Dissertations / Theses on the topic 'Separation of ionogenic compounds'

Under the framework of this PhD project, a study on chromatographic behaviour of selected azaarenes on octadecylsilica stationary phases during their separation by reversed phase liquid chromatography was developed. The main goal was aimed at an application of the basic RPLC principles on the retention behaviour of azaarenes including the effects such as peak tailing, peak broadening, irreproducible retention or strong retention on a stationary phase. This study was particularly focused on basic azaarenes, also called acridines, which belong to a group of persistent organic pollutants providing mutagenic and/or carcinogenic activity. The retention patterns were studied on a group of eight acridines that are often present in environmental samples polluted with polycyclic aromatic compounds. The mixture of acridines was separated under isocratic elution conditions on three octadecylsilica stationary phases using two different binary mixtures as a mobile phase. Evaluation of the surface properties of the selected stationary phases was firstly performed, followed by the separation experiments. The structure of the experiments was designed to evaluate the effect of three different factors on the retention behaviour of acridines: properties of a stationary phase, composition of a mobile phase and physical-chemical properties of acridines. The treatment of the results was based on the capacity factor values providing the best fitting and repeatability of data, but other parameters that evaluate thermodynamic and kinetic aspects of the separation process were also given. Simple correlations between the three factors and a character of retention mechanism of acridines on the given separation system were found. Application of this knowledge simplifies an optimisation process and helps to solve common separation problems for acridines, but also for other basic analytes displaying similar physical-chemical properties (Mr and pKa).

Current prostate cancer (PCa) diagnosis based on prostate-specific antigen (PSA) has been gradually losing its credibility over the last decade due to contradictory results in published literature and clinical practice. Recently, a group of potential PCa biomarkers in urine, particularly sarcosine, was found to increase significantly as the cancer progressed to metastasis. In Chapter 2, we report a simple, robust, and reproducible capillary electrophoresis–electrospray ionization–tandem mass spectrometry (CE–ESI-MS/MS) method for the determination of sarcosine and other representative potential biomarkers in pooled urine. A solid phase extraction (SPE) technique was optimized for maximum recovery of sarcosine. With no derivatization step, excellent resolution between sarcosine and its isomers (α-alanine and β-alanine) was achieved. A separate non-SPE method was also developed for quantitative determination of highly concentrated urinary metabolites. Precision for intra- and inter-day standard addition calibration of sarcosine were found to be within 15%, whereas intra-day precisions for the rest of the metabolites varied from 0.03 to 13.4%. Acceptable intra-day and inter-day accuracies, ranging from 80 to 124%, were obtained for sarcosine and the other metabolites. The second part of the thesis takes on a more challenging task. The importance of chiral separation in pharmaceutical, agriculture, and food industries has driven separation scientists to develop more powerful methodologies in conjunction with the structural capabilities of mass spectrometry. In Chapter 3, chiral separation of D- and L-tryptophan was compared on a bare-fused silica capillary and a PEI-coated capillary. Although a higher resolution was observed for uncoated capillaries, analytes were found to migrate slower resulting to longer analysis times (tm > 20 min). With shorter migration times (tm < 10 min) and acceptable resolution, further investigations on different factors that could affect enantioseparation were conducted on a coated capillary. Highly-sulfated cyclodextrins (HS-CDs), a group of charged CD derivatives, were also utilized for the separation of several racemic amino acids. Resolution with HS-CDs was found to be superior to using native CDs. Unfortunately, due to time constraint, no MS work was presented as the chiral CE/MS work is still currently in progress.

Microporous materials play an important role in a variety of industrial and domestic applications. While a diverse range of microporous materials have been identified, this thesis focuses on porous organic cages (POCs) because they have received much attention as synthetically tunable, solution processable, microporous materials. After introducing the latest developments in POC synthesis and the general application of microporous materials as selective sorbents, this thesis presents three developments in organic cage chemistry: a high-throughput workflow for the discovery of POCs, which yielded a novel organic cage compound; the measurement of selective adsorption by POCs, wherein the first instance of chiral selectivity by a POC was recorded; and the first instance of applying POCs as stationary phases for gas chromatography, which produced columns that separate racemic mixtures, alkylaromatic isomers, and alkane isomers. Chapter 2, discovering novel organic cages, presents attempts to use high-throughput and in-silico techniques to accelerate the discovery of novel organic cages. These methods were utilised to isolate a novel organic cage, CCX-S, which is characterised and discussed. Chapter 3, organic cages as selective sorbents, presents the development of approaches for measuring selective adsorption. These methods were used to identify the first reported instance of enantioselective adsorption by an organic cage. Further measurements to explain this separation behavior are also presented. Chapter 4, chromatographic separations with organic cages, presents one method of practically leveraging the presented separation behavior. In Chapter 4, the coating of capillary columns with CC3 is presented. These columns were used to successfully perform gas chromatographic separations, the first recorded instance of using a POC to do so. The columns were further improved by modifying the coating method and using prefabricated CC3 nanoparticles. This modification enabled difficult separations to be performed using the column; for example, the separation of hexane’s five isomers.

