Soil Science Society of America Journal 67:540-543 (2003)
© 2003 Soil Science Society of America
DIVISION S-3—SOIL BIOLOGY & BIOCHEMISTRY
Changes in Composition of Nitrogen-15-Labeled Gases during Storage in Septum-Capped Vials
Ronald J. Laughlin and
R. James Stevens*
Dep. of Agriculture and Rural Development, Agricultural and Environmental Science Division, Newforge Lane, Belfast BT9 5PX, UK
* Corresponding author (jim.stevens{at}dardni.gov.uk)
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ABSTRACT
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We tested the reliability of 12-mL Exetainers (Labco Ltd., High Wycombe, UK) fitted with bromobutyl rubber septa for the storage of a gas sample containing 15N-labeled N2O and N2. The gas mixture was analyzed monthly over a 1-yr storage period by continuous-flow isotope-ratio mass spectrometry (IRMS). After a year the N2O concentration had decreased by 30% but the change in 15N enrichment of N2O was not detectable. Accurate determination of the N2O concentration was possible up to 1 yr if a calibration gas was stored and analyzed along with the test mixture. This technique has wide applicability because diffusive loss of N2O as a fraction of starting concentration was predicted by Fick's Law to be almost independent of concentration above 3 µL L-1. The rate of decrease in the enrichment of N2 as measured by the ratio differences for 29/28 and 30/28 was slow. Decreases were not significantly different from time zero values after storage for 8 wk. After 1 yr of storage the losses of 29N2 and 30N2 significantly lowered the calculated value for the fraction of the N2 from the labeled pool (d) by about 2% of the initial value, but had no effect on the calculated value for the enrichment of the labeled pool (15XN). The diffusivity of N2O, calculated from loss rate and concentration gradient between Exetainer and atmosphere using Fick's Law, was 40 times higher than that of 29N2 or 30N2. Nitrous oxide may have been lost from the Exetainer because of adsorption by the septum as well as diffusion through the septum.
Abbreviations: aD, the enrichment of the pool from which the labeled N2O is derived • d, the fraction of the N2 derived from the labeled source • I, ion current • IRMS, isotope-ratio mass spectrometry • LSD, least significant difference • R, molecular ratio • 15XN, the enrichment of the pool from which the labeled N2 is derived • 
29R, difference between the molecular ratios for 29N2/28N2 in the enriched test sample and normal atmosphere • 30R, difference between the molecular ratios for 30N2/28N2 in the enriched test sample and normal atmosphere
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INTRODUCTION
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DENITRIFICATION RATES in the field are often measured by the flux chamber method (Hauck, 1986). With this method the fluxes of N2 and N2O can be determined simultaneously if the NO3 pool in soil is labeled with 15N (Mosier and Klemedtsson, 1994). Isotope-ratio mass spectrometry is the method of choice for the analysis of 15N in these gases (Mulvaney, 1993). The IRMS technique has evolved from manual systems (Siegel et al., 1982) to fully automated continuous-flow systems (Prosser et al., 1991; Stevens et al., 1993). Gas samples are usually stored in septum-capped vials that are accessed directly by the gas-handling system of the IRMS. There is no published work on the efficacy of septum-capped vials for the storage of 15N-labeled gases. In practice, headspace samples are transferred directly to septum-capped vials using a gas-tight syringe and may be stored weeks or months depending on the availability of the IRMS facility.
Previous studies have tested the reliability of glass vials fitted with seals made from different materials for storage of N2O. Butyl rubber seals offer the best potential for preventing diffusion, uptake or production of N2O (Covert et al., 1995; Mosier and Klemedtsson, 1994; Scott et al., 1999; Segschneider et al., 1997). Our aim in this study was to test the reliability of screw-cap 12-mL glass Exetainers fitted with bromobutyl rubber septa for storage of samples containing 15N-labeled N2 and N2O. The isotopic composition of the N2 and the concentration and isotopic composition of the N2O in the vials were determined by automated continuous-flow IRMS at monthly intervals over 1 yr.
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MATERIALS AND METHODS
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The storage vials were Exetainers, which are 16 x 100 mm glass, screw-top, septum-capped tubes. Black bromobutyl septa were selected for their low gas permeability. The septa were 14-mm in diameter by a 3-mm thickness, and had a hardness of 45 shore A, a specific gravity of 1.21 g cm-2, and an ash content of 32%. Although the Exetainers were supplied pre-evacuated (Isochem, Crewe, UK), they were alternately filled with He (0.12 MPa) and evacuated (<100 Pa) three times on the day of use.
