Using Orbitrap mass spectrometry to assess the isotopic compositions of individual compounds in mixtures

The isotopic compositions of individual chemical species are routinely used by the geochemical, environmental, forensic, anthropological, chemical, and biomedical communities to elucidate the conditions, sources, and reaction pathways of the molecules in question. Mass spectrometric methods of measuring isotopic compositions of individual compounds generally require that analytes be pure to yield precise, accurate results, yet most applications examine materials that are mixtures of multiple components. Various methods of chemical purification, e.g., chromatography, are used to isolate analytes from mixtures prior to mass spectrometric analysis. However, these techniques take time and specialized instrumentation, both of which could potentially be obviated via the use of ultra-high-resolution mass spectrometry. Here we report on the use of Orbitrap™-based Fourier-transform mass spectrometry to perform isotope ratio measurements of single species within mixtures delivered to the mass spectrometer (MS) without prior chromatographic separation. We demonstrate that instrument biases (attributed here to space charge effects) within the Orbitrap mass analyzer can cause the measured ¹³C/¹²C ratio of a molecular ion in the presence of non-analyte-derived ‘contaminating’ species to spuriously decrease relative to the ¹³C/¹²C ratio measured for the same ion in a pure analyte. We observe that the decrease in ¹³C/¹²C is proportional to the relative concentrations of the additional ‘contaminating’ components. We then recommend several strategies by which this effect can be mediated such that accurate isotope ratios can be obtained

Using Orbitrap mass spectrometry to assess the isotopic compositions of individual compounds in mixtures

The isotopic compositions of individual chemical species are routinely used by the geochemical, environmental, forensic, anthropological, chemical, and biomedical communities to elucidate the conditions, sources, and reaction pathways of the molecules in question. Mass spectrometric methods of measuring isotopic compositions of individual compounds generally require that analytes be pure to yield precise, accurate results, yet most applications examine materials that are mixtures of multiple components. Various methods of chemical purification, e.g., chromatography, are used to isolate analytes from mixtures prior to mass spectrometric analysis. However, these techniques take time and specialized instrumentation, both of which could potentially be obviated via the use of ultra-high-resolution mass spectrometry. Here we report on the use of Orbitrap™-based Fourier-transform mass spectrometry to perform isotope ratio measurements of single species within mixtures delivered to the mass spectrometer (MS) without prior chromatographic separation. We demonstrate that instrument biases (attributed here to space charge effects) within the Orbitrap mass analyzer can cause the measured ¹³C/¹²C ratio of a molecular ion in the presence of non-analyte-derived ‘contaminating’ species to spuriously decrease relative to the ¹³C/¹²C ratio measured for the same ion in a pure analyte. We observe that the decrease in ¹³C/¹²C is proportional to the relative concentrations of the additional ‘contaminating’ components. We then recommend several strategies by which this effect can be mediated such that accurate isotope ratios can be obtained

Methane Clumped Isotopes: Progress and Potential for a New Isotopic Tracer

The isotopic composition of methane is of longstanding geochemical interest, with important implications for understanding petroleum systems, atmospheric greenhouse gas concentrations, the global carbon cycle, and life in extreme environments. Recent analytical developments focusing on multiply substituted isotopologues (‘clumped isotopes’) are opening a valuable new window into methane geochemistry. When methane forms in internal isotopic equilibrium, clumped isotopes can provide a direct record of formation temperature, making this property particularly valuable for identifying different methane origins. However, it has also become clear that in certain settings methane clumped isotope measurements record kinetic rather than equilibrium isotope effects. Here we present a substantially expanded dataset of methane clumped isotope analyses, and provide a synthesis of the current interpretive framework for this parameter. In general, clumped isotope measurements indicate plausible formation temperatures for abiotic, thermogenic, and microbial methane in many geological environments, which is encouraging for the further development of this measurement as a geothermometer, and as a tracer for the source of natural gas reservoirs and emissions. We also highlight, however, instances where clumped isotope derived temperatures are higher than expected, and discuss possible factors that could distort equilibrium formation temperature signals. In microbial methane from freshwater ecosystems, in particular, clumped isotope values appear to be controlled by kinetic effects, and may ultimately be useful to study methanogen metabolism

