Environ. Sci. Technol. 2000, 34, 2337-2341
Rapid and Precise Determination of the δ18O of Dissolved and Gaseous Dioxygen via Gas Chromatography-Isotope Ratio Mass Spectrometry BRIAN J. ROBERTS,† MARY E. RUSS,‡ AND N A T H A N I E L E . O S T R O M * ,‡ Department of Ecology and Evolutionary Biology, E305 Corson Hall, Cornell University, Ithaca, New York 14853-2701, and Department of Geological Sciences, 206 Natural Sciences Building, Michigan State University, East Lansing, Michigan 48824-1115
Despite the importance of O2 in biogeochemical processes, relatively few environmental studies have incorporated stable isotope information to assess the origins and cycling of this gas. A major limitation to the routine use of δ18O has been the cost and complexity associated with traditional off-line preparation, dual-inlet techniques. A gas chromatograph-isotope ratio mass spectrometry (GC-IRMS) technique providing rapid and precise δ18O-O2 values is presented. The procedure utilizes a 5-Å molecular sieve column held at a constant temperature of 50 °C to separate O2 and N2 in time. A precision (( SD) of (0.3‰ or better for δ18O-O2 is demonstrated on gaseous and dissolved samples spanning an environmentally relevant range in size of 20-700 µM. The potential for utilizing the technique for δ17O-O2 analysis (precision of (0.5‰) and δ15N-N2 analysis (precision of (0.2‰) on air samples is also demonstrated. Preliminary results from two unique environments, the subtropical North Pacific and Cornell University experimental ponds, are presented to demonstrate potential applications of the technique.
Introduction Dioxygen (O2) is the second most abundant gas in the earth’s atmosphere (20.9%) and is intimately involved in the biosphere through the processes of photosynthesis and respiration. O2 has long been recognized as an important component of biogeochemical processes on land, in air, and in water (1, 2). While changes in the concentration of O2 have been routinely used in biogeochemical studies, relatively few studies have attempted to utilize stable isotopes to evaluate rates of the biogeochemically important processes involving dioxygen (3-9). The isotopic composition of atmospheric O2 was first characterized over 60 years ago. Dole (10) and Morita (11) independently observed that the isotopic composition of atmospheric O2 is enriched (by approximately 20‰) in 18O with respect to average ocean water [O stable isotope ratios * Corresponding author e-mail:
[email protected]; phone: (517)355-4661; fax: (517)353-8787. † Cornell University. ‡ Michigan State University. 10.1021/es991109d CCC: $19.00 Published on Web 04/18/2000
2000 American Chemical Society
are expressed in per mil (‰) notation: δ18O ) [(Rsample/ Rstandard) - 1] × 1000 where R is the abundance ratio of the heavy to light isotope. The internationally recognized standard for O is Vienna-Standard Mean Ocean Water (V-SMOW), which by definition has a per mil value of 0.]. This difference, which has become known as the “Dole effect”, was later refined to an enrichment of 23.5‰ (12). Isotopic fractionations associated with numerous processes contribute to this imbalance (13-15) including respiration (13, 3, 4, 16, 17), photosynthesis (16, 18), air-water O2 exchange (19, 20), and evapotranspiration (21, 22). In addition to understanding the present day Dole effect, changes in the isotopic imbalance between atmospheric O2 and water have been examined over geologic time scales by extracting and determining the δ18O values of atmospheric O2 from ice cores (23, 24, 15). Despite knowing the approximate magnitude of the Dole effect for many years, our ability to understand the relative proportions of the processes contributing to the unique isotopic composition of O2 is still lacking. Bender et al. (15) attempted to quantitatively account for the magnitude of the Dole effect (23.5‰) based on our current understanding of the aforementioned oxygen isotope fractionations, terrestrial and marine rates of photosynthesis and respiration, exchange rates between air and water, and stratospheric photochemistry. This exercise was only able to account for 20.8‰ of the known 23.5‰ enrichment in atmospheric O2 relative to ocean water (15) demonstrating a need for further clarification of the rates and associated fractionation factors for the biogeochemical processes involving O2. The above discussion of the potential usefulness of δ18OO2 in determining rates of processes involving O2 (3-9) combined with our demonstrated inability to account for the Dole effect (15) point to an essential need for more studies examining the isotopic