Thesis (PhD)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: Separation of the metal ions Cu2+, Pb2+, Cd2+ Mn2+, Cr3+, Fe3+ and Zn2+ with poly ethylenediamine tetra-acetic acid (EDTA), ethylenediamine disuccinic acid ([S,S']- EDDS) and ethylenediamine dimalonic acid (EDDM) was performed by Capillary Electrophoresis. The electropherograms obtained by Capillary Electrophoresis were validated with a speciation model Joint Expert Speciation System (JESS). Excellent agreement was observed for the speciation diagrams for these species with the pH curves determined by capillary electrophoresis with those determined by speciation modelling using JESS. The ligands EDDS and EDDM are readily biodegradable. They have been proposed as substitute ligands for EDTA. Detection of the above species was performed using both an electrochemical and a UV detector. The obtained electropherograms were used for the determination of the detection limits of these species. The electrochemical detector has a lower detection limit for these species than the UV detector in conformity with reported literature for these detectors.
AFRIKAANSE OPSOMMING: Skeiding van die metaal ione Cu2+, Pb2+, Cd2+, Cr3+, Fe3+ en Zn2+ met behulp van etileendiamientetra-asyn suur (EDTA), etileendiamiendisuksien suur ([S,S']-EDDS) en etileendiamiendimaleen suur (EDDM) was gedoen deur kapilêre elektroferose. Die elektroferogramme, verkry deur kapilêre elektroferose was gebruik om die spesiasie model "Joint Expert Speciation System (JESS)" te valideer. Uitstekende ooreenstemming was waargeneem vir die verspreidings diagramme van hierdie spesies as 'n funksie van pH wat verkry was deur kapilêre elektroferose met die' wat vasgestel was deur die spesiasie modeleeringsproses van JESS. Die ligande EDDS en EDDM toon onmiddelike biodigradeerbaarheid. Hulle was voorgehou as alternatiewe ligande vir EDTA. Bepaling van die bogenoemde spesies was ook gedoen deur beide elektrochemiese en UV deteksie. Deur gebruik te maak van hierdie detektore was die deteksie limiete van hierdie spesies bepaal. Die elektroochemiese detektor het 'n laer deteksie limiet vir hierdie spesies in vergelyking met die UV detector. Dit stem ooreen met die literatuur vir hierdie detektore.

Thesis (PhD)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: Separation of the metal ions Cu2+, Pb2+, Cd2+ Mn2+, Cr3+, Fe3+ and Zn2+ with poly ethylenediamine tetra-acetic acid (EDTA), ethylenediamine disuccinic acid ([S,S']- EDDS) and ethylenediamine dimalonic acid (EDDM) was performed by Capillary Electrophoresis. The electropherograms obtained by Capillary Electrophoresis were validated with a speciation model Joint Expert Speciation System (JESS). Excellent agreement was observed for the speciation diagrams for these species with the pH curves determined by capillary electrophoresis with those determined by speciation modelling using JESS. The ligands EDDS and EDDM are readily biodegradable. They have been proposed as substitute ligands for EDTA. Detection of the above species was performed using both an electrochemical and a UV detector. The obtained electropherograms were used for the determination of the detection limits of these species. The electrochemical detector has a lower detection limit for these species than the UV detector in conformity with reported literature for these detectors.
AFRIKAANSE OPSOMMING: Skeiding van die metaal ione Cu2+, Pb2+, Cd2+, Cr3+, Fe3+ en Zn2+ met behulp van etileendiamientetra-asyn suur (EDTA), etileendiamiendisuksien suur ([S,S']-EDDS) en etileendiamiendimaleen suur (EDDM) was gedoen deur kapilêre elektroferose. Die elektroferogramme, verkry deur kapilêre elektroferose was gebruik om die spesiasie model "Joint Expert Speciation System (JESS)" te valideer. Uitstekende ooreenstemming was waargeneem vir die verspreidings diagramme van hierdie spesies as 'n funksie van pH wat verkry was deur kapilêre elektroferose met die' wat vasgestel was deur die spesiasie modeleeringsproses van JESS. Die ligande EDDS en EDDM toon onmiddelike biodigradeerbaarheid. Hulle was voorgehou as alternatiewe ligande vir EDT A. Bepaling van die bogenoemde spesies was ook gedoen deur beide elektrochemiese en UV deteksie. Deur gebruik te maak van hierdie detektore was die deteksie limiete van hierdie spesies bepaal. Die elektroochemiese detektor het 'n laer deteksie limiet vir hierdie spesies in vergelyking met die UV detector. Dit stem ooreen met die literatuur vir hierdie detektore.

