Mass Spectrometric Measurement of Oxygen-18 in Small Carbon Dioxide Samples
Sir: The atom fraction of oxygen-18 in a compound is generally determined by converting the compound to a gas and then analyzing the gas by mass spectrometry. Carbon dioxide is the gas usually preferred (2) and several methods for the conversion of the oxygen of organic and inorganic compounds to carbon dioxide have been described (4, 9, IO)-also see ( 2 ) and references cited there. A problem sometimes encountered in stable isotope tracer investigations, particularly in physiological and biochemical studies, is the need t o determine the atom fraction of oxygen-18 in very small samples of carbon dioxide; it may be tedious in certain types of investigations, or perhaps even impossible, to obtain large quantities of material for mass spectrometric measurements. Usually, one-micromole samples of gas present no special analytical difficulties ( 3 ) . However, the mass spectrometric measurement of isotope abundance in gas samples smaller than one micromole may require special instrumental adaptations which are not always convenient or may have shortcomings or disadvantages (3, 3, 7 ) . During the course of a recent investigation (6), it became desirable to determine the oxygen-18 content of submicromole quantities of carbon dioxide by mass spectrometry. Isotope abundance of very small samples could be measured accurately and precisely by the simple procedure of adding an inert gas-sufficient in quantity t o raise the total gas pressure to that equivalent to about one micromole-to the carbon dioxide. EXPERIMENTAL
Apparatus. Carbon dioxide was analyzed for oxygen-18 content in a Consolidated Electrodynamics Corp., Pasadena, Calif., model 21-130 recording mass spectrometer. Normal operating conditions recommended b y t h e manufacturer were followed; the ionizing current was 20 pa, and the ionizing voltage was 68 volts. I n this instrument, the gas to be analyzed is first introduced into a 3-liter expansion reservoir equipped with an electricallyoperated micromanometer with a pressure-sensitive diaphragm. According to specifications of the manufacturer ( 5 ) , gas pressure from 1 to 150 microns of mercury can be measured with a reproducibility better than 1%. Reagents. Carbon dioxide labeled with oxygen-18 was prepared b y equilibrating samples of water enriched with oxygen-18 with t a n k carbon dioxide of natural isotopic composition prepurified by pumping off the vapors not condensable at liquid nitro-
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ANALYTICAL CHEMISTRY
MICROMANOMETER 200
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Figure 1. Effect of inlet reservoir pressure on measured atom fraction of oxygen-1 8 in carbon dioxide
gen temperatures. Enriched water was purchased from Y E D A Research and Development Co., Ltd., Rehovoth, Israel. T h e exchange reaction was catalyzed by the enzyme carbonic anhydrase (Sigma Chemical Co., St. Louis, Mo.) and was carried out in a glass manifold vacuum system; the detailed Drocedure has been described (6). Procedure. After the inlet reservoir gas pressure was recorded, the carbon dioxide was introduced into t h e analyzer. T h e atom fraction of oxygen-18 in a sample of carbon dioxide was calculated according t o formulas derived by Klein ( 8 ) , which describe the atom fraction of oxygen-18 as a function of ratios of peak heights 44, 45, 46, 47, and 48. Peak heights were recorded b y scanning and were normalized for (very small) pressure changes which occurred during the scanning process. When necessary, corrections for residual background were made, Early experience suggested that introduction of carbon dioxide into the mass spectrometer a t high pressures resulted in background peaks which persisted for several hours. T o make this source of error negligibly small, the data shown in Figures 1 and 2 apply to samples analyzed successively from lower to higher pressures. RESULTS A N D DISCUSSION
Peak heights for carbon dioxide could be measured with a precision of & 0.2% when the gas pressure in the inlet expansion reservoir was approximately 5 microns of mercury or higher; 5 microns of pressure corresponded to approximately 0.9 pmole of carbon dioxide. Also, the atom fraction of oxygen-18 in carbon dioxide samples of the order ' oxygen-18 and lower could of 5 atom %
be measured with an accuracy of 0.01 atom % a t inlet reservoir pressures of 5 microns or higher; this estimate of accuracy was based on results obtained with samples of carbon dioxide in air, and with a sample of carbon dioxide of known oxygen-18 enrichment (purchased from and analyzed by the YEDA Research and Development Co., Ltd., Rehovoth, Israel). However, when the gas pressure in the inlet reservoir was less than 5 microns, then the oxygen-18 isotope abundance which was measured for any given sample of carbon dioxide deviated from the values obtained at pressures higher than 5 microns, and these deviations increased as the pressure in the reservoir decreased, as shown in Figure 1. Also, a considerable degree of uncertainty (see error bars in Figure 1) accompanied the erroneous results for samples of carbon dioxide smaller than 0.9 pmole; as the inlet reservoir pressure was decreased, the peak heights of the less abundant masses became progressively smaller and could not be measured precisely. The results for only one sample of carbon dioxide (4.79 atom yo oxygen-18) are shown in Figure 1; however, similar results were obtained for every sample of carbon dioxide which was analyzed, ranging in enrichment from normal abundance of oxygen-18 to 73.0 atom % oxygen-18. The erroneous results obtained for the atom fraction of oxygen-18 when carbon dioxide pressures in the inlet reservoir were less than 5 microns could be corrected by adding an inert gas like nitrogen or helium to the carbon dioxide. Figure 2 shows the measured atom fractions of oxygen-18 in carbon dioxide
