526
Anal. Chem. 1990, 62, 526-529 M. V., Ed.; ACS Sym oslum Series 359; American Chemical Society:
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Washington, DC, pp 84-59. Wang, M.; Marshall, A. G. Anal. Chem. 1989, 61, 1288. Wang, M.; Marshel, A. G. Promedings of the 36th Annual Conference On Mass Spectrmby and Allied Topics, Mami, FL ; American Society for Mass Spectrometry: 1989; RPA 9. Hanson, C. D.; Castro, M. E.; Russell, D. H. Proceedings of the 37th Annual Conference on Mess Spectrometry and Allled Topics Miami, FL ; American Society for Mass Spectrometry: 1989; RPA 34. Sommer, H.; Thomas, H. A.; Hipple, J. A. phys. Rev. 1949, 76, 1877. Sommer, H.; Thomas, H. A.; Hipple, J. A. Phys. Rev. 1950, 80, 487. Sommer, H.; Thomas, H. A.; Hipple. J. A. Phys. Rev. 1951, 82, 697. Shockiey, W. J. Appl. Phys. l M 8 , 9 , 635. McIver, R. T.; Hunter, R. L.; Ledford, E. B.;Locke, M. J.; Franci, T. J. Int. J . Mass Spectrom. Ion Processes 1981, 3 9 , 65. Remple, D. L. proceedings of the 35th Annual Conference on Mass Spectrometry and A M Topics, Denver, CO; American Society for Mass Spectrometry: 1987, p 1124. ~
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(30) Remple, D. L.; Gross, M. L. Proceedings of the 37th Annual Conference on Mass Spectromeby end A W Topics, Mami. FL : American Society for Mass Spectrometry: 1989; RPA 5. (31) Ijames, C. F.; Wilkins, C. L. J . Am. Chem. Soc. 1988, 110, 2687. (32) Smalley, R. E., Department of Chemistry, Rice University, Houston, Tx, private communication. (33) Hunt, D. F.; Shabanowitz, J.; Yates, J. R.; Zhu, N.-2.; Russell, D. H.; Castro, M. E. Roc. Natl. Acad. Sci. 1987, 8 4 , 620.
RECEIVED for review August 8,1989. Accepted November 27, 1989. This work was supported by the National Science Foundation (CHE-8418457). We gratefully acknowledge the Texas A&M University Office of University Research Services and the College of Science for providing a portion of the funds for purchase of the Nicolet FTMS 1000 mass spectrometer.
Combustion Tube Method for Measurement of Nitrogen Isotope Ratios Using Calcium Oxide for Total Removal of Carbon Dioxide and Water Carol Kendall* and Elizabeth Grim U.S. Geological Survey, 431 National Center, Reston, Virginia 22092
The nltrogen isotope ratlos of several organlc and Inorganic materials have been analyzed by a sealedtube combustion method requiring on-line cryogenic purllcatlon and by a new sealed-tube combustlon technlque using CaO for the quantltatlve removal of CO, and water. Samples purlled cryogenlcaliy are enriched In 16Nby an average of 0 . l l k relatlve to samples prepared with CaO. The enrlched values of samples p d b d cryoornicaWy probaMy resutl from the larger amounts of reddual contaminants In samples prepared wlthout CaO. Because samples prepared wlth the CaO technlque requlre no addltlonal purllcatlon, the technlque Is Ideal for use wlth multlsample mass spectrometer Inlet systems.