The zirconium/hafnium separation, as currently practiced, is mostly carried out by solvent extraction in which the aqueous chloride solution of metals is contacted with an immiscible organic phase containing a reagent that removes hafnium leaving zirconium in the aqueous solution. Hafnium is extracted as its thiocyanate complex using methyl isobutyl ketone (MIBK). There are environmental consequences of this system that were acceptable when the process was developed but should become a serious problem as legislation becomes more stringent. These problems are primarily the high aqueous solubility of MIBK and the potential for toxic gas generation. This work has tested four organophosphorous reagents to separate zirconium from hafnium without the need of thiocyanate, avoiding toxic gas formation. The extractants and diluent have very low aqueous solubility, removing effluents control problems. We see these features as representing a "zero waste" process. Additional advantages over MIBK process are lower vapor pressure and higher flash point (lower fire risk). This work presents the lab-scale experimental results using mainly two reagents designated by Cytec Canada Ltd as Cyanex 923 and Cyanex 925. The key feature of this work is replacement of MIBK and thiocyanate with one of a family of organophosphorous extractants that were unavailable in the 1950's at the start of the MIBK process. This work shows they can be efficiently applied to hafnium/zirconium separation in aqueous chloride solution, with minimum adjustments of the present industrial practice.

The project has demonstrated the use of both poly(styrene-divinylbenzene) PS-DVB and silica monolithic columns for the separation of nitro-compounds. Methods were developed with PS-DVB and ODS silica packed columns for the separation of these compounds. The first part of the project was the preparation of the monolithic stationary phases prepared from PS-DVB of (250 μm I.D. × 70 mm) functionalized with methacrylate by in-situ polymerisation. The alkylated PS-DVB then was used successfully for the first time in the separation of three aryl nitro-compounds (2-NA, 1,4-DNB and 4-NT) on micro-HPLC. However, the efficiency of this column was poor N = 318 (4675/m).The second part of the thesis used a commercial column (Chromolith Performance from Merck), with different diameters for the separation of nitro-compounds. Nitrocompounds were analysed on both Chromolith Performance 3 mm I.D. column and Chromolith Performance 4 mm I.D. column by HPLC/UV. Van Deemter plots showed that the 3 mm I.D. column gave higher efficiencies at higher flow rates than the 4.6 mm I.D. column. The plate number was 8216 (H = 0.0121 mm) at a flow rate of 0.4 ml/min (1.0206 mm/sec) and for Chromolith 4.6 mm I.D. it was 9436 (H = 0.0105 mm) at a flow rate of 0.8 ml/min (0.8577 mm/sec). The nitro-compounds analysed in this study were nitroaromatic, nitramines and nitrate esters which are used in the manufacture of explosives. These compounds were analysed for the first time using a Chromolith Performance 3 mm I.D. column on LCMS using both ESI and APCI in negative ionization modes. The sensitivity was higher in the APCI than the ESI mode in terms of higher intensity and lower background noise especially for nitroaromatic compounds. The LC-ESI-MS method was evaluated by injection of samples of pentaerythritol tetranitrate (PETN) in different concentrations. Calibration curves were constructed over the range of 1-1000 pg/μl with a correlation coefficient of (R2 = 0.9986) and with a concentration range between 1-200 ng/μl with a correlation coefficient of (R2 = 0.9971) and were found to be linear. The limit of detection (LOT) for pentaerythritol tetranitrate (PETN) was 5 pg/μl at a signal-to-noise ratio (S/N) of 3:1 and the limit of quantification (LOQ) was 10 pg/μl at a signal-to-noise ratio of 10:1. The applicability of the monolithic column for the LC-ESI-MS method was evaluated by injection of samples of the commercial explosives, P9 and Semtex 1H. The results showed that Semtex 1H contains 35% PETN using calibration curve 1-200 ng/μl and was much higher than in P9 0.0082% using calibration curve 1-1000 pg/μl.

Kyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第14634号
工博第3102号
新制||工||1461(附属図書館)
26986
UT51-2009-D346
京都大学大学院工学研究科材料化学専攻
(主査)教授 大塚 浩二, 教授 木村 俊作, 教授 松原 誠二郎
学位規則第4条第1項該当

Dissertation (PhD(Eng))--University of Stellenbosch, 2001.
ENGLISH ABSTRACT: Coal pyrolysis liquors are a major source of phenolic compounds. The separation of the phenolic compounds from the neutral oils and nitrogen bases also present in the pyrolysis liquors is difficult due to low relative volatilities and the formation of azeotropes. The desired phenolic recovery and phenolic product purity of 99.5 % can therefore not be achieved by means of conventional separation processes. Alternative processes such as liquid-liquid extraction with various low-boiling solvents, mixtures of high-boiling solvents and extractive distillation have been investigated. Disadvantages of these processes include the high solvent ratios required, low recovery of the higher substituted phenolic compounds, inability to treat a wide-boiling feedstock in one process step and complex post-purification of the phenolic product. A solvent system consisting of a selective solvent, water as a co-solvent, and hexane as a countersolvent, is proposed. An industrial heavy naphtha stream was analysed and the most prevalent phenolic compounds, neutral oils and nitrogen bases identified. Three synthetic feed streams were compiled to represent the industrial stream, namely: 1. phenol + benzonitrile + aniline + mesitylene + 5-et-2-me-pyridine 2. m-cresol + o-tolunitrile + o-toluidine + pseudocumene + undecane + indene 3. 2,4-xylenol + 3,5-xylenol + 3,4-xylenol + indane + dodecane + naphthalene The stream containing phenol was used as a basis for solvent selection, with emphasis on the separation of phenol from benzonitrile. A variety of molecules containing hydroxyl and ether functional groups were identified as potential solvents by means of computer-aided molecular design using a genetic algorithm. Of the commercially available solvents tested on batch extraction scale, triethylene glycol achieved the highest phenol-benzonitrile, phenol-aniline and phenol-5-et-2-me-pyridine separation factors as well as the highest phenol recovery. It was concluded from the solvent selection process that effective solvents for the problem under investigation were those containing hydroxyl groups positioned on the molecule backbone in such a way as to facilitate hydrogen bonding with more than one phenolic molecule at a time. Two commercially unavailable solvents, 1,3-(ethoxy-2- hydroxy)-propane-2-01 and 1,3-(diethoxy-4-hydroxy)-propane-2-01 were therefore synthesised from ethylene glycol and diethylene glycol respectively. The molecular structures of these two solvents are analogous to that of triethylene glycol, and contain an additional hydroxyl group. The performance of the synthesised solvents was evaluated and compared to that of triethylene glycol on the basis of m-cresol-otolunitrile, 2,4-xylenol - o-tolunitrile, and 2,4-xylenol - o-toluidine separation factors and phenolic recoveries achieved by means of batch extraction tests. 1,3-(Diethoxy-4- hydroxy)-propane-2-01 yielded higher phenolic recoveries, but lower separation factors than did triethylene glycol. Triethylene glycol was therefore selected for further process development as it is commercially available. A series of batch extractions were carried out on each of the synthetic feed streams using the proposed solvent system. For phenol and m-cresol, recoveries in excess of 99% were obtained in a single stage. Recoveries in excess of 98% were obtained for the xylenol isomers. It was found that the recoveries of the xylenol isomers were more sensitive to changes in the solvent ratios. The separation of phenolic compounds from paraffins, naphthalene, indene, indane and the alkyl-substituted benzenes was trivial using the proposed solvent system. Highly satisfactory separation of the phenolic compounds from pyridines and aromatic nitriles was achieved. The separation of phenol from aniline, although satisfactory, was not as good. The optimum solvent to feed, water to solvent and hexane to feed ratios were identified as being 3.0, 5.0 and 0.25 respectively. Binary interaction parameters for the NRTL equation were obtained by regression of the equilibrium data from the batch extraction tests. The NRTL model fitted the equilibrium data satisfactorily. The proposed solvent system was tested on pilot plant scale. The performance of the extraction column was optimised using a synthetic feed stream consisting of m-cresol, p-cresol, aniline and o-tolunitrile. The optimum solvent ratios and operating parameters were then implemented in further tests on an industrial heavy naphtha stream. A phenolic product purity of 99.75% was achieved for this stream. The corresponding phenolic recovery was in excess of 91 %. The proposed separation process, including solvent recovery was simulated using the NRTL model with the experimentally determined interaction parameters. A single stream consisting of all the components used in the batch extraction tests was specified as the feed stream to the simulated process. A final simulated phenolic product purity of 99.5% and recovery in excess of 94% was obtained after solvent recovery. The optimum solvent to feed, hexane to feed and water to solvent ratios were determined as being 3.0, 5.0 and 0.25 in both the pilot plant tests and the simulated extraction process. It can be concluded that the proposed separation process is successful in recovering high purity phenolic compounds from tar liquors. Further development of the process has commenced in industry.
