Biodegradability screening of soil amendments through coupling of wavelength‐scanned cavity ring‐down spectroscopy to multiple dynamic chambers

A system was developed for the automatic measurements of ¹³CO₂ efflux to determine biodegradation of extra carbon amendments to soils. The system combines wavelength‐scanned cavity ring down laser spectroscopy (WS‐CRDS) with the open‐dynamic chamber (ODC) method. The WS‐CRDS instrument and a batch of 24 ODC are coupled via microprocessor‐controlled valves. Determination of the biodegradation requires a known δ¹³C value and the applied mass of the carbon compounds, and the biodegradation is calculated based on the ¹³CO₂ mixing ratio (ppm) sampled from the headspace of the chambers. The WS‐CRDS system provided accurate detection based on parallel samples of three standard gases (¹³CO₂ of 2, 11 and 22 ppm) that were measured simultaneously by isotope ratio mass spectrometry (linear regression R² = 0.99). Repeated checking with the same standards showed that the WS‐CRDS system showed no drift over seven months. The applicability of the ODC was checked against the closed static chamber (CSC) method using the rapid biodegradation of cane sugar – δ¹³C‐labeled through C4 photosynthesis. There was no significant difference between the results from 7‐min ODC and 120‐min CSC measurements. Further, a test using samples of either cane sugar (C4) or beetroot sugar (C3) mixed into standard soil proved the target functionality of the system, which is to identify the biodegradation of carbon sources with significantly different isotopic signatures. Copyright © 2011 John Wiley & Sons, Ltd.     

Continuous analysis of δ18O and δD values of water by diffusion sampling cavity ring‐down spectrometry: a novel sampling device for unattended field monitoring of precipitation,ground and surface waters

A novel sampling device suitable for continuous, unattended field monitoring of rapid isotopic changes in environmental waters is described. The device utilises diffusion through porous PTFE tubing to deliver water vapour continuously from a liquid water source for analysis of δ¹⁸O and δD values by Cavity Ring‐Down Spectrometry (CRDS). Separation of the analysed water vapour from non‐volatile dissolved and particulate contaminants in the liquid sample minimises spectral interferences associated with CRDS analyses of many aqueous samples. Comparison of isotopic data for a range of water samples analysed by Diffusion Sampling‐CRDS (DS‐CRDS) and Isotope Ratio Mass Spectrometry (IRMS) shows significant linear correlations between the two methods allowing for accurate standardisation of DS‐CRDS data. The internal precision for an integration period of 3 min (standard deviation (SD) = 0.1 ‰ and 0.3 ‰ for δ¹⁸O and δD values, respectively) is similar to analysis of water by CRDS using an autosampler to inject and evaporate discrete water samples. The isotopic effects of variable air temperature, water vapour concentration, water pumping rate and dissolved organic content were found to be either negligible or correctable by analysis of water standards. The DS‐CRDS system was used to analyse the O and H isotope composition in short‐lived rain events. Other applications where finely time resolved water isotope data may be of benefit include recharge/discharge in groundwater/river systems and infiltration‐related changes in cave drip water. Copyright © 2011 John Wiley & Sons, Ltd.     

Concentration effects on laser-based δ18 O and δ2 H measurements and implications for the calibration of vapour measurements with liquid standards

Recently available isotope ratio infrared spectroscopy can directly measure the isotopic composition of atmospheric water vapour (δ¹⁸O, δ²H), overcoming one of the main limitations of isotope ratio mass spectrometry (IRMS) methods. Calibrating these gas‐phase instruments requires the vapourisation of liquid standards since primary standards in principle are liquids. Here we test the viability of calibrating a wavelength‐scanned cavity ring‐down spectroscopy (CRDS) instrument with vapourised liquid standards. We also quantify the dependency of the measured isotope values on the water concentration for a range of isotopic compositions. In both liquid and vapour samples, we found an increase in δ¹⁸O and δ²H with water vapour concentration. For δ¹⁸O, the slope of this increase was similar for liquid and vapour, with a slight positive relationship with sample δ‐value. For δ²H, we found diverging patterns for liquid and vapour samples, with no dependence on δ‐value for vapour, but a decreasing slope for liquid samples. We also quantified tubing memory effects to step changes in isotopic composition, avoiding concurrent changes in the water vapour concentration. Dekabon tubing exhibited much stronger, concentration‐dependent, memory effects for δ²H than stainless steel or perfluoroalkoxy (PFA) tubing. Direct vapour measurements with CRDS in a controlled experimental chamber agreed well with results obtained from vapour simultaneously collected in cold traps analysed by CRDS and IRMS. We conclude that vapour measurements can be calibrated reliably with liquid standards. We demonstrate how to take the concentration dependencies of the δ‐values into account. Copyright © 2010 John Wiley & Sons, Ltd.     

