Emily R Stuchiner1,2, Zachary D Weller3, Joseph C von Fischer1,2. 1. Department of Biology, Colorado State University Fort Collins, CO, 80523, USA. 2. Graduate Degree Program in Ecology, Colorado State University Fort Collins, CO, 80523, USA. 3. Department of Statistics, Colorado State University Fort Collins, CO, 80523, USA.
Abstract
RATIONALE: Technological advances have motivated researchers to transition from traditional gas chromatography/isotope ratio mass spectrometry to rapid, high-throughput, laser-based instrumentation for N2 O isotopic research. However, calibrating laser-based instruments to yield accurate and precise isotope ratios has been an ongoing challenge. To streamline the N2 O isotope research pipeline, we developed the calibration protocol for laser-based analyzers described here. While our approach is targeted at laboratory soil incubations, we anticipate that it will be broadly applicable for diverse types of stable isotope research. METHODS: We prepared standards diluted from USGS52 and from a commercial cylinder to develop a calibration curve spanning from 0.3 to 300 ppm N2 O. To calibrate over this broad range, we binned each isotopocule (N2 O, N15 NO, 15 NNO, and NN18 O) into low, medium, and high concentration ranges and then used mathematically similar polynomial functions to calibrate the isotopocules within each concentration range. We also assessed the temporal stability of the instrument and the capacity for our calibration approach to work with isotopically enriched gas samples. RESULTS: Our calibration approach yielded generally accurate and precise data when isotopocules were calibrated in concentration ranges, and the measurements appeared to be temporally stable. For all isotopocules at natural abundance, the residual percentage error was smallest in the medium N2 O range. There was more noise in the corrected isotopomers and isotopologue at natural abundance in samples with the lowest and highest N2 O concentrations. Corrected isotopomer results from isotopically enriched samples were very precise. CONCLUSIONS: Developing our calibration strategy involved learning several key lessons: (1) calibrate isotopocules in distinct concentration ranges, (2) use mathematically similar models to calibrate the isotopocules in each range, (3) calibrated N2 O concentrations and δ values tend to be most accurate and precise in the medium N2 O range, and (4) we encourage users to take advantage of isotopic enrichment to capitalize on laser-based instrument strengths.
RATIONALE: Technological advances have motivated researchers to transition from traditional gas chromatography/isotope ratio mass spectrometry to rapid, high-throughput, laser-based instrumentation for N2 O isotopic research. However, calibrating laser-based instruments to yield accurate and precise isotope ratios has been an ongoing challenge. To streamline the N2 O isotope research pipeline, we developed the calibration protocol for laser-based analyzers described here. While our approach is targeted at laboratory soil incubations, we anticipate that it will be broadly applicable for diverse types of stable isotope research. METHODS: We prepared standards diluted from USGS52 and from a commercial cylinder to develop a calibration curve spanning from 0.3 to 300 ppm N2 O. To calibrate over this broad range, we binned each isotopocule (N2 O, N15 NO, 15 NNO, and NN18 O) into low, medium, and high concentration ranges and then used mathematically similar polynomial functions to calibrate the isotopocules within each concentration range. We also assessed the temporal stability of the instrument and the capacity for our calibration approach to work with isotopically enriched gas samples. RESULTS: Our calibration approach yielded generally accurate and precise data when isotopocules were calibrated in concentration ranges, and the measurements appeared to be temporally stable. For all isotopocules at natural abundance, the residual percentage error was smallest in the medium N2 O range. There was more noise in the corrected isotopomers and isotopologue at natural abundance in samples with the lowest and highest N2 O concentrations. Corrected isotopomer results from isotopically enriched samples were very precise. CONCLUSIONS: Developing our calibration strategy involved learning several key lessons: (1) calibrate isotopocules in distinct concentration ranges, (2) use mathematically similar models to calibrate the isotopocules in each range, (3) calibrated N2 O concentrations and δ values tend to be most accurate and precise in the medium N2 O range, and (4) we encourage users to take advantage of isotopic enrichment to capitalize on laser-based instrument strengths.