INTRODUCTION In the years since Stump and Frazer (1) first proposed a dry combustion technique for the liberation of nitrogen, combustion techniques have become the preferred method for the analysis of organic and inorganic nitrogen samples for nitrogen isotopic composition. Most commonly, samples are combusted inside sealed tubes (2,3),the tubes are cracked into a vacuum line, the contents are purified cryogenically to remove H20and C02,sometimes further purified by cycling through various furnaces (4), and then the nitrogen gas is concentrated before introduction into the mass spectrometer or into a sample vessel by using a Toepler pump (41, by freezing with He (5,6),or by trapping onto a molecular sieve cooled by liquid nitrogen (7-9). The sample vessels may then be mounted on the mass spectrometer inlet system for isotope ratio measurement. Alternatively, samples may be combusted under vacuum in furnaces connected to the mass spectrometer and the purified gas frozen into a small inlet volume (10). All of these methods require considerable time and labor for the various purification and concentration steps. The recent availability of isotope ratio mass spectrometers equipped with multiple automated tube crackers makes sealed-tube tech-
niques that produce pure gas especially attractive. Such techniques have been developed for preparation of water for 6D determination (11). In their classic papers, Fiedler and Proksch (2,12)describe their techniques and apparatus for the first multisample semiautomated inlet system for a nitrogen isotope ratio mass spectrometer. Their system enabled 80 samples to be processed per day. Samples were combusted in Pyrex tubes with copper and copper oxide, and CaO was added to absorb the resultant H20, COz, HC1, and other products. The low reaction temperature required by Pyrex produced incomplete yields (85-90%)) which impaired precision and accuracy, making the technique unsuitable for natural abundance measurements. The authors rejected the use of quartz tubes because of cost, because of difficulty of handling, and because they found that quartz reacted with CaO a t temperatures above 600 "C and cracked due to formation of calcium silicates. We have combined their use of CaO with our current 850 "C sealed-tube technique, which is modified after Macko (8)) and have experienced no problem with reaction of CaO with Vycor tubes. The use of CaO results in complete removal of COP and H20 and, hence, is ideal for use with a multiport tubecracking inlet system. This study compares our former and new sealed-tube combustion techniques for precision, accuracy, and ease of use. Samples prepared by either method are cracked directly into the inlet system of the mass spectrometer. However, samples prepared without CaO require cryogenic purification on-line prior to analysis, whereas samples prepared with CaO may be directly introduced into the mass spectrometer without further purification.
EXPERIMENTAL SECTION Apparatus. Samples are combusted in 9-mm Vycor tubes inside hollow nickel tubes in a muffle oven. The nickel tubes protect adjacent tubes from exploding if one tube explodes. After combustion, a custom-made temperature controller cools the samples for 17 h by automatically decreasing the temperature at
This article not subject to U.S. Copyright. Published 1990 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 5, MARCH 1, 1990
a rate of 50 "C per hour. A Finnigan Model 59670 12-port tube-cracker module designed for breaking 6-mm tubes inside glass cracker tubes by flexing of ball joints was modified to accept both 6-mm and 9-mm tubes. To use the tube cracker, each tube is scored about 5 cm from the tip, a metal spring is dropped inside the cracker tube, and the sample tube is loaded into the cracker tube. The spring has two functions: to hold the Vycor tube above the base of the cracker tube so that the cracker is not strained during automatic tube cracking, and to allow tubes of various lengths to be used. The length of the spring is adjusted prior to insertion so that the score on the Vycor tube lines up with the base of the ball joint. The ball joint is liberally lubricated with Apiezon N grease, assembled, and held securely with a metal pinch clamp. Reagents. Our normally used reagents and reagent-cleaning methods are as follows: Copper oxide wire (Baker Analyzed) is purified prior to use by heating in air to 600"C for 3 h, then sieved through no. 45 mesh to remove dust. Copper metal accelerator granules (Alpha Resources, Inc., Stevensville, MI) are used without additional cleaning. CaO (Fisher Scientific) is "activated" by baking