Although gas and liquid chromatography have emerged as dominant separation techniques in environmental analytical chemistry, these methods do not allow for the concurrent analysis of chemically diverse groups of persistent organic pollutants (POPs). There are also a small number of compounds which are not easily amenable to either of these traditional separation techniques. The main objective of this thesis was to address these issues by demonstrating the applicability of packed column supercritical fluid chromatography (pSFC) coupled to mass spectrometry (MS) in various aspects of environmental chemistry. First, pSFC/MS analytical methods were developed for legacy POPs (PCDDs, PCDFs, and PCBs) as well as the emerging environmental contaminant Dechlorane Plus (DP), and issues relating to the ionization of target analytes when pSFC was coupled to MS were explored. Novel APPI and APCI reagents (fluorobenzene and triethylamine) were optimized and real samples (water and soil) were analyzed to demonstrate environmental applicability. The possibility of chiral and preparative scale pSFC separations was then demonstrated through the isolation and characterization of thermally labile hexabromocyclododecane (HBCDD) stereoisomers. The analytical pSFC separation of the α-, β-, and γ-HBCDD enantiomers as well as the δ and ε meso forms was shown to be superior to results obtained using a published LC method. Finally, technical mixtures of phosphorus flame retardants (RBDPP, BPA-BDPP, and DOPO; a group of related compounds which are challenging to analyze concurrently) were examined using multiple analytical techniques and pSFC was found to be the only method which facilitated the accurate determination of the components of all 3 mixtures. This thesis confirms the potential of pSFC/MS as a fast, green, and cost effective means of separating and analyzing environmental contaminants.

In this project the photodegradation of 3,5-diamino-6-chloro-N-(2-(methylamino)ethyl)pyrazine-2-carboxamide was studied. A hypothetical degradation pattern for the compound was proposed and the aim of the project was to study the formed secondary photodegradants and to, if possible, structure elucidate some of these compounds. In order to do this, the parent compound was photodegraded in two steps, where a primary photodegradant was isolated using semi-preparative supercritical fluid chromatography (SFC) and then further degraded into the secondary photodegradants. The photodegradation was first carried out in aqueous solution, where the parent compound was irradiated in UV-A light of 300-400 nm. This resulted in a primary photodegradant with a molecular ion of m/z = 227, where the chloride in position 6 of the pyrazine group had been replaced by a hydroxyl group. During the large scale photodegradation, prior to the preparative purification, the yield of primary photodegradant was very low due to the photodegradation being dependent on both sample volume and concentration and due to the primary photodegradant also being unstable in aqueous solution at room temperature. Due to the above mentioned difficulties the parent compound was photodegraded in methanol instead of water in order to avoid the freeze-drying process where a lot of the primary photodegradant was lost. This resulted in a primary photodegradant with a molecular ion of m/z = 241, where the chloride had been replaced by a methoxy group instead of a hydroxyl group. This compound was more stable which allowed workup by rotary evaporation, instead of freeze-drying, before the preparative purification. This primary photodegradant was isolated using semi-preparative SFC on a Viridis® BEH Prep OBD TM column (250 x 30 mm, 5 µm) and a Luna HILIC column (250 x 30 mm, 5 µm) with MeOH/NH3 100/1 v/v as organic modifier. About 1.2 mg material was isolated and further photodegradation tests in ordinary water and 18O-water were conducted. Some secondary photodegradants were observed in LC-MS analyses, and their element compositions were proposed by accurate mass results. Fundamental structures for these compounds were proposed. Further structural investigational analyses are needed for confirmation in the future.