Doubly labeled water analysis using cavity ring-down spectroscopy

The doubly labeled water method provides an objective and accurate measure of total energy expenditure in free‐living subjects and is considered the gold‐standard method for this measurement. Its use, however, is limited by the need to employ isotope ratio mass spectrometry (IRMS) to obtain the high‐precision isotopic abundance analyses needed to optimize the dose of expensive ¹⁸O‐labeled water. Recently, cavity‐ring down spectroscopy (CRDS) instruments have become commercially available and may serve as a less expensive alternative to IRMS. We compared the precision and accuracy of CRDS with those of IRMS for the measurement of total energy expenditure from urine specimens in 14 human subjects. The relative accuracy and precision (SD) for total body water was 0.5 ± 1% and for total energy expenditure was 0.5 ± 6%. The CRDS instrument displayed a memory between successive specimens of 5% for ¹⁸O and 9% for ²H. The memory necessitated carefully ordering of specimens to reduce isotopic disparity, performance of several injections of each specimen to condition the analyzer, and use of a mathematical memory correction on subsequent injections. These limited the specimen throughput to about one urine specimen per hour. CRDS provided accuracy and precision for isotope abundance measurements of urine that were comparable with those of IRMS. The memory problems were easily recognized by our experienced laboratory staff, but future efforts should be aimed at reducing the memory of the CRDS so that it would be less likely to result in poor reproducibility in laboratories using doubly labeled water for the first time. Copyright © 2010 John Wiley & Sons, Ltd.     

Can we use the CO2 concentrations determined by continuous‐flow isotope ratio mass spectrometry from small samples for the Keeling plot approach?

A common method to estimate the carbon isotopic composition of soil‐respired air is to use Keeling plots (δ¹³C versus 1/CO₂ concentration). This approach requires the precise determination of both CO₂ concentration ([CO₂]), usually measured with an infrared gas analyser (IRGA) in the field, and the analysis of δ¹³C by isotope ratio mass spectrometry (IRMS) in the laboratory. We measured [CO₂] with an IRGA in the field (n = 637) and simultaneously collected air samples in 12 mL vials for analysis of the ¹³C values and the [CO₂] using a continuous‐flow isotope ratio mass spectrometer. In this study we tested if measurements by the IRGA and IRMS yielded the same results for [CO₂], and also investigated the effects of different sample vial preparation methods on the [CO₂] measurement and the thereby obtained Keeling plot results. Our results show that IRMS measurements of the [CO₂] (during the isotope analysis) were lower than when the [CO₂] was measured in the field with the IRGA. This is especially evident when the sample vials were not treated in the same way as the standard vials. From the three different vial preparation methods, the one using N₂‐filled and overpressurised vials resulted in the best agreement between the IRGA and IRMS [CO₂] values. There was no effect on the ¹³C‐values from the different methods. The Keeling plot results confirmed that the overpressurised vials performed best. We conclude that in the cases where the ranges of [CO₂] are large (>300 ppm; in our case it ranged between 70 and 1500 ppm) reliable estimation of the [CO₂] with small samples using IRMS is possible for Keeling plot application. We also suggest some guidelines for sample handling in order to achieve proper results. Copyright © 2008 John Wiley & Sons, Ltd.     

