-ml syringe to a glass-stoppered centrifuge tube. The aqueous phase is washed three times with successive 2-ml portion:; of hexane and the combined hexane ___
(7) E. G. Bligh and W. J. Dyer, Can. J . Biochem. Physiol., 37, 912 (1959).
wash and supernatant are washed twice with 2 ml of water. After each wash, the bottom aqueous phase is removed by syringe and discarded, the hexane layer is then dried over anhydrous sodium sulfate. The hexane solution is decanted into a 15-ml graduated centrifuge tube and the sodium sulfate is washed with 2 ml of hexane. The hexane solution approximately 10 ml in volume is concentrated to 6 ml and 1 ml chloroform, 1 ml of methanol, and 2 grams of the anion exchange resin are added. The fatty acids are adsorbed on the resin. The resin is washed with 3 successive 2-ml portions of chloroform-methanol-hexane (1: 1:6). The wash solution can be analyzed for cholesterol. Transmethylation of Phospholipids. The phospholipids are transmethylated directly on the silicic acid. Methanolysis is carried out by adding 4 ml of 0.3N sodium methoxide to the silicic acid and mixing the slurry on a Vortex mixer, then allowing the excess hexane to separate as a thin surface layer. The centrifuge tube is flushed with prepurified nitrogen; this serves to remove the hexane and to establish a nitrogen atmosphere. The centrifuge tube is closed with a glass stopper and heated at 60" C for 45 minutes. The reaction mixture is cooled, 4 ml of water are added, and the solution is acidified to Methyl Red and several drops in excess of 2N hydrochloric acid are added. Three milliliters of hexane are added, the mixture is stirred on the Vortex mixer, the phases are allowed to separate, and the upper hexane layer is transferred by syringe to a glass-stoppered test tube. The aqueous phase is washed three times with 2-ml portions of hexane. The combined hexane solution is washed twice with 2-ml of water and after each wash the lower aqueous phase is removed by syringe. The hexane phase is dried over anhydrous sodium sulfate and the dried solution is transferred to a graduated centrifuge tube and concentrated to C.5 ml. A suitable aliquot is used for the analysis by gas chromatography. Esterification of Fatty Acids. The esterification of fatty acids is carried out directly on the anion exchange resin. A small-glass-fritted filter stick attached to a water aspirator is used to suck traces of solvent from the resin. Three milliliters of anhydrous methanol are added, the mixture is stirred on the Vortex mixer, and the methanol is removed using the filter stick. Eight milliliters of the anhydrous methanol-hydrochloric acid solution are added to the resin. The mixture of resin plus methylating agent is allowed to stand for 10-15 minutes, 2 ml of water are added, and the slurry is extracted with 3 ml of hexane. The hexane layer is transferred by syringe to a glass-stoppered test tube. The slurry is washed three times with 2-ml portions of hexane. The combined hexane solution is washed once with distilled water. The hexane solution is dried over anhydrous sodium sulfate, transferred to a graduated centrifuge tube, and concentrated to 0.5 ml. An appropriate aliquot is taken for the analysis by gas chromatography. Gas Chromatographic Analysis of Methyl Esters. An F & M Model 810 chromatograph equipped with a hydrogen flame ionization detector was used. The liquid phase was 10 w/w (DEGS) poly (diethylene glycol succinate), coated on 80-100 mesh Gas Chrom P (Applied Science Laboratory) packed into a 6-foot X 1/8-inchstainless steel column. Helium at 20 ml/minute was the carrier gas. The conditions of operation were such that 1 pg of methyl stearate gave approximately full scale deflection on a 1-mv recorder with 10-inch chart span. RESULTS AND DISCUSSION
Choudhury and Arnold (3) removed phospholipids from a chloroform solution of vegetable oils by adsorption on activated silicic acid prior to the determination of the neutral oil content. Carlson ( 4 ) removed phospholipids in a similar fashion from a chloroform extract of serum lipids and then determined the serum triglyceride by analyzing the supernatant VOL. 39, NO. 3, MARCH 1967
353
Table I. Per Cent Composition of the Triglyceride Fatty Acids from Beef Muscle Tissue Using (A) The Procedure Described, and (B) A Standard TLC Separation of Total Lipids, Followed by Analysis of the TG Fraction Total fatty acid, Z Fatty Acida A B c120
c 1 4 ' c 1 4
Cl42= C16O C16l" c 1 7 '
Cl,'= ClSO c1s1=
c1s2-
0.01 3.98 1.05 0.93 31.21 3.94 1.65 0.70 20.20 34.46 1.87 100.00
0.01 4.09 0.99 0.93 31.81 4.00 1.73 0.70 20.67 34.21 0.86 100. 00
Superscript represents the number of double bonds in the molecule, the subscript the number of carbon atoms. 5
