Development and evaluation of a 2D-LC-IRMS hyphenation for the use of conventional reversed-phase HPLC in compound-specific stable isotope analysis

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Compound-specific stable isotope analysis (CSIA) is a method for determining the ratio of naturally occurring isotopes in single molecules. The coupling of liquid chromatography (LC) with an isotope ratio mass spectrometer (IRMS) allows the determination of carbon isotope ratios in complex samples and is used in various fields, including the determination of the origin and fate of organic pollutants in the environment and authenticity analysis in food monitoring. LC-IRMS coupling uses wet chemical oxidation of the analytes, in which all oxidisable carbon is converted to CO2. For this reason, any organic additives as eluents or buffers must be avoided as they can falsify the carbon isotope determination. However, as 90% of established LC methods use organic solvents, the application of LC-IRMS has been very limited to date. To overcome this obstacle of CSIA by LC-IRMS, the proposed project shall develop and validate the concept of two-dimensional LC (2D-LC) for stable isotope analysis. To that end, established separations from literature will be used in the first dimension that utilize organic solvents. In the second dimension, the organic solvent is separated from the analyte before the latter is oxidised to CO2 in the LC-IRMS interface. Different methods such as the sample loop method, the at-column dilution method and the trap method will be investigated. Here, the analytical parameters solvent compatibility, sensitivity and integrity of isotope signatures are the focus of the validation. Besides the separation of analytes from the solvent peak coeluting compounds from the first dimension shall also be separated by selecting suitable separation mechanisms in the second dimension. In the context of the investigation of the trap method the use of thermoresponsive phases in the trap column will be additionally investigated to allow the transfer of the analyte from 1D to 2D via a moderate temperature change. In the final part of the project, the knowledge gained will be used to demonstrate the applicability of the developed methods in various examples of the characterisation of chemical substances and transformation processes.