The suitability of nitrophenyl boronates as derivatives for GC and HPLC analysis has been studied. Samples of ortho, meta and para nitrophenyl boronic acids were prepared and purified, the purity of these products was examined by GC. The derivatization performance of these acids was studied employing a number of bifunctional model compounds such as: diols, aminoalcohols, hydroxy acids etc. The derivatives formed were studied to determine their stability, speed of formation, quantitative analysis and overall chromatographic behavior. The derivatization reactions were found to be very fast and essentially quantitative, reaching equilibrium in just a few minutes at room temperature. The boronates formed are of different stabilities depending on their structure, the single most important stability factor is the boronate ring size, this is followed by the alkyl substitution present and the position of the nitro group on the aromatic ring. Excellent quantitative performance was obtained with flame ionization and electron capture detectors. Detection limits in the femtomole range were achieved with the EC detector. Solvent background and adsorption problems were found at the picomole level in some cases. The applicability of nitrophenyl boronates to HPLC analysis is seriously limited by the column activity due to residual silanol groups, this tends to decompose all but the most stable boronates. However, excellent results were obtained in the case of 2,4-pentandiol boronates, reaching detection limits in the picomole level with a variable wavelength ultraviolet detector. Nitrophenyl boronic acids can also derivatize ortho disubstituted aromatic molecules containing hydroxyl, amino and carboxylic acid groups. Molecules which contain the reactive groups on the side chain attached to an aromatic ring can present a steric obstruction when reacting with the ortho nitrophenyl boronic acid. This same problem was found in the case of 1,3-propandithiol which reacts only with the meta and para nitro phenyl boronic acids. / Ph. D.

The modification of a gas chromatograph for capillary supercritical fluid was demonstrated. The resulting instrument was used to characterize the performance of capillary columns manufactured specifically for SFC applications. The columns were tested for inertness, efficiency, and immobilization before and after extraction with the supercritical CO₂ solvent. Stable deactivation layers were obtained with polyethylene glycol pyrolysis of Carbowax 20M and Superox 20M. Crosslinking was evaluated with dicumyl peroxide (DCP) and azo-t-butane (ATB) as free radical initiators with OV-1701 as a stationary phase. ATB was found to yield more efficient crosslinking and had less effect on column polarity. Quantitative reproducibility of SFC was evaluated for alkanes, alcohols and acids on columns which were deactivated by polyethylene glycol pyrolysis or by mixed cyclic siloxanes. Both gave better reproducibility than an untreated column but the overall reproducibility of the polyglycol deactivation was better than the cyclic deactivation. Liquid CO₂ extraction was performed on a variety of spices and food products. This sample preparation proved useful in isolating CO₂ soluble materials from complex matrices which were insoluble in CO₂. / Ph. D.

The purpose of this investigation was to employ newly improved qualitative and quantitative chromatographic techniques to obtain purified myosin from 1 G and 3 G chick embryos and to determine if muscle myosin production either follows or precedes the unparallel bone growth during chronic acceleration as reported by several investigators.

Four mechanisms were considered as possible causes of the loss in resolution for a Dionex CG2 ion chromatography resin: 1) presence of inorganic ions strongly bound to the active sites; 2) adsorption of organic species; 3) physical alterations; and 4) chemical alterations. The instrumental analyses used to gather data were ICP, FT-IR, SEM, solid C-13 NMR and IC. Based on the results, no metal ions are bound to the resin, no organic species are held onto the resin, and no physical change was observable. The cause for the loss of resolution is a strong reduction in the number of active sites in the resin as confirmed by elemental analysis for the sulfur in the sulfonic acid present in the active sites.

The reaction between nitrogen-containing organic compounds (pyridines and amines) and alumina was studied through the use of chromatographic titrations and elemental analysis. The aluminum-nitrogen bond was the basis for the preparation of a series of new chromatographic adsorbents. A series of amines and pyridine homologs were bound to alumina and evaluated as chromatographic stationary phases. The surface coverage was determined using elemental analysis. The adsorption properties of the stationary phases were investigated by determining differential enthalpies, entropies, and free energies of adsorption for a selected group of organic moieties. Rate theory was used to examine further the interaction between the molecular probes and the tailored supports. Relative peak broadening was measured both as a function of flow rate and temperature to provide a chromatographic evaluation of molecular probe-adsorbent interaction occurring during the chromatographic process. Results indicated that the single-molecule moieties used as tailoring agents served as excellent deactivating agents but produced no significant changes in the selectivity of the adsorbents. Pellicular beads were synthesized by using 4-vinylpyridine as a linking agent between the alumina substrate and a series of polymers. The polymers used in this study were polystyrene, polymethylmethacrylate, and polyacrylonitrile. Each type of polymer-coated bead was prepared at different loading levels. Scanning electron microscopy was used to examine the gross change in the surface and elemental analysis used to determine the polymer loading. The adsorption properties of the polymeric pellicular supports were studied by determining the aforementioned thermodynamic quantities and by rate theory. The relative peak broadening was mentioned as a function of polymer type, loading, flow rate, and temperature. The chromatographic behavior of the selected molecular probes on the pellicular supports was sensitive to polymer type and loading. It was established that alumina can be modified with amines and pyridines and that a molecule possessing a nitrogen atom and a polymerization site can be used as an adhesive interface to chemically link polymeric phases to alumina. Currently, there are no commercially available modified aluminas. It has been demonstrated here that the preparation of such supports is feasible and can lead to chromatographically useful products.

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Inthis work we aim to put into place a system to separate or immobilise biomacromolecules by means ofimmobilised transition metal ions like nickel(II) or copper(II). Although the concept of immobilised metal affmity chromatography (IMAC) has been around since the early 1960's, the metal ions were always immobilised by covalent modification of the support matrix. Recently the concept of IMAC was applied to membranes, and again the metal ion was immobilised by covalent modification of the membrane surface. Inthis study we covalently modified the support matrix by attaching a linear, EDTA type ligand to the hydroxy end groups of a tri-block copolymer (polyethylene oxide (PEO)m = 129 - polypropylene oxide (PPO)n = 56 - polyethylene oxide (PEO)m= 129), Pluronic® F108. The middle block of this polymer, which is hydrophobic, will non-covalently adsorb onto the membrane surface through hydrophobic interaction. The hydrophilic outer blocks, with the ligand modified end groups, will associate with the aqueous substrate exposing the chelated metal ion for interaction with the bio-macromolecules. This affords a system which is recyclable, without replacing the membranes, simply by stripping the expired ligand modified-polymer and adsorbing fresh polymer. A series of model ligands and their complexes were synthesised and characterised, to study the coordination of the ligand around the metal ions. The model compounds were also essential in characterising the final product - the ligand modified Pluronic. Finally the ligand modified Pluronic was tested for its metal binding capabilities. This was done in aqueous solution by qualitatively comparing the UV-VIS spectra of the modified Pluronic with that of the model ligands and complexes. The spectra indicate that metal coordination does take place. / AFRIKAANSE OPSOMMING: In die studie het ons beoog om 'n sisteem daar te stel vir die skei en immobilisering van bio-makromolekules deur middel van oorgangsmetale soos nikkel(II) en koper(II). Alhoewel die beginsel van geïmmobiliseerde Metaal-ioon Affmiteits Chromatografie (IMAC) reeds sedert die vroeë 1960's bekend is, is die metaal ione geïmmobiliseer deur kovalente modifikasie van die draermaterial. Onlangs is die beginsel van IMAC uitgebrei na membrane en ook hierin is die metaalione geïmmobiliseer deur kovalente modifikasie van die membraanoppervlaktes. In die projek het ons die draermateriaal kovalent gemodifiseer deur 'n lineêre EDT A-tipe ligand te koppel aan die hidroksie eindgroepe van 'n tri-blok ko-polimeer (poli-etileen oksied (PEO)m = 129 - poli-propileen oksied (PPO)n = 56 - poli-etileen oksied (PEO)m = 129), Pluronic® FI08. Die middelste blok van die polimeer, wat hidrofobies is, sal nie-kovalent aan die membraan oppervlakte adsorbeer d.m. v hidrofobiese interaksie. Die hidrofiliese buite blokke, met die ligand-gemodifiseerde eindgroepe, sal assosieer met die waterige substraat en die metal ioon blootstel vir interaksie met die biomakromolekules. Dit stel dus 'n sisteem in plek wat herbenut kan word, sonder om die membrane te vervang, deur eenvoudig die ligand-gemodifiseerde polimeer wat verval het te stroop en te vervang met nuwe polimeer. 'n Reeks modelligande en hul komplekse was gesintetiseer en gekarakteriseer om die koördinasie van die ligande rondom die metaal ione te bestudeer. Dié model verbindings was van groot belang in die karakterisering van die finale produk - die ligand-gemodifiseerde Pluronic. Laastens is die ligand-gemodifiseerde Pluronic getoets vir sy metaal bindings vermoeë. Dit is gedoen deur die UV-VIS spectra van die gemodifiseerde Pluronic kwalitatiefte vergelyk met die spectra van die modelligande en komplekse, in waterige oplossings. Die spectra dui aan dat metaalbinding wel plaasvind.

