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Two dimensional liquid chromatography and its applications to environmental problematics and natural samples
Ramia Al Bakain, doctorante. Crédits : ESPCI ParisTech The social demand to characterize complex samples containing several hundred components becomes stronger in both environmental and public health. Thus, it is necessary to develop more efficient analytical tools able to address ever-increasing difficult issues. This purpose has been one of the research axes of the Laboratoire des Sciences Analytiques, Bioanalytiques et Miniaturisation (LSABM) from ESPCI ParisTech which has a solid experience in the separation of organic compounds at trace levels in various environmental matrices by developing several approaches for the samples treatment.
Complementary to selective sample handling, when an overall characterization of the sample is looked for, multidimensional separation techniques appears as the solution to increase dramatically the separation power.
This thesis aimed at a better understanding of LC×LC coupling with UV and mass spectrometry detection, the studies on orthogonality evaluation, and the coupling in on-line and off-line mode. Van Deemter and Knox curves were also used as tools to evaluate the performance of stationary phases in the second dimension. The objective was to develop a comprehensive two-dimensional liquid chromatographic approach (LC×LC) for the analysis of complex mixtures with weakly volatile compounds, which cover a wide array of environmental or biological samples. To achieve this objective, the thesis included several steps:
- First, by using model compounds from environmental and biological fields, we studied the combinations of mechanisms in reverse phase mode to achieve favorable couplings in terms of orthogonality. This work, which necessitated a rigorous statistical characterization, was performed in close collaboration with the Équipe de Statistique Appliquée (ESA) de l’ESPCI.
- Second, we optimized the conditions of our system in order to achieve a rapid and efficient separation in the second dimension.
- Third, we achieved an effective LC×LC coupling in on-line chromatographic systems by adapting existing tools or developing new ones that were lacking. The tests were performed by using synthetic solutions containing neutral, acidic and basic compounds. In
parallel with a separation in on-line mode, we performed an off-line separation to compare the performances obtained in the two modes in terms of peak capacities. - Finally, to manage the large amount of data generated by the LC×LC analysis, chemometric and statistical strategies for the treatment of the results were used.
Examples of applications coupled with UV and mass spectrometry, applied to synthetic mixtures, confirmed the relevance of our approach and the potential of the LC×LC technique.