Membrane permeation continuous‐flow isotope ratio mass spectrometry for on‐line carbon isotope ratio determination

Gaseous membrane permeation (MP) technologies have been combined with continuous‐flow isotope ratio mass spectrometry for on‐line δ¹³C measurements. The experimental setup of membrane permeation‐gas chromatography/combustion/isotope ratio mass spectrometry (MP‐GC/C/IRMS) quantitatively traps gas streams in membrane permeation experiments under steady‐state conditions and performs on‐line gas transfer into a GC/C/IRMS system. A commercial polydimethylsiloxane (PDMS) membrane sheet was used for the experiments. Laboratory tests using CO₂ demonstrate that the whole process does not fractionate the C isotopes of CO₂. Moreover, the δ¹³C values of CO₂ permeated on‐line give the same isotopic results as off‐line static dual‐inlet IRMS δ¹³C measurements. Formaldehyde generated from aqueous formaldehyde solutions has also been used as the feed gas for permeation experiments and on‐line δ¹³C determination. The feed‐formaldehyde δ¹³C value was pre‐determined by sampling the headspace of the thermostated aqueous formaldehyde solution. Comparison of the results obtained by headspace with those from direct aqueous formaldehyde injection confirms that the headspace sampling does not generate isotopic fractionation, but the permeated formaldehyde analyzed on‐line yields a ¹³C enrichment relative to the feed δ¹³C value, the isotopic fractionation being 1.0026 ± 0.0003. The δ¹³C values have been normalized using an adapted two‐point isotopic calibration for δ¹³C values ranging from ?42 to ?10‰. The MP‐GC/C/IRMS system allows the δ¹³C determination of formaldehyde without chemical derivatization or additional analytical imprecision. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatography/mass spectrometry stable isotope analysis of dissolved organic carbon in stream and soil waters

A commercial interface coupling liquid chromatography (LC) to a continuous‐flow isotope ratio mass spectrometry (CF‐IRMS) instrument was used to determine the δ¹³C of dissolved organic carbon (DOC) in natural waters. Stream and soil waters from a farmland plot in a hedgerow landscape were studied. Based on wet chemical oxidation of dissolved organics the LC/IRMS interface allows the on‐line injection of small volumes of water samples, an oxidation reaction to produce CO₂ and gas transfer to the isotope ratio mass spectrometer. In flow injection analysis (FIA) mode, bulk DOC δ¹³C analysis was performed on aqueous samples of up to 100 μL in volume in the range of DOC concentration in fresh waters (1–10 mg C.L^–1). Mapping the DOC δ¹³C spatial distribution at the plot scale was made possible by this fairly quick method (10 min for triplicate analyses) with little sample manipulation. The relative contributions of different plot sectors to the DOC pool in the stream draining the plot were tentatively inferred on the basis of δ¹³C differences between the hydrophilic and hydrophobic components. Copyright © 2011 John Wiley & Sons, Ltd.     

Oxygen isotope analysis of phosphate: improved precision using TC/EA CF‐IRMS

Oxygen isotope values of biogenic apatite have long demonstrated considerable promise for paleothermometry potential because of the abundance of material in the fossil record and greater resistance of apatite to diagenesis compared to carbonate. Unfortunately, this promise has not been fully realized because of relatively poor precision of isotopic measurements, and exceedingly small size of some substrates for analysis. Building on previous work, we demonstrate that it is possible to improve precision of δ¹⁸O_PO4 measurements using a ‘reverse‐plumbed’ thermal conversion elemental analyzer (TC/EA) coupled to a continuous flow isotope ratio mass spectrometer (CF‐IRMS) via a helium stream [Correction made here after initial online publication]. This modification to the flow of helium through the TC/EA, and careful location of the packing of glassy carbon fragments relative to the hot spot in the reactor, leads to narrower, more symmetrically distributed CO elution peaks with diminished tailing. In addition, we describe our apatite purification chemistry that uses nitric acid and cation exchange resin. Purification chemistry is optimized for processing small samples, minimizing isotopic fractionation of PO₄^?3 and permitting Ca, Sr and Nd to be eluted and purified further for the measurement of δ⁴⁴Ca and ⁸⁷Sr/⁸⁶Sr in modern biogenic apatite and ¹⁴³Nd/¹⁴⁴Nd in fossil apatite. Our methodology yields an external precision of ± 0.15‰ (1σ) for δ¹⁸O_PO4. The uncertainty is related to the preparation of the Ag₃PO₄ salt, conversion to CO gas in a reversed‐plumbed TC/EA, analysis of oxygen isotopes using a CF‐IRMS, and uncertainty in constructing calibration lines that convert raw δ¹⁸O data to the VSMOW scale. Matrix matching of samples and standards for the purpose of calibration to the VSMOW scale was determined to be unnecessary. Our method requires only slightly modified equipment that is widely available. This fact, and the demonstrated improvement in precision, should help to make apatite paleothermometry far more accessible to paleoclimate researchers. Copyright © 2009 John Wiley & Sons, Ltd.     