Exetainers were held in racks on a Gilson 222 Autosampler (Anachem, Luton, UK), which was interfaced to a PDZ Europa Scientific 20-20 Stable Isotope Analyzer using a Europa Scientific Trace Gas Preparation System (PDZ Europa, Crewe, UK). The concentration and 15N content of the N2O and the 15N content of the N2 in each 12-mL vial were determined as described by Stevens et al. (1993), but with automation of source switching and valve settings so that N2 and N2O were analyzed in the same sample. For N2O, the ion currents (I) at m/z 44, 45, and 46 enabled concentrations and molecular ratios 45R (45I/44I) and 46R (46I/44I) to be measured. The enrichment of the labeled N2O (aD) was then calculated using 45R and 46R according to (Arah, 1997). For N2, the ion currents at m/z 28, 29, and 30 enabled molecular ratios 29R (29I/28I) and 30R (30I/28I) to be determined. Differences between the molecular ratios in the enriched test sample and normal atmosphere were calculated as 29R and 30R. Values for the enrichment of the pool from which the labeled N2 was derived (15XN), and the fraction of the N2 which was derived from the labeled source (d), were calculated from 29R and 30R according to Mulvaney and Boast (1986).
A mixture of N2O and N2 labeled at 20 atom% excess in 15N was produced by adding alkaline NaOBr (Hauck, 1982) to (NH4)2SO4 containing 15N at the same enrichment. The reaction produces >97% N2 and <3% N2O (Clusius and Rechnitz, 1953). This procedure for generating N2O and N2 was described by Stevens et al. (1993). Briefly, a 1-mL aliquot of a solution of enriched (NH4)2SO4 (200 mg) was placed in an Exetainer and sealed with a septum-capped lid. A needle (5 cm by 0.63-mm-o.d.) connected to a 20-mL disposable syringe was passed through the septum and the Exetainer was evacuated by inserting another needle attached to a vacuum pump. Sodium hypobromite (10 mL) was injected slowly into the evacuated Exetainer and the generated gases were collected in the empty syringe. A 5-mL sample was transferred from the syringe to a 1-L gas bag filled with air to produce the test gas mixture. The test mixture contained 78% (v/v) N2 with a 29R value of 0.001416 and a 30R value of 0.000184. It also contained 18.4 µL N2O L-1 enriched at 20.4 atom% 15N.
A batch of 60 Exetainers was filled with 12-mL aliquots of the test mixture. On the same day another batch of 60 Exetainers was filled with 12-mL aliquots of a N2O standard gas mixture containing 100 µL L-1 of N2O at natural abundance in 15N (Bedfont, Upchurch, UK). After 0, 4, 8, 12, 16, 22, 26, 31, 37, 41, 46, and 50 wk of storage at 20°C ± 2, five Exetainers were randomly selected from each batch. These Exetainers, together with five Exetainers freshly filled with 12 mL of the standard N2O mixture and five Exetainers freshly filled with 12 mL of air, were analyzed in one analytical run by IRMS. The concentration and enrichment of the N2O in the Exetainers containing the test mixture and the stored standard were calculated as described by Stevens et al. (1993), using data for 45R and 46R from the analyses of the Exetainers freshly filled with the standard N2O mixture for calibration. Then the concentration and enrichment of the N2O in the Exetainers containing the test mixture were calculated using data for 45R and 46R from the analyses of the Exetainers containing the stored standard N2O mixture for calibration. The enrichment of the N2 in the stored Exetainers was calculated as 29R and 30R relative to data for the analysis of Exetainers freshly filled with normal air.
Analysis of variance was conducted using Genstat (Lawes Agricultural Trust, 1993) to determine the significance of storage time on 29R, 30R, d, 15XN, aD, and N2O concentration. When effects were significant at P < 0.05, the least significant difference (LSD) between means was calculated for P = 0.05.
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RESULTS AND DISCUSSION
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The N2O concentration in the test mixture or stored standard did not decrease significantly (P > 0.05) for 4 wk, but then the concentrations decreased linearly with time (Fig. 1a and 1b) . After 50 wk of storage the N2O concentration had decreased by 35% in the test mixture, and by 34% in the standard mixture. A value for the diffusivity of N2O was calculated from the loss from the Exetainer and the concentration change during one time interval of 50 wk using Fick's Law (Hillel, 1998).
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Fig. 1. The effect of storage time in Exetainers on (a) the concentration of nitrous oxide in a test mixture, (b) the concentration of nitrous oxide in a standard gas mixture, and (c) the enrichment of the labeled nitrous oxide (aD) in the test mixture. (LSD values are for comparing any two means at P = 0.05).
