Novel concepts and techniques in capillary electrochromatography

Capillary electrochromatography (CEC) has been realised as a combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) allowing the separation of analytes based on their differing adsorption and migration behaviour. There is a considerable interest in developing new methodologies for CEC, which uses chromatographic beads or monoliths as stationary phases or alternatively employs open tubular (OT) capillaries relying on surface modifications. Besides the bulk flat flow profile of the mobile phase in CEC columns allowing an enhanced resolution, due to the influence of the applied electrical field there are other possibilities to provide a higher resolution with better detection. As an additional newly means for peak sharpening and sample enrichment isotachophoresis and a “sweeping mechanism” could be envisaged, which both take advantage of the inhomogeneous electric field pattern in the capillary by choosing appropriate leading and terminating background electrolytes (BGEs). Transient isotachophoresis would be another technique to provide an increased plate height in the separations, using an optimized sample matrix, e.g. containing a relatively high acetonitrile content, with the same leading and terminating BGEs. In this thesis, a new method to generate a “sharpening zone”, which is similar to the concept of transient isotachophoresis, was established using an aqueous sample matrix containing an organic solvent. Target analytes within this zone could be concentrated whilst they migrated through the capillary. A novel technique was developed that used the sharpening zone for sweeping and concentrating several unresolved analyte peaks allowing an enhanced peak resolution. As the aim was to concentrate only some part of the analyte zone containing compounds of interest, the position and width of the sharpening zone needed to be well controlled. Various techniques to tune the width and position of the sharpening zone were thus developed, involving modifying the organic modifier content in the BGE, applying pressure additionally to the electroosmotic flow (EOF) and modifying the inner surface of the capillaries. This work also critically evaluated some related existing knowledge and theoretical approaches in CEC including the concept of tuneable system eigenpeaks (noncomigrating or ghost peaks caused by waves of an electric field in the capillary). Although peak broadening caused by eigenpeaks (which often suppresses resolution in CEC) has been well described for aqueous or pure non-aqueous BGEs, a systematic study of eigenmobility in aqueous/acetonitrile BGEs had not been reported. The experiments related to the eigenmobility led to a new approach to tune the eigenpeak position in chromatograms whereby a selective sample peak sharpening mechanism was elaborated using the sharpening zone. The novel approach was demonstrated for the separation of various peptides in the CE mode and for the resolution of incompletely resolved chiral amino acids in CEC. These investigations furthermore led to the development of an appropriate stationary phase, which was produced using techniques for an in-column synthesis of a molecular imprinted polymer (MIP). Well prepared MIPs are capable of specifically enhancing the retention of target analytes, herein focusing on enantio-selective isolation of chiral molecules. Since such a MIP was rather rigid, which often caused capillary clogging when used as a conventional monolithic stationary phase, this MIP was incorporated into porous layer open tubular (PLOT) capillaries. Numerous experimental difficulties were overcome with respect to the vinylisation of long fused-silica capillaries and for the polymerisation of the MIP within using thermal- and photo-polymerisations. A method for the characterization of such MIP-PLOT capillaries was elaborated involving the Langmuir adsorption theory. The new columns were evaluated using chromatography, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and CEC analysis. All experiments were designed according to the principles of Green Chemistry. These concepts involved the use of small scale analysis, simulation studies reducing the number of performed experiments, systematic studies with low amounts of solvents and materials, and optimisation of synthesis methods to reduce time, energy and reagent consumption. The theories, methods and results presented in this thesis will, in the future, allow further targeted developments of novel analytical tools for a green, sensitive, selective and improved resolution analysis of chemical and biological compounds including chiral compounds, peptides and proteins

Identification of Volatile Compounds and Selection of Discriminant Markers for Elephant Dung Coffee Using Static Headspace Gas Chromatography—Mass Spectrometry and Chemometrics

Elephant dung coffee (Black Ivory Coffee) is a unique Thai coffee produced from Arabica coffee cherries consumed by Asian elephants and collected from their feces. In this work, elephant dung coffee and controls were analyzed using static headspace gas chromatography hyphenated with mass spectrometry (SHS GC-MS), and chemometric approaches were applied for multivariate analysis and the selection of marker compounds that are characteristic of the coffee. Seventy-eight volatile compounds belonging to 13 chemical classes were tentatively identified, including six alcohols, five aldehydes, one carboxylic acid, three esters, 17 furans, one furanone, 13 ketones, two oxazoles, four phenolic compounds, 14 pyrazines, one pyridine, eight pyrroles and three sulfur-containing compounds. Moreover, four potential discriminant markers of elephant dung coffee, including 3-methyl-1-butanol, 2-methyl-1-butanol, 2-furfurylfuran and 3-penten-2-one were established. The proposed method may be useful for elephant dung coffee authentication and quality control

Thermally Sensitive Behavior Explanation for Unusual Orthogonality Observed in Comprehensive Two-Dimensional Gas Chromatography Comprising a Single Ionic Liquid Stationary Phase

In this study, a theoretical concept and method to achieve a degree of orthogonality in comprehensive two-dimensional gas chromatography (GC × GC) for separation of fatty acid methyl esters (FAME) by using a single ionic liquid (IL) stationary phase (1-phase-GC × GC) were established. The 1-phase system comprises a long IL column and shorter IL column of the same phase before and after the modulation region, operated under temperature-programmed conditions. Initial isothermal experiments employing six commercial IL columns were conducted at different temperatures. On the basis of the temperature-dependent linear solvation energy relationship (LSER) concept, SLB-IL111 exhibited the greatest thermal sensitivity and degree of difference over the tested temperature (<i>T</i>) range, so it was selected for investigation of the 1-phase-GC × GC mode. With the same temperature program, a significantly high degree of orthogonality was observed for the experiment, varied with column lengths. The switchable separation result, which inverts the retention of saturated and unsaturated FAME on the downstream column (²D), was achieved by varying column diameters and surface thicknesses of the IL-coated layers. These results were explained according to the corresponding LSER principles. Also, the time summation model was applied for the simulation of the observed 1-phase-GC × GC results

In Silico Modeling of Hundred Thousand Experiments for Effective Selection of Ionic Liquid Phase Combinations in Comprehensive Two-Dimensional Gas Chromatography

The selection of the best column sets is one of the most tedious processes in comprehensive two-dimensional gas chromatography (GC × GC) where a multitude of choices of column sets could be employed for an individual sample analysis. We demonstrate analyte/stationary phase dependent selection approaches based on the linear solvation energy relationship (LSER), which is a reliable concept for the study of interaction mechanisms and retention prediction with a large database pool of columns and compounds. Good correlations between our predicted results, with experimental results reported in the literature, were obtained. The developed approaches were applied to the simulation of 157 920 individual experiments in GC × GC, focusing on the application of 30 nonionic liquid and 111 ionic liquid (IL) stationary phases for separation of some example sets of model compounds present in practical samples. The best column sets for each sample separation could then be extracted according to maximizing orthogonality, which estimates the quality of separation

Switchable Enantioselective Three- and Four-Dimensional Dynamic Gas Chromatography–Mass Spectrometry: Example Study of On-Column Molecular Interconversion

A novel hybrid online enantioselective four-dimensional dynamic GC (<i>e</i>4D-<i>D</i>GC) approach to study reversible molecular interconversion through specific isolation of a diastereo and enantiopure oxime, 2-phenylpropanaldehyde oxime, from prior multidimensional separation, is described. It incorporates a pre-enantioseparation step that applies comprehensive two-dimensional GC (GC × GC), prior to multiple microfluidic (Deans) switching for selection of components of a diastereomeric (<i>E</i>,<i>Z</i>) and enantiomeric (<i>R</i>,<i>S</i>) oxime into a third reactor column where isomerization occurs. This is followed by <i>E</i>/<i>Z</i> separation in a fourth analytical column. The enantioselective first dimension (¹D_enant) yields enantioseparation of <i>E</i>(<i>R</i>), <i>Z</i>(<i>R</i>), <i>E</i>(<i>S</i>), and <i>Z</i>(<i>S</i>) isomers, with a characteristic interconversion zone between the <i>E</i> and <i>Z</i> isomers. However, these are contaminated with underlying stereoisomers. Selected separation regions were then modulated and separated using a second dimension (²D) column via GC × GC, resolving the interfering stereoisomers. Individual pure enantiomers were then selectively heart-cut from within the 2D separation space, cryofocused, then eluted on a ³D reactor column for <i>E</i> ⇌ <i>Z</i> isomerization under controlled oven temperature and flow. Heart-cuts taken over the resulting interconversion distribution were cryotrapped at the inlet of a ⁴D column, on which achiral separation allows precise quantification of each <i>E</i> and <i>Z</i> isomer of the enantiomer. From peak areas and isomerization time, the forward and backward rate constants (<i>k</i>_{<i>E</i>→<i>Z</i>} and <i>k</i>_{<i>Z</i>→<i>E</i>}) were determined. The described methodology is suited to other configurationally labile molecules (for instance, hydrazones and imines), which exhibit isomerization, and can be used to isolate individual compounds from multicomponent samples, without requiring pure compound synthesis, or complex mathematical models or in-silico simulations

Two-Dimensional Retention Indices Improve Component Identification in Comprehensive Two-Dimensional Gas Chromatography of Saffron

Comprehensive two-dimensional gas chromatography hyphenated with accurate mass time-of-flight mass spectrometry (GC × GC-accTOFMS) was applied for improved analytical accuracy of saffron analysis, by using retention indices in the two-dimensional separation. This constitutes 3 dimensions of identification. In addition to accTOFMS specificity, and first dimension retention indices (¹<i>I</i>), a simple method involving direct multiple injections with stepwise isothermal temperature programming is described for construction of isovolatility curves for reference alkane series in GC × GC. This gives access to calculated second dimension retention indices (²<i>I</i>). Reliability of the calculated ²<i>I</i> was evaluated by using a Grob test mixture, and saturated alkanes, revealing good correlation between previously reported <i>I</i> values from the literature, with R² correlation being 0.9997. This essentially recognizes the retention property of peaks in the GC × GC 2D space as being reducible to a retention index in each dimension, which should be a valuable tool supporting identification. The benefit of ²<i>I</i> data, in supplementing ¹<i>I</i> and MS library matching, was clearly demonstrated by the progressive reduction of the number of possible compound matches for peaks observed in saffron. 114 analytes were assessed according to ¹<i>I</i> and ²<i>I</i> values within ±20 index unit of reference values, and by MS spectrum matching above a match statistic of 750 (including mass accuracy of the molecular ion <20 ppm) and their possible identities derived. The described method provides a new avenue to utilize the full capability of the two-dimensional separation (GC × GC), in combination with MS library matching in complex sample analysis, to provide improved component identification