No description available.

No description available.

High Pressure Liquid Chromatography (HPLC) is the process of separating multiple analytes by differing affinities toward the stationary phase. The selectivity of a stationary phase is dependent on the surface functional groups, though the majority of research only characterises new phases on a mesoscopic level. Solid-state NMR is a non-invasive technique which can provide detailed analysis on these materials on a molecular level. The technique can be used to determine not only whether a functional group has been successfully incorporated but can analyse whether this group is on the pore surface, able to interact with analytes, or trapped within the pore wall. Furthermore, we can assess whether the organic moiety is homogeneously distributed throughout the stationary phase and the interconnectivity of the silica framework. Analysing new stationary phases on a molecular level is essential to determining their chromatographic applications. The synthesis of three bi-functionalised PMOs under acidic conditions using combinations of bridging (-CH2CHr, -CH=CH-) and tether (-CH2C6HS, -C6Hs) functionalities is presented. By incorporating the organic groups into the silica framework we have produced new reversed phase columns containing a high carbon content. The synthesis of each periodic mesoporous organosilica (PMO) was optimised by varying the acid content and analysing the effect on a mesoscopic scale. The bi- functionalised silicas were then assessed on a molecular level to determine the mobility and the homogeneity of the functionalities using 2-D experiments such as 1H_13C Wideline (WISE) NMR and 1H_29Si Heteronuclear correlation (HETCOR) spectroscopy. The most viable stationary phases were analysed chromatographically using PAH and Tanaka tests and the results were compared to commercially available columns. There are many columns available, though even those containing the same functionality can produce very different separations due to the different synthetic procedures. We have compared two commercially available silicas, 1.7 urn Syncronis (containing monorneric . -C18) and Spherisorb ODS2 (containing polymeric -C18). Although both stationary phases contain the same functionality we have shown that on a molecular level they are very different. We have tried to relate these differences to the selectivity of the two phases. Using techniques such as 1H Nuclear Overhauser Effect Spectroscopy (NOESY) we have been able to compare how a toluene analyte interacts with two -C18 functionalised stationary phases. By also analysing the interactions between a -CH2CHr/ -CH2C6Hs functionalised PMO and toluene we have shown the way in which the analyte interacts with the stationary phase is dependent on the functionality incorporated. The synthesis of a new sphere-in-sphere stationary phase to improve the selectivity of larger biomolecules is presented. By sintering silica nanoparticles to the surface of large pore silica the mechanical strength and surface area of the material is improved though the large pores remaining can still facilitate the mass transfer of high molecular weight compounds. We have shown that by grafting -C12 moieties onto the surface of the silica the shorter elution time of smaller molecules compared to larger compounds is observed, whereas classical size exclusion chromatography (SEC) elutes larger analytes first. This suggests that the separation may be effected by some reversed- phase characteristics.

The current study has demonstrated that CN phases have poor stability and should not be operated above pH 5.7. Nonetheless, a number of protocols have been developed for the characterization of CN phases that include a measurement of silanophilic interaction determined in mobile phases with pH > 5.7. The aim of this study was to perform the chromatographic characterization of commercially available CN phases using two distinct approaches.

The retention mechanism of polar compounds in hydrophilic interaction chromatography (HILIC) was investigated in order to analyse many polar compounds in pharmaceutical and biological samples. HILIC uses a polar stationary phase in conjunction with an aqueous mobile phase containing a high concentration of acetonitrile (60- 95%). The use of bare silica, silica based bonded phases and polymer based bonded phases were investigated for the separation of catecholamines (dopamine, epinephrine and norepinephrine) in HILIC and per aqueous liquid chromatography (PALC) mode. A bonded phase column provided complete resolution of catecholamines in comparison to a bare silica column. The retention mechanisms for these polar basic compounds were found to be ionic interaction, partitioning and adsorption. The use of a polymer based ZIC-HILIC column proved that some of the ionic interaction (which is less on a silica based ZIC-HILIC column) is due to sulfonic groups on the column. The HILIC contribution is higher at higher buffer concentration on both polymer and silica based columns. High buffer concentration reduces ionic interaction but also may absorb more water resulting in a thicker aqueous layer on the column surface for solute partitioning. An overloading study of various basic and a quaternary ammonium compound was performed in the HILIC mode with respect to various pH and % ACN in the mobile phase using different colunms including bare silica (Atlantis HILIC, Zorbax Rx SIL, Ascentis HILIC) and bonded phase (ZIC-HILIC, Diol), The results of the study were complex and the results difficult to interpret. However, it was possible to draw some general conclusion. In general the loading capacity of the bare silica and bonded phase column was of the same order. However, there were variations in the comparative loading capacities with pH. The partitioning mechanism was investigated in the context of the octanol-water distribution coefficient Dow of the solute, A good correlation (R) was achieved between log D and the retention factor (k) of 29 compounds (acidic, basic, quaternary ammonium and neutral) on six different HILIC columns comprising bare silica, amide, dial. zwitterionic and a mixed-mode dial/hydrophobic phase. The correlation of k with log D was found to be improved for each column when the aqueous content of the mobile phase was increased from 5- 15% and by changing the pH from 6.0 to 3.0. It is possible that adsorption on to the polar groups of the stationary phase at low water content and ionic interact ion at higher pH (ionised silica) are competing mechanisms. The effect of pH, % ACN, temperature and stationary phase on selectivity was studied by comparing the correlation of plots of k using one condition vs a second condition. The higher the deviation from linearity, the greater is the effect of the condition change on selectivity. Changing the stationary phase was shown to be a powerful tool in influencing selectivity. HILIC was successfully used for bioanalytical purposes for the analysis of catecholamines in urine using electrochemical detection and gives a very good result in order to analyse polar antibiotics in human serum using mass spectrometry detection. Further, HILIC can be used for the analysis of other polar metabolites in biological samples for drug discovery and metabolomics study

No description available.

This project has focused on the study of hybrid phases at low percentage of organic modifier and pure water using high temperature in liquid chromatography (HTLC). It examined the effect of temperature on the retention of a range of test solutes and phenol homologues. The hybrid columns all showed linear van't Hoff relationship at low percentages of methanol but at higher temperature non linear van't Hoff curves were observed. Non·linear van't Hoff curves were observed with pure water on hybrid phases. This was thought to be because the retention mechanism changed at a discontinuity point above 100 ·C due to change in the properties of the mobile phase as well as changes in the entropy. The methylene selectivity decreased with temperature and increase with increasing pressure. XTerra phenyl and XBridge phenyl columns were stable up to 200 ·C and also in different flow rates without distortion in peaks. The efficiency of both columns was significantly decreased at low linear velocities due the domination of the B-term but at higher linear velocities the C-term dominated the separation. XBridge phenyl showed a flattened van Deemter curve indicates that high flow rate enabled a better separation. The application of high temperature on hybrid phases was studied for the separation of the selected steroids on XTerra MS C18 at low percentages methanol and pure water. A high flow rate was used to decrease the retention on XTerra MS C18 column at low percentage of methanol. In this project, variable back-pressure was used as an external parameter at constant flow rate and temperature to study the effect of pressure on retention in liquid chromatography. Hydrophobicity and shape selectivity, which depend on retention factor, increased due to increases in the pressure.

This study focused on the capabilities of elevated temperature chromatography and its potential applications. The effects of separation temperature on compound retention, selectivity and efficiency were studied. The change in retention with temperature was explored using a number of test compound solutions. A detailed investigation of the importance of system variables like dead volume, mobile phase composition, different stationary phases, and temperature control of column and eluent was performed. The effects of different eluents and different means of temperature control on the separation selectivity and efficiency were investigated and results were compared.

Natural products or traditional medicine has been used for centuries to treat various ailments by mankind. The World Health Organization estimates that in recent times 80% of people in emerging economies rely on traditional medicine as their primary health care [1]. However, unlike pharmaceutical products which have methods in place for quantification of the active compounds traditional medicine still requires a lot of focus in this area. Moringa oleifera is one of those species that have been used traditionally to cure various ailments for centuries. Even though extensive phytochemical and pharmacological studies have been conducted on the different parts of the plant, there is still no analytical method that enables the quantification of the compounds in the Moringa products in the market. The aim of this research was to develop an HPLC separation method that can be used to quantify the compounds found in the Moringa oleifera products. Compounds were extracted from the seeds of Moringa oleifera using the maceration method with a mixture of water and ethanol (1:1 v/v). The compounds were isolated using preparative HPLC. The structures of the compounds were elucidated using FTIR, NMR and MS. An HPLC separation method for quantification of the isolated compounds was developed and validated. The method was applied to the crude extract to quantify the isolated compounds in the extract. The following compounds 3-caffeoylquinic acid, 4-(α-L-Rhamnosyloxy) benzyl isothiocyanate, O-ethyl-[4-(α-L-Rhamnosyloxy) benzyl] thiocarbamate and O-butyl-[4-(α-L-Rhamnosyloxy) benzyl] thiocarbamate were isolated at percentage purities ranging from 90 to 99%. The HPLC separation method for quantification showed a linear relationship between peak area and concentration of the compounds with regression coefficients ranging from 0.9977 to 0.9994. The method is also precise with % RSD values between 0.01 and 1.16%. The method was shown to be specific to the compounds of interest. The percentage distribution of compounds in 50 mg of the seeds extract was between 0.25 and 1.10 % w/w. This study successfully developed and validated an HPLC separation method for four compounds found in the seeds of Moringa oleifera and quantified them in the crude extract of the seeds found in Zambia. This method can be used for identification and quantification of these four compounds in any of the Moringa products. As far as it could be ascertained this is the first time that such a method has been developed for these compounds / Chemistry / M. Sc. (Chemistry)

An environmentally friendly, rapid and cost effective analytical procedure based on dispersive liquid-liquid microextraction was developed for the determination of six tetracyclines (TCs) in meat destined for human consumption. Meat extracts were analyzed for TCs using a sensitive and selective analytical technique, liquid chromatography tandem mass spectrometry. Various influencing factors on the extraction, separation and determination of TCs such as pH of mobile phases, type and volume of disperser solvent, type and volume of extraction solvent and sample pH were optimized. Validation parameters such as calibration function, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCα), decision limit (CCβ), accuracy and precision were established according to EU commission decision 2002/657/EC. Linearity in the range of 25-200 μg kg-1 was obtained with regression coefficients ranging from 0.9991 to 0.9998. Recoveries of spiked blank muscle samples at three levels (i.e. 50, 100 and 150 μg kg-1) ranged from 80 to 101% and reproducibility was between 2 and 7%. The LODs and LOQs ranged from 2.22 to 3.59 μg kg-1 and from 7.38 to 11.49 μg kg-1 respectively. The CCα ranged from 105 to 111 μg kg-1 while CCβ ranged from 107 to 122 μg kg-1. The proposed method compared well with the dispersive solid phase extraction method and was successfully applied to the determination of TCs in meat samples. Some of the thirty bovine muscle samples obtained from local abattoirs and butcheries were found to contain two tetracycline antibiotics residues (chlortetracycline and oxytetracycline) with oxytetracycline being the most commonly detected. The concentration levels of the TC residues detected in the eleven bovine muscle samples were between 12.4 and 68.9 μg kg-1, levels that are lower than the European Union set maximum residue level (MRL) of 100 μg kg-1 hence the meat was fit for human consumption. / Chemistry / M. Sc. (Chemistry)