A Combined Gas Chromatographic-Mass Spectrometric Methods for Identifying II - and Branched-Chain Alkanes in Sedimentary Rocks Vincent E. Modzeleski, William D. MacLeod, Jr., and Bartholomew Nagy University of California at San Diego, La Jolla, Calif. 92037
THE OCCURRENCE of trace quantities of alkanes in Recent sediments and in sedimentary rocks has been known for several years, and the pertinent information in this connection has even been discussed in standard textbooks (1, 2). Following the work of Dean and Whitehead (3) and Bendoraitis, et a[. (4) on isoprenoid hydrocarbons in petroleum and on their possible biological precursors, several studies were undertaken t o search for these compounds in nature. The presence of the isoprenoid hydrocarbons, 2,6,10714-tetramethylpentadecane (pristane) and 2,6,10,14-tetramethylhexadecane (phytane) has been reported in sedimentary rocks of various ages, including those of Precambrian age (>lo9 years), (5,6). Pristane also has been described in Recent marine sediments (9, zooplankton (8), beef liver (9), likely laboratory contaminations (IO, I I ) , etc. These isoprenoid hydrocarbons appear t o be ubiquitous in nature. It may be of interest t o note that Gazzarrini and Nagy (12) found alkanes even in human arterial tissues and plaques. Isoprenoid and certain other saturated hydrocarbons may indicate biological activity in early geological times. They may also throw light on yet unknown metabolic processes in living systems; hence, it is necessary that they be identified with certainty. This, unfortunately, has not always been possible in the past because it is usually difficult t o sufficiently fractionate the complex hydrocarbon mixtures or even t o interpret with certainty the mass spectra of individual compounds separated by gas chromatography. McCarthy, et al. (13) have found that the mass spectra of the two Czl hydrocarbons, 2,6,10,14-tetramethylheptadecaneand 2,6,10,15-tetramethylheptadecane are very similar which suggests caution in interpretation of the mass spectra of isoprenoids when samples introduced in the mass spectrometer contain (1) I. A. Breger, Ed., “Organic Geochemistry,” Pergamon Press, New York (1963). (2) B. Nagy and U. Colombo, Ed., “Fundamental Aspects of Petroleum Geochemistry,” Elsevier Publishing Co., Amsterdam, (1967). (3) R. A. Dean and E. V. Whitehead, Tetrahedron Lett., 21, 768 (1961). (4) J. G. Bendoraitis, B. L. Brown, and L. S.Hepner, ANAL.CHEM., 34,49 (1962). (5) J. Oro, D. W. Nooner, A. Zlatkis, S. A. Wikstrom, and E. S. Barghoorn, Science, 148,77 (1965). (6) W. E. Robinson, J. J. Cummins, and G. U. Dinneen, Geochim. Cosmochim. Acta, 29, 249 (1965). (7) M. Blumer and W. D. Snyder, Science, 150, 1588 (1965). (8) M. Blumer, M. M. Mullin, and D. W. Thomas, Zbid., 140, 974 (1963). (9) B. Nagy, V. E. Modzeleski, and W. D. MacLeod, Jr., unpublished data (1967). (10) J. Oro, personal communication (1966). (1 1) T. C . Hoering, personal communication (1966). (12) F. Gazzarrini and B. Nagy, Arch. Biochem. Biophys., 113, 245 (1966). (13) E. D. McCarthy, W. Van Hoeven, andM. Calvin, Tetrahedron Lett., 45, 4437 (1967).