at 1000 "C for 1 h to remove absorbed water and stored in a sealed container. The Vycor tubes are baked for 4 h at 600 "C before use. The normal cleaning method described above was compared with a higher temperature method modified after Minagawa et al. (13) where the tubes and copper oxide are heated to 850 OC for 2 h, and the copper is baked under vacuum to 750 "C for 2 h and then reduced with dry hydrogen. The Alpha copper and a finer-grained copper (Mallinckrodt, Inc., St. Louis, MO) were tested for relative amounts of carbon contaminants by using the two cleaning methods. Procedure. The following procedure is used in both combustion methods: 3 g of cupric oxide wire pieces is put into the 23 cm X 9 mm 0.d. Vycor tube using a long-tipped funnel, followed by enough sample to produce 6-60 pM of N2,another 4 g of cupric oxide, and 5 g of copper granules. These amounts of copper and copper oxide could probably be reduced by 50%. For samples that might react with the Vycor, such as sodium nitrate or samples on glass filters, the sample is placed inside a prebaked 6-mm 0.d. Vycor tube which is then placed inside the 9-mm tube (13). If CaO is used, 10-500 mg is added after the sample and before the remaining reagents. The copper granules prevent sample and CaO powders from migrating into the vacuum system during evacuation. The tubes are evacuated and sealed to a length of about 18 cm. The tubes are shaken and vibrated with an engraving pen to mix the sample with the reagents and then combusted for 2 h at 850 "C, followed by slow cooling. Samples prepared without CaO must be purified cryogenically. A 6-mm-0.d. stainless steel manually operated inlet system of the mass spectrometer was designed so that each sample can be purified on-line while the previously purified sample is being measured by the mass spectrometer. About two samples per hour can be purified and measured with this system. The tubes are loaded into a tube cracker (modified after Des Marais and Hayes (I4),which is permanently mounted to the inlet system, and the system is evacuated. The tubes are frozen in liquid nitrogen for 15 min before loading, cracked open with the Dewar in place, and expanded into a 12-cm-longsteel U-trap cooled in liquid nitrogen for an additional 15 min. These freezing times have proved necessary for total removal of H20 and CO, from 60 pM N, gas samples. A t this point, the previous sample in the mass spectrometer is pumped away, the handvalve to the inlet system is opened, and the purified N2 is expanded into the mass spectrometer for analysis. Samples prepared with CaO require no further purification and are loaded in groups of up to 12 onto the multisample inlet system for computer-controlled analysis. After all the tube crackers are loaded and evacuated, the computer is started and the sample tubes are automatically cracked and analyzed under computer control. The CaO dust does not migrate out of the tubes; however, as a precaution, balls of copper wire turnings are stuffed into the inlet system between the tube cracker and pneumatic valves to protect the valve seats. Isotope ratio measurements are performed on a Finnigan 251 EM triple-collecting mass spectrometer. After each analysis, a scan is made of the mass spectrum of the sample gas to determine
527
Table I. 616NValues Relative to Air' of Samples Purified Cryogenically and with CaO material potassium nitrate ammonium sulfate thiourea peptoneb corn leaves nitrogenous coalc N-Id N-2d
cryogenic purification
CaO purification
A6I5N
+3.53 f 0.10 (n = 15) +3.49 f 0.05 (n = 14)
0.04
+0.57 f 0.12 (n = 12) +0.49 f 0.07 (n = 15)
0.08
-1.46 f 0.10 (n = 9) +6.72 f 0.18 (n = 4) +3.44 f 0.14 (n = 3) +7.83 f 0.66 (n = 7)
-1.53 i 0.04 (n = 12) +6.34 i 0.17 (n = 6) +3.36 f 0.16 (n = 5) +7.70 f 0.05 (n = 3)
0.07 0.29 0.08 0.13
+0.58 i 0.02 (n = 7) +0.45 f 0.05 (n = 10) +20.39 i 0.07 (n = 3) +20.35 f 0.02 (n = 3)
0.13 0.04
Reported in per mil (960) using values of -1.63 f 0.03960 for N-SVEC relative to NBS-14and -2.81960 for N-SVEC relative to Air. bType 1 peptone. 'Black band coal from Durham Basin, NC; obtained from M. D. Krohn, USGS. International Atomic Energy Agency ammonium sulfate reference samoles.
its purity. The peak heights at masses 18, 30,32,40, and 44 are automatically measured on the minor collector and recorded. One tube containing 120 pM of the NBS standard N-SVEC prepared by using a modification of Coplen and Kendall's (15) procedure is loaded each day to serve as a working standard and to determine the fractionation between capillaries. The precision of multiple measurements of the same gas is better than kO.02L.