Stable isotopic analysis of pyrogenic organic matter in soils by liquid chromatography–isotope‐ratio mass spectrometry of benzene polycarboxylic acids

Pyrogenic organic matter (PyOM), the incomplete combustion product of organic materials, is considered stable in soils and represents a potentially important terrestrial sink for atmospheric carbon dioxide. One well‐established method of measuring PyOM in the environment is as benzene polycarboxylic acids (BPCAs), a compound‐specific method, which allows both qualitative and quantitative estimation of PyOM. Until now, stable isotope measurement of PyOM carbon involved measurement of the trimethylsilyl (TMS) or methyl (Me) polycarboxylic acid derivatives by gas chromatography–combustion–isotope ratio mass spectrometry (GC‐C‐IRMS). However, BPCA derivatives can contain as much as 150% derivative carbon, necessitating post‐analysis correction for the accurate measurement of δ¹³ C values, leading to increased measurement error. Here, we describe a method for δ¹³ C isotope ratio measurement and quantification of BPCAs from soil‐derived PyOM, based on ion‐exchange chromatography (IEC‐IRMS). The reproducibility of the δ¹³ C measurement of individual BPCAs by IEC‐IRMS was better than 0.35‰ (1σ). The δ¹³ C‐BPCA analysis of PyOM in soils, including at natural and artificially enriched ¹³ C‐abundance, produced accurate and precise δ¹³ C measurements. Analysis of samples that differed in δ¹³ C by as much as 900‰ revealed carryover of <1‰ between samples. The weighted sum of individual δ¹³ C‐BPCA measurements was correlated with previous isotopic measurements of whole PyOM, providing complementary information for bulk isotopic measurements. We discuss potential applications of δ¹³ C‐BPCA measurements, including the study of turnover rates of PyOM in soils and the partitioning of PyOM sources based on photosynthetic pathways. Copyright © 2011 John Wiley & Sons, Ltd.     

Reducing and correcting for contamination of ecosystem water stable isotopes measured by isotope ratio infrared spectroscopy

Concern exists about the suitability of laser spectroscopic instruments for the measurement of the (18)O/(16)O and (2)H/(1)H values of liquid samples other than pure water. It is possible to derive erroneous isotope values due to optical interference by certain organic compounds, including some commonly present in ecosystem-derived samples such as leaf or soil waters. Here we investigated the reliability of wavelength-scanned cavity ring-down spectroscopy (CRDS) (18)O/(16)O and (2)H/(1)H measurements from a range of ecosystem-derived waters, through comparison with isotope ratio mass spectrometry (IRMS). We tested the residual of the spectral fit S(r) calculated by the CRDS instrument as a means to quantify the difference between the CRDS and IRMS δ-values. There was very good overall agreement between the CRDS and IRMS values for both isotopes, but differences of up to 2.3‰ (δ(18)O values) and 23‰ (δ(2)H values) were observed in leaf water extracts from Citrus limon and Alnus cordata. The S(r) statistic successfully detected contaminated samples. Treatment of Citrus leaf water with activated charcoal reduced, but did not eliminate, δ(2)H(CRDS) - δ(2)H(IRMS) linearly for the tested range of 0-20% charcoal. The effect of distillation temperature on the degree of contamination was large, particularly for δ(2)H values but variable, resulting in positive, negative or no correlation with distillation temperature. S(r) and δ(CRDS) - δ(IRMS) were highly correlated, in particular for δ(2)H values, across the range of samples that we tested, indicating the potential to use this relationship to correct the δ-values of contaminated plant water extracts. We also examined the sensitivity of the CRDS system to changes in the temperature of its operating environment. We found that temperature changes ≥4 °C for δ(18)O values and ≥10 °C for δ(2)H values resulted in errors larger than the CRDS precision for the respective isotopes and advise the use of such instruments only in sufficiently temperature-stabilised environments.     

