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

化合物特异性稳定同位素分析(CSIA)是一种测定单分子中天然存在的同位素比例的方法。液相色谱(LC)与同位素比值质谱仪(IRMS)联用，可以测定复杂样品中的碳同位素比值，被广泛应用于各种领域，包括环境中有机污染物的来源和去向的确定以及食品监测中的真实性分析。LC-IRMS联用使用分析物的湿化学氧化，在该过程中所有的氧化碳被转化为二氧化碳。因此，必须避免使用任何有机添加剂作为淋洗剂或缓冲剂，因为它们会影响碳同位素测定。然而，由于现有的LC方法中90%使用有机溶剂，LC-IRMS的应用到目前为止一直非常有限。为了通过LC-IRMS克服CSIA的这一障碍，拟议的项目将开发和验证用于稳定同位素分析的二维LC(2D-LC)的概念。为此，将在使用有机溶剂的第一个维度中使用从文献中确定的分离。在第二个维度中，有机溶剂与分析物分离，然后分析物在LC-IRMS界面上被氧化成二氧化碳。将研究不同的方法，如样品循环法、柱上稀释法和捕捉法。在这里，分析参数溶剂兼容性、灵敏度和同位素签名的完整性是验证的重点。除了分析物与溶剂峰的分离外，第一维的洗脱化合物也应通过选择合适的第二维分离机制来分离。在研究捕集方法的背景下，还将研究捕集色谱柱中热响应相的使用，以允许分析物通过适度的温度变化从一维转移到二维。在该项目的最后部分，所获得的知识将用来证明所开发的方法在各种化学物质和转化过程的表征实例中的适用性。

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.