RESULTS AND DISCUSSION We have experienced no problems caused by weakening of either Vycor or quartz tubes by reaction of CaO with the walls of the tube. With our tube crackers, sample tubes are scored a t the opposite end of the tube from the CaO so that if the tubes were weakened by reaction, they are not strained additionally. With the sample tubes being cracked inside the automated tube crackers, samples are not lost even if the tubes shatter upon cracking. The nitrogen isotopic compositions of a variety of samples prepared with both the cryogenic and CaO purification techniques are given in Table I. Both techniques produce pure nitrogen with 44/29 and 18/29 mass ratios generally less than 0.03. The analytical precisions (la) range from *0.02%0 for reagents to *O.66%0 for cryogenic purification of a coal; the lower precision for natural samples is probably due to insufficient homogenization and trace contaminants. The average analytical precisions for samples prepared with and without CaO are 0.08 and 0.17k, respectively. The mean values for cryogenically purified samples are consistently enriched in 15Nrelative to samples prepared with CaO by 0.04 to 0.2%~. The relative enrichment in 15N for peptone samples prepared by the cryogenic purification method is twice the difference seen with any other type of sample. Peptone is often used as a standard because its organic complexity provides a more rigorous test of the performance of a technique than simpler materials (IO). There is no correlation between the amount of isotopic fractionation and the d15N values of these samples. Fiedler and Proksch (2) suggested that the amount of CaO used should be twice the amount theoretically required for absorption, or about 5 mol of CaO per mole of CH20. We used potassium nitrate, ammonium sulfate, and thiourea to determine the amount of CaO actually required to remove CO, and H20 quantitatively, and to check the effect of excess CaO on the 6I5N value (Table 11). These three nitrogen-bearing reagents where chosen because they contain, respectively, no carbon or hydrogen, only hydrogen, and both carbon and hydrogen. Sample sizes were chosen to produce 60 KMof N, (about 5-10 mg of sample). The critical factor in determining the required amount of CaO to use is the amount of carbon rather than the amount of hydrogen (Table 11). Use of 0.8 mol of CaO/mol of H 2 0
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 5, MARCH 1, 1990
15N by up to 0.2% compared to samples prepared with activated CaO. However, when identical samples were left a t room temperature for several weeks between combustion and analysis, all the water and COz was absorbed by the CaO and the 6I5N values were within 0.1'3~of the expected value. These limited data suggest that aging of combusted samples may increase absorption and permit use of less CaO. X-ray diffraction analysis of reacted CaO indicates the formation of Ca(OHIzand calcite; the relative amounts of COz and water removed by chemical reaction versus absorption are unknown. There is a slight inverse correlation between sample size and 615N for ammonium sulfate samples prepared with the CaO purification technique (Table 111). These two sets of samples, ranging from 6 to 60 pM in size, were prepared by using the same size sample tube and quantity of reagents regardless of sample size. Because of the poor correlation and the small effect on sample 615N, we have not corrected our results for this possible source of contamination. Table IV shows the results of an investigation of whether differences in the amounts of trace contaminants might be responsible for the enrichment of cryogenically purified samples relative to samples prepared by using CaO and for the slight enrichment of small samples relative to larger ones. For this experiment, the combustion tubes were identical in length and in weights of reagents, except for the presence of CaO. Two cleaning procedures and two varieties of granular copper were tested. Mass spectral data are all reported as ratios of peak heights, rather than as absolute intensities, to facilitate comparison with spectra produced by different mass spectrometers. Absolute amounts of Nz or other gases in blanks were not calculated because of the difficulties associated with linear extrapolations of the relationships between ion intensities and amount and type of gas a t low intensities. The main contaminants that can affect the 615N of a sample are as follows: nitrogen oxides, indicating incomplete reduction of the nitrogen with resultant isotope fractionation; carbon monoxide, with masses 28 and 29; and carbon dioxide, which produces daughter fragments with masses 28 and 29. Mass 30 (NO) is barely above background with both methods; hence, nitrogen oxide contamination is insignificant. The critical factors in evaluating the merits of different procedures and reagents are (1) the absolute amount of the mass 28 background in the blanks and (2) amounts of masses 28 and 29 that are not due to NP Because mass 14 is entirely produced from Nz of mass 28, all peak heights in Table IV are reported relative to it. The best procedure is the one producing blanks (tubes with reagents but no sample) with the lowest absolute amount of mass 28 and the lowest ratios of other masses relative to mass 14. Blanks prepared with CaO were substantially cleaner than blanks prepared without CaO and introduced into the mass
Table 11. Amount of CaO Required for Removal of C02 and H 2 0 and Its Effect on P N Value" 6l5Na;,
amt of CaO, mg 5
10 50
100 250 500
potassium nitrate
ammonium sulfate
n.a.b
n.a.