Nicotine,acetanilide and urea multi‐level 2H‐, 13C‐ and 15N‐abundance reference materials for continuous‐flow isotope ratio mass spectrometry

Accurate determinations of stable isotope ratios require a calibration using at least two reference materials with different isotopic compositions to anchor the isotopic scale and compensate for differences in machine slope. Ideally, the δ values of these reference materials should bracket the isotopic range of samples with unknown δ values. While the practice of analyzing two isotopically distinct reference materials is common for water (VSMOW‐SLAP) and carbonates (NBS 19 and L‐SVEC), the lack of widely available organic reference materials with distinct isotopic composition has hindered the practice when analyzing organic materials by elemental analysis/isotope ratio mass spectrometry (EA‐IRMS). At present only L‐glutamic acids USGS40 and USGS41 satisfy these requirements for δ¹³C and δ¹⁵N, with the limitation that L‐glutamic acid is not suitable for analysis by gas chromatography (GC). We describe the development and quality testing of (i) four nicotine laboratory reference materials for on‐line (i.e. continuous flow) hydrogen reductive gas chromatography‐isotope ratio mass‐spectrometry (GC‐IRMS), (ii) five nicotines for oxidative C, N gas chromatography‐combustion‐isotope ratio mass‐spectrometry (GC‐C‐IRMS, or GC‐IRMS), and (iii) also three acetanilide and three urea reference materials for on‐line oxidative EA‐IRMS for C and N. Isotopic off‐line calibration against international stable isotope measurement standards at Indiana University adhered to the ‘principle of identical treatment’. The new reference materials cover the following isotopic ranges: δ²H_nicotine ?162 to ?45‰, δ¹³C_nicotine ?30.05 to +7.72‰, δ¹⁵N_nicotine ?6.03 to +33.62‰; δ¹⁵N_acetanilide +1.18 to +40.57‰; δ¹³C_urea ?34.13 to +11.71‰, δ¹⁵N_urea +0.26 to +40.61‰ (recommended δ values refer to calibration with NBS 19, L‐SVEC, IAEA‐N‐1, and IAEA‐N‐2). Nicotines fill a gap as the first organic nitrogen stable isotope reference materials for GC‐IRMS that are available with different δ¹⁵N values. Comparative δ¹³C and δ¹⁵N on‐line EA‐IRMS data from 14 volunteering laboratories document the usefulness and reliability of acetanilides and ureas as EA‐IRMS reference materials. Published in 2009 by John Wiley & Sons, Ltd.     

Methods and limitations of ‘clumped’ CO2 isotope (Δ47) analysis by gas‐source isotope ratio mass spectrometry

The geochemistry of multiply substituted isotopologues (‘clumped‐isotope’ geochemistry) examines the abundances in natural materials of molecules, formula units or moieties that contain more than one rare isotope (e.g. ¹³C¹⁸O¹⁶O, ¹⁸O¹⁸O, ¹⁵N₂, ¹³C¹⁸O¹⁶O₂^2?). Such species form the basis of carbonate clumped‐isotope thermometry and undergo distinctive fractionations during a variety of natural processes, but initial reports have provided few details of their analysis. In this study, we present detailed data and arguments regarding the theoretical and practical limits of precision, methods of standardization, instrument linearity and related issues for clumped‐isotope analysis by dual‐inlet gas‐source isotope ratio mass spectrometry (IRMS). We demonstrate long‐term stability and subtenth per mil precision in 47/44 ratios for counting systems consisting of a Faraday cup registered through a 10¹² Ω resistor on three Thermo‐Finnigan 253 IRMS systems. Based on the analyses of heated CO₂ gases, which have a stochastic distribution of isotopes among possible isotopologues, we document and correct for (1) isotopic exchange among analyte CO₂ molecules and (2) subtle nonlinearity in the relationship between actual and measured 47/44 ratios. External precisions of ～0.01‰ are routinely achieved for measurements of the mass‐47 anomaly (a measure mostly of the abundance anomaly of ¹³C‐¹⁸O bonds) and follow counting statistics. The present technical limit to precision intrinsic to our methods and instrumentation is ～5 parts per million (ppm), whereas precisions of measurements of heterogeneous natural materials are more typically ～10 ppm (both 1 s.e.). These correspond to errors in carbonate clumped‐isotope thermometry of ±1.2 °C and ±2.4 °C, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Testing the use of septum‐capped vials for 13C‐isotope abundance analysis of carbon dioxide