AFRIKAANSE OPSOMMING: Die pirolise van steenkool is 'n belangrike bron van fenoliese verbindings. Die skeiding van die fenoliese komponente vanuit die neutrale olies ook teenwoordig in die pirolise mengsel word bemoeilik deur lae relatiewe vlugtighede en die vorming van aseotrope. Dit is dus nie moontlik om die gewenste hoë fenoliese herwinning en fenoliese produk suiwerheid van 99.5% d.m.v. konvensionele distilleerprosesse te behaal nie. Alternatiewe prosesse soos ekstraktiewe distillasie en vloeistof-vloeistof ekstraksie met verskeie laagkokende oplosmiddels en mengsels van hoogkokende oplosmiddels is al ondersoek. Die hoë oplosmiddel verhoudings wat benodig word, lae herwinning van die hoërgesubstitueerde fenoliese verbindings, onvermoë om in een prosesstap 'n prosesstroom met In wye kookgebied te behandel en die ingewikkelde suiwering van die fenoliese produk tel onder die nadele van hierdie prosesse. 'n Oplosmiddelsisteem wat In selektiewe oplosmiddel, water as polêre oplosmiddel en heksaan as teenoplosmiddel bevat is as 'n alternatiewe proses voorgestel. 'n Industriële swaar nafta prosesstroom is ge-analiseer en die fenoliese verbindings, neutrale olies en stikstofbasisse met die hoogste konsentrasies daarin geïdentifiseer. Drie sintetiese strome is op grond van hierdie analise saamgestelom die industriële stroom te verteenwoordig: 1. fenol + benzonitriel + anilien + mesitileen + 5-et-2-me-piridien 2. m-kresol + o-tolunitriel +o-toluïdien + pseudokumeen + undekaan + indeen 3. 2,4-xilenol + 3,5-xilenol + 3,4-xilenol + indaan + dodekaan + naftaleen Die fenolbevattende stroom is as basis vir oplosmiddelkeuring gebruik, met die klem op die skeiding van fenol vanuit benzonitriel. Verskeie molekules wat hidroksie- en eter funksionele groepe bevat is as potensiële oplosmiddels uitgeken d.m.v. rekenaargesteunde molekulêre ontwerp met In genetiese algoritme. Hierdie oplosmiddels is d.m.v. enkellading ekstraksie toetse geëvalueer. Die kommersieel beskikbare oplosmiddel wat die hoogste fenol-benzonitriel, fenol-anilien en fenol-5-et-2-me-piridien skeidingsfaktore, sowel as die hoogste fenol herwinning op enkellading ekstraksie toetsvlak gelewer het, was triëtileenglikol. Vanuit die proses vir die keuse van 'n oplosmiddel was dit duidelik dat, vir hierdie skeidingsprobleem, die mees effektiewe oplosmiddels dié is met hidroksiel groepe wat so geposisioneer is in die oplosmiddel molekuul dat dit waterstofbindings kan vorm met meer as een fenoliese molekuul. Twee oplosmiddels wat nie kommersiëel beskikbaar is nie, 1,3-(etoksie-2-hidroksie)-propaan-2-01 en 1,3-(diëtoksie-4-hidroksie)-propaan-2- ol, is gesintetiseer vanuit etileenglikol en diëtileenglikol onderskeidelik. Die molekulêre strukture van hierdie twee oplosmiddels is analoog aan dié van triëtileenglikol en bevat 'n addisionele hidroksielgroep. Die effektiwiteit van die gesintetiseerde oplosmiddels is geëvalueer en met dié van triëtileenglikol vergelyk op grond van rn-kresol-o-tolunltriel, 2,4-xilenol - o-tolunitriel en 2,4-xilenol - o-toluïdien skeidingsfaktore en fenoliese herwinning behaal d.m.v. enkellading ekstraksie toetse. Hoër fenoliese herwinning en laer skeidingsfaktore is behaal met 1,3-(diëtoksie-4-hidroksie)-propaan-2-01as met triëtileenglikol. Triëtileenglikol is dus gekies vir verdere prosesontwikkeling aangesien dit kommersiëel beskikbaar is. Enkellading ekstraksie toetse is op elk van die sintetiese voerstrome uitgevoer met die voorgestelde oplosmiddelsisteem. Fenol- en m-kresol herwinning van meer as 99% en xilenol herwinning van meer as 98% is behaal. Die skeiding van fenoliese verbindings vanuit paraffiene, naftaleen, indeen, indaan en die alkielgesubstitueerde benseenverbindings is triviaal met die voorgestelde oplosmiddelsisteem. Hoogs aanvaarbare skeiding van fenoliese verbindings van die piridiene en aromatiese nitriele is vermag. Die skeiding van fenol en anilien is nie so goed nie, maar is nog steeds aanvaarbaar. Die optimum oplosmiddel tot voer, water tot oplosmiddel en heksaan tot voer is as 3.0, 5.0 en 0.25 vasgestel. Binêre interaksie parameters vir die NRTL vergelyking is verkry d.m.v. regressie van die ewewigsdata wat deur die enkelladingstoetse gegenereer is. Die NRTL model het die ewewigsdata goed gepas. Die voorgestelde oplosmiddelsisteem is op loodsaanlegvlak getoets. Die werking van die ekstraksie kolom is ge-optimeer met In sintetiese voerstroom wat uit m-kresol, pkresol, anilien en o-tolunitriel bestaan. Die optimum oplosmiddel verhoudings en bedryfstoestande is verder toegepas op 'n industriële swaar naftastroom. 'n Fenoliese suiwerheid van 99.75% is behaal met hierdie stroom. Die ooreenkomstige fenoliese herwinning was groter as 91%. Die voorgestelde skeidingsproses, insluitende oplosmiddelherwinning is gesimuleer met die NRTL model wat op die eksperimentele data gepas is. 'n Enkele stroom wat bestaan het uit al die komponente wat in die enkelladingstoetse gebruik is, is as die voerstroom tot die gesimuleerde proses gespesifiseer. 'n Finale gesimuleerde fenoliese produksuiwerheid van 99.5% en herwinning groter as 94% is na oplosmiddelherwinning behaal. Die optimum oplosmiddel tot voer, heksaan tot voer en water tot oplosmiddel verhoudings is vasgestel as 3.0, 5.0 en 0.25 onderskeidelik vir beide die gesimuleerde proses en die loodsaanleg toetse. Die voorgestelde skeidingsproses kan dus 'n hoogs suiwer fenoliese produk uit pirolisestrome herwin. Verdere ontwikkeling van die proses is in die industrie begin.

