N e w Catalyst and Technique for Analyzing Fatty Acids, Their Esters, and Long-chain Compounds by Carbon-Skeleton Chromatography SIR: Carbon-skeleton chromatography (2, 4-6) helps determine the structure of compounds a t the microgram level by catalytically stripping from a molecule functional groups containing oxygen, nitrogen, sulfur, and halogen and giving the parent hydrocarbon and/or the next lower homolog, which products are identified by their retention times. The palladium catalyst used in previous studies (5) gives very low yields of hydrocarbons from carboxylic acids and their esters and usually gave no product response from compounds with chain lengths longer than 20 carbon atoms. A modified catalyst and the use of shorter catalyst beds have overcome these defects and broadened the scope of carbon-skeleton chromatography to include the analysis of compounds previously not analyzable by the technique. The catalyst has been especially useful for identifying fatty acids and their esters.
with the Chromosorb P support in a Rinco rotating evaporator until the preparation is dry. The catalyst is activated the day before intended use by passing hydrogen through the bed for 30 minutes a t 125' C . , 30 minutes at 200' C., and over-night a t the test temperature. The analytical set-up is essentially the same as that already reported ( 2 , 5 ) . An F & M Scientific Co. Model 609 flame-ionization gas chromatograph was used with the carbon-skeleton determinator produced by the Xational Instruments Laboratory, Rockville, Md. (4). The hydrogen carrier gas and air were purified by passing them through a 5A molecular sieve. Hydrogen flow
CI
rate was 20 ml./min., catalyst temperature, 300" C. A few micrograms of compound are injected in the carbon-skeleton determinator filled with the new catalyst and the products are identified by retention time in the usual a ay. Catalyst beds shorter than the full 9-inch length are prepared by filling the catalyst tube with acid-washed Chromosorb P to any desired length-e.g., half way-and then adding enough catalyst to fill the tube (just short of the hydrogen inlet port). Temperature programming and shorter catalyst beds are used in the analysis of higher fatty acids, their esters, and higher molecular weight compounds (those above C14).
c17
C
M ETHYL LINOLENATE
METHYL STEARATE
STEARIC ACID
EXPERIMENTAL
The catalyst is the "neutral-type" palladium (by weight as the metal) on 99% acid-washed 30- to 60-mesh Chromosorb P (Johns-Manville, New York, N. Y.). (Non-acidwashed Chromosorb P is not satisfactory.) It is prepared by dissolving palladium chloride in 5% aqueous acetic acid, adding sufficient sodium carbonate to neutralize the chloride anion (5), and evaporating the solution in contact
(6) 1%
C
A I
ESTER MIXTURE
$1
1
cI1 ,13,c15,1c7, c
M E T H Y L STEARA'IE
Table 1. Hydrocarbon Products from Compounds with N e w Catalyst
Principal prodType Compound uct" Acid 12-Hydroxystearic acid C17, Ct* Alcohol 1-Dodecanol C11, ClZ 1-Octadecanol Cll, ClS Aldehyde Dodecanal C11, ClZ Amide h',A',-dimethyltetraC14,C13 decanamide Amine Hexadecyl amine Cl6, Cl6 Ester Methyl linoleate Cll, Cl8 Ether Allyl hexadecyl ether C16,CL6 Halide Octadecyl bromide CI8 Sitrile Tridecanenitrile C13, ClZ Sulfide 1-Dodecanethiol ClZ a Product listed first is obtained in greater abundance.