Studying ecosystem processes in the context of carbon cycling and climate change has never been more important. Stable carbon isotope studies of gas exchange within terrestrial ecosystems are commonly undertaken to determine sources and rates of carbon cycling. To this end, septum‐capped vials (‘Exetainers’) are often used to store samples of CO₂ prior to mass spectrometric analysis. To evaluate the performance of such vials for preserving the isotopic integrity (δ¹³C) and concentration of stored CO₂ we performed a rigorous suite of tests. Septum‐capped vials were filled with standard gases of varying CO₂ concentrations (～700 to 4000 ppm), δ¹³C values (approx. ?26.5 to +1.8‰_V‐PDB) and pressures (33 and 67% above ambient), and analysed after a storage period of between 7 and 28 days. The vials performed well, with the vast majority of both isotope and CO₂ concentration results falling within the analytical uncertainty of chamber standard gas values. Although the study supports the use of septum‐capped vials for storing samples prior to mass spectrometric analysis, it does highlight the need to ensure that sampling chamber construction is robust (air‐tight). Copyright © 2010 John Wiley & Sons, Ltd.     

Direct analysis of carbon isotope variability in albumins by liquid flow-injection isotope ratio mass spectrometry

We demonstrate the high precision C isotopic analysis of a series of purified albumins by liquid chromatography-combustion isotope ratio mass spectrometry (IRMS) by using direct aqueous liquid injection. Albumins from 18 species and albumens from chicken and turkey egg were obtained from a commercial source and shown to be of > 98% purity by capillary zone electrophoresis and high-performance liquid chromatography. One microliter of an aqueous protein solution with a total of < 40-pmol protein (2. 5 µg), which contained about 150-nmol C, was injected directly into a flowing stream of high-performance liquid chromatography grade water. The solution passed through a pneumatic nebulizer, was sprayed onto a moving wire, passed through a drying oven, and was combusted in a furnace. After the water of combustion was removed, the resulting CO₂ gas was directed to a high precision IRMS instrument operated in continuous flow mode. The average precision across the 20 samples analyzed was SD(δ ¹³C)=0.45%., and the average accuracy was δ¹³C < 0.4%. compared to aliquots analyzed by conventional preparation by using combustion tubes and dual inlet analysis. The observed isotope ratio range was about ?22.5%. < δ ¹³C_PDB < ?16%. as expected for modern materials from a natural source. These results demonstrate rapid, high precision, and accurate C isotopic analysis of untreated macromolecules in an aqueous stream by liquid source IRMS.     

Comparison of different mass spectrometry techniques in the measurement of L‐[ring‐13C6]phenylalanine incorporation into mixed muscle proteins

Precise measurement of low enrichment of stable isotope labeled amino‐acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L‐[ring‐¹³C₆]phenylalanine and a bolus dose of L‐[ring‐¹³C₆]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC‐combustion‐isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring‐¹³C₆]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R² = 0.9962 and R² = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra‐assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter‐assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L‐[ring‐¹³C₆]phenylalanine tracer enrichment in low abundance and in small quantity samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Calcite‐CO2 mixed into CO2‐free air: a new CO2‐in‐air stable isotope reference material for the VPDB scale

