Table 11. Combustible Carbon in Meteorites sample Carbon content, Meteorite size, mg Measured Reported (Ref.) Pueblito de Allende 48.49 0.28 86.71 0.29 0.3 (10) 43.75 0.31 Murchison 9.56 2.3 2.0 (11) Mokoia 28.70 0.97 0.57-0.84 (12, 13) Murray 14.34 2.6 2.78 (14) 8.45 3.2 (12) Karoonda 18.82 0.10 0.10
Table I. Analysis of Calibration Standards Carbon content, Samplea NBS Measured 10 g 0.240 0.234 1 0 . 0 0 8 12h 0.407 0.428 i 0.008 1 . 0 7 z k 0.008 16 e 1.09 a Numbers correspond to NBS nomenclature.
deviation. The precision within a given series corresponds to rt 3 % and the mean error is also within 3 % of the NBS accepted value. The maximum relative error, corresponding t o sample 12h, i? within 5 % of the accepted value. Results of the determination of the combustible carbon content of 5 carbonaceous chondrites are summarized in Table IT. Sample sizes ranged between 8 and 87 mg, and the measured values are in good agreement with the values reported by other investigators. Since carbonaceous chondrites are nonhomogeneous rare samples, too valuable t o fragment large pieces to obtain a truly representative sample, the agreement between the values shown in Table I1 and those reported previously is good. Measurements o n selected samples of Allende indicate that the repeatability of the method for meteorite analysis is within + 4 % of the average value. One of the major disadvantages of the method proposed here is the relatively long time required for an individual analysis. When two separate furnaces are used, one to condition a new reactor and a second to perform the combustion, it is possible to analyze one sample every 24 hours. However,
the accuracy of these techniques for the analysis of small samples justifies the use of a low pressure combustion bomb followed by a chromatographic determination of the product gases.
RECEIVED for review August 28, 1972. Accepted November 6, 1972. This research was conducted under the McDonnell Douglas Independent Research and Development Program. (10) E. A. King, Jr., E. Schonfeld. D. A. Richardson, and J. S. Eldridge, Science. 163,928 (1969). ( 1 1) K. Kvenvolden, J. Lawless, K. Pering, E. Peterson, J. Flores, C. Ponnamperuma, I. Kaplan, and C. Moore, Nature, 228, 923 (1 970). (12) C. B. Moore and C. Lewis, Science, 149, 317 (1965). (13) G. Boato, Geocliim. Cosmocliim Acta, 6, 209 (1954). (14) H. B. Wilk, ibid.,9,279 (1956).
Py roIys is -Gas Chromatography of Perf Iuoro-n-pentane Raymond R. Rogers, Gordon S. Born, Wayne V. Kessler, and John E. Christian Department of Bionucleonics, School of Pharmacy and Pharmacal Sciences, Purdue Unioersity, West Lafayette, Ind. 47907 RECENTSTUDIES in our laboratories required a rapid and efficient method for the separation and identification of the 358980 "C pyrolysis products of perfluoro-n-pentane (n-C5FI?). It is the purpose of this paper to report the pyrolysis of n-C5F,? using a Curie point pyrolyzer and the subsequent separation and identification of the pyrolysis products by gas-solid and gas-liquid chromatography. A survey of the literature revealed little information regarding the pyrolysis of n-C5FI2. The pyrolysis of n-C5FI?was reported in 1951 by Rogers and Cady ( I ) . The n-C5F12was pyrolyzed in a chamber constructed of a 1-liter borosilicate glass flask with a platinum wire coil at the center. The products of n-C5Flppyrolysis were separated and identified by making an analytical fractional distillation and measuring the vapor densities and boiling ranges of the different cuts. Unsaturated product was determined by chlorination of the C3 cut. Temperatures of the platinum wire ranged from 840 to 1325 "C; however, pyrolysis products of the n-C5Flpwere not detected below 900 "C. When n-C5FI?was subjected to higher temperatures, the pyrolysis products identified by ( 1 ) G . C. Rogers and G. H. Cady. J . Amer. Cliem. Soc.. 73, 3523 ( 1951 ).
