A new automated method for high-throughput carbon and hydrogen isotope analysis of gaseous and dissolved methane at atmospheric concentrations

Rationale: The dual isotope ratio analysis, carbon ( δ 13 C value) and hydrogen ( δ 2 H value), of methane (CH 4 ) is a valuable tracer tool within a range of areas of scientific investigation, not least wetland ecology, microbiology, CH 4 source identification and the tracing of geological leakages of thermogenic CH 4 in groundwater. Traditional methods of collecting, purification, separating and analysing CH 4 for δ 13 C and δ 2 H determination are, however, very time consuming, involving offline manual extractions. Methods: Here we describe a new gas chromatography, pyrolysis/combustion, isotope ratio mass spectrometry (IRMS) system for the automated analysis of either dissolved or gaseous CH 4 down to ambient atmospheric concentrations (2.0 ppm). Sample introduction is via a traditional XYZ autosampler, allowing either helium (He) purging of gas or sparging of water from a range of suitable, airtight bottles. Results: The system routinely achieves precision of <0.3 ‰ for δ 13 C values and <3.0 ‰ for δ 2 H values, based on long-term replicate analysis of an in-house CH 4 /He mix standard (BGS-1), corrected to two externally calibrated reference gases at near atmospheric concentrations of methane. Depending upon CH 4 concentration and therefore bottle size, the system runs between 21 (140-mL bottle) and 200 samples (12-mL exetainer) in an unattended run overnight. Conclusions: This represents the first commercially available IRMS


| INTRODUCTION
Atmospheric methane (CH 4 ) is the second most abundant and potent greenhouse gas after carbon dioxide (CO 2 ). 1 Methane concentrations in the atmosphere have more than doubled (to 1858 ppb in 2018) since pre-industrial times, 2-5 with a current growth rate in the atmosphere of 10 ppm/year. 1 Therefore, understanding CH 4 formation pathways and sources of emission is critical to informing effective mitigation strategies and limiting the role of CH 4 in future climate change. 1 Important natural emission sources include shallow wetlands and water-saturated soils, 4,6,7 the digestive systems of ruminants and termites, and natural geological sources. 4 However, changes in the flux associated with anthropogenic sources represent the major factor responsible for the significant post-industrial CH 4 increase, with approximately 60% of CH 4 emissions attributed to human activities, both direct and indirect. 4 These include energy production (gas and coal), biomass burning, agriculture 8 (including rice farming), waste management, and leakages associated with subsurface natural gas extraction, 9 gas storage and piped delivery. 10 Evaluation of their relative contributions to the global CH 4 budget is, however, complex to determine and requires a robust set of geochemical tools to accurately identify methane sources and sinks. 5,11 Alongside aiding global warming within the atmosphere, high concentrations of dissolved CH 4 in groundwater degas rapidly and can build up to cause risk of explosion or asphyxiation when confined. 12 Recent investments in the shale gas and coal bed CH 4 industries have meant that fingerprinting CH 4 sources within groundwater is becoming a critical concern for operators and regulators alike. 9 Under natural or "baseline" conditions the majority of UK groundwater CH 4 is bacterially derived, 12 but leakages of thermogenic gas associated with hydraulic fracturing of shale deposits have been identified as a potential polluter of groundwater supplies within the USA, 13 as have leakages associated with gas wells 9 and buried pipework. 14 With future development of the shale gas industry in some regions, this issue requires careful monitoring pre-, during and post-hydraulic extraction. 9,12,15,16 The carbon (δ 13 C-CH 4 ) and hydrogen (δ 2 H-CH 4 ) isotope compositions of CH 4 can help identify mechanisms and sites of CH 4 formation and destruction both in atmospheric and in dissolved gas samples. 5 Stable isotope composition is a powerful tracer tool because of the unique isotope fractionations imparted during the different CH 4 production pathways. 5,8,17,18 CH 4 production is derived from three main sources, biogenic, thermogenic and pyrogenic (mainly biomass burning), with different average isotope ratios for each process, allowing for source attribution. 5,11 Biogenic production occurs either through the reduction of CO 2 or by the fermentation of reduced carbon substrates such as acetate and methanol. 18,19 The reduction of CO 2 to CH 4 discriminates against the heavier 13 C and 2 H isotopes, producing CH 4 with distinct, isotopically low δ 13 C-CH 4 (< −110‰) and δ 2 H-CH 4 signatures (−150 to −250‰). The fermentation of acetate and methylated substrates also causes kinetic fractionation, this time more pronounced in the δ 2 H-CH 4 composition: δ 13 C-CH 4 (< −50 to −60‰) and δ 2 H-CH 4 (−300 to −400‰). 18 It should be noted that recent work has demonstrated that some reduction processes, such as that during nitrogen fixation by nitrogenase, can lead to small amounts of CH 4 produced with much lower δ 2 H-CH 4 values (−560‰). 20 Differences in the δ 2 H-CH 4 values of microbially derived CH 4 can be used to distinguish CH 4 source, where terrestrial sources (marsh and glacial tills) have lower values than those for CH 4 derived from marine environments; much of this is associated with the δ 2 H composition of the surrounding water. 17 Post CH 4 formation, secondary processes of CH 4 consumption (both aerobic and anaerobic) can cause isotopic fractionations, resulting in the 13 C and 2 H enrichment of the residual CH 4 pool. 18 These processes can lead to the misinterpretation of residual CH 4 isotope ratios as this residual pool can reflect isotopic composition more characteristic of thermogenic CH 4 . Thermogenic CH 4 is derived from diagenesis which produces gas with relatively high δ 13 C-CH 4 values (−45 to −55‰). 11 This δ 13 C-CH 4 range observed in thermogenic methane 11 is controlled by a combination of factors including the precursor kerogen δ 13 C composition, the thermal maturity, the contribution of coal bed microbial methanogenisis 11 and the source rock type. The CH 4 becomes progressively enriched in 13 C with increasing thermal maturity, eventually approaching the original δ 13 C composition of the source organic material. 17,18,21 Further scrutiny of the δ 2 H-CH 4 composition of thermogenic CH 4 has been used on a site-specific basis to help to differentiate between the original organic matter sources of thermogenic CH 4 17 and to show heterogeneities in this source material. 21  (2) Approximately 20 mL of sample water is removed and helium (He) gas introduced via a gas-tight syringe system, creating 20 mL of inert gas headspace for dissolved CH 4 to degas into, the bottles are reweighed, and the exact volume of sample remaining is calculated.

