<p> An inexpensive method was developed for the conversion of a high performance liquid chromatography (HPLC) system for use with 1 mm I.D. microbore columns. Chromatographic performance of the system was tested under both isocratic and gradient elution conditions, using a standard mixture of 16 polycyclic aromatic hydrocarbons (PAH).</p> <p> The microbore column HPLC was also coupled to a mass spectrometer equipped with a moving belt interface. Chromatographic performance under isocratic and gradient elution and mass spectral performance under scanning and selected ion monitoring modes were tested using the PAH standard.</p> <p> A marine sediment extract was subjected to qualitative and quantitative analysis for PAH. Qualitative results on the sample were obtained from a combination of retention indices, mass spectra, and retention times. Quantitation was performed by microbore column liquid chromatography-mass spectrometry (LC/MS) in the selected ion monitoring mode of operation. The method of calibration used was external calibration.</p> <p> The microbore column HPLC system exhibited good chromatographic behavior. Resolution, peak shape and short term retention time reproducibility were good, although, long term retention time fluctuations, due to changing mobile phase flow rates, were noted.</p> <p> The combination of microbore column HPLC with a moving belt interface and mass spectrometer gave excellent results. Problems commonly encountered with conventional column (4.6 mm I.D.) LC/MS, such as backstreaming, droplet formation, and splattering were greatly reduced, resulting in no apparent loss of chromatographic integrity and stable mass spectrometer operating conditions. These operating conditions proved to be most advantageous in the quantitative analysis of the marine sediment extract by selected ion monitoring.</p> / Thesis / Master of Science (MSc)

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No description available.

A balanced density slurry-packing apparatus was developed and used to pack sixteen HPLC columns having different lengths and internal diameters with 10 µm Lichrosorb Sl-100. The evaluation of these columns led to several conclusions: the column efficiency, expressed in plates per meter, is a function of the ratio of column internal diameter to column length and increases as this ratio increases; a new definition for preparative efficiency, Time Yield Factor (TYF), is proposed. This factor differentiates among HPLC columns with different lengths and internal diameters using samples of the same volume and concentration; the column that showed the highest TYF value was used for the preparative separation and identification of three positional isomers of high molecular weight (634): 3,4-di(p-bromophenyl)-1,2,5-triphenylcyclo-2,4-pentadien-1-methyl ether; 2,3-di(p-bromophenyl )-1,4,5-triphenylcyclo-2,4-pentadien-1-methyl ether; and 1,2-di(p-bromophenyl )-3,4,5-triphenylcyclo-2,4-pentadien-1-methyl ether, obtained in the synthesis work. / Ph. D.

Two on-line multidimensional HPLC systems are described; one coupling two liquid chromatographic columns (LC/LC), and one coupling liquid and gas chromatography (LC/GC). Theoretical equations relating the reproducibility, accuracy and transfer efficiency to system operating variables such as flow rate, retention time, column efficiency and transfer volume have been developed. These effects are explained and verified using experimental systems. Three major applications are shown for each of the systems. For the LC/LC systems, these include; the analysis of caffeine and theophylline in biological fluids; the analysis of hydrocarbon group types in fuels and oils and the analysis of catecholamines in urine with electrochemical detection. For the LC/GC system, the applications include; the analysis of pesticides in butter, the analysis of hydrocarbon group types in fuels and the analysis of polycyclic aromatic hydrocarbons in petroleum related samples. / Ph. D.

Means of achieving rapid, efficient separations of analytes are explored in detail, with particular emphasis on the use of chromatographic and electrophoretic theory as an aid in system design and optimization. The benefits of miniaturization of chromatographic systems are assessed. First, the utility of semi-micro Ion Chromatography is explored by the manufacture of 2mm ID analytical and suppressor columns and a micro-conductivity cell. The quality of the columns and detector cell are evaluated by the separation of a test mixture and the calculation of peak variance contribution of the detector cell. The use of readily available analytical scale instrumentation for semi-micro IC is demonstrated. Next, a further downsizing of the IC system is described, in which 530μm ID fused silica tubing is utilized for column manufacture. In this case, a suppressor column is not used and UV detection is employed in the analysis of nucleoside monophosphates. Again, column performance characteristics are measured and noted. Application of this system to the separation of a hydrolysed nucleic acid sample demonstrates the feasibility of the technique to the analysis of volume-limited samples in low concentration with notable sensitivity. The benefits of a miniaturized liquid chromatographic system under pressure-driven flow is studied and the improved permeability of micropacked capillary columns is exploited in the manufacture of several 25 to 30cm columns which achieve high efficiencies with relatively low pressure drops. Van Deemter plots illustrate the performance characteristics of the columns. Finally, electroosmotic flow is studied as the motive force for liquid chromatographic separations. This combination of two techniques, packed capillary liquid chromatography and capillary electrophoresis, results in a system which achieves good resolving power through maximization of selectivity and efficiency. / Ph. D.