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Figure 1. Gas chromatogram of Little Osage shale extract, normal alkane fraction Molecular distillate at 25” C-200” C/lmm Hg. 200-ft X 0.020inch i.d. Apiezon L. S.C.O.T.column (3 X lo4 theoretical plates), 3 ml/min. flow rate of He. The relative quantities of the n-alkanes at the low and high molecular weight ends may be reduced by the evaporation of solvents with N2and by molecular distillation
traces of impurities. Similar observations were made in this laboratory, and consequently an investigation was undertaken in order t o define and correct sources of possible analytical errors. EXPERIMENTAL
All solvents used were freshly distilled and all glassware was acid-cleaned with hot, conc. H z S O ~conc. H N 0 3 (85 : 15 v/v). No rubber or plastic implements were permitted t o come into contact with the solvents or the solutions. 60.1 grams of the Little Osage shale (middle Pennsylvanian age from Kansas; part of the Fort Scott limestone formation of the Marmaton group) was Soxhlet extracted with 300 ml of benzene and methanol 6 :4 (v/v) for a period of six hours. The methanol contained 10% by weight KOH t o saponify any acidic components that might have been present. The benzene layer contained the nonsaponifiable components and was washed with distilled water and evaporated under water-pumped N2 filtered by a Matheson gas purifier, Model No. 450, with Type A cartridge. The nonsaponifiable fraction (401.7 mg) was molecularly distilled up to 200” C at 1 mm H g pressure for a period of approximately 30 minutes using a short-path micro still. The distillate (137.0 mg) was dissolved in benzene and extracted with 25 ml of 10% HC1 and then washed with water. The nonextractable distillate (108.5 mg) was then chromatographed with benzene on a basic alumina (Woelm, grade 1) column, extensively prewashed with benzene, and having a n adsorbent t o sample ratio of 300:l (w/w). The eluate was rechromatographed with n-hexane on a silica gel (Davison grade 923) column, again prewashed extensively with n-hexane and also having a 300:l (w/w) adsorbent t o sample ratio (14). The n-hexane residue was refluxed in benzene for one week over 5A Linde molecular sieve pellets (previously activated at 200” C for two weeks) using a 50: 1 sieve t o sample ratio. After the (14) P. Hamway, M. Cefola, and B. Nagy, ANAL.CHEM.,34, 43 (1962). VOL 40, NO. 6, M A Y 1968
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Figure 2. Gas chromatogram of Little Osage shale extract, branched and cyclic alkane fraction Molecular distillate at 25' C-200" C/1 mm Hg. 300-ft X 0.01-inch i.d. Apiezon L column (7 X lo4theoretical plates), 2 CC/& Bow rate of He
supernatant benzene solution was decanted, the molecular sieve was rinsed with fresh benzene, and finally refluxed with a fresh aliquot of benzene for two hours. The benzene fractions were combined and evaporated to yield a branchedchain hydrocarbon fraction (47.2 mg or 85% of the hexane eluate). The straight chain fraction (5 mg or 9%) was obtained by n-hexane extraction of the molecular sieve by refluxing for a period of one week. Both the n-alkane and the branched/cyclic fractions were analyzed by combined capillary gas chromatography and mass spectrometry on a Perkin-Elmer Model No. 226 gas chromatograph (hydrogen-flame detector) connected to a Hitachi RMU-6E mass spectrometer by a heated length of stainless-steel capillary (4-foot X 0.010-inch) leading to a Watson-Biemann type (15) molecular separator. The samples were injected into a Hamilton Model No. 86800 low-deadvolume inlet attached to the gas chromatograph. Carrier gas (He) flow was maintained constant during temperature programming with a Brooks Instrument Model No. 8743 regulator. Approximately 5 % of the stream emerging from the columns was diverted to the hydrogen-flame detector and the remaining 95 % proceeded to the mass spectrometer. Millivolt servorecorders registered the chromatograms simultaneously from the hydrogen-flame detector and the ion monitor. Mass spectral scans were timed according to the ion monitor signal and required approximately 3 sec. Background scans were taken before and after the peak scans and were subtracted from the peak scans. Complete procedure blanks were run on both the n-alkane and the branched/cyclic fractions. DISCUSSION
The n-hexane residue from molecular sieve fractionation contained the n-alkanes (CE-28). They were well resolved on a gas chromatographic capillary (Figure 1) and readily identified by their mass spectra. The few small peaks visible between the n-alkanes may be due to minor amounts of branched/cyclic hydrocarbons adsorbed by the binder of the molecular sieve. The branched/cyclic hydrocarbon residue from the benzene fraction of molecular sieve treatment was considerably more complex (Figure 2) with the well defined Cis, Cls, c18, Clg, and CZoisoprenoids as the dominant components. The CIS, C19, and Cz0isoprenoid mass spectra from the Little Osage shale compare very well with those of authentic farnesane, pristane, and phytane, respectively (Figures 3-5). Gas chromatographic retention times of the C16, (15) J. T. Watson and R. Biemanii, ANAL.CHEM., 36,1135 (1964).