and linolenic acid in 2 ml of the mixed chloroform-hexanediethyl ether solvent was carried through the initial phospholipid separation step and both the solvent and the adsorbent were analyzed by TLC for FFA. All FFA were found in the supernatant and no FFA were adsorbed by the silicic acid. In the conversion of the fatty acid portions of the triglycerides to methyl esters, the triglycerides are saponified and the fatty acids are adsorbed on the anion exchange resin. The supernatant at this point contains the cholesterol and can be used for cholesterol analysis. This system of saponification followed by esterification is preferred because direct methanolysis would require, prior to the use of GC, a chromatographic separation of the cholesterol from the methyl esters. Hornstein et a/. (5) reported that adsorption of FFA on an anion exchange resin (50-100 mesh) and subsequent conversion to methyl esters could be made quantitative only if an internal standard such as n-heptadecanoic acid was used. Bills, Khatri, and Day (9) also reported that the FFA were not adsorbed quantitatively and that an internal standard was necessary. Odd-numbered acids are the preferred internal standards as these generally do not occur to any great extent in lipids. At the level of the sensitivity of a flame detector, however, odd-numbered acids are usually detected in small amounts. It was considered preferable to omit the internal standards if the adsorption of FFA from the mixed solvent, and their subsequent methylation could be made quantitative. This was accomplished by using a 20C-400 mesh anion exchange resin. To check quantitative recoveries, 40 mg of a mixture of stearic and oleic acids in 10 ml of solvent was adsorbed on 2 grams of the anion exchange resin and the filtrate was analyzed for free fatty acids; no fatty acids were recovered from the filtrate. The conversion of FFA adsorbed on the resin to methyl esters, using methanol-hydrochloric acid at room temperature, was essentially complete after 10 minutes, Recoveries were 9 5 z or better and no improvement resulted from the use of longer esterification periods. The fatty acid composition of the triglyceride fraction is subject to some error as the fatty acid portions of mono- and diglycerides and cholesterol esters are reported as triglyceride fatty acids. The extent of this error for intramuscular lipids was determined by separating the total lipid extract by TLC on silicic acid, scraping the triglyceride fraction from the plate, and transmethylating with sodium methoxide in methanol, then comparing the fatty acid analysis obtained by this TLC method with the triglyceride fatty acid analysis obtained by the method described in this paper. The results are given in Table I. Except for linoleic acid the results agreed witnin i3 of the amount present, in each case an acceptable figure for fatty acid determinations by GLC. Minor fractions separated by TLC when analyzed for fatty acid composition by GLC did not yield any additional linoleic acid.
for glycerol. Kuchmak and Dugan (6), however, reported that they did not obtain a clean separation of phospholipids from a chloroform extract of pork muscle lipids using activated silicic acid as the adsorbent in a batch procedure. No phospholipids were found in the unadsorbed lipid fraction but neutral fats were present in the phospholipid fraction. They used adsorption on silicic acid from chloroform to provide a preliminary separation and then purified the phospholipids by silicic acid chromatography. We also found that silicic acid adsorption of phospholipids from a chloroform extract containing muscle lipids did not give an adequate separation of phospholipids. The latter could, however, be quantitatively removed, free from other lipid material, if the polarity of the solvent was increased. The separation from a solution of chloroform-hexane-diethyl ether ( 2 :1 :1) gave best results as determined by preliminary investigations. That the separation of the phospholipids was quantitative and clean was shown as follows: (a) An aliquot of the solution was separated by TLC on silicic acid using hexane-diethyl ether-acetic acid (75 :25 :1) as the developer. Phospholipids remain at the origin using this solvent system. No phospholipid spot was observed when the developed plate was sprayed with 5 0 x sulfuric acid and heated at 180°C. (b) The phospholipids adsorbed on the activated silicic acid were eluted with chloroform-methanol (1 : l), the extract was concentrated and an aliquot spotted on silicic acid, and the thin layer plate was developed as above. In this case, only the phospholipid spot was observed. As a further check, phosphorus was determined by a modification of the method of Chen, Toribara, and Warner (8). No phosphorus was found in the triglyceride fraction and the phospholipid phosphorus corresponded to that expected from the determination of phosphorus in the original solution. To check the possibility that free fatty acids might be adsorbed on the silicic acid and not be separated by TLC, a mixture of 10 mg each of stearic, oleic,
RECEIVED for review July 25, 1966. Accepted December 27, 1966.
(8) P. S. Chen, T. Y. Toribara, and H. Warner, ANAL.CHEM.,28, 1756 (1956).
(9) D. D. Bills, L. I. Khatri, and E. A. Day, J. Dairy Sci., 46, 1342 (1963).
354
ANALYTICAL CHEMISTRY