This thesis exemplifies the strength in the combination of inductively coupled plasma (ICP) spectrometry and electrospray ionization tandem mass spectrometry ESI-MS/MS as detection techniques for liquid chromatography (LC) in the search for and investigation of compounds that bind or can bind hetero-atoms. Furthermore, some aspects involved in the coupling of LC and ICP spectrometry and quantification without identical standards have been studied.

The importance of using a separation step in combination with ICP spectrometry was shown for urine and blood plasma samples from patients treated with boron neutron capture therapy. In addition to the carrier molecule used in the therapy, one major and a few possible minor metabolites were found in the urine samples. One fragment mass of the major metabolite was obtained with LC-ESI-MS/MS. Liquid chromatography coupled to ICP-MS was also shown to be a valuable tool for fingerprinting metal-binding compounds in complex matrices, such as siderophores (iron-complexing compounds) in soil. The presence of at least two siderophores in a field soil solution sample could be revealed by LC-ICP-MS. Their identities could thereafter be determined by LC-ESI-MS/MS.

The non-UV-absorbing o-carboranylalanine could be quantified in relation to its degradation products by LC-ICP-AES, which provided information about the mechanism behind the degradation. Moreover, LC-ICP spectrometry was shown to provide an accurate quantification of biomolecules (bias < 10 %) when evaluated from external calibration graphs based on inorganic elemental standards.

Finally, the causes of the large decrease in boron signal seen when adding acetonitrile to the LC mobile phase in LC-ICP-MS was investigated in some detail. Space charge effects might explain a large part of the depression from carbon species on the boron signal.

Many industrial processes handling organic solvents produce volatile organic compounds (VOCs). These VOCs not only cause environmental pollution, but also represent an economic loss. VOC removal and recovery have become a big issue that needs to be addressed. Traditional techniques for VOCs removal include carbon adsorption, condensation, and absorption, and none is efficient enough to meet every need. Membrane separation has emerged as an excellent alternative or complementary technology for VOC separation. Separation of VOCs from nitrogen by composite hollow fiber membranes is studied in this thesis. Microporous hollow fiber membranes were spun from polyvinylidene fluoride (PVDF) using the phase inversion method, and the hollow fibers were coated with a thin layer of poly(ether block amide) (PEBA), thereby forming composite membranes. PVDF was chosen as the substrate material because of its excellent thermal and chemical stabilities and good mechanical strength, and PEBA was selected as the active separating layer because of its good permselectivity and film forming properties. In PEBA polymer, the hard polyamide blocks provide high mechanical strength, and the soft polyether blocks provide flexibility and elasticity. This study is focused on the preparation and characterization of PEBA/PVDF composite hollow fiber membranes. The membranes were tested for the removal of representative VOCs including hexane, heptane and cyclohexane, which are the main components of gasoline, and dimethyl carbonate (DMC), ethanol, methanol, and methyl t-butyl ether (MTBE) that are the oxygenates and octane number enhancers of gasoline. The separation of gasoline vapor from nitrogen was also investigated. It was found that the PEBA/PVDF composite hollow fiber membranes are effective for the separation of hydrocarbon vapors from nitrogen. The effects of hollow fiber membrane preparation conditions on the membrane performance were studied, and the separation performance of the composite hollow fiber membranes at various operating conditions (e. g. feed concentration, operating temperature) was evaluated.