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ANALYTICAL CHEMISTRY
Figure 1 . Typical chromatograms with improved catalyst in carbon-skeleton determinator Temperature programmed 9'/min. starting a t 6 5 ' C. A . Methyl stearate 99.8%, Appl. Science Lab. (ASL), State College, Pa. 6. Methyl linolenate (99.8%, ASL) C. Stearic acid (tech. grade) D . Standard methyl ester mixture (ASL) (2.52% Cln, 4.1 8% C16, 7.31 % CM, 13.64% Czo, 25.35% Czz, 47.00% C d E. Commercial-alpha-olefin mixture, Clo-Cz1, used as retention-time standard for n-Clo-Czl hydrocarbons. Column a t 1 9 5 ' C. (isothermal) F. Methyl stearate (99.8%) Column a t room temp. 4 min., then raised to 1 9 5 " (2.3 min.) and held G. Methyl stearate (99.8%) Analytical column is 6-foot, 3/16"-o.d.-copper tube containing 5% SE-30 on acid-washed Chromosorb W. Each division under chromatograms equals 2 minutes. About 0.02 PI. of the pure compounds was injected, 0.1 PI.of the methyl ester and olefin mixtures.
RESULTS A N D DISCUSSION
Typical chromatograms are shown in Figure 1. Products derived from a variety of typical compounds are given in Table I. Identifying a gas chromat’ographic peak of a fatty acid or ester is complicated by the fact that t,he compound may possess branched chains, double bonds of cis or trans configuration, and even cyclic structures. As a consequence, retention times alone provide insufficient proof of a peak’s identity and other evidence must generally be sought (9, 10, I S ) . Carbon-skeleton chromatography is useful for such identifications, but the catalysts previously used ( 2 , 5 ) are incapable or barely capable of analyzing the higher fatty acids and esters-e.g., 50 ,ug. of stearic acid was required to give a very small Cii peak. Figure 1 shows that yields of hydrocarbon from about 20 pg. of these compounds are a t least 10 to 100 times greater with the new catalyst, stearic acid (Figure lC), methyl stearate (Figure I d ) , and linolenate (Figure I n ) giving heptadecane (C17)and some octadecane (CB). The parent hydrocarbon of acids and their esters, not obtained with previous catalysts, comes through in appreciable quantity and this datum can be helpful in structure determination. Once the carbon chain is identified, the structure of t,he fatty acid or ester can be more reliably deduced from relative retention time data using one of several procedures---e.g., equivalent chain length (11, 12), carbon number (15) the relative retention volume grid ( 8 ) , or, in the case of straight-chain esters . with methylene-interrupted double bonds, by the application of separation factors ( I ) . With other types of compounds, appropriate retention time increments based on Kovats’ Index ( 7 ) may be added to the retention time of the carbon skeleton to help identify compounds. As shown in Figure 1D mixtures of fatty acid esters may be analyzed. The pink Chromosorb P has been reported to be more adsorptive than the white supports [Gas Chrom P (Applied Science Laboratories, State College, Pa.) and Chromosorb W ] previously used (14 ) . This adsorption causes slow desorption of the hydrocarbon products of acids, acid derivrttives, and other compounds from the catalyst bed. Thus, with the analytical column run isothermally, methyl stearate gives a Ci7 and a C18 peak which tail badly (because of slow elution from the catalyst) and exhibit a delayed retention time (Figure 1F). I n a temperature programmed run of the same compound (Figure 1A ) , the heptadecane and octadecane accumulate a t the beginning of the analytical column ~
(column may be held at low temperature before programming) and do not move until the temperature rises sufficiently; then the peaks come through on schedule with practically no tailing. (Retention times of peaks agree with those of the saturated hydrocarbons resulting from alpha-olefin mixture, Figure 1E.) Temperature programming is therefore necessary in these analyses. When temperature programming is not available, the procedure giving the chromatogram illustrated in Figure 1G may be helpful in eliminating tailing and delayed retention times. h compound is injected and a few minutes allowed for the products to elute onto a column a t room temperature; the temperature of the column is then raised rapidly to a higher value and continued isothermally. Sharp, well-separated peaks come through. The temperature