988
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Figure 3. Mass spectra of authentic farnesane and of the Little Osage shale C15 isoprenoid gas chromatographic fraction Ionizing voltage 80V. Multiplier voltage lzOOV
A U T H E N T I C PRISTANE
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Figure 4. Mass spectra of authentic pristane and of the Little Osage shale Clgisoprenoid gas chromatographic fraction. Ionizing voltage 80V. Multiplier voltage 1200V
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C19, and CZOisoprenoids, identical with those of authentic farnesane, pristane and phytane, respectively, add to the certainty of the identification. The completely straight line gas chromatograms of the procedure blanks showed that the samples were not contaminated during analysis. Because some seemingly ambiguous identifications of isoprenoid hydrocarbons have appeared in the literature, it must be emphasized that reliable identification of these compounds can be accomplished only if the gas chromatographicmass spectrometric analysis is preceded by an extensive wet chemical fractionation. It was found in this laboratory, that (a) saponification to remove acids and esters, followed by (b) molecular distillation to remove nonvolatiles, (c) dilute HC1 extraction to remove bases, (d) elution chromatography on alumina to remove nonhydrocarbon compounds, and (e) on silica gel to remove aromatic compounds, and finally (f) fractionation of the resulting hydrocarbons into straight and branched/cyclic fractions with molecular sieves accomplished this end. The molecular distillation step appears to be beneficial because it has been found (14) that high molecular weight components in chromatographic samples reduce the separation efficiency and may retard the elution of the com-
ponents from alumina or silica gel beyond their normal point of emergence. These same considerations may also apply to capillary gas chromatography, which, even without undue sample compositional problems, is sufficiently difficult to optimize for tandem mass spectrometry. The mass spectra (Figures 3-5) demonstrate that by careful performance of all these steps, quite rigorous mass spectral identifications can be obtained for isoprenoid hydrocarbons even where isomers are known to give similar spectra. ACKNOWLEDGMENT
The authors wish to thank Donald Baker of Rice University, Houston, Texas, for supplying the rock sample; Eugene D . McCarthy of the University of California at Berkeley for providing the farnesane and phytane standards which he synthesized and Harold C. Urey of the University of California at San Diego for his advice. RECEIVED for review January 8, 1968. Accepted February 26, 1968. This study was supported by the National Aeronautics and Space Administration, under research grants NsG-541 and NGR-05-009-043.
Analysis of Tall Oil by Gel Permeation Chromatography Teh-Liang Chang Research Seruice Department, Central Research Division, American Cyanamid Co., Stamford, Conn. 06904
THECONVENTIONAL ASTM method ( I , 2) for determining resin acids in tall oil is by titration following preferential esterification of fatty acids. The fatty acid is determined by substracting the resin acid content from total acid content. This method requires a skillful technique and is often subject to a large error. \ A more sophisticated method studied extensively recently employs gas chromatography (3, 4). The acids are esterified and then separated into the individual acids. The high separability by gas chromatography unnecessarily complicates an analysis where only total resin acids and fatty acid data are required. An accurate and simple method for tall oil analysis certainly is desirable. It is known that separation by the gel permeation chromais based tography (GPC), technique developed by Moore (3, on molecular size (6). GPC, then, appears t o be a promising tool for tall oil analysis. Because the major constituents are fatty acid, resin acid, their dimers and trimers, it should be possible to separate them by their sizes. This possibility was also implied in a review article by Bartosiewicz (7). A careful investigation was therefore undertaken. A similar work was also performed by Zinkel(8). An agreeable result
(1) Am. SOC.Testing Mater., D 803 ASTM Std., Part 18 (1965). (2) Am. SOC.Testing Mater., D 1240 ASTM Std., Part 20 (1967). 39, 1118 (3) F. H. Max Nestler and D. F. Zinkel, ANAL.CHEM., (1967). (4) R. G. Ackman, J . Gas Chromatog., 1(6), 11 (1963). ( 5 ) J. C. Moore, J . Polymer Sci., 2,835 (1964). (6) R. L. Pecsok and D. Saunders, Sep. Sci., 1, 613 (1966). (7) R. Bartosiewicz, J. Paint Technol., 39, 28 (1967). (8) D. F. Zinkel, U.S.D.A., Madison, Wisconsin, private communication, Dec. 1967.
was obtained even though different column substrates and eluents were used. It was found that the GPC method has several advantages over other methods. The analysis is carried out at room temperature and a complete analysis of acid components is made in a single experiment. Tall oil is analyzed for fatty acids, resin acids, fatty acid dimer, resin acid dimers and resin acid trimers. No pretreatment of the sample, such as esterification, is required. The original constituents of the sample are not destroyed and can be recovered for subsequent identification by established techniques. EXPERIMENTAL
Apparatus. A multicolumn unit was used. It consisted of four 4-ft X 0.25-inch stainless steel columns, two of which were packed with Bio-bead S-X2 gel and the other two, with Bio-bead S-X8 gel (Bio-rad Laboratories). Another type of column substrate examined was Sephadex LH-20 gel (Pharmacia Fine Chemicals). A differential refractometer (Waters Associates) was employed as the detector. The temperature was maintained at 30.0' =t0.1 C by a Lauda Circulator Model K-2 (Lauda Instruments Division of Brinksman Instruments, Inc.). Tetrahydrofuran (THF) was the eluent and the solvent. The eluent flow rate was maintained at 40 ml/hr with a Lapp LS-20 Microflo Pulsefeeder Pump (Lapp Insulator Inc.). A pressure release valve was attached to the system to regulate the operational pressure. The flow rate was first controlled by the operational and then finely adjusted by a needle valve. The operational pressure was approximately 8 psi/ft of column. Reagents. Tall oils, fatty acids, resin acids, fatty acid dimers, wood rosin and gum rosin were supplied by the Arizona Chemical Co. All other chemicals were obtained from Eastman Kodak Co. O
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