2644
Anal. Chem. 1985, 57, 2644-2646
Determination of Oxygen Stable Isotope Ratios in Organic Matter Containing Carbon, Hydrogen, Oxygen, and Nitrogen A r n d t Schimmelmann* and Michael J. DeNiro Department of Earth a n d Space Sciences, University of California, Los Angeles, California 90024
A method for determining the re0/160 ratio of organic matter composed of carbon, hydrogen, oxygen, and nltrogen Is described. The precision of measurements of 6"O values Is *0.2%&
Stable oxygen isotope ratios of modern and fossil organic matter bear potentially valuable geochemical, environmental, and climatic information (2-3). Two types of analytical approaches have been used to determine oxygen isotope ratios in substrates composed of carbon, hydrogen, and oxygen. In the first, samples are subjected to exhaustive chlorination via reaction with mercury(I1) chloride at 550 OC (4-6). The other approach involves pyrolysis of a sample in nickel tubing (7-10). In both methods, oxygen in the sample is converted to carbon dioxide and carbon monoxide. After disproportionation of CO to C02, either via electrical discharge or in contact with nickel, the oxygen isotope ratio of the total carbon dioxide is determined mass spectrometrically. Organic substrates consisting of carbon, hydrogen, oxygen, and nitrogen cannot be analyzed by previously available methods in which the disproportionation of CO to C 0 2 was accomplished using a high-voltage discharge between platinum electrodes (11). During the discharge process it is necessary to exclude all traces of nitrogen in order to prevent the formation of nitrogen oxides, because I4Nl6O2,with molecular mads 46, interferes with the mass spectrometric measurement shifting P O values by as much of COz (mass 46 for 12C180160), ~1s10%0 at NOz concentrations of only 50 ppm (12). One attempt has been made to determine oxygen isotope ratios in organic matter with elemental composition more complex than carbon, hydrogen, and oxygen. Libby and Pandolfi (23) pyrolyzed wood in the presence of HgClz at 550 "C, collected the COPgenerated in this reaction (discarding CO and other oxygen-containing gases that were also generated), and measured 1sO/160 ratios on this COz fraction. However, calculation of oxygen stable isotope fractionation factors between C 0 2 and CO in thermodynamic isotopic equilibria predicts isotopic differences of about l%o in 6l80 values between the two gases at the pyrolysis temperatures (IO). Our experimental observations of values of CO from 0.8 to 3.5% (average 2.0 f 0.8%; n = 20) more positive than those of COZin cellulose samples that had been pyrolyzed for 5 h at 550 "C and cooled to room temperature confirm that isotopic heterogeneity exists between the two gases and is preserved a t lower temperatures. Depending on the sample type, we found 5-20% of the total oxygen of a pyrolyzed sample in the form of CO. Thus the oxygen pools in COPand CO must be combined prior to mass spectrometric analysis in order to avoid introducing isotopic artifacts. We describe here a method for determining the stable oxygen isotope ratios of compounds containing carbon, hydrogen, oxygen, and nitrogen using the HgClz pyrolysis method at 550 O C (aRer ref 4). The volatile products of the reaction are COz, (XI, N2, HCl, and small amounts of low-molecular weight, partly chlorinated oxygenated organics (6). However, oxygen yield experiments performed in this study and by other
workers using various methods and substrates (5, 14, 15) indicate that these organics do not contain significant amounts of the total oxygen from the samples. The CO and N2 are cryogenically separated from the COz, HCI, and volatile organics. After COz is separated from HC1 and volatile organics, it is combined with CO and Nz.The CO is then quantitatively disproportionated to COz on metallic nickel, according to the reaction
3Ni
+ 2CO
-
Ni,C
+ C02
(1)
(8,16-18). No nitrogen oxides form when this reaction takes place in the presence of N2. After Nzis removed, the total COB fraction is used for mass spectrometric determination of its l8O/l6O ratio. Our modification of the Rittenberg and Ponticorvo ( 4 ) method expands its applicability from C, H, 0 compounds to C, H, 0, N compounds. We note that the method cannot be used for compounds containing sulfur. Despite this limitation, the method is suitable for the determination of ls0/l6O ratios in many biological and biogeochemical substrates.
EXPERIMENTAL SECTION Cellulose, D-glucosamine hydrochloride (GlcN-HCl),mercury(I1) chloride, coconut charcoal, and 5,6-henzoquinoline were purchased from Sigma, Eastman Kodak, Mallinckrodt, Fisher, and Aldrich, respectively. Ammonium chloride (Baker) and aniline hydrochloride (Baker) were both twice sublimated in vacuo to eliminate traces of sorbed or crystal waters. Reduced nickel powder was prepared by a modified version of the technique described in ref 19. Aqueous 0.25 M nickel(I1) chloride hexahydrate solution (200 mL) was added to a solution of 112 g of KOH in 500 mI, of deionized water with vigorous stirring. The green precipitate was washed 5 times with 150 mI, of hot water containing 0.5 g of ammonium nitrate and each time recovered by centrifugation. Amounts of 0.8 g of freeze-dried product were reduced with dry hydrogen gas for 4 days at 215 "C to yield finely dispersed metallic nickel. When hot, the nickel tended to be pyrophorous on contact with air and required special precautions. The nickel reduction vessel was therefore first evacuated for 2 h at 275 "C to desorb excess hydrogen. After cooling to room temperature, the vessel was aerated with dry oxygen-free nitrogen or helium gas and the nickel was transferred to storage in sealed ampules under an inert gas or under vacuum. Even when the nickel is cold, prolonged contact with air must be avoided. Preliminary experiments required pure carbon monoxide, which was prepared by reacting concentrated sulfuric acid with 98% formic acid at approximately 40 "C (20)in a previously evacuated system and by passing the CO through a trap immersed in liquid nitrogen. Aliquots of 30-170 pmol of CO were separated with a Toepler pump and volumetrically measured with a precision of k3%.
The regular procedure of obtaining oxygen from organic compounds in the form of carbon dioxide was modified after those described in ref 4 and 5 and is described as follows. First, 5-10 mg of dried substrate was loaded into a Vycor breakseal ampulc (length 15 cm, outer diameter 9 mm) together with 0.37 f 0.02 g of HgCl2. Prior to sealing, ampules were evacuated for 2 h while being immersed in a boiling water bath. Batches of 15 sealed ampules were then heated to 550 "C for 5 h and left in the furnace to cool to room temperature over 12 h. The vacuum system used for further processing of samples is shown in Figure 1.
0003-2700/85/0357-2644$01.50/00 1985 American Chemical Society
qALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
2845
Table I. COz Yields in Quantitative Disproportionation of
co
amt of nickel, mg
residual incondensables, @mol
amt of COP, pmol
31
5.9
96.8
32 34 48 78
105.0
153
8.3 30.0 40.0 30.0
106.7 102.0
165
20.0