In order to generate a reliable and long‐lasting stable isotope ratio standard for CO₂ in samples of clean air, CO₂ is liberated from well‐characterized carbonate material and mixed with CO₂‐free air. For this purpose a dedicated acid reaction and air mixing system (ARAMIS) was designed. In the system, CO₂ is generated by a conventional acid digestion of powdered carbonate. Evolved CO₂ gas is mixed and equilibrated with a prefabricated gas comprised of N₂, O₂, Ar, and N₂O at close to ambient air concentrations. Distribution into glass flasks is made stepwise in a highly controlled fashion. The isotopic composition, established on automated extraction/measurement systems, varied within very small margins of error appropriate for high‐precision air‐CO₂ work (about ±0.015‰ for δ¹³C and ±0.025‰ for δ¹⁸O). To establish a valid δ¹⁸O relation to the VPDB scale, the temperature dependence of the reaction between 25 and 47°C has been determined with a high level of precision. Using identical procedures, CO₂‐in‐air mixtures were generated from a selection of reference materials; (1) the material defining the VPDB isotope scale (NBS 19, δ¹³C = +1.95‰ and δ¹⁸O = ?2.2‰ exactly); (2) a local calcite similar in isotopic composition to NBS 19 (‘MAR‐J1’, δ¹³C = +1.97‰ and δ¹⁸O = ?2.02‰), and (3) a natural calcite with isotopic compositions closer to atmospheric values (‘OMC‐J1’, δ¹³C = ?4.24‰ and δ¹⁸O = ?8.71‰). To quantitatively control the extent of isotope‐scale contraction in the system during mass spectrometric measurement other available international and local carbonate reference materials (L‐SVEC, IAEA‐CO‐1, IAEA‐CO‐8, CAL‐1 and CAL‐2) were also processed. As a further control pure CO₂ reference gases (Narcis I and II, NIST‐RM 8563, GS19 and GS20) were mixed with CO₂‐free synthetic air. Independently, the pure CO₂ gases were measured on the dual inlet systems of the same mass spectrometers. The isotopic record of a large number of independent batches prepared over the course of several months is presented. In addition, the relationship with other implementations of the VPDB‐scale for CO₂‐in‐air (e.g. CG‐99, based on calibration of pure CO₂ gas) has been carefully established. The systematic high‐precision comparison of secondary carbonate and CO₂ reference materials covering a wide range in isotopic composition revealed that assigned δ‐values may be (slightly) in error. Measurements in this work deviate systematically from assigned values, roughly scaling with isotopic distance from NBS 19. This finding indicates that a scale contraction effect could have biased the consensus results. The observation also underlines the importance of cross‐contamination errors for high‐precision isotope ratio measurements. As a result of the experiments, a new standard reference material (SRM), which consists of two 5‐L glass flasks containing air at 1.6 bar and the CO₂ evolved from two different carbonate materials, is available for distribution. These ‘J‐RAS’ SRM flasks (‘Jena‐Reference Air Set’) are designed to serve as a high‐precision link to VPDB for improving inter‐laboratory comparability. a Copyright © 2005 John Wiley & Sons, Ltd.     

A system for high‐quality CO2 isotope analyses of air samples collected by the CARIBIC Airbus A340‐600

In 2007, JRC‐IRMM began a series of atmospheric CO₂ isotope measurements, with the focus on understanding instrumental effects, corrections as well as metrological aspects. The calibration approach at JRC‐IRMM is based on use of a plain CO₂ sample (working reference CO₂) as a calibration carrier and CO₂‐air mixtures (in high‐pressure cylinders) to determine the method‐related correction under actual analytical conditions (another calibration carrier, in the same form as the samples). Although this approach differs from that in other laboratories, it does give a direct link to the primary reference NBS‐19‐CO₂. It also helps to investigate the magnitude and nature for each of the instrumental corrections and allows for the quantification of the uncertainty introduced. Critical tests were focused on the instrumental corrections. It was confirmed that the use of non‐symmetrical capillary crimping (an approach used here to deal with small samples) systematically modifies δ¹³C(CO₂) and δ¹⁸O(CO₂), with a clear dependence on the amount of extracted CO₂. However, the calibration of CO₂‐air mixtures required the use of the symmetrical dual‐inlet mode. As a proof of our approach, we found that δ¹³C(CO₂) on extracts from mixtures agreed (within 0.010‰) with values obtained from the ‘mother’ CO₂ used for the mixtures. It was further found that very low levels of hydrocarbons in the pumping systems and the isotope ratio mass spectrometry (IRMS) instrument itself were critical. The m/z 46 values (consequently the calculated δ¹⁸O(CO₂) values) are affected by several other effects with traces of air co‐trapped with frozen CO₂ being the most critical. A careful cryo‐distillation of the extracted CO₂ is recommended. After extensive testing, optimisation, and routine automated use, the system was found to give precise data on air samples that can be traced with confidence to the primary standards. The typical total combined uncertainty in δ¹³C(CO₂) and δ¹⁸O(CO₂) on the VPDB‐CO₂ scale, estimated on runs of CO₂‐air mixtures, is ±0.040‰ and 0.060‰ (2‐σ values). Inter‐comparison with MPI‐BGC resulted in a scale discrepancy of a similar magnitude. Although the reason(s) for this discrepancy still need to be understood, this basically confirms the approach of using specifically prepared CO₂‐air mixtures as a calibration carrier, in order to achieve scale unification among laboratories. As important practical application and as a critical test, JRC‐IRMM took part in the passenger aircraft‐based global monitoring project CARIBIC ( http://www.caribic‐atmospheric.com ). In this way, reliable CO₂ isotope data for the tropopause region and the free troposphere were obtained. From June 2007 to January 2009, approximately 500 CARIBIC air samples have been analysed. Some flights demonstrated a compact correlation of both δ¹³C(CO₂) and δ¹⁸O(CO₂) with respect to CO₂ concentration, demonstrating mixing of tropospheric and stratospheric air masses. These excellent correlations provide an independent, realistic data quality check. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous analysis of 13C‐glutathione as its dimeric form GSSG and its precursor [1‐13C]glycine using liquid chromatography/isotope ratio mass spectrometry