Using a model system of glycosylated and aglycon standards consisting of rutin and quercetin respectively, and a series of pre-packed solid phase extraction cartridges, including C18 Extra-Clean, DSC-18, DPA-6S, Oasis HLB and Amberlite XAD-2. The experimental findings also showed that use of a commercial hesperinidase preparation, resulted in adequate hydrolysis of the glycosylated standard rutin. Based on these findings, the phenolic compounds and flavonoids from maple sap and syrup were separated using the Amberlite XAD-2 column, where the glycosylated fractions eluted with 60% aqueous methanol solution and the aglycon fractions eluted with a methanol:acetonitrile mixture (1:1, v/v). The recovered glycosylated fractions were subjected to enzymatic hydrolysis using the hesperinidase preparation and the liberated phenolic compounds and flavonoids, as well as the sugar components were analyzed by high-performance liquid chromatography (HPLC).

In this study a method was developed to identify and quantify platinum(II) complexes of the type [PtCl4-n(OH)n]2- and [PtCl4-n(H2O)n]2-n. Separation of the various species was achieved with the aid of a hyphenated reversed phase HPLC-ICP-MS technique coupled with an ion-pairing reagent, HMHDCl2. The adsorption of HMHD2+ onto a C-18 column was investigated by generating a series of breakthrough curves. It was found that the selectivity for high charge density anions originates from its low surface coverage relative to TBA+, which on the other hand could not separate Pt(II) complexes. The peaks in the chromatographic traces were assigned by following the stepwise ligand substitution of [PtCl4]2- in hydroxide medium with UV/Vis spectrophotometry and HPLC-ICP-MS simultaneously. A computer program was written by the author to analyse chromatographic data by deconvoluting the chromatogram into its individual components and calculating the mole fraction of each component. The validity of the consecutive pseudo-first order model was validated by constructing 3D Mauser diagrams with the raw spectrophotometric data (A1 vs A2 vs A3). Additional software was used to simulate the raw spectrophotometric data and processed chromatographic data. The pseudo-first order rate constants obtained in both cases were in agreement with each other. Hence, peaks were assigned to [PtCl4]2-, [PtCl3(OH)]2-, [PtCl2(OH)2]2-, [PtCl3(H2O)]-. The molar extinction coefficient spectra of [PtCl3(OH)]2- and [PtCl2(OH)2]2- were obtained by simulating the spectrophotometric data at wavelengths from 280 to 450 nm. The reaction of [PtCl4]2- with sodium hydroxide was investigated with UV/Vis spectrophotometry at 25 °C. A rate constant consisting of a first and second order term was obtained. The first order term agreed with what has been reported in the literature for aquation of [PtCl4]2- at 25 degrees C. The influence of temperature was established by conducting the experiment at different temperatures. It was found that the reaction proceeds essentially via aquation at elevated temperatures.

The retention behaviour of 16 phenothiazines and structurally-related drugs on a $ beta$-cyclodextrin-bonded phase column was studied with respect to pH, mobile phase composition and column temperature. Both isocratic and gradient-elution separations of these compounds were investigated.
The enantiomers of twelve racemic dinitrophenyl amino acid derivatives were separated on a $ beta$-cyclodextrin-bonded phase column. The effects of pH, methanol and triethylammonium acetate (TEAA) buffer concentrations on the retention and resolution were investigated. The chiral recognition mechanism was studied by means of UV-visible, circular dichroism and proton nuclear magnetic resonance spectroscopic methods.
A multiple-interaction type of chiral stationary phase was developed by bonding $ beta$-cyclodextrin to silica gel and modifying the cyclodextrin cavity by flexibly capping its primary hydroxyl or small side. These modified $ beta$ cyclodextrin stationary phases contain a hydrophobic cavity, capable of inclusion complexation; aromatic groups, capable of $ pi$-$ pi$ interaction; and polar hydrogen-bonding sites, capable of forming hydrogen-bonding with the polar functional groups of the solutes. These stationary phases exhibit a high stereoselectivity toward a wide variety of chiral compounds. The preparation and properties of these modified $ beta$-cyclodextrin stationary phases are described. The enantiomeric separation of amino acids and their derivatives, of carboxylic acids, of phenothiazine drugs, and of other chiral compounds are reported. The effects of mobile phase composition on the retention and resolution are discussed.