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 | [1] |
where Jg is the diffusive flux of gas, D is the diffusion coefficient, c is the concentration, x is the distance, and dc/dx is the concentration gradient. Rearranging Eq. [1] gives
 | [2] |
where D/dx is the diffusivity. Then this value for diffusivity was used to predict the relationship between diffusive loss and the starting concentration of N2O (Fig. 2)
. The predicted losses for the stored gases will be approximately correct but higher (42%) than the actual losses (34%) because the relative concentration decrease during the single time interval is large. Exact values could only be obtained by numerical simulation with time intervals approximating to zero. Diffusive loss of N2O from Exetainer to atmosphere over 50 wk was predicted to be almost constant for all concentrations above 3 µL L-1 but to decrease abruptly with concentration below this value. It was therefore valid to calibrate for N2O using the data from the stored standard (100 µL L-1 at time zero) to calculate the starting N2O concentration in the stored test mixture (18.4 µL L-1 at time zero). When the values for the starting concentration of the test mixture were calculated in this way they were not significantly different from 18.4 µL L-1 (data not shown).
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Fig. 2. The relationship between the nitrous oxide concentration in an Exetainers and diffusive loss as predicted from Fick's Law.
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The 15N enrichment of labeled N2O was not significantly different from the enrichment at time zero (P > 0.05) over the 50 wk of storage (Fig. 1c). The loss processes lowering the concentration of N2O had therefore no effect on its enrichment. Using the calculated value for the diffusivity of N2O and Eq. [1] to predict the diffusive loss of 45N2O and 46N2O at starting concentrations from 100 to 0.33 µL L-1 confirmed that diffusive loss has no affect on aD.
The effect of time on the measured values of 29R and 30R in N2 stored in Exetainers is shown in Fig. 3a and 3b
, respectively. The values of each ratio difference decreased with time, the decreases being significant (P > 0.05) after 8 wk. From the linear regressions for all data, the value of 29R decreased by 0.16%, and the value for 30R decreased by 1.09% over 50 wk. Diffusion of 30N2 would have been faster than 29N2 because of the larger concentration gradients between Exetainer and atmosphere. Values for the diffusivity of 29N2 and 30N2 were calculated from the loss from the Exetainer and the concentration change during one time interval of 50 wk using Eq. [2]. The diffusivities of 29N2 and 30N2 were similar but about 40 times lower than the diffusivity calculated for N2O. Theoretically the calculated values for the diffusivities of N2O and N2 should be similar if diffusion is the only loss process. The most likely reason for the difference was adsorption of N2O by the butyl rubber septum.
The diffusive losses of N2 had a significant effect on the calculated values of d but not on the values of 15XN (Fig. 4)
. Values for 15XN were not significantly different (P > 0.05) from 20.4 atom% 15N, apart from at Week 50 when the value was considered to be an outlier. The values for the diffusivity of N2 and Eq. [1] were used to predict the diffusive loss of 29N2 and 30N2 over 50 wk at different starting values of d (Table 1). At the end of 50 wk the new values of 29R and 30R were used to calculate 15XN (Table 1) according to Mulvaney and Boast (1986). Diffusive losses were low when compared with the loss of N2O at a similar concentration because of the higher diffusivity and concentration gradient between Exetainer and atmosphere for N2O. The percentage losses decreased with d, but the losses had no effect on the measurement of the enrichment of the labeled N2.
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Table 1. Predicted diffusive loss of 29N2 and 30N2 for different starting values of d during one time interval of 50 wk, and the predicted enrichment of the labeled N2 after 50 wk.
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The efficacy of septum-capped containers for storing gas samples has been tested previously for the determination of N2O concentration but not for the determination of 15N in N2O or N2. The composition, size, and thickness of the septum can affect the rate of loss by diffusion or absorption. Segschneider et al. (1997) and Scott et al. (1999) concluded that a butyl rubber septum was a reliable seal for 7 d of storage. A reliable storage period of 4 wk was reported using Vacutainers fitted with thick seals (Covert et al., 1995). No uptake of gas was reported from glass serum bottles fitted with bromobutyl rubber stoppers (Mosier and Klemedtsson, 1994).
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CONCLUSIONS
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Exetainers fitted with 3-mm thick bromobutyl septa can be used to store gas samples for the analysis of N2O concentration and 15N in N2O and N2. Significant losses, however, occurred during a 50-wk storage period and affected the determination of concentration but not enrichment. Losses as a fraction of starting concentration were greater for N2O than for N2 because of its larger concentration gradient between Exetainers and atmosphere, and because adsorption was probably occurring onto the septum as well as diffusion through the septum. Using Fick's Law, diffusive loss of N2O as a fraction of starting concentration was predicted to be almost independent of concentration above 3 µL L-1. Above this concentration, accurate determination of the concentration of N2O in samples can therefore be achieved by using a stored standard mixture with >3 µL N2O L-1 for calibration. Losses of 29N2 and 30N2 decreased the value for d by about 2% after 50 wk but had no effect on the value for 15XN.
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ACKNOWLEDGMENTS
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We thank Michael Nicholson (Department of Agriculture and Rural Development, Belfast, UK) for his technical assistance, and Reinhard Well (University of Göttingen, Germany) for his assistance with calculations of diffusive loss using Fick's Law.
Received for publication January 15, 2002.
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REFERENCES
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ABSTRACT
INTRODUCTION