Rogers and Cady ( I ) were CF4, C2F6, C3F8, C3F6, carbon, and one o r more forms of C4F8. The authors also indicated the presence of a high polymer on the walls of the pyrolysis flask. In 1952, Steunenberg and Cady ( 2 ) reported a study of the pyrolysis of fluorocarbons, including n-CSFI2. The pyrolysis vessel used for this study was constructed of gold plated copper tubing and a platinum filament. The methods used for separating and identifying the n-CjFip pyrolysis products were similar to those described by Rogers and Cady ( I ) . Steunenberg and Cady ( 2 ) identified C2F6, C3F8, C3F6, C4F8, carbon, and a -CF- polymer as pyrolysis products of n-C6F12. Campbell and Gudzinowicz (3) reported the separation of fluorocarbon and sulfur-fluoride compounds by gas-liquid chromatography in 1961. The separaton of CF4, C Z F ~CsF6, , cyclic-C4F8,and i-C4F8was achieved on a column consisting of 33 No. 3 Kel-F polymer oil. Greene and Wachi ( 4 ) investigated the separation of some C1-C4 perfluorocarbons on a gas-solid column of 45/60 mesh ( 2 ) R. K. Steunenberg and G. H. Cady, ibid.,74,4165 (1952). (3) R. H. Campbell and B. J. Gudzinowicz, ANAL.CHEM.,33,
842 (1961). (4) S . A. Greene and F. M. Wachi; ibid.,35,928 (1963). ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
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Table I. Chromatographic Conditions Column Gas-solid Gas-liquid Parameter (Deactigel) (30% DFHA) Column temperature, "C 185 (isothermal) 0 (isothermal) Injector temperature, "C 205 75 Detector temperature, "C 205 200 Helium flow, ml/min 25 25 Hydrogen flow, ml/min 25 25 Air flow, ml/min -400 -400
Table 11. Micrograms of n-C6Flz Pyrolysis Products Detected. Pyrolysis temperature, "C Pyrolysis product 358 480 510 610 770 980 b b b c 0.4 66 C2F6 b b e 0.6 3.1 428 CzF4 b b c 2.6 104 C3Fs b b c 0.5 1.1 101 C3F6 b c 4.3 137 rt-CdFio ' a 1.5 p1 samples of n-C5F12subjected to pyrolysis at each temperature. No pyrolysis products detected. Trace amount detected.
Table IV. Retention Indices of Standard Perfluorocarbons and n-C5F12Pyrolysis Products on 30 % DFHA Retention Retention index of index of Standard standard pyrolysis per fluorocarbon per fluorocarbon products CZF6 160.0 160.9 C2Fi 254.9 254.9 CaFa 269.2 275.1b t?-C3F6 369.2 II-C~FIO 363.6 369.4 cyclic-C4F8 413.6 ... C4Fa-2 ( t r a ~ s ) 440.5 ... 450.31~ C4Fs-2 (cis) ... II-C~FIZ 467.8 467,8 a Appeared as a shoulder on 440.5 peak. Appeared as a shoulder on 254.9 peak.