Supplemental Information Tables for Carbon Isotope SOM Model for Meteorites

These are the carbon isotope ratio in delta space (d13C) for measurements and predictions the soluble organic material on meteorites assuming production by the integrated aldehyde network (Strecker synthesis, aldehyde oxidation, and reductive amination), Fischer Tropsch type synthesis, and Formose type chemistry

Supplemental Tables for Deuterium Isotope Model

Data for all deuterium measurements used in moiety model for thesis Chapter 5 (Laura Chimiak). Includes measurement data, references, moiety assignments, and predictions. Table S1 presents data for organic mater in the Murchison meteorite and Table S2 presents data for organic mater in Murchison and other meteorites

Prebiotic Fingerprints

Meteorites contain organic compounds that occur in all known life. These compounds, commonly referred to as prebiotic compounds, include α-amino acids and are most prevalent on carbonaceous chondrites. As carbonaceous chondrites are pristine samples from early in the solar system that have not had living organisms on them, we can study the chemistry that produced α-amino acids on them to better understand the processes by which they might have formed on early Earth or on other bodies. Multiple syntheses have been put forth as routes to form amino acids on meteorites and include ice-grain chemistry on interstellar ices and Strecker synthesis in meteorite parent bodies. Prior measurements of molecular-average carbon isotope ratios (¹³C/¹²C) have found ¹³C enrichments of up to 53‰ in certain α-amino acids and molecular-average hydrogen isotope ratios (D/H) have found enrichments of 100s of ‰. With this data, it has been suggested that Strecker synthesis—a synthesis in which an aldehyde or ketone reacts with ammonia and cyanide to produce an α-aminonitrile that is hydrolyzed into an α-amino amide and then an α-amino acid—is the primary pathway to produce α-amino acids on aqueously altered meteorites. Here, we develop an instrument that can measure site-specific isotope ratios (SSIR) for carbon — that is the ¹²C/¹³C at each site in a molecule — and use it to first constrain the site-specific isotope effects associated with Strecker synthesis and then the carbon SSIR of an alanine sample extracted from the Murchison meteorite. The instrument, the Q-Exactive Orbitrap, is a Fourier Transform Mass Spectrometer that has resolution of 240,000 full width-half maximum and can measure site-specific carbon isotope ratios on samples as small as 1 picomole. When we use it to measure the carbon SSIR in multiple samples of alanine produced by Strecker synthesis, we find a -20 ‰ equilibrium isotope effect between the product alanine's C-2 site (amine carbon, ¹³C-depleted) reactant acetaldehyde’s carbonyl carbon (¹³C-enriched), a potential -15 ‰ kinetic isotope effect on the C-1 site (eventual carboxyl carbon) for the first hydrolysis of α-aminopropanenitrile (¹³C-enriched) into alaninamide (¹³C-depleted), and a -15.4 ‰ kinetic isotope effect on the C-1 carbon for the second hydrolysis step in which α-alaninamide (¹³C-enriched) becomes alanine (¹³C-depleted). Through conventional isotope ratio mass spectrometry, we also measure a +56.4 ‰ equilibrium isotope effect between ammonia (¹⁵N-depleted) and the amine site on alanine (¹⁵N-enriched). When we measure the sample of alanine from the Murchison meteorite, we find site-specific carbon isotope ratios of -29 ± 10 ‰, 142 ± 20 ‰, and -36 ± 20 ‰ for the C-1, C-2, and C-3 (methyl) sites, respectively. This pattern agrees with the hypothesis that Strecker synthesis created alanine in Murchison. Combining these data with the isotope effects found for Strecker synthesis, we find initial site values of -7 ± 10 ‰, 162 ± 20 ‰, and 36 ± 20 ‰ for the C-1, C-2, and C-3 sites, respectively. With these values, we create a model of potential organic synthesis on the Murchison parent body that predicts the molecular-average δ¹³C values of 19 other prebiotic compounds. Finally, we create a model that uses the previously measured molecular average carbon and deuterium isotope ratios for organics on Murchison to create models that predict site-specific and molecular average isotope ratios for organic compounds. This model finds that organic compounds with have methyl sites that are enriched in deuterium by up to 3000 ‰ relative to other sites in the compound and that the degree of enrichment scales both with a compound class’s solubility in water and with a sample’s degree of aqueous alteration and terrestrial weathering. These patterns suggest that a primordial ISM-derived deuterium signal exchanges with water and that the methyl site hosts the highest amount of this enrichment due to its low acidity. The carbon model demonstrates that using only the aldehyde and cyanide values measured on Murchison and isotope effects inferred from other studies, we can predict 59 of 82 organic compounds on it (72%) that have δ¹³C values spanning over 149 ‰ with an average residual of 6 ‰. To achieve this level of prediction, the model combines Strecker synthesis, reductive amination, and oxidation of aldehydes to create straight-chain α-H hydroxy and amino acids, amines, and monocarboxylic acids with subsequent formaldehyde addition to these compounds to create branches.</p

