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In modern chemical laboratories, high performance liquid chromatography (HPLC) has become the technique of choice amongst separation scientists as evidenced by the huge body of published literature that employ the technique as the pre-eminent analytical method. Increasingly analytical chemists are seeking to determine the chemical signature (or fingerprint) of complex and perhaps even simple samples. The analysis of environmental samples, natural products, such as essential oils, the evaluation of illicit drugs, or the quality assurance of pharmaceuticals are just four areas where chemical fingerprinting may be important. The chemical classification of these types of mixtures can be undertaken using chromatographic methods of analysis coupled with detection processes that provide identification ie. mass spectroscopy (MS), infrared spectroscopy (IR), atomic spectroscopy (ICP) and nuclear magnetic resonance spectroscopy (NMR). While these techniques find widespread use and their applications are growing at a rapid rate, their limitations as routine applications for determining chemical signatures, is limited by the initial purchase price and the high associated running costs; especially for hyphenated liquid chromatographic systems. In general, a high degree of operator expertise is also required to ensure correct operation and interpretation of the results. While complete chemical specification may be undertaken by using multiple hyphenated methods, none of the above mentioned techniques provide for a rapid means of detection on a routine or continuous mode of operation. This can limit their application. However, an alternative to monitor and evaluate the chemical signature of complex samples is to employ multidimensional separations. In the expanded multidimensional separation space the probability that two species will elute with exactly the same retention time in both separation dimensions decreases compared to the one-dimensional separation. This probability further decreases as the separation mechanisms become more divergent and the two-dimensional separation space is maximised. Hence, the uniqueness of a two-dimensional retention time increases and thereby the multidimensional separation approach becomes a means of chemical fingerprinting. Hence 2D-HPLC technique is a powerful and less expensive method of separation compared to hyphenated techniques. This thesis describes the development of a stand-alone 2D-HPLC system for the separation of complex samples. A comprehensive 2D-HPLC software package has been developed for data acquisition, hardware control, data processing and graphical presentation. Two data acquisition hardware modules have also been developed. These hardware and software modules have been integrated with existing equipment in our laboratory. The performance of this newly developed 2D-HPLC system has been successfully evaluated. All these details have been described in the five chapters of this thesis. / Master of Science (Honours)

A novel analytical method is developed to examine the chemical composition of plant oils by electrospray ionisation high-resolution Fourier transform ion cyclotron resonance mass spectrometry in both positive- and negative-ion modes. To date, this is the first reported application of this technique for the study of macadamia nut oil. Samples of macadamia nut oil from the Macadamia Integrifolia- Proteaceae family (smooth shell) are examined. The fatty acid profile of the oil is obtained by this mass spectrometric examination of the transesterified and hydrolysed oil samples. The Fourier transform ion-cyclotron resonance mass spectrometry results are compared to those obtained from similar samples using gas chromatography-mass spectrometry techniques. High performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry are used to separate and assign the isomers present in the methanol extract of the oils in separate experiments. Significant results in this study include: - The first observation and identity of a number of oxidised triacylglycerols in macadamia oil samples. - The first observation of oxidised and free fatty acids, measured directly in hydrolysed oil and in the methanol extract of macadamia oil. - High resolution Fourier transform ion cyclotron resonance mass spectrometry in broadband mode which enables isobars to be observed. - Esterified oil Fourier transform ion cyclotron resonance mass spectrometry results are consistent with our gas chromatography-mass spectrometry results and with the results of similar studies on macadamia oil in the literature. - A number of fatty acids with odd number of carbon atoms are observed in the oil. - In electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry of oils, the sample preparation is straightforward. The sample is dissolved in methanol or acetonitrile and the solution is introduced to the electrospray source directly. Introducing oil samples to the gas chromatograph-mass spectrometer needs the oils to be esterified prior to the analysis. - In this work, state-of-the-art mass spectrometry demonstrates distinct advantages in comparison to gas chromatography measurements such as direct identification of free fatty acids in oil samples, whereas this is not possible in gas chromatography-mass spectrometry due to the required esterification step prior to the analysis. - High performance liquid chromatography fraction collection is combined with Fourier transform ion cyclotron resonance mass spectrometry in off-line mode and found to improve the sensitivity, selectivity and signal to noise levels due to the lower number of compounds in each high performance liquid chromatography fraction compared to the methanol extract of macadamia oil sample. Also isomers of monoacylglycerols have been resolved using the high performance liquid chromatography technique.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 149-167).

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Dissolved organic matter (DOM) is comprised of a complex array of molecular constituents that are linked to many globally-relevant processes and yet this material is still largely molecularly uncharacterized. Research presented here attempted to probe the molecular complexity of this material from both Arctic and temperate climates via multifaceted and novel approaches. DOM collected from remote Arctic watersheds provided evidence to suggest that permafrost-disturbed systems contain more photochemically- and biologically-labile material than undisturbed systems. These results have large implications for predicted increasing temperatures where widespread permafrost melt would significantly impact stores of organic carbon in polar environments. In attempting to address the complexities and reactivity of DOM within global environments, more information at the molecular-level is necessary. Further research sought to unravel the molecularly uncharacterized fraction via use of nuclear magnetic resonance (NMR) spectroscopy in conjunction with hyphenated and varied analytical techniques. Directly hyphenated high performance size exclusion chromatography (HPSEC) with NMR was explored. This hyphenation was found to separate DOM into structurally distinct fractions but proved limited at reducing DOM heterogeneity. Of the many high performance liquid chromatography (HPLC) techniques tested, hydrophilic interaction chromatography (HILIC) was found the most effective at simplifying DOM. HILIC separations utilizing a sample from Florida resulted in fractions with highly resolved NMR signals and substantial reduction in heterogeneity. Further development with a 2D-HILIC/HILIC system to achieve additional fractionation was employed. This method produced fractions of DOM that were homogenous enough to produce excellent resolution and spectral dispersion, permitting 2D and 3D NMR experiments to be performed. Extensive NMR analyses of these fractions demonstrated strong evidence for the presence of highly oxidized sterols. All fractions, however, provided 2D NMR spectra consistent with oxidized polycyclic structures and support emerging data and hypotheses suggesting that cyclic structures, likely derived from terpenoids, are an abundant, refractory and major component of DOM. Research presented within this thesis demonstrates that HILIC and NMR are excellent co-techniques for the analysis of DOM as well as that oxidized sterols and other cyclic components with significant hydroxyl and carboxyl substituents are major constituents in DOM.

University of Macau / Institute of Chinese Medical Sciences

Zirconia is one of the most promising column packing materials for High Performance Liquid Chromatography (HPLC). The perfect HPLC support material should be energetically homogenous, have a high surface area on which different chemical species can reversibly attach and be physically and chemically stable over a wide range of pH, temperature and solvent conditions. Most existing supports do not have all of these properties. This project is also focused on a proteomics study. Zirconia, hafnium oxide and titanium oxide which are some of the more promising materials currently available, can be used for the separation and analysis of phosphorylated proteins. Adenosine triphosphate, Adenosine diphosphate and Adenosine monophosphate were used as prototypes for phosphorylated proteins. Separation, absorption, fluorescence and SEM studies were performed to determine the adsorption of Adenosine phosphates species at a particular pH on Zirconia. Zirconia was also used for the purification of Fibrinogen Growth Factor (FGF) protein, which are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique was used to analyze the off-column purification and separation of this protein. This research suggests that, at acidic conditions, adenosine monophosphate has more favorable absorption on the Zirconia surface. On the other hand, the separation study suggests that basic conditions are more favorable for the absorption of ATP, ADP and AMP when mixed together on Zirconia 500. Furthermore, it was found that Zirconia is a very promising material for the purification of FGF protein.

There are two purposes in this research, one is the development of the new method which can be used for detection and quantification of triarylmethanes in fish tissues. The other is that we confirmed validation and utility of triarylmethanes by the method that is according to Commission Decision 2002/657/EC. Homogenized fish tissues were extracted twice with acetonitrile and defatted with n-hexane. HPLC separation was conducted with the RP-18 column. The mobile phases consisted of 0.5 mM ammonium acetate buffer (pH 3.8, adjusted with acetic acid)¡V ACN (contained 0.1% formic acid) solution. Triarylmethane was determined by LC-ESI-MS/MS in positive mode. The correlation coefficients of calibration curves with triarylmethane in fish tissues were 0.998 ~ 0.999. The decision limits (CC£\) were 0.16 ¡Ó 0.07 £gg/kg(MG), 0.15 ¡Ó 0.04 £gg/kg(LMG), 0.20 ¡Ó 0.13 £gg/kg(CV) and 0.23 ¡Ó 0.12 £gg/kg(LCV), and detection capabilities (CC£]) were 0.20 ¡Ó 0.09 £gg/kg(MG), 0.18 ¡Ó 0.05 £gg/kg(LMG), 0.24 ¡Ó 0.16 £ggkg-1(CV) and 0.29 ¡Ó 0.15 £gg/kg(LCV).

Thesis (M.S.)--Eastern Illinois University, 2009. / Includes bibliographical references (leaves [117]-125).