satisfactory separation of air and CF4 as some of the advantages of this method. EXPERIMENTAL
silica gel and a gas-liquid column of 3 3 x lH,lH,7H-dodecafluoroheptyl acrylate (DFHA). During the gas-solid separation, the temperature was programmed from room temperature to 180 "C. The gas-solid separation of eleven C1-C4 perfluorocarbons was completed in sixty minutes. The cis and trans isomers of GF8-2 were not separated on the gassolid column. The gas-liquid separation described by Greene and Wachi ( 4 ) was carried out isothermally a t 0°C. Eleven CI - Ci perfluorocarbons were separated in about eighteen minutes. The cis and trans isomers of C4F8-2were separated on the gasliquid column ; however, CyCk-CsFf, and n-C3F6could not be resolved. Wright and Askew (5) recently reported the separation of C1-Cgperfluoroalkanes into molecular weight classes by temperature programmed gas-solid chromatography. The gassolid columns employed for this investigation consisted of 30/60 mesh silica gel. A dual-column system was used to eliminate the base-line drift observed in earlier studies (5). The authors listed no overlapping of peaks, no tailing, and a
Apparatus and Reagents. The gas chromatograph employed for this study was a Varian Aerograph Model 204-1B equipped with flame ionization detectors, a Packard Model 891 Curie Point Pyrolyzer, and a Westronics Model L D l l A strip chart recorder. Hydrogen was supplied to the detectors by a Varian Aerograph Model 9652 Elhygen hydrogen generator. Air was supplied to the detectors by two Oscar's 55 air pumps. Helium was supplied to the chromatograph from a cylinder without treatment. The carrier gas line was connected to the pyrolysis head with an l/g-in. Swagelok T. The other part of the T was equipped with a n injection septum to permit injection of volatile materials into the carrier gas stream. The columns used in this study were similar to those used by Greene and Wachi (4). The gas-solid column consisted of a 12-ft X lig-in. 0.d. stainless steel tube packed with SOjlOO mesh Deactigel (purchased from Applied Science Laboratories, Inc., State College, Pa.). The Deactigel was previously acid washed according to the method described by Thornsberry ( 6 ) . The Deactigel column was conditioned for 24 hours at 200 "C with a helium carrier gas flow of 25 ml/min. The gas-liquid column consisted of a 25-ft X ',kin. 0 . d . copper tube packed with 3 0 z D F H A (purchased from PCR, Inc., Gainesville, Fla.). The gas-liquid column was conditioned for 2.5 hours a t 50 "C with a helium carrier gas flow of 25 ml/min. The perfluorocarbons used in this investigation were obtained from various sources. The C ~ F SC3Fs, , n-CaF6, cyclicC4Fg, C4F8-2, and n-CjFle were purchased from PCR, Inc., Gainesville, Fla. The C?F4was obtained by the pyrolysis of cyclic-C4Fg(4, 7). The n-C4Flowas purchased from Marshallton Research Laboratories, Inc., West Chester, Pa. The n-C5F was purified by preparative gas-solid chromatography before it was used in the pyrolysis study. Infrared and mass spectra of the purified n-C5F12were identical to standard spectra. Procedure. Samples of n-C5FI2 were injected into the helium carrier gas stream at the point where it was connected to the pyrolysis head. Three seconds after the sample was injected, the pyrolysis wire was heated at its Curie point. The sample was swept through the pre-heated pyrolysis head, over the hot wire, and into the chromatograph. The residence time of the parent compound in the pyrolysis zone was 0.2 second at a helium carrier gas flow rate of 25 ml/min.
(5) J. R. Wright and W. C. Askew. J . Chromurogr. Sei., 9, 651 (1971).
(6) W. L. Thornsberry, ANAL.CHEM., 43, 452 (1971). (7) J . N. Butler, J . Anler. Chen?.SOC., 84, 1393 (1962).
Table 111. Retention Indices of Standard Perfluorocarbons and n-C6Fl2Pyrolysis Products on Deactigel Retention Retention of Standard of standard pyrolysis perfluorocarbon perfluorocarbon products 181.7 181.7 c2F6 210.2 210,2 CiFi 274.2 274,3 CaFs /z-C~F~ 324.7 324.3 tI-CqFio 359.3 360.6 364.9 ... cyclic-C4Fa C4Fs-2(cis & trans) 391.4 ... t~-CaFiz 440.0 443.5
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Figure 1. Typical 980 "C pyrogram of nCsF12on Deactigel
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Figure 2. Typical 980 " C pyrograrn of nCsFlzon 30 DFHA
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The amount of parent compound Dvrolvzed with this svstem ranged from no-detectable pyrolysis-at iower temperatuies to 33 Z at 980 "C. The columns used for this investigation are described in the Apparatus and Reagents section. The chromatographic conditions are listed in Table I. Samples of C1 to C 6 normal alkanes were chromatographed before and after the pyrolysis experiment. Elution times of the normal alkanes and the pyrolysis product peaks were determined. All peaks observed in each pyrogram were converted to retention indices following the method of Kovats (8). Samples of eight Cl-C4 perfluorocarbons were chromatographed individually and as a mixture. Chromatographic conditions were identical to those described in Table I . The retention indices of C2F6, C3F8, n-C3F6, cyc1ic-C4F8,C4F8-2, n-C4Flo, and n-C5FIz were determined on both columns. CZF4 was formed when cyclic-C4F8was pyrolyzed and its retention index was determined on both columns.
RESULTS AND DISCUSSION No reaction of the sample with any of the Curie point wires was observed other than pyrolysis of n-CSF12. No pyrolysis (8) E. sz. Kovats, "Advances in Chromatography," Vol. I, J. C. Giddings and R . A. Keller, Ed., Marcel Dekker. New York, N.Y., 1965, pp 229 -47.
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products were detected at pyrolysis temperatures of 480 and 358 "C. Pyrolysis products were detected when n-CsF12was pyrolyzed at 980, 770, 610, and 510 "C. The 980, 770, 610, and 510 "C pyrograms varied only in the amounts of pyrolysis products formed at the different temperatures. The amounts of n-C5F12pyrolysis products formed at each temperature are listed in Table 11. Reproducibility at each pyrolysis temperature was within 5 %. Pyrolysis of n-CjF12followed by gas-solid chromatographic separation of the pyrolysis products on the Deactigel column yielded five peaks plus the un-pyrolyzed n-CSF1?peak. The retention indices of the five pyrolysis products detected and the retention indices of nine standard perfluorocarbons are listed in Table 111. The n-C4FIoand the cyclic-C4Fs peaks were not resolved when the mixture of standard perfluorocarbons was chromatographed. Also, the cis and trans forms of C4Fs-2 were not separated on the gas-solid column. Figure 1 is a typical 980 "C pyrogram of n-CjF12on the Deactigel column. Pyrolysis of n-C5FIzfollowed by gas-liquid chromatographic separation of the pyrolysis products on the 3 0 z D F H A column yielded four peaks plus the un-pyrolyzed n-CjF~:!peak. C2F4 and C3F8 were not completely resolved under these conditions. The C3Fspeak was observed as a shoulder on the C2F, peak. The n-C3F6 and n-C,FlO perfluorocarbons were not resolved on the 3 0 z D F H A column. The retention indices of the four pyrolysis product peaks observed and the ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
569
retention indices of nine standard perfluorocarbons are listed in Table IV. Figure 2 is a typical 980 "C pyrogram of n-C5F12 on the 3 0 z D F H A column. C2F6, CzF4, C&, n-C86, and n-C4Flo were identified as n-CSF12 pyrolysis products by comparing their retention indices with those of standard perfluorocarbons on the two different columns. Separation of the n-C5F12pyrolysis products required less than 15 minutes on each column. Rogers and Cady ( I ) suggested that if CZF,is formed during n-C5Flz'pyrolysis, it disappears because of rapid polymerization to yield one or more forms of C4Fs and (CFJ. The detection of C2F4 and n-C4Flo in our study lends support to their suggestion. Rogers and Cady ( I ) reported small amounts of CF4present at higher pyrolysis temperatures. Steunenberg and Cady (2) did not list CF4as a n-CsF12pyrolysis product. CF4was not detected in our study; however, this may be a result of the
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~
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~~
very low response of the flame ionization detector to this perfluorocarbon (9). Thermistor and electron capture detectors give less varied responses to perfluoroalkanes than flame ionization detectors. However, concurrent investigations and performance characteristics warranted the use of the flame ionization detector for our studies. RECEIVED for review July 27, 1972. Accepted November 14, 1972. This investigation was conducted under the auspices, of the Institute for Environmental Health, Purdue University, and was supported in part by PHS Training Grant No. 5T 0 1 - E S 0 0 0 7 1 from the National Institute of Environmental Health Sciences. (9) W. C. Askew and K. D. Maduskar, J. Chromatogr. Sci., 9, 702 (1971).
~
Deuterium Substituted Amphetamine as an Internal Standard in a Gas Chromatographic/Mass Spectrometric (GC/MS) Assay for Amphetamine Arthur K. Cho, Bjorn Lindeke, Barbara J. Hodshon, and Donald J. Jenden Department of Pharmacology, UCLA School of Medicine, Los Angeles, Calif, 90024
GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS) is a technique that couples the high resolving power of gas chromatography with the sensitivity and specific ion detecting capability of the mass spectrometer. These features are especially important when small quantities of drugs and metabolites are to be measured in biological fluids, and the technique has found numerous applications in the qualitative identification of drugs ( I ) . An additional modification has been described by Gaffney et a!. (2) in which stable isotope substituted compounds are used as internal standards for the quantitative analysis of drugs. Similar methods have been described for prostaglandins (3), homovanillic acid (59, and acetylcholine (5). This paper describes a GC/MS assay for the quantitative analysis of amphetamine in biological fluids in which three deuterium substituted amphetamines have been synthesized and evaluated as internal standards. A preliminary account of this research has been presented (6). The criteria used to evaluate stable isotope labeled internal standards for GC/MS analysis are somewhat different from those for the more conventional gas chromatography technique. Quantitation is achieved by comparing the response of the mass spectrometer at two nominal masses, one char~~
~
( I ) N. C. Law, V. Aandahl, H. M. Fales, and G. W. A. Milne, C h i . Cliitn.Acta. 32, 221 (1971). (2) T. E. Gaffney, G. C. Hammar, B. Holmstedt, and R. E. McMahon, ANAL.CHEM., 43,307 (1971). (3) 9.Samuelsson. M. Hamberg, and C. C. Sweeley. Anal. Biocliem., 38, 301 (19:O). (4) 9.Sjoqvist, E. Anggdrd, B. Fyro, and C. T. Sedual, Proc. I t i t . Pliurmacol. Cotigr.. J t h , 1972,1291. (5) D. J. Jenden, Fed. Proc., Fed. Amer. So?. Exp. Biol. 31, 5 1 5 (1972). (6) A. K Cho, B. Lindeke, R J. Hodshon, and D. J. Jenden, Proc. I t i t . Pliarmacol. Cotigr.. 5th, 1972, 41.
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
acteristic of the internal standard and the other characteristic of the substance to be analyzed. Since the internal standard is an isotopic variant of the compound analyzed, the fragmentation patterns and retention times will be the same. However, some fragments will contain one o r more atoms of an isotope such as deuterium rather than the more abundant natural isotope, and will therefore appear at masses shifted by one or more mass units from the compound to be analyzed. These different mje values are the basis for the assay and should be chosen with care. An ion of high relative abundance should be selected to maximize sensitivity, and should be isolated on the mass scale from other ionic fragments SO that each of the two masses selected is as far as possible representative of one isotopic variant, and is not produced by the other. Since any unlabeled variant present in the internal standard will appear as a blank, it is likely to limit the sensitivity of the method by providing a statistically variable background, and the internal standard should therefore have the maximum possible isotopic purity. This is particularly important because with picomole levels of amphetamine the internal standard also serves as a carrier to minimize mechanical losses and may be present in 100- to 1000-fold excess. Resolution of the mass spectrometer will generally not be limiting even with a low resolution instrument such a s a quadrupole, because the need for low contamination of the internal standard with the unlabeled variant makes it desirable to prepare a standard with more than one labeled atom, and the masses of the fragment ions selected for measurement would thus differ by 2-3 mass units. The isotopically substituted compounds used in this work are shown below. The synthesis (7) and mass spectral analy(7) B. Lindeke and A. K. Cho. Acta Pliurm. Suecicn, 9,363 (1972).