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine ¹³C‐glutathione as its dimeric form (GSSG) and its precursor [1‐¹³C]glycine in a small volume of erythrocytes in one single analysis. After having transformed ¹³C‐glutathione into its dimeric form GSSG, we determined both the intra‐erythrocytic concentrations and the ¹³C‐isotopic enrichment of GSSG and glycine in 150 µL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of µmol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 µmol/mL. The ¹³C‐isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3‰) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants. Copyright © 2009 John Wiley & Sons, Ltd.     

High-temperature reversed-phase liquid chromatography coupled to isotope ratio mass spectrometry

Compound‐specific isotope analysis (CSIA) by liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) has until now been based on ion‐exchange separation. In this work, high‐temperature reversed‐phase liquid chromatography was coupled to, and for the first time carefully evaluated for, isotope ratio mass spectrometry (HT‐LC/IRMS) with four different stationary phases. Under isothermal and temperature gradient conditions, the column bleed of XBridge C₁₈ (up to 180 °C), Acquity C₁₈ (up to 200 °C), Triart C₁₈ (up to 150 °C), and Zirchrom PBD (up to 150 °C) had no influence on the precision and accuracy of δ¹³C measurements, demonstrating the suitability of these columns for HT‐LC/IRMS analysis. Increasing the temperature during the LC/IRMS analysis of caffeine on two C₁₈ columns was observed to result in shortened analysis time. The detection limit of HT‐RPLC/IRMS obtained for caffeine was 30 mg L^–1 (corresponding to 12.4 nmol carbon on‐column). Temperature‐programmed LC/IRMS (i) accomplished complete separation of a mixture of caffeine derivatives and a mixture of phenols and (ii) did not affect the precision and accuracy of δ¹³C measurements compared with flow injection analysis without a column. With temperature‐programmed LC/IRMS, some compounds that coelute at room temperature could be baseline resolved and analyzed for their individual δ¹³C values, leading to an important extension of the application range of CSIA. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of low‐concentration gas samples with continuous‐flow isotope ratio mass spectrometry: eliminating sources of contamination to achieve high precision

Developments in continuous‐flow isotope ratio mass spectrometry have made possible the rapid analysis of δ¹³C in CO₂ of small‐volume gas samples with precisions of ≤0.1‰. Prior research has validated the integrity of septum‐capped vials for collection and short‐term storage of gas samples. However, there has been little investigation into the sources of contamination during the preparation and analysis of low‐concentration gas samples. In this study we determined (1) sources of contamination on a Gasbench II, (2) developed an analytical procedure to reduce contamination, and (3) identified an efficient, precise method for introducing sample gas into vials. We investigated three vial‐filling procedures: (1) automated flush‐fill (AFF), (2) vacuum back‐fill (VBF), and (3) hand‐fill (HF). Treatments were evaluated based on the time required for preparation, observed contamination, and multi‐vial precision. The worst‐case observed contamination was 4.5% of sample volume. Our empirical estimate showed that this level of contamination results in an error of 1.7‰ for samples with near‐ambient CO₂ concentrations and isotopic values that followed a high‐concentration carbonate reference with an isotope ratio of ?47‰ (IAEA‐CO‐9). This carry‐over contamination on the Gasbench can be reduced by placing a helium‐filled vial between the standard and the succeeding sample or by ignoring the first two of five sample peaks generated by each analysis. High‐precision (SD ≤0.1‰) results with no detectable room‐air contamination were observed for AFF and VBF treatments. In contrast, the precision of HF treatments was lower (SD ≥0.2‰). VBF was optimal for the preparation of gas samples, as it yielded faster throughput at similar precision to AFF. Copyright © 2009 John Wiley & Sons, Ltd.