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The three factors of the resolution (Rs)equation(see Equation 1.1)were explored in this thesis. During the course of the research project, an important aim was to explore separation processes that would lead to an increase in productivity without sacrificing Rs. To that end, an increase in the retention factor (k)to enhance Rs was deemed detrimental to the cycle time, hence the production rate, particularly when preparative separations are involved. Consequently the primary objectives were to (i)prepare more efficient columns and (ii)investigate new strategies in manipulating selectivity. The significance of the work contained in this thesis is highlighted in 27th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2003)held in Nice, France between 15-19 June, 2003. Many of the papers presented significantly compared to chapters contained in this research / Doctor of Philosophy (PhD)

This thesis describes a developmental process required for the construction and optimisation of two-dimensional liquid chromatographic systems. The text encompasses much of the difficulties and provides a frame work for achieving positive results. The hardware requirements and the concepts for successful completion of heart-cutting and comprehensive modes of separation are addressed. Low molecular weight polystyrenes play an essential role in providing a model framework or theme for the development of two-dimensional liquid chromatographic systems. Low-molecular weight oligostyrenes were initially used to provide a theoretical assessment of combinations of liquid chromatographic systems. Interpretation of theoretical results then allowed for the development of a heart-cutting two-dimensional liquid chromatographic system for the separation of oligostyrene complex samples. This allowed for practice to be compared with the theoretical results, which are presented and provided direction as to what attributes of a two-dimensional liquid chromatographic system are beneficial. Petroleum oils are one of the most complex mixtures found in the natural environment and are included to provide a real-world application that demonstrates the potential power of comprehensive two-dimensional liquid chromatographic systems. Orthogonality is a central theme throughout the whole thesis. Separation correlation is shown to be advantageous in certain instances in the evaluation of hybrid stationary phases, as shown in the analysis of a low molecular weight polystyrene sample. The thesis finishes by the incorporation of monolithic separation media as an alternative second dimension stationary phase support. A method is discussed that allows for a substantial increase in the volume and concentration loading of two-dimensional liquid chromatographic systems that employ monolithic chromatography columns in the second dimension. This is important since volume overloading and concentration overloading in the second dimension can reduce separation performance and hence the final chapter provides a new direction for research. / Doctor of Philosophy (PhD)

Thesis (M. Sc. (Chemistry) -- University of Pretoria. 2006. / Includes bibliographical references (leaves 111-117).

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2009 Aug 16

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 140-153). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

 Proteomics is the systematic study of the proteome: the total protein expression of a cell or tissue under specified conditions. The multiplicity and complexity of proteins in cells requires sensitive, selective, and comprehensive methodologies for their distinction and characterization. Multidimensional liquid chromatography (MDLC) coupled with biological tandem mass spectrometry (MS/MS) is uniquely suited to fulfill those requirements and has become an indispensable tool in MS-based proteomics. Our laboratory has developed an online high-/low-pH reversed-phase/reversed-phase (RP–RP) LC system exhibiting fully automatable and reproducible performance. It is a promising alternative to the strong cation exchange/reversed-phase (SCX–RP) system commonly used in high-throughput comprehensive proteomics analyses. The first part of this Thesis (Chapter 2) describes the development of a variant of the high-/low-pH RP–RP platform—RP–SCX–RP—that integrates an additional SCX trap column between the two RP columns to enhance sample recovery. This new system allows the detection of larger numbers of hydrophilic peptides. Indeed, in the analyses of a lysate of Arabidopsis chloroplast proteins, it identified approximately 25% more non-redundant proteins than those identified using the previous version of the RP–RP system. The modified platform has been extended for the online removal of sodium dodecyl sulfate and other excess interference chemicals used in Isobaric Tags for Relative and Absolute Quantification (iTRAQ) reactions, thereby avoiding the need for time-consuming offline SCX clean-up prior to RP–RP separation in the quantitative proteomics analyses of crude biological samples at low-microgram levels. A novel online three-dimensional liquid chromatography (3DLC) system was derived from the RP–SCX–RP design, exhibiting remarkably enhanced orthogonality, resolution, and peak capacity. Peptides were separated in the first-dimension high-pH RP column based on their hydrophobicity, followed by sub-fractionation in the second-dimension SCX column, primarily based on charge; the third dimension was a typical low-pH RP separation, prior to MS analysis. The overall performance of the system was evaluated through analysis of a cell lysate of mouse embryonic fibroblasts. Relative to the two-dimensional high-/low-pH RP–RP system, the new 3D system yielded significant increases in the number of unique peptides and proteins identified, making it a good alternative to SCX–RP and high-/low-pH RP–RP as an efficient automated MDLC platform for high-throughput shotgun proteomics. An optimized and miniaturized variant of the three-dimensional LC platform was also developed. Its simplified setup and operation, by decreasing the number of six-port switching valves (from three to two) and the number of SCX fractionation steps, minimized both the potential sample loss and the total analysis time (by ca. 30%). Thus, a variety of novel, automatable, and robust RP–SCX–RP-based MDLC platforms have been developed for high-throughput qualitative and quantitative analysis. The performance of these systems complements conventional MDLC systems, with enhanced quality, quantity, reproducibility, and throughput of protein identification and quantification. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

No description available.

No description available.