+3.53 f 0.03 ( n = 4) +3.53 f 0.01 ( n = 2) +3.47 f 0.03 ( n = 2) +3.44 f 0.01 ( n = 2) +3.48 f 0.07 (n = 4)
+0.53 f 0.04 ( n = 3) +0.48 f 0.04 ( n = 3) +0.48 f 0.06 ( n = 3) +0.48 f 0.05 ( n = 3) +0.57 f 0.08 ( n = 3)
thiourea -0.12' -0.25' -1.55 f 0.05 ( n = 3) -1.54 i 0.03 ( n = 3) -1.52 f 0.04 ( n = 3) -1.52 f 0.06 ( n = 3)
a All samples produced 60 pM of Nz (5-10 mg of sample). Theoretically required amounts of CaO to absorb all the C 0 2 and HzO produced by combustion of KNO,, (NH4)+3O4,and thiourea (as calculated from Fiedler and Proksch, 1972) are, respectively, 0,67, and 34 mg of CaO. *Not analyzed. 'No H20 (mass 18) present; however, the amount of C 0 2 (mass 44) was more than 100 times normal background.
Table 111. Relationship between 6"N and Nitrogen Yield (aM) for the CaO Technique total range in P N , k
no. of samples
corr coeff
0.15 0.30
n=6 n=6
-0.540 -0.532
linear regression
61SN = -0.00017 pM 615N = -0.00074 pM
+ 0.49 + 0.49
appears sufficient to remove all HzO. On the basis of the results in Table 11, we recommend using at least 15 mol of CaO/mol of carbon to ensure that all carbon is removed. There is no correlation between the amount of excess CaO and the 615Nvalue for the samples tested (Table 11). However, the differences in 615N values between groups of samples prepared with different amounts of CaO are often greater than the analytical uncertainty within a group. Analytical scatter for samples prepared with 500 mg of CaO generally is slightly greater than that for samples prepared with less CaO. Activation of the CaO improves its absorption efficiency. Fiedler and Proksch (2) suggested using CaO that was freshly activated at lo00 "C; however, we experienced no differences in absorption efficiency between freshly prepared CaO and CaO that had been stored in a bottle for 3 months. Samples containing 50 mg of unactivated Ca0/5 mg of thioura, measured on the mass spectrometer the day they were combusted, contained 50-200 times more water (mass 18), contained 2 times more COP (mass 44), and had 615N values enriched in
Table IV. Amounts of Contaminants in Blanks Prepared with anId without CaO, Using Different Types of Copper and Reagent-Cleaning Methods4
CaO amt, mg
copper grain sizeb
reagent cleaning method'
0 0 0
coarse coarse coarse fine fine coarse coarse
normal higher T normal/ frozen normal higher T normal higher T
50 50 50
50
magnitude of mass 28 peak," %
29/14
28/14
44/14
12/14
1.7 1.1
0.7
0.41 0.33 0.15
0.7 0.4 0.5 0.4
266 376