Cette thèse vise à démontrer la pertinence de la chromatographie en phase supercritique (SFC) et de son couplage à la spectrométrie de masse tandem haute résolution (HRMS/MS) quant à l’étude de produits naturels en mélange complexe. Pour y parvenir trois projets ont été menés en parallèle.Une méthode d’analyse structurale ciblée sur les acétogénines d’Annonaceae a été développée. Le recours à une cationisation post-colonne par des sels de lithium a permis d’obtenir les informations structurales recherchées. Des études ont également été menées quant à l’influence de la nature du cation sur la conformation de l’adduit formé et les voies de fragmentation observées.Une séparation de composés polaires et ionisables a également été mise au point à travers l’étude des nucléosides modifiés de l’ARN. La SFC-HRMS permet la séparation et l’étude d’un grand nombre de modifications et constitue donc un nouvel outil d’analyse pour le biologiste.Pour finir, une étude en lipidomique globale a également été entreprise. Bien que préliminaires, les premiers résultats semblent très prometteurs avec pour but de développer des méthodes d’analyses non ciblées associés à des outils d’annotation automatique.Pour chaque projet, la pertinence de nos approches a été vérifiée par l’étude d’échantillons réels et complexes permettant de tirer des conclusions réalistes quant aux possibilités de la SFC-HRMS/MS pour l’étude de composés naturels. / This PhD work aims to demonstrate the relevance of supercritical fluid chromatography (SFC) and its hyphenation to high resolution tandem mass spectrometry (HRMS/MS) in the field of natural product analysis. Three different research projects were carried out.A new strategy for structural analysis of Annonaceous acetogenins was developed by SFC and targeted HRMS/MS including post-column lithium cationisation to give access to the relevant structural information. Investigations were conducted to observe the influence of cations onto the gas-phase conformation and fragmentation pathways.A separation of polar and ionisable compounds was also developed using modified nucleosides from RNA as models. The SFC-HRMS method led to the separation and analysis of several tens of modifications and demonstrated to be a new performant analytical tool for biologists.Finally, a global lipidomic approach was optimized. Preliminary results look compatible with the development of untargeted approaches using automatic annotation tools.For each project, the relevance of our work was evaluated analyzing complex samples to obtain a realistic point of view of the capabilities for SFC-HRMS/MS systems for natural product studies.

Supercritical fluid chromatography (SFC) is an expanding chromatographic technique that is part of the evolving field of green chemistry due to its short run times and use of non-toxic solvents in combination with recycled CO2. The use of a suitable column is essential for separation science in general and there is a wide variety of SFC compatible columns available on the market. In collaboration with the AstraZeneca Gothenburg Separation Science Laboratory (SSL), an investigation of 25 stationary phases in combination with four different mobile phases was conducted in order to evaluate interactions between the mobile phase, stationary phase and analytes. The aim was to find a new column set, able to operate with a wide range of molecules at both analytical and preparative scale. The 36 compounds analysed were chosen to represent the chemical space suitable for current and foreseen compounds of interest within AstraZeneca. Furthermore, to mitigate the risk of degradation of sensitive compounds in basic conditions, different combinations of basic stationary phases and neutral mobile phases were investigated.The study was performed using a Waters Acquity UPC2 system equipped with a photodiode array (PDA) detector and a single quadrupole detector (SQD). The data was gathered by Empower 3 and analysed using statistical methods to evaluate corelations and orthogonality with the help of Python. Visualisations were produced using Pandas, XLstat and SIMCA.The results evaluated retention time, symmetry factor, distribution of retention times of compounds over the columns, and DMSO retention. The results showed that MeOH and the basic additive NH3 included in the mobile phase composition provided the shortest retention times, best peak symmetry and distribution of compounds. It was concluded that Kromasil 2EP, Kromasil Diol, DC Pak PBT, and Kromasil NH2 columns represent a diverse set for the intended chemical space. They all also operate with good results without additive, for the compounds used. / Superkritisk vätskekromatografi (SFC) är en växande kromatografisk teknik som tack vare sina snabba analyser, användandet av icke toxiska lösningsmedel, samt återvinningsbar CO2 utgör en del av den allt mer populära “gröna” kemin. Det finns en mängd kolonner utvecklade för SFC på marknaden, och att använda rätt sorts stationär fas är en viktig del för separationen av olika ämnen. I samarbete med Separation Science Laboratory på AstraZeneca i Göteborg, har 25 olika stationära faser i kombination med fyra mobila faser studerats för att analysera interaktionerna mellan faserna och analyterna. Målet är att hitta en uppsättning av kolonner som kan användas vid upprening av substanser med en stor variation av kemiska egenskaper/deskriptorer. För den här studien har 36 molekyler som representerar den kemiska rymd som AstraZeneca jobbar i använts. För att minimera risken av nedbrytning av känsliga molekyler i basiska miljöer, är även basiska kolonner med neutrala mobilfaser inkluderade. Under studien användes SFC systemet Acquity UPC2, utrustad med en Diode array detektor, en Single quadrapole detektor samt Empower 3 för insamling av data. Den statistiska analysen utfördes i Python och visualiseringen gjordes med hjälp av Pandas, Xlstat och Simca.Resultaten utvärderades med avseende på retentionstid, symmetrifaktorn, fördelningen av substanser över kolonnerna och retention av DMSO. Resultaten visade att MeOH med NH3 som additiv i mobilfasen gav kortast retentionstid, bäst symmetri och bredast fördelning av substanser. Slutsatserna var att kolonnerna Kromasil 2EP, Kromasil Diol, DC Pak PBT, and Kromasil NH2 tillsammans representerar den diversa uppsättning av kolonner som passar den kemiska rymnd som användes i studien. Samtliga kolonner kan även köras med goda resultat i neutrala mobila faser, med de betingelser som använts.