Several phenyl alkyl bonded phases for liquid chromatography were synthesized and characterized by liquid chromatography, gas chromatography and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The chromatographic physical parameters investigated include a quantitative determination of the mobile phase volume and the stationary phase volume. The stationary phase volume was determined to be a function of the bonded moiety chain length and the chromatographic solvent system employed. The interpretation of the stationary phase volume is discussed in terms of the porous nature of the silica gel support. The chemical parameters determined include a quantitative determination of the mobile phase and stationary phase composition (which were different from each other). The selectivity of the chromatographic separations was dependent on the chemical composition and the volume of both the stationary and mobile phases. Carbon 13 NMR spectroscopy provided information about the environment of the bonded moiety in the stationary phase. The liquid-like nature of the bonded moiety was influenced by the chain length of the attached species, the choice of organic modifier, and the chemical composition of the solvents. Temperature did not appear to play a role in the line widths under the experimental conditions examined. The separation of several peptide diastereoisomers on different commercially available hydrocarbon bonded adsorbents is also reported. Select diastereoisomers of arginine vasopressin and oxytocin are extremely sensitive to differences in the composition of the stationary phase. The selectivity and elution order were dependent upon the choice of adsorbent and solvent system employed. The addition of a second organic modifier provided a method for the dynamic modification of the stationary phase. The ability to dynamically modify the stationary phase can enhance the selectivity for the separation of selected peptide diastereoisomers.

The effect of the physical state and chemical composition on molecular interactions has been studied for a number of chemically modified adsorbents. During the course of the study a reaction scheme was put together for the synthesis of a particular substituted crown ether. The synthesized allyl-benzo-15crown-5, which is not reported in the literature to date, was silylated and immobilized on a silica surface. The bonded phase was characterized by UV spectroscopy and by chromatography under both "normal" and "reverse" phase conditions. UV spectroscopy was also used to elucidate the physical state of several other phenyl alkyl bonded phases. Chromatographically, the bonded crown ether phase was found to be more polar than a C₈ stationary phase. A comparison of the selectivity of the two phases revealed that the former has a better selectivity towards a homologous series of alkyl benzenes under different reverse phase conditions. The selectivity of the crown ether phase was found to be dependent on the nature of the organic modifier in the mobile phase. This dependence was considered to be added evidence for the universality of the dynamic solvated stationary phase model. Both normal and reverse phase chromatographic conditions indicated an acid-base type of interaction between the crown ether and a number of substituted phenols. This was reflected in an increase in the retention of these probes as a function of their increasing acidity. A dramatic temperature effect observed on the crown ether stationary phase under aqueous THF mobile phase, but not under aqueous MeOH, was attributed to a temperature and/or solvent-induced phase change. A hysteresis effect, also seen only with aqueous THF, indicated that the crown ether phase undergoes a solvent-assisted conformational change. Further evidences for such a change was found spectroscopically in the abrupt break in the UV absorbance of these molecules as a function of temperature, as well as the irreversibility of the absorbance of the n- π* band on cooling. UV spectroscopy of bonded phenyl alkyls showed that there are about two monolayers of water molecule strongly adsorbed to the surface and totally impermeable to lypophilic species. Evidence for the existence of a solvated crystal, or liquid crystal, like clusters was rationalized with a cooperative sorption effect which may be dependent on the reaction conditions during immobilization. Despite a significant increase in the liquid character observed as the chain length is increased to 4-7 methylene groups, the bonded clusters still appear to preserve a fairly ordered environment. The physical state of the immobilized species was found to change with the experimental conditions and the change was reflected on the selectivity of the system.

A new theory has been proposed for stationary phase formation of chemically modified chromatographic adsorbents. This theory consists of a model in which the bonded hydrocarbon moiety, silica substrate, and their respective solvation layers all participate in stationary phase formation. Stationary phase formation was found to be dependent on three parameters: (1) Solvent strength of the mobile phase components for the bonded organic moiety and the silica substrate; (2) the type of organic moiety covalently bound to the surface; and (3) the bound moiety density or surface coverage. Binary aqueous-organic mobile phases were investigated for LiChrosorb RP-8 and RP-18. For RP-8 the solica substrate played a more important role in stationary phase formation. Whereas, for RP-18 the longer bound hydrocarbon chain dominated stationary phase formation. With different organic modifiers in the mobile phase, the modifier with the larger solvent strength for the bound hydrocarbon was selectively enriched in the stationary phase solvation layer for RP-18. Ternary mobile phase systems were also investigated for RP-18. The second modifier was found to exert a large influence on stationary phase formation. Temperature's role in stationary phase formation was studied with a ternary mobile phase of 40/45/15 methanol, water, THF with RP-18. In this specific case, changing the temperature of the system did not impact on stationary phase formation. A new type of column structure was investigated. This structure involved a totally porous silica gel as compared to a column packed with totally porous silica microparticles. These silica gel columns were characterized both thermodynamically and kinectically. Under Normal Phase chromatographic conditions the silica gel column was found to have a higher selectivity but poorer efficiency for the separation of aniline from nitrobenzene than a packed column. The silica gel can be chemically modified by silane reaction and its bonded phase characteristics were investigated. The gel also showed ion-exchange properties which were investigated using sodium nitrite.

Environmental and safety concerns have prompted an active quest for ―green‖ alternatives to / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2009.