| INSTRUMENT DESIGN
The CryoGas is a versatile gas sample preparation module coupled to a 20-22 isotope ratio mass spectrometer (CG-2022), configured in this case for the high-precision isotope analyses of CO 2 or H 2 ( Figure 1). The following sections describe sample introduction, the automated separation of CH 4 from other gas components, conversion into CO 2 or H 2 , and isotope measurement, developed from the designs of "lab built" instruments. 22
The sample bottle is flushed with lab grade 99.9% pure He (approx.

| Switching between carbon and hydrogen mode
Switching from carbon to hydrogen mode involves manually replacing the cryotraps T2 and T3 with short packed GC columns,

| Hydrogen (δ 2 H) mode
As the sample is purged from the bottle, it passes through a CO 2 (EMASorb, Sercon PN: SC0236) and chemical water scrubber    to be used at any time to automatically condition/re-condition the pyrolysis tube.
Once conditioned the tube is stable without the requirement for regular re-conditioning. If, however, the pyrolysis tube has been

| Isotope ratio, 17 O and H 3 + corrections
Callisto software (Sercon) automatically applies the necessary ion corrections for 17 O in CO 2 26 and calculates the peak area and the isotope ratio of the peaks. The software allows two modes of isotope F I G U R E 5 Standard MS peak showing CO 2 reference gas injection peak (purple box) 5 s baseline integration intervals for both reference and sample (yellow bars) and sample peak (grey box) for a δ 13 C-CH 4 measurement. The stable baseline highlights efficient contaminant trapping, showing no baseline disturbance pre-or post-peak and the sharp sample peak indicates effective cryofocusing post-combustion F I G U R E 6 A, Stable δ 13 C-CH 4 and B, δ 2 H-CH 4 data (raw vs reference gas) for samples between 100 and 0.8 nmol CH 4 (atmospheric values in 12 mL of air 23 ) highlighting the limit of detection of the CG-2022 system ratio characterisation. First, a single-point calibration can be applied automatically against one of the reference gases within the run. This is achieved by characterising this gas as a "Standard" within the run sheet and attributing the known isotope ratio to this standard gas.
The Callisto system then corrects all "unknowns" including other reference gases and samples to this single calibration gas. The second, and preferred method, is to run all the reference and sample gases as "unknowns", with the Callisto system correcting these to the working reference gas delta value (tank CO 2 or H 2 ). This allows for an initial calculation of the isotope ratio vs working gas within the software.
Assignment of the isotope ratio vs calibrated standard materials is then undertaken offline, following blank and drift correction via a two-point calibration to AL-high and AL-low with BGS-1 acting as a check standard, for both within-run and long-term accuracy, and precision characterisation. Especially for natural CH 4 samples this two-point correction is highly recommended due to the wide range of possible δ 13 C-CH 4 and δ 2 H-CH 4 values found in natural gases and the need to bracket this range as well as possible with references.
For H 2 , the H 3 + factor is determined via one of two methods.
First, this can be done by running a series of different sized reference gas peaks spanning the range of the samples and plotting the beam area incidence in nano amps against the uncorrected 2/1 ratio. in <150 mL of air as a sensible trade-off between sample collection/ storage in the field and analytical complexity. As suggested in section 2, for this system (based on significant prototype testing) we recommend collection of 140 mL of air for both δ 13 C-CH 4

| Carryover
Large variations in methane isotope ratios are seen within natural samples, >10s of ‰ in δ 13 C-CH 4 values and >100s of ‰ in δ 2 H-CH 4 values. 1,27 It is therefore fundamental that instruments regularly analysing CH 4 to attribute the source, either of gas or of dissolved gas, are able to cope with a large dynamic isotope range with minimal carryover effects between samples. To test this for the CG-2022 system we analysed our external standards and internal reference gas to ascertain the extent of carryover from one sample to the next across a large isotope ratio range. Figures 7A and 7B show the results for δ 13 C-CH 4 values and δ 2 H-CH 4 values, respectively, all run against the instrument working gas. Ordering of gases in this manner (ALhigh, AL-low, BGS-1) enabled the largest possible isotope jumps to be made based on the available gases; no obvious indication of sample carryover is observed, with each gas falling within the stated error range of the instrument (section 6.1).

| CONCLUSIONS
The CG-2022 IRMS system offers a versatile "off the shelf" option for the dual measurement of δ 13 C-CH 4  where a wide range of CH 4 isotope ratios could be expected.