This thesis aims at a deeper understanding of Supercritical Fluid Chromatography (SFC). Although preparative SFC has started to replace Liquid Chromatography (LC) in the pharmaceutical industry - because of its advantages in speed and its less environmental impact - fundamental understanding is still lacking. Therefore there is no rigid framework to characterize adsorption or to understand the impact of changes in operational conditions.   In Paper I we demonstrated, after careful system verification, that most methods applied to determine adsorption isotherms in LC could not be applied directly in SFC. This was mainly due to operational differences and to the fact that the fluid is compressible which means that everything considered constant in LC varies in SFC.   In Paper II we showed that the most accurate methods for adsorption isotherm determination in LC, the so called plateau methods, do not work properly for SFC. Instead, methods based on overloaded profiles should be preferred.   In Paper III a Design of Experiments approach was successfully used to quantitatively describe the retention behavior of several solutes and the productivity of a two component separation system. This approach can be used to optimize SFC separations or to provide information about the separation system.   In Paper IV severe peak distortion effects, suspected to arise from injection solvent and mobile phase fluid mismatches, were carefully investigated using experiments and simulations. By this approach it was possible to examine the underlying reasons for the distortions, which is vital for method development.   Finally, in Paper V, the acquired knowledge from Paper I-IV was used to perform reliable scale-up in an industrial setting for the first time. This was done by carefully matching the conditions inside the analytical and preparative column with each other. The results could therefore provide the industry with key knowledge for further implementation of SFC. / This thesis aims at a deeper understanding of Supercritical Fluid Chromatography (SFC). Although preparative SFC has started to replace Liquid Chromatography (LC) in the pharmaceutical industry - because of its advantages in speed and its less environmental impact - fundamental understanding is still lacking. Therefore there is no rigid framework to characterize adsorption or to understand the impact of changes in operational conditions.   In Paper I-II it was demonstrated why most methods applied to determine adsorption isotherms in LC could not be applied directly for SFC. Methods based on extracting data from overloaded profiles should be preferred.   In Paper III a Design of Experiments approach was successfully used to quantitatively describe the behavior of several solutes in a separation system. This approach can be used to optimize SFC separations or to provide information about the separation system.   In Paper IV severe peak distortion effects often observed in SFC were carefully investigated and explained using experiments and simulations.   Finally, in Paper V, the prerequisites for performing reliable and predictable scale-up of SFC were investigated by small and large scale experiments. / <p>Paper 4 ("Evaluation of scale-up from analytical to preparative...") ingick som manuskript med samma titel i avhandlingen. Nu publicerad. </p>

