Multidimensional chromatography: new techniques and applications
Creators: name: Pandohee, Jessica
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 340109
subject: Separation science

description: The development of more robust, powerful and accurate analytical techniques have enabled scientists to study an increasingly intricate range of samples by providing novel ways to resolve and identify their chemical entities with greater confidence in a complex sample. The work presented in this thesis explores new analytical capabilities to enhance sample preparation, separation science especially multidimensional chromatography and detection technologies, and their applicability in food, metabolomics and forensic science.<br><br>Many parts of the work involved food products that have had undergone some kind of processing; whether by irradiating them with UV (mushrooms) or inserting a new gene to enhance their nutritional content. According to our knowledge no one has performed a metabolomics investigation on these new foods. Previous studies on the mushrooms focussed on optimising the irradiation duration and intensity to maximise the amount of vitamin D, neglecting its effect on other metabolites. The transgenic plants have been engineered to produce omega-3 long chains in 2015; therefore they have not been studied at all. Moreover the entire process of profiling and quantifying metabolites using 2DLC is novel since most metabolomics studies to date use NMR spectroscopy and Gas chromatography.<br><br>Solid phase extraction is most commonly utilised for quantitative extraction and pre-concentration of analytes and/or sample cleanup. The development of micro solid phase extraction cartridges is very recent; the system tested in this study was only brought to market in 2014 and have never been investigated for on-column derivatisation methods before. This proof of principle demonstrates the possibility of performing on-column derivatisation of fatty acids. Our results show that on-column derivatisation suitable for metabolomics and lipidomics studies was possible with the micro SPE system and that the method allowed fast and accurate analysis with minimal sample separation.<br><br>Two-dimensional gas chromatography is a mature separation method known to provide high resolution and peak capacity required for the study of complex petrochemicals. The work in our laboratory has shown that the developed method enabled the distinction of various petroleum products available in the market. The simulated weathering experiments are also looking promising in providing an estimate of the time of fire. Additionally, this will lead to analysis of more complex samples and problems that forensic police are facing potentially the identification of suspects using individual evidence containing petroleum such as lipstick. The work demonstrates an optimised method for petrochemical products so as to provide forensic scientists a robust, fast and sensitive technique of characterisation during their analysis.<br><br>The development of a two-dimensional liquid chromatography method is more challenging as fundamental factors such as column selection, sampling rate, compatible solvent systems among others need to be considered and optimised for a successful analysis. The development of an offline two-dimensional liquid chromatography methodology for metabolites profiling is demonstrated. Agaricus bisporus mushrooms irradiated with UV light are used as an example to test this method and to show the viability of the method to detect changes in metabolite concentration. The detection of up to 150 peaks was achieved, of which the concentrations of 72 were found to increase and 47 compounds to decrease. Although the developed method took several hours per sample, the increase in peak capacity resulted in the separation of a greater number of compounds than 1D-HPLC, NMR spectroscopy or GC-MS based analysis and without the need for chemical derivatisation with economic costs being roughly equal to the other methods.<br><br>This research involved the development and assessment of new multidimensional chromatographic approaches for the analysis of complex natural samples. The optimised methodologies will be applicable applied to many diverse areas of research such as metabolomics, lipidomics, food and forensic science. Although not yet widespread the increased separation power of two-dimensional chromatography is of great potential in a large variety or areas, particularly for compounds that are too sensitive for mass spectrometry but contained in matrices too complex for standard LC analysis. The combinations of new column chemistries that could be used are many and varied and the use of size exclusion and chiral columns in the first dimension offers up many further opportunities for novel applications.