containing 5.34 atom % oxygen-18, at partial pressures of carbon dioxide above 5 microns as well as below 5 microns. For carbon dioxide samples of partial pressures less than 8 microns, sufficient helium gas was added to the carbon dioxide so that the total pressure was 5 microns or higher. Mixtures of carbon dioxide and helium were prepared with a glass vacuum manifold system with attached mercury manometer. I n contrast t o the results shown in Figure 1, it is apparent from Figure 2 that the same values for the atom fraction of oxygen-18 were measured a t partial pressures of carbon dioxide above as well as below 5 microns-even for partial pressures as low as 0.1 micron. The corrective influence of the addition of an inei t gas was tested for several samples of carbon dioxide ranging in enrichment from normal abundance of oxygen-18 to 73.0 atom yo oxygen-18 and the results were similar to those in Figure 2. The results indicate that the oxygen18 isotope abundance of very small samples of carbon dioxide can be measured accurately and precisely if a n inert gas like helium is added to the carbon dioxide; in the absence of the inert gas, incorrect oxygen-18 values were obtained for very small samples of carbon dioxide. For the Consolidated Electrodynamics Corp. model 21-130 mass spectrometer, the critical inlet reservoir pressure is about 5 microns of mercury, which corresponds l o about 0.9 pmole of carbon dioxide; accurate and precise measurements of the oxygen-18 content of quantities of carbon dioxide less than this amount can be obtained if the inlet reservoir is brought to the equivalent of 0.9 pmole or higher by the addition of an inert gas. I n our experience, the smallest possible amount of carbon dioxide mhich is needed to carry out an analysis of isotope abundance depends on the atom fraction of oxygen-18 in the sample. Samples of carbon dioxide as small as 0.01 pmole could be analyzed if the oxygen-18 content was higher than 10 atom % oxygen-18. For cases in which samples as small as these are obtained by conversion of oxygen of organic or inorganic compounds to carbon di-
MICROMANOMETER
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Figure 2. Effect of addition of helium to submicromole quantities of carbon dioxide on the measured atom fraction of oxygen-1 8 in carbon dioxide.
0 Carbon
dioxide x Carbon dioxide and helium The abscissa indicates the partial pressure of carbon dioxide; sufficient helium was a d d e d to the carbon dioxide so that the total pressure was 5 microns or higher
oxide, it is apparent that procedures must be adapted to ensure the removal of impurities which might contribute to the peak heights of carbon dioxide. It is possible that the inert gas exerts its corrective influence on the background, but further work would be needed to verify this possibility. This technique of analyzing a small sample for its isotope abundance by adding a n inert gas has not been recorded in the literature, with the exception of a very brief report (1) concerned with the analysis of small quantities of hydrogen in the presence of neon or argon. T h a t this technique of extending the limit of reliable performance of the mass spectrometer need not be restricted to carbon dioxide, but may be used for other gases when samples of limited size only are available, appears probable. LITERATURE CITED
(1) Anbar, M., Meyerstein, D., Israel At. Energy Research Laboratories Semiannual Rept., No. IA 900, JanuaryJune (1963); also, J . Phys. Chem. 68, 1713 (1964). (2) Beynon, J. J., “Mass Spectrometry and its Application to Organic Chemistry,” Elsevier Publishing Co., Amsterdam, 1960. (3) Biemann, K., “Mass Spectrometry, Organic Chemical Applications,” McGraw-Hill Book Co., New York, 1962.
(4) Boyer, P. D., Graves, D. J., Suelter, C. H., Dempsey, M. E., ANAL.CHEM. 33, 1906 (1961). (5) Consolidated Electrodynamics Corp., “Operation and Maintenance Manut! for Type 21-130 Mass Spectrometer, Pasadena, Calif. ( 6 ) Fritz, G. J., Han, I., Ellis, W. H., Intern. J . A p p l . Radiation Isotopes,
16, 431 (1965). (7) Hintenberger, H., Naturwissenschaften 51, 473 (1964). (8) Klein, F. S., in “Stable Isotopes of Oxygen, Technical Data and Price Information Catalogue,” YEDA R e search and Development Go., Ltd., Rehovoth, Israel. (91 Lee. J. S.. ANAL.CHEM.34.835(1962). (16) Rihenberg, D., Ponticorvo, L., Intern. J . A p p l . Radiation Isotopes 1, 208 (1956).
INGUNH A N ~ GEORGE J. FRITZ Departments of Nuclear Engineering and Botany University of Florida Gainesville, Fla. SUPPORTED in part by the U. S. Atomic Energy Commission, Contract No. AT(40-1)-2834, by a grant (G-21410) from the National Science Foundation. and also by Sigma Xi-RESA grant-in-a{d to one of us (G.J.F.). Present address, Department of Physics, State University of New York, College of Fredonia, Fredonia, N. Y.
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