composition of O2. One of the reasons for the relative lack of emphasis on the isotopic composition of O2 has been the complexity of analyzing O2 isotopically by traditional methods. Most studies have analyzed the isotopic composition of O2 by first converting it to CO2 and then analyzing the ratio of mass 46 to mass 44. The procedure is described in detail in Kroopnick (25) and with modifications in Bender and Grande (5). Briefly, a sample is released into a vacuum line and cryogenically purified of CO2 and H2O with a liquid N2 trap. The remaining gases are initially collected onto a molecular sieve column at liquid N2 temperature and then released and circulated over a graphite rod with a Pt wire catalyst heated to approximately 800 °C. The O2 present in the sample reacts with the C in the graphite rod to form CO2 that is subsequently analyzed within the dual-inlet of the mass spectrometer. The quantitative conversion of O2 to CO2 is crucial as isotopic segregation during incomplete conversion can result in highly inaccurate determinations of δ18OO2 (12). Over the past 10 years, a second method has been developed using the dual-inlet of the mass spectrometer for determination of δ18O-O2 (26, 27, 7, 8). This procedure involves the initial removal of CO2 and H2O by cryogenic purification. The remaining gases are condensed into a steel finger using liquid He. The finger is removed allowing the gases to equilibrate at room temperature for several hours (4 in 27) prior to introduction to the mass spectrometer for isotopic analysis of O2 in the presence of N2 and Ar. The measurement of δ18O (masses 32 and 34) in dual-inlet approaches is very sensitive to the concentration of N2 (masses 28 and 29); therefore, careful measurement of the O2:N2 ratio is also necessary (27). This approach can be VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Schematic representation of the GC-IRMS system used for stable isotopic analysis of dissolved and gaseous O2. Arrows indicate direction of flow. See text for detailed description. complicated by a nonlinearity isotope effect associated with the analysis of sample gas mixtures that differ greatly in O2: N2 ratios from the standard gas (28). This nonlinearity isotope effect can be accounted for (7) but does complicate analysis of samples with a wide range of O2:N2 ratios (e.g., differing greatly in productivity). A major advantage of the use of gas chromatography in determination of the δ18O-O2 is that there is no need to correct isotope values for variation in O2:N2 ratios since the isotopic analysis is performed on pure gases. Both off-line preparation techniques can provide very accurate and precise measurements of δ18O-O2 when the proper precautions are taken [e.g., Emerson et al. (26) state a precision of replicate samples of (0.04‰]. However, the time involved in preparation for dual-inlet analysis combined with laboratory equipment costs is prohibitive. The goals of this paper are to (a) present a gas chromatograph-isotope ratio mass spectrometer technique for analysis δ18O of dissolved and gaseous O2, (b) demonstrate the accuracy and precision of the technique, and (c) present preliminary data demonstrating the utility of this technique in biogeochemical studies.
Methods Field Sampling Procedure. Collection of samples for determination of the δ18O-O2 followed the procedure of Emerson et al. (26, 27). Samples were collected in preevacuated 200-mL glass vessels fitted with a glass high-vacuum stopcock (Chemglass, 1/4 in.) and 3/8 in. o.d., 90° arm inlet at one end. Prior to evacuation, 1 mL of a saturated mercuric chloride (HgCl2) was added to each vessel and dried. In the field, a 20-cm piece of Tygon tubing was placed on the vessel inlet and thoroughly flushed with CO2 gas flowing through a 3/16 on. o.d. tubing. Water was collected using either a 12-L poly(vinyl chloride) (PVC) “Niskin-type” water sampler (subtropical North Pacific) or a 3.2-L Beta Bottle (Wildco) horizontal sampler (Cornell Experimental Ponds). Flow was initiated from the sampler through a 3/16 in. o.d. plastic tubing inserted into the Tygon tubing on the glass vessel. The CO2 line was then removed. Water was allowed to overflow the Tygon tubing on the glass vessel, and any trapped bubbles of CO2 were removed by gently tapping. At this point, the stopcock on the vessel was gradually opened, allowing a stream of water to enter the vessel by vacuum at a slow rate such that water continued to overflow the Tygon tube. Once the vessel was approximately half full, the stopcock was closed. For storage, the neck of the vessel was flushed with CO2 gas that was then maintained in place by a rubber serum 2338
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septum (Bittner Inc.). In this manner, vessels can be stored for several weeks without compromising data quality. Upon return to the laboratory, gases within the headspace of the vessels were equilibrated in a constant temperature water bath (28 °C) for 8 h with continuous agitation. At the end of 8 h, the vessels were inverted, and the water was removed by vacuum until only 1 mL remained (enough to prevent evacuation of the headspace). The neck of the vessel was then filled with CO2 gas until analysis could be performed. Dissolved O2 concentrations were determined via the Winkler technique as modified by Carpenter (29). GC-IRMS Analysis Procedure. Determination of δ18O of O2 was accomplished using a gas chromatograph (HewlettPackard 5890) interfaced to a Micromass Prism stable isotope ratio mass spectrometer. The sample vessel is connected to an inlet system on the gas chromatograph that consists of ascarite (12 mm × 8 cm) and LiOH (6 mm × 8 cm) traps to remove CO2 and water; a 6-port, 2-position sampling valve (valve 1, Valco Instruments Co. Inc.); a 3-mL gas sampling loop; a 4-port, 2-position sampling valve (valve 2, Valco Instruments Co. Inc.); a vacuum isolation valve; and a vacuum pump (Figure 1). In practice, we have found that LiOH removes CO2 more efficiently than water and that ascarite removes water more efficiently than CO2; nonetheless, the use of both LiOH in addition to ascarite is probably not necessary. Initially, the inlet system is completely evacuated and then isolated from vacuum. The sample is then released and immediately expanded throughout the inlet system. During this process, water and CO2 are absorbed. After 1015 s of equilibration, the valves on the gas sampling loop are rotated to initiate He flow through the loop and push sample gases onto the GC column. A 5 m × 1/8 in. o.d. molecular sieve 5-Å column (Alltech) held at a constant temperature of 50 °C is used with a He head pressure of 50 psi to separate N2 and O2. Any residual water or CO2 entering the gas sampling loop is very efficiently trapped onto the molecular sieve column and removed later by heating. The effluent of the gas chromatograph is allowed to enter the mass spectrometer (via an open split to prevent over pressurizing the mass spectrometer), and the isotope ratio is determined in comparison to a pulse of calibrated O2 gas standard within the reference bellows of the mass spectrometer. The chromatographic conditions used do not result in separation of O2 and Ar; however, these gases are separated within the magnetic field of the mass spectrometer, and we have not observed any deterioration in data quality as a result of coelution. Sensitivity and linearity were optimized at a source
trap current of 600 µA. The total time for the sample analysis is approximately 150 s. All isotope values are corrected for isotopic segregation during exchange between gaseous and dissolved phases during equilibration in the constant temperature water bath (20).
Results and Discussion Demonstration of Accuracy and Precision of Technique. The accuracy of the technique was tested by analysis of calibrated tank standards or atmospheric air samples injected into the inlet system. These tests demonstrated that samples with a peak height equal or above 6 × 10-9 A yielded δ18O-O2 measurements equal to atmospheric O2 (23.5% relative to V-SMOW) with a precision generally better than (( SD) of (0.3‰ (Table 1). This level of precision is readily obtained for samples with O2 concentrations as low as 30 µM. A wide range of sample sizes can be accommodated by adjusting the interface helium dilution (Figure 1). This dilution can be increased (to ensure that high O2 concentration samples do not saturate the collectors) or decreased (to ensure that sufficient O2 enters the IRMS) to optimize the peak height for isotopic analysis. Dissolved O2 samples containing between 20 and 700 µM O2 have been accurately measured using our system. This range of sample sizes suggests that the technique is suitable for most environmental applications. We estimate that the quantity of O2 introduced to the inlet of our system ranges between 0.5 and 2.0 µmol; however, detection limits can be lowered by adjustment to source tuning parameters, which we rarely found necessary. Furthermore, the size of the sampling loop (currently 3 mL) can be increased or decreased in size to accommodate samples containing extreme amounts of O2 (i.e., less than 20 µM or greater than 700 µM). The GC-IRMS technique also provides isotopic data on the δ17O of O2 for each sample analyzed. The precision (( SD) of δ17O-O2 of calibrated tank standard air and lab air samples is (0.5 ‰ (Table 1). We have found that to attain this level of precision requires that considerable effort be expended in the optimization of source tuning parameters that is not normally done on a routine basis when only δ18O is of interest. Additionally, the separation of O2 from N2 in the molecular sieve column can also be utilized to provide δ15N values of N2. Replicate analysis of calibrated tank and atmospheric air samples yielded δ15N-N2 values with a precision of (0.2‰ (Table 1). While the level of precision for δ17O may not have great utility for natural abundance studies (30), the precision obtained for the δ15N of N2 is suitable for both natural abundance (31, 32) and enrichment (33) level studies of denitrification. Preliminary Field Data. Comparison of GC-IRMS to Traditional Dual-Inlet IRMS Technique. A depth profile of δ18O-O2 samples was collected from the oligotrophic subtropical North Pacific at Hawaii Ocean Time-Series (HOT) Station ALOHA (22°45′ N, 158°00′ W) during research cruise HOT 101 (January 1999; R/V Moana Wave) and analyzed using the methods detailed above. Station ALOHA (A LongTerm Oligotrophic Habitat Assessment) is located 100 km north of the island of Oahu, HI, and has a water column depth of 4500 m. Approximately 9 years earlier, during HOT cruise 13 (January 1990), a depth profile of δ18O-O2 samples was collected at this location using the dual-inlet technique discussed in the Introduction (7, 8, 26, 27). Dissolved O2 concentration and δ18O data for both profiles is shown in Figure 2 and allows a comparison of data determined by both the dual-inlet (HOT13; Steve Emerson and Paul Quay, unpublished data) and GC-IRMS techniques (HOT101) from the same station, although separated in time by 9 years. Dissolved O2 concentration ranged from 21.4 to 224.6 µM during HOT 101 with the highest concentrations occurring in the top 200 m of the water column and the minimum at
TABLE 1. Precision and Accuracy of Gas Chromatograph Isotope Ratio Method for δ18O, δ17O, and δ15N of Purified Gas Standards or Atmospheric Aira sample
δ18O
δ17O
July 13, 1998
δ15N
major beam height (amp × 10-8)
N25431-1 N25431-2 N25431-3 N25431-4 N25431-5 mean ( SD
-3.00 -3.06 -2.63 -2.90 -3.04 -2.93 ( 0.18
1.7 1.5 1.9 1.7 1.5
UP air-1 UP air-2 UP air-3 UP air-4 UP air-5 UP air-6 mean ( SD
-0.12 -0.26 -0.27 -0.28 -0.24 -0.23 -0.23 ( 0.06
1.6 1.4 1.2 1.1 1.6 1.4
October 1, 1998
UP air-1 UP air-2 UP air-3 UP air-4 UP air-5 mean ( SD
-0.54 0.78 -0.44 0.15 -0.12 0.04 -0.19 -0.37 -0.10 -0.01 -0.28 ( 0.20 0.12 ( 0.42
air-1 air-2 air-3 air-4 air-5 air-6 air-7 air-8 air-9 mean ( SD
0.07 0.14 -0.14 0.29 -0.34 0.46 -0.13 -0.11 -0.20 0.00 ( 0.26
1.0 1.0 0.75 0.76 0.66
October 30, 1998
ALOHA air-1 ALOHA air-2 ALOHA air-3 ALOHA air-4 mean ( SD
0.63 0.57 1.50 1.50 1.50 1.50 1.40 1.40 1.40
0.15 0.14 0.31 0.38 0.24 ( 0.12
1.5 1.3 1.2 1.2
December 17, 1998
-0.10 -0.00 0.05 -0.08 0.35 0.07 -0.33 -0.01 ( 0.21
air-1 air-2 air-3 air-4 air-5 air-6 air-7 mean ( SD
0.87 0.87 0.87 1.35 0.85 0.51 0.38
December 19, 1999 air-1 0.10 air-2 0.14 air-3 -0.10 air-4 0.07 air-5 -0.04 mean ( SD 0.01 ( 0.11
1.95 1.95 1.90 1.93 1.96
a The δ15N of the standard N25431 has previously been determined to be -2.92‰. The δ18O and δ17O for air and Ultrapure air (UP air, Scott Marin Inc.) are reported with respect to atmospheric air and on this scale are equal to 0‰. Samples labeled ALOHA air are atmospheric air samples collected at station ALOHA. No entry indicates that the isotope value was not determined.
approximately 800 m (Figure 2b). δ18O-O2 values for HOT101 ranged from 22.2 to 36.6 ‰ with the maximum values associated with the O2 minimum region (Figure 2a). In the upper 200 m of the water column, O2 concentrations are at a maximum, and δ18O-O2 values are at a minimum reflecting contributions of O2 from photosynthesis and atmospheric exchange (5, 7, 8, 16). Higher δ18O-O2 values lower in the water column are consistent with the fractionation associated with respiratory O2 consumption (6, 17). Isotopic composition VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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a
FIGURE 3. Diel patterns of dissolved [O2] (closed circles) and δ18OO2 (open circles) in pond 223 at the Cornell Experimental Ponds Facility in Ithaca, NY. Time 0 represents midnight on September 24, 1998, and time 24 represents midnight on September 25, 1998. Sunrise on September 24 and 25 was at approximately 0630, and sunset was at 1900.
b
FIGURE 2. Depth profiles of (a) δ18O-O2 and (b) dissolved O2 concentration taken at HOT station ALOHA during HOT 13 (open circles) and HOT 101 (closed circles). δ18O-O2 samples collected during HOT 13 (January 1990) were analyzed using a dual-inlet technique (26, 27, 7, 8) (n ) 38), while samples collected during HOT 101 (January 1999) were analyzed using the GC-IRMS technique detailed here (n ) 33). and concentration data of dissolved O2 collected during research cruises HOT13 and HOT101 are remarkably similar (Figure 2). The similarity of these profiles collected within the same month (January) indicates that the dual-inlet and GC-IRMS techniques provide comparable data and suggests that the concentration and isotope dynamics of O2 at station ALOHA have been stable over the 9-year period separating their analysis. This stability is undoubtedly a consequence of the oligotrophic nature of this environment. Demonstration of Potential Applicability of Technique. Bender and Grande (5) and Quay et al. (7, 8) have used natural abundance δ18O-O2 values in conjunction with dissolved O2 concentrations in order to estimate gross primary production in aquatic ecosystems. In September 1998, replicate samples were collected for δ18O-O2 and dissolved O2 concentration every 4 h for 24 h in two ponds at the Cornell Experimental Ponds Facility in Ithaca, NY (9). The preliminary pattern in pond 223 (Figure 3) demonstrates that δ18O-O2 decreased approximately 4‰ (from 25‰ to 21‰) during daylight hours 2340
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(7-19 h) while O2 concentration increased 60 µM (from 220 to 280 µM). The inverse pattern is seen at night. This pattern of variation is consistent with the release of isotopically depleted O2 from H2O via photosynthesis during the day (16) and increases in δ18O-O2 during the night as a result of isotopic fractionation during respiration (17). The reproducibility (( SD) of samples taken between 0.25 and 0.5 h apart was (0.1‰ (n ) 14). Slight differences in the pairs of samples may reflect isotope shifts occurring during the time between sampling replicates of 0.5 h. These data demonstrate that the technique yields isotopic measurements of more than sufficient accuracy and precision to study in situ O2 cycling in aquatic ecosystems. The GC-IRMS technique for analyzing δ18O of dissolved and gaseous O2 permits rapid, accurate, and precise isotopic determinations. The findings presented here illustrate the potential for this technique to be utilized in a wide variety of environmental applications. Wassenaar and Koehler (34) have recently analyzed the δ18O-O2 by continuous flow following injection of samples using a gas-tight syringe into a elemental analyzer interfaced to the isotope ratio mass spectrometer. There are minor but important differences in the two techniques. Our approach utilizes an evacuated sample loop that avoids problems associated with cryogenic trapping (used in dual-inlet techniques) and the tendency for syringes and septum to fractionate and leak. In addition, the method presented here is better optimized for subsurface water samples as it does not require that the sampling vessel be immersed for collection. It is important to note that both methods need to correct for isotopic discrimination during headspace equilibration (20). Despite these differences in the techniques, the independent development of two rapid techniques employing the principles of gas chromatographic separation to analyze the isotopic composition of O2 with good precision ((0.3‰ or better) will greatly facilitate the use of δ18O-O2 in a wide variety of studies.
Acknowledgments This work has greatly benefited by assistance and input provided from Bob Howarth, Peggy Ostrom, Tim Bergsma, and two anonymous reviewers. Funding was provided by National Science Foundation Research Training Grant in Biogeochemistry and Environmental Change at Cornell University (B.J.R.) and the National Science Foundation (OCE 9817064 to N.E.O.). We greatly appreciate the efforts of the officers and crew of the recently decommissioned R/V Moana Wave and researchers of the HOT program; notably Dave Karl, Louie Tupas, and Dan Sadler. The HOT program is
supported by National Science Foundation Grants OCE 9301368 (Dave M. Karl, Principal Investigator) and OCE 9303094 (Roger Lukas, Principal Investigator). We thank Steve Emerson and Paul Quay for sharing their data from HOT13 and Matthew Emmons for the initial development of our inlet system.
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Received for review September 28, 1999. Revised manuscript received March 6, 2000. Accepted March 7, 2000. ES991109D
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