Mestrado em Biotecnologia - Industrial e Ambiental
Tipicamente, apenas um dos enantiómeros é responsável pelo efeito pretendido de um fármaco, sendo que o outro pode levar a respostas menos potentes ou até mesmo indesejadas. As entidades reguladoras praticam políticas restritas em relação à comercialização de fármacos como misturas racémicas. Assim, a indústria farmacêutica tem enfrentado desafios relacionados com o desenvolvimento de métodos para produção de fármacos oticamente puros. No entanto, e considerando a dificuldade acrescida na produção de enantiómeros puros por síntese direta, a síntese de misturas racémicas seguida da sua purificação surge como uma alternativa mais barata, simples e flexível. Os sistemas aquosos bifásicos (SABs) e os sistemas de duas fases sólida-líquida (SDFSL) são técnicas alternativas mais biocompatíveis que têm sido utilizados como técnicas de separação enantiosseletiva de fármacos e/ou aminoácidos com enantiosseletividades bastante promissoras. Para além disso, apresentam benefícios de custo, rapidez, simplicidade e versatilidade de operação e possibilidade de aumento de escala. Este trabalho foca-se no desenvolvimento de SABs e SDFSL constituídos por seletores quirais que possam atuar simultaneamente como solvente. Numa primeira abordagem o objetivo foi desenvolver novos SABs quirais, mais biocompatíveis, simples e eficientes. Para tal, SABs constituídos por açúcares, aminoácidos e líquidos iónicos quirais foram aplicados na resolução enantiomérica de ácido mandélico racémico. O sistema mais promissor, composto por [C1Qui][C1SO4] + K3PO4, obteve um excesso enantiomérico de -33.4%. Numa segunda abordagem, foi possível criar uma alternativa mais simples e mais eficiente recorrendo a SDFSL. Com estes sistemas, foi obtido o valor mais elevado de excesso enantiomérico deste trabalho, de 49.0%, através da precipitação enantiosseletiva do R-ácido mandélico por interação com [N4444][D-Phe].
Conventionally, only one of the enantiomers is responsible for the intended effect of a drug, whilst the other may lead to a less potent or even undesired response. Regulation entities are very strict regarding the commercialization of racemic drugs. Thus, pharmaceutical industry has been facing challenges related to the creation of methods to produce optically active drugs. However, considering the increased difficulty in the production of pure enantiomers by direct synthesis, the synthesis of racemic mixtures followed by their purification appears as a cheaper, simpler and more flexible alternative. Aqueous biphasic systems (ABS) and solid-liquid biphasic system (SLBS) are more biocompatible alternatives that have been used to separate racemic drugs and amino acids with promising enantioselectivities. Furthermore, these are cost-effective, quick, simple and operationally flexible. This work intended to develop ABS and SLBS using chiral selectors that can simultaneously act as solvents. In a first attempt, chiral ABS of better biocompatibility, simplicity and efficiency were developed. For that purpose, ABS constituted by sugars, amino acids and chiral ionic liquids (CILs) were applied for chiral resolution of racemic mandelic acid (MA). The most promising ABS was a system composed of [C1Qui][C1SO4] + K3PO4 which yielded the maximum enantiomeric excess of -33.4%. In a second approach, it was possible to create a simpler and more efficient technique resorting to SLBS. The enantiomeric excess value of 49.0% was achieved, by the enantioselective precipitation of the R-MA caused by interactions with [N4444][D-Phe].

Metal membranes are useful for hydrogen separation from mixed gas streams. They can exhibit perfect selectivity for hydrogen. However, in order to be commercially viable, in addition to providing high hydrogen fluxes, they must also be resistant to poisoning, possess long operating lifetimes and be cost effective. Many types of metal membranes such as pure metals, disordered alloys and amorphous metals have been studied for this application. In this work, we aim to identify intermetallic stoichiometric compounds of two or more metals that could be used as potential membrane materials for hydrogen separation. In the past, first principle calculations combined with Monte Carlo methods have been developed that can accurately predict H₂ fluxes through metal membranes at different hydrogen pressures and temperatures. Although these models are accurate, they are computationally intensive. In this work, we use these methods and develop screening criteria based on calculated properties that enable us to perform detailed calculations on a diