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Determination of the Concentration and Stable Isotopic Composition of Oxygen in Organic Matter Containing Carbon, Hydrogen, Oxygen, Nitrogen, and Sulfur Michael J. DeNiro*Jp2and Samuel Epstein Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California 91125 sample diffused into the atmosphere. The products that remained in the reaction vessel were COz and CO. After spark discharge was used to disproportionate the CO to COP plus C, all of the 0 in the cellulose had been converted to COz, which was analyzed mass spectrometrically. The other method used by Epstein et al. (5) involved pyrolysis of cellulose in the presence of HgClZ,a t 550 OC in Vycor tubes, a modification of a procedure introduced by Rittenberg and Ponticorvo (11) to measured l80contents of isotopically spiked samples. The products of the pyrolysis reaction were COz, CO, and HCl. After the HCl was removed by reaction with benzoquinoline to form benzoquinoline hydrochloride, the CO and COz were processed as for the Ni tube pyrolysis method prior to mass INTRODUCTION spectrometric analysis. Stable isotope ratios of modern, historic, and fossil organic In 1985, Schimmelmann and DeNiro (12) published a paper matter record biological and environmental information of in which the HgClz pyrolysis technique was modified to allow potential interest to a variety of researchers (e.g., ref 1-31, ratio determinations of glucosamine, a CHON for 180/160 Extraction of this information requires the use of analytical compound. The products of the pyrolysis reaction were COz, procedures that permit quantitative conversion of an element CO, Nz, and HCl. After HC1 was removed with benzoquinoline in an organic sample to a gas, which can then be analyzed mass (as above), the COz,CO, and Nz were sealed into a Vycor tube spectrometrically. Almost all of the published work utilizing containing finely dispersed Ni powder. The tube was heated oxygen isotope ratios in this manner has involved organic to 275 O C , causing the CO to disproportionate to COz and C. matter consisting of carbon, hydrogen, and oxygen, such as The Nz did not react. The COz in the tube, which now consucrose or cellulose (e.g., ref 4-6). Two reports of 180/160 tained all the 0 from the glucosamine, was separated cryoratios of a CHON compound, glucosamine, isolated from genically from the Nz prior to mass spectrometric determiarthropod exoskeletons, have appeared (7,8).However, the nation of the 6 l 8 0 value. bulk of naturally occurring organic matter, both modern and The HgC1, technique introduced by Epstein et al. (5) for fossil, consists of carbon, hydrogen, oxygen, nitrogen, and analysis of CHO compounds does not work for organic comsulfur. Such compounds have not been subjected to oxygen pounds that contain s. Milburn and DeNiro (13) demonisotopic analysis because the available techniques for deterstrated that pyrolysis of methionine, a CHONS compound, ratios in organic matter do not work when mining l80/l8O in the presence of HgClz produced substantial amounts of CSz, sulfur is present. Santrock and Hayes (9) have reported a COS, and SOz, along with COP,CO, and Nz. The presence ratios in CHONS comprocedure for determining 180/160 of oxygen in two sulfur-containing gases would render oxygen pounds, but, as discussed below, this procedure requires the isotope ratios based on analysis of oxygen in CO and COz,done use of special equipment and the application of a number of as discussed above, artifactual even if the CO and COz were correction factors. In this paper we report a method that separated from the COS and SOzprior to the isotopic analysis. permits quantitative conversion of oxygen in CHONS comSantrock and Hayes (9) showed that a modified version of pounds to COP,which can then be analyzed manometrically the Unterzaucher procedure can be used for determining P O and mass spectrometrically to determine both the concenvalues in organic matter. Organic matter is pyrolyzed, the ratio of the oxygen. tration and l80/l6O pyrolysis products are equilibrated with elemental C a t 1060 We review briefly below the analytical procedures that have OC to produce CO, and the CO is oxidized with Iz05 to produce been used to determine 180/180 ratios in organic matter. COz,which is analyzed in the mass spectrometer. Corrections Three methods for high precision (i0.5%) measurements must be made for contributions of oxygen to the final COz of P O values (see Experimental Section for definition of P O from an oxygen blank, oxygen from previous samples, and values) in organic matter consisting of C, H, and 0 have been oxygen from Iz05(since half the oxygen in COz produced from developed. The first (IO), which involved pyrolysis of carCO comes from the iodine pentoxide). Evaluation and bohydrates (CHO compounds), in the presence of diamonds, monitoring of the correction factors require analysis of has not been used since its introduction, to our knowledge. multiple standards in parallel with the samples. Moreover, Epstein et al. (5) introduced two other methods that have been achievement of reproducible results practically requires the widely used to measure P O values of cellulose, a CHO comuse of automated equipment. If hundreds of samples are to pound. One involved pyrolysis of the sample at 1100 OC in be processed at a rate of 20 per day or more, the method has a nickel reaction vessel, through which all of the H in the advantages of speed and convenience. Otherwise, a procedure requiring lower investments in calibration and equipment may Also Department of Earth and Space Sciences and Institute of be favored. Geophysics and Planetary Physics, University of California, Los We report here a method that permits quantitative coneles, CA 90024. version of 0 in CHONS compounds to COz, which can then An'Current address: Department of Earth Sciences, University of California, Santa Barbara, CA 93106. be analyzed mass spectrometrically. We found that pyrolysis
A method for the quantltatlve productlon of carbon dloxlde from oxygen In organlc matter conslstlng of carbon, hydrogen, oxygen, nitrogen, and sulfur has been developed. Manometric and mass spectrometric analysis of the C02 permit direct determlnation of the concentratlon and isotopic composition of oxygen In CHONS compounds with accuracy comparable to that previously achlevable for CHO and CHON compounds. Samples contalnlng as lfflle as 187 pmol of oxygen with S/O ratlos as hlgh as 0.16 have been analyzed by using thls method.
0003-2700/89/0361-1887$01 SO/O
@ 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 17, SEPTEMBER 1, 1989
of CHONS compounds in nickel reactions vessels, as was done for CHO compounds by Epstein e t al. (5), converts all the oxygen in the sample to C 0 2 and CO. The other gaseous product of this reaction is N2. Apparently the S in the sample reacts with the nickel pyrolysis vessel to form nickel sulfide, while the H in the sample diffuses out through the nickel reaction vessel into the atmosphere. COz was separated from CO and N2 by cryogenic condensation. Since CO cannot be quantitatively disproportionated to COz and C by spark discharge in the presence of N2 because NOPforms as well (141, we used a nickel catalyst described by Clayton and Epstein (15)to accomplish the disproportionation. Nz does not react in this system. Analysis of a CHONS compound and of a CHO compound doped with a CHNS compound indicates that the precision and accuracy of the P O values determined with the method presented here are as good as that determined for CHO and CHON compounds with previously available techniques.
EXPERIMENTAL SECTION Glucose, cysteine, and thioguanine were purchased from Sigma. Nickel oxide was purchased from Baker. Organic samples (cysteine, glucose, or glucose and thioguanine mixtures) were pyrolyzed according to the technique introduced by Epstein et al. (5), which is described in detail in ref 16. Briefly, the sample was weighed in a platinum boat, which was then inserted into a reaction vessel made of nickel 200, which was closed at one end and had an internal volume of -20 cm3, After the vessel was evacuated, it was heated at 100 "C for 6 h while being pumped. A piston was then lowered in vacuo, crushing a gold gasket and sealing off the interior of the nickel reaction vessel from the vacuum system. The vessel was heated at 1100 "C for 3 h and then allowed to cool to 350 "C over 2 h. With the vessel then maintained at 350 "C, the piston was raised, breaking the gold gasket seal and allowing the gaseous pyrolysis products to pass into the vacuum system. The gases were passed through a trap cooled with liquid nitrogen, in which C 0 2 condensed out. If the sample did not contain N (for glucose, a CHO compound), the noncondensible gas (CO) was admitted to a high-voltage discharge chamber (IO), in which CO was quantitatively disproportionated to C 0 2 and C. The discharge chamber was cooled with liquid nitrogen, which caused the COz t o condense out as it formed. If the sample contained N (for cysteine or for glucose doped with thioguanine), the noncondensible gases (CO and N2) were collected with a Toepler pump and then circulated through a tube made of nickel 200 maintained at 400 "C containing finely divided nickel powder (which was prepared by passing hydrogen through nickel oxide powder at 450 "C as described in ref 15) and through a liquid nitrogen cooled trap until disproportionation of CO to COP and C was complete (20 min or less for all samples reported here). The noncondensible gas (N2)that remained after the disproportionation was pumped away. The two COz aliquots (that formed directly during pyrolysis and that formed during the disproportionation of CO) were combined. The amount of C 0 2 was determined manometrically, after which the C 0 2 was analyzed mass spectrometrically. Isotopic compositions are given in the 6 notation relative to the V-SMOW standard (Vienna Standard Mean Ocean Water) as
RESULTS AND DISCUSSION Mass spectral scans indicated no or negligible amounts of S-containing gases (COS, CS2,SO2)present in the C 0 2samples generated from CHONS compounds or mixtures. In the worst case, the largest peak a t a mass for any of these three gases was