A novel chromatographic method was devised that makes use of the superb group separation power of normal phase supercritical fluid chromatography (SFC) combined with a fast second separation by a resistively heated gas chromatograph (GC). The SFC was operated isothermally with stopped flow to provide the time required for the GC analysis. The GC analysis had a typical cycle time of 1 minute. During this time the GC column was independently heated at a rate of 450°C/min to 250°C and actively cooled down again to -50°C before the next GC injection takes place. This was achieved with an in-house designed, resistively heated, temperature programmable gas chromatograph. Various temperature measurement circuits were also evaluated. An interface was developed that allows transfer between the SFC and the GC in such a way that the entire eluent from the first separation is analysed by the second separator. Chromatographic resolution was not lost during the transfer process from the first to the second separation stages. The interface also allows for the exchange of the carrier gas used in the second gas chromatographic separation to provide for the maximum separation speed. In the first separation, a silica gel packed column and the novel application of a silica gel porous layer open tubular capillary column was used for SFC group separation. The SFCxGCftp was applied to petrochemical samples and essential oils and the results were compared to that obtained with a commercially available GCxGC system. / Thesis (PhD)--University of Pretoria, 2003. / Chemistry / unrestricted

(EN) Cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase was used for separation of selected 24 analytes. Enantioseparations were realized using two systems, high performance liquid chromatography and supercritical fluid chromatography. Effect of mobile phase composition was studied. Five different aditives (isopropylamine, diethylamine, triethylamine, trifluoroacetic acid, isopropylamine combined with trifluoroacetic acid) and their influence on enantioseparation were tested. Influence of two different modifiers (methanol, propan-2-ol) combined with all aditives was also tested in supercritical fluid chromatography system. The aim of this work was to find optimized composition of mobile phase which was suitable for separation of the analytes studied and to compare separation potential among all mobile phases and also between used separations systems. The supercritical fluid chromatography was shown to yield better results, i.e. better resolution in shorter analysis time. However examples of analytes better resolved under optimized conditions in high performance liquid chromatography system have also been found. Keywords (EN) Chirality, enantiomers, enantioselective separation, chiral stationary phase, high performance liquid chromatography, supercritical fluid chromatography.

(EN) Proteinogenic amino acids are key components of living organisms. Thus, the latest metabolomics research has focused on developing fast and sensitive methods for the determination of amino acids. In this context, this thesis contains two studies describing development of high-performance separation techniques for the quantification of amino acids. In the first study, a capillary electrophoresis method was developed for the determination of free amino acids in tobacco plants, particularly focusing on optimizing the extraction of amino acids from solid plant materials. The extraction procedure was optimized using design of experiments (DoE) to obtain the highest possible extraction yield of amino acids. Factors such as volume and concentration of the extraction solvent (hydrochloric acid) were assessed as the most significant. Subsequently, the optimal values of these factors were determined using response surface methodology (RSM). Lastly, proteinogenic amino acids were quantified using capillary electrophoresis with contactless conductivity detection and calibration with internal standard, which improved the precision of the method. The second study aimed at developing a supercritical fluid chromatography method for the determination of free proteinogenic amino acids in human plasma. The most...

Raman spectroscopy, laser-induced fluorescence, and sum-frequency generation (SFG) spectroscopy are used to investigate the behavior of compressible mobile phases and stationary phases under a variety of chromatographic conditions. Efforts to understand and optimize separations employing compressible mobile phases have been limited by a lack of understanding of the mobile phase density gradient. Mobile phase compressibility leads to gradients in linear velocity and solute retention and affects separation speed and efficiency, especially in packed columns. This work describes on-column density measurement of CO2, a common carrier fluid for SFC and SGC, in packed capillary columns using Raman microspectroscopy. On-column detection by laser-induced fluorescence is used to observe the effect of the mobile phase density gradient on separation speed and efficiency, and experimental efficiency is compared to a theoretical model. Additionally, SFG spectroscopy allows for probing the structure of model monomeric and polymeric C18 stationary phases under pressure; this provides a basis for correlating selectivity with pressure-induced structural changes in stationary phase materials. Together, this work provides a more complete understanding of the role of column pressure and fluid compressibility on the speed, efficiency, and selectivity of chemical separations.

Orientador: Carol Hollingworth Collins / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T07:24:16Z (GMT). No. of bitstreams: 1 Silva_CarlaGrazieliAzevedoda_D.pdf: 4489958 bytes, checksum: f1c0299f6db7beed410c437dcdfc5886 (MD5) Previous issue date: 2013 / Resumo: Este trabalho apresenta o desenvolvimento de fases estacionárias (FE) para cromatografia líquida de alta eficiência em fase reversa (CLAE-FR) e cromatografia com fluido supercrítico (CFS) a partir da imobilização térmica de poli(metiloctadecilsiloxano) (PMODS) sobre suportes de sílicas metalizadas com zircônio e titânio. O processo de imobilização térmica do PMODS foi otimizado aplicando-se planejamento composto central. O polímero, os suportes e as fases estacionárias foram caracterizados por testes físicoquímicos e cromatográficos. As fases estacionárias Si(PMODS), Si-Zr(PMODS) e Si-Ti(PMODS) apresentaram os melhores resultados quando foi aplicada a temperatura de 120 °C por 16 horas. As melhores FE foram submetidas ao processo de capeamento. Estas FE mostraram eficiências entre 82.000 e 90.000 pratos m e boas separações com picos simétricos para compostos apolares e picos com simetria adequada, segundo parâmetros estabelecidos pela Farmacopéia Americana (United States Pharmacopeia), para compostos polares, avaliados pelos testes de Engelhardt, Tanaka, Neue, SRM 870 e pelo modelo dos parâmetros de solvatação utilizando CFS. A presença do óxido metálico no suporte resultou em FE com maior estabilidade química em condições drásticas de fase móvel (pH 1,7 e pH 10 a 50 °C), quando comparada com fases similares baseadas em sílica nua. A reação de capeamento melhorou a estabilidade química das FE e diminuiu o número de silanóis residuais. As FE Si-Zr(PMODS), Si-Ti(PMODS), Si-Zr(PMODS)ec e Si- Ti(PMODS)ec apresentam potencialidade na separação de fármacos psicoativos, filtros ultravioleta (UV), xantinas e hidrocarbonetos policíclicos aromáticos (HPA), utilizando CLAE e CFS. / Abstract: This work presents the development of stationary phases (SP) for high performance liquid chromatography in the reversed phase (RP-HPLC) and for supercritical fluid chromatography (SFC) prepared by the sorption and thermal immobilization of poly(methyloctadecylsiloxane) (PMODS) onto silica and metalized silica supports modified with zirconium and titanium. The immobilization process was optimized using central composite design for determination of the best conditions of time and temperature. The polymer, supports and stationary phases were characterized with physico-chemical and chromatographic tests. The stationary phases Si(PMODS), Si-Zr(PMODS) and Si-Ti(PMODS) presented the best results when a temperature of 120 °C for 16 hours was applied. The best SP were submitted to endcapping processes. These SP showed efficiencies between 82,000 and 90,000 plates m and good separations with symmetric peaks for apolar and polar compounds evaluated by the Engelhardt, Tanaka, Neue and SRM 870 tests and the solvation parameter model using supercritical fluid chromatography (SFC). The presence of metallic oxide on the supports resulted in stationary phases with better chemical stability under drastic conditions of mobile phase (pH 1.7 and pH 10 at 50 °C), when compared to similar stationary phases based on bare silica. The endcapping reaction improved the chemical stability. The SP Si-Zr(PMODS), Si- Ti(PMODS), Si-Zr(PMODS)ec e Si-Ti(PMODS)ec presented potentialities for the separation different pharmaceutical compounds used as psychotropic drugs, ultraviolet (UV) filters, xanthines, and polycyclic aromatic hydrocarbons (PAH), using separations in HPLC and SFC. / Doutorado / Quimica Analitica / Doutor em Ciências