conditions must be repeatable in order that retention times be reproducible and therefore meaningful. The trapping arrangement previously advocated for eliminating tailing (3, 5 ) should be used for lower acids or acid derivatives (usually those giving hydrocarbons smaller than C13 from acid portion). Need for the trap depends on the analytical column and its temperature schedule at the outset of the analysis. The hydrocarbon products obtained with the new catalyst are the same as those obtained from previous ones (2, 4-S), except that more parent hydrocarbon is produced and less cracking of the carbon chain occurs. I n the analysis of compounds above the Ci6 level it is desirable to use catalyst beds shorter than the full 9-inch length. I n general, the longer the carbon chain being analyzed, the shorter the catalyst bed should be to cut down the residence time of the less volatile molecules on the catalyst bed. A 4-inch-long catalyst bed performs well with compounds having carbon chains between C12 and Cao. Aishorter catalyst tube or a suitable spacer in the present catalyst tube may be used to obtain shorter catalyst beds., Immediately after preparation the new catalyst is very active and may crack long-chain hydrocarbon products, usually those above C18. For this reason the catalyst was not used until the day following activation. Several injections of long-chain compounds, especially fatty acids, tend to overcome cracking, when encountered, probably by reacting with or covering the cracking sites. Sufficient time for high-molecular-weight products to elute from the catalyst bed should be allowed. Results R ith known compounds ill indicate whether unknowns may be analyzed with confidence. If longchain products do not come through, or come through in small yield, a shorter
catalyst bed is indicated. Lowering the catalyst temperature tends to cut down the cracking of long-chain hydrocarbon products. The analysis of halides was avoided because they generate acid ( 5 ) ,which can cause cracking. Shorter beds of the “neutral-type” 1% palladium on Gas Chrom P may be used for the analysis of long-chain compounds, but the new catalyst is very much better for analyzing fatty acids and esters. It should be noted that a tube full of 1% palladium on Chromosorb P (w./w.) contains twice as much palladium as one with 1% of the metal on Gas Chrom P because the specific gravity of Chromosorb P is about twice that of Gas Chrom P. With the nem catalyst, products have been obtained as sharp peaks from steroids (cholesterol acetate), long chain hydrocarbons (e.g., C28),a hydrogenated triterpene (squalane) , and phenols. For best results in analyzing amines the catalyst should be the basic type described in reference 6. If sufficient substance is available, the larger size carbon-skeleton determinator [for use with thermal-conductivity gas chromatographs (6)] may be used to collect enough material for spectral analysis, a procedure that would make identifications more certain. LITERATURE CITED
(1) Ackman, R. G., J . A m . Oil Chemists’ SOC.40, 564 (1963). (2) Beroza, M., ASAL. CHEM.34, 1801
11962). (3j Beroza, M., J . Gas Chromatog. 2 , 330 (1964). (4) Beroza, M., Acree, F., Jr., J . Assoc. Ofic. Agr. Chemists 47, 1 (1964). (5) Beroza. 51.. Sarmiento. R.. ANAL. CHEW35. 1353 (1963). ’ (6) Ibid., 36, 1744 (1964). (7) Ettre, L. S.,Ibzd., 36, 31A (1964). (8) James, A . T., J . Chromatoa. 2. 552 (1959). ( 9 ) James, A. T., Webb, J., Biochem. J . 66. 515 119.57). , (lOi‘Kishimoto, B. T.,Radin, Y . S., J . Lipid Res. 4, 437 (1963). (11) Miwa, T. K., J . A m . Oil Chemists’ SOC. 40, 30 (i963). (12) Miwa, T. K., Mikolajczak, K. L., Earle, F. R., Wolff, I. A., ANAL.CHEM. 32, 1739 (1960). (13) Piikellyj J. G., Zbid., 36, 2245 (1964). (14) Ottenstein, D. M., J . Gas Chromatog. 1, 11 (1963). (15) Woodford, F. P., van Gent, C. M., J . Lipid Res. 1, 168 (1960). MORTON BEROZA SARMIENTO RAFAEL Entomology Research Division Agricultural Research Service U. S. Department of Agriculture Beltsville, Md. Presented in part at Chicago Gas Chromatography Discussion Group LIeeting, Chicago, Ill., January 21, 1963, and in full at the 149th Xational ACS Meeting, Detroit, hfich., A4pril6, 1963. Afention of proprietary products does not constitute endorsement by the U. S.Department of Agriculture. I
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