The advantages and disadvantages of packed capillary columns for high performance liquid chromatography are examined. Historically, the advantages are smaller sample and phase consumption, enhancement in sensitivity, easy column synthesis, higher obtainable efficiency, and easier interfacing to other techniques. These points are explored through experiments in microbore ion chromatography, packed fused silica columns, and capillary zone electrophoresis. These studies also address the disadvantages of microscale HPLC which are stringent instrument design, brittle or weak columns, poor column stability, and the lack of commercial instrumentation. The results of these investigations indicate the following. First, the purported sensitivity enhancement is really attributable to solute focusing and not to column miniaturization. Second, column synthesis is still a difficult procedure that requires experience. Third, higher efficiencies are realized, especially when the column diameter to particle diameter ratio is optimized. Fourth, interfacing to other techniques is simplified because of the lower volumetric flow rates. Finally, the only real disadvantages are stringent instrument design and brittle columns in some instances. This thesis offers means for circumventing these difficulties. / Ph. D.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: In this study, the analysis of ammo acids usmg High Performance Liquid Chromatography (HPLC) with pre-column derivatisation was optimised. The derivatisation reagents include o-phthaldialdehyde (OPA), 9- fluorenylmethylchloroformate (FMOC) and iodoacetic acid (IDA). Detection was performed using UV and fluorescence in series. The developed method was utilised for the analysis and quantitation of amino acids in eighteen wines. The application of chemometric data evaluation was initiated. / AFRIKAANSE OPSOMMING: Hierdie ondersoek behels die optimisering van die aminosuuranalise deur gebruik te maak van Hoë Druk Vloeistof Chromatografie (HDVC) in kombinasie met pre-kolom derivatisering. Die derivativatiserings reagense sluit in o-phthaldialdehied (OPA), 9- fluorenielmetielchloroformaat (FMOC) en jodoasynsuur (IDA). Deteksie is gedoen deur gebruik gemaak van 'n ultraviolet (UV) en 'n fluorosensie detektor in serie. Die metode sodoende ontwikkel is gebruik vir die analise en kwantifisering van aminosure in agtien wyne. Die toepassing van chemometriese data evaluasie is ook geïnisieer.

M. Tech. (Biomedical technology, Faculty of Applied and Computer Science), Vaal University of Technology / Approximately 1% of the world’s population has epilepsy, the second most common neurological disorder after stroke. In South Africa almost 1 in every 100 people has epilepsy, affecting all ages. Levetiracetam (LEV), marketed as Keppra® is an anticonvulsant drug used in the treatment of epilepsy. The daily dosage is 500 mg twice daily with a maximum of 3000 mg. The therapeutic range of LEV is between 12-46 μg/ml. Therapeutic drug monitoring (TDM) should be considered for LEV in patients with poor seizure control or long term treatment. TDM depends on accurate drug concentration measurements. In order to provide an accurate measurement, the High performance liquid chromatography (HPLC) method was developed, compared with a commercially available kit, and the stability of the samples was investigated. Ethical approval was obtained from the Human Research Ethics Committee (Medical), VUT (Ethics reference number: 2015024.4). The study was conducted from January to October 2015. This study involved three groups of volunteers who gave written consent. The first group were fifteen healthy MTech students in the Biomedical Technology Department at the Vaal University of Technology (VUT). Their blood samples were used for the analytical validation of the method and for the stability studies over a 4 weeks period. The second group were six patients from Pathcare Laboratories in Potchefstroom, Klerksdorp and Vereeniging who used Levetiracetam. Their blood samples were used to investigate the influence of different collection tubes as well as the handling and storage of samples on the LEV concentration. The third group were forty four patients from Pathcare Laboratories, Cape Town. Their blood samples were transported to Clinical Pharmacokinetic Laboratory (CPL) for routine therapeutic drug monitoring analysis of LEV and used to compare the newly developed HPLC method and the Commercial kit. The HPLC method was successfully developed and validated to determine LEV in human plasma/serum samples. The calibration curves showed good linearity (r2 = 0,999) over the concentration range of 1 – 60 μg/ml. Accuracy, mean extraction recovery, lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were 98-112%, 97,15% (±1,57), 0,5 and 1,0 μg/ml respectively, in plasma standards. The method was shown to be simple and fast, reproducible and effective for routine laboratory analyses in the future. The agreement between the newly developed method and the ClinRep® HPLC complete commercial kit was the same and there was a statistical significant correlation between the two methods (average r=0.999; p-value < 0.0001, F-test with a true value =0). The method was much cheaper than the commercial kit, used less sample (100 μl) and had a longer running time (15 minutes) to ensure no endogenous interference. The costs of the developed method was 71-82% lower than the three commercial kits available in South Africa. Stability experiments were performed to evaluate the stability of LEV in human plasma/serum, simulating the same conditions which occurred during study samples’ analyses. The % RSD was lower than 5% under all the conditions: freeze, fridge, room temperature and auto sampler over the 4 week period. The results showed that both LEV and the I.S (internal standard) were stable in human serum/plasma under all these conditions. The influence of five different collection tubes, Gold (SST Gel), Red, Purple (EDTA)Green (Heparin) and Blue (Sodium Citrate) was investigated. In two patients, decreased levels were observed in tubes containing blue (sodium citrate) and Green (Heparin). The decrease was not statistically significant. This is an important observation and is an indication that anticoagulants may cause some problems due to drug-protein binding and interference in the matrix effect. A cost effective and reliable HPLC-method with minimal sample preparation time for the routine determination of LEV in plasma/serum samples was developed. It was also shown that the plasma/serum samples were stable at different temperatures over a time period. The only collection tubes that may interfere with the concentrations were the Green (Heparin) and Blue (Sodium Citrate) tubes.

High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.