Prebiotic Synthesis on Meteorite Parent Bodies: Insights from Hydrogen and Carbon Isotope Models

Several mechanisms could produce the biorelevant compounds in carbonaceous meteorites. These include radiation-driven reactions in the interstellar medium, gas-phase mineral-catalyzed reactions in the solar nebula, and aqueous chemistry in meteorite parent bodies. The ratio of heavy-to-light isotopes in a compound can constrains its formation history: a reaction’s substrates, mechanisms, and physiochemical conditions impact isotope ratios. Studies of the stable isotope compositions of meteoritic organic compounds have focused on sample- and molecular-average isotope measurements and have interpreted those data via qualitative or semi-quantitative models. Here we create quantitative models (i.e., explicitly fit to measurements) for hydrogen and carbon isotope compositions of organic compounds in primitive carbonaceous meteorites and use these models to reach broader conclusions regarding the environments, substrates, and chemical processes that contributed to pre- and early-solar-system organic synthesis. The hydrogen model fits measured molecular-average deuterium concentrations in a compound class (e.g., amines, carboxylic acids) as linear combinations of hydrogens with similar chemical environments. In the chondrites studied, methyl hydrogens are amongst the most deuterium-enriched moiety and hydrogens attached to α-carbons are the least. Deuterium enrichment is inversely related to both a compound class’s water solubility and a meteorite sample’s degree of aqueous alteration and terrestrial weathering. These values suggest that ISM-sourced compounds reacted to form deuterium-enriched molecules on meteorites’ parent bodies and the enrichments were attenuated through exchange with water during aqueous alteration on the parent body and subsequent terrestrial processing. The carbon model fits the δ13CVPDB of products from various reaction mechanisms by applying isotope effects to reactant δ13C measurements. The model with the most accurate δ13C fits of the compounds in the Murchison meteorite (62 % of previous measurements fit by model) and the lowest average residuals (5 ‰) uses the integrated aldehyde network (oxidation, reductive amination, and Strecker synthesis on aldehydes and ketones) to produce straight-chain compounds that undergo formaldehyde addition to create branched-chain compounds. Formaldehyde addition has not been previously considered in prebiotic chemical reaction networks, but the best-fit network’s ability to fit compounds that span over 100 ‰ in carbon isotope abundances makes it an attractive chemistry to explore

Isotope heterogeneity in ethyltoluenes from Australian condensates, and their stable carbon site-specific isotope analysis

Low-molecular-weight (LMW) aromatic compounds from petroleum fluids have not been widely studied for fluid-source correlations due to their volatility and their relatively low abundances in source rocks. However, LMW aromatics are important components in fluids, including condensates which lack biomarkers (molecular fossils typically used for correlation studies). Here, we have investigated the distribution of ethyltoluenes (o-ET, m-ET and p-ET; ortho, meta and para, respectively) in fluvial-deltaic condensates which contain relatively high abundances of the meta- isomer. The meta-selectivity found in these petroleum fluids is consistent with a mineral catalytic effect on the molecular distribution of these compounds, as it occurs during the clay-catalyzed synthesis of ethyltoluenes. Isomers differ up to 6‰ in δ^(13)C values. Condensates from the Northern Carnarvon Basin, North West Shelf of Australia (NWS), have been analyzed by compound specific isotope analysis (CSIA) by GC-ir-MS, and site-specific isotope analysis (SSIA) using a Q-Exactive-GC Orbitrap^(TM)-based mass spectrometer. The SSIA revealed a ^(13)C enrichment at the methyl end of the ethyl branch of m-ET, following a normal kinetic isotope effect during thermal maturation (cleavage). Continuous development of this first SSIA application will make possible high resolution analysis of light aromatics to outline the evolution of the organic matter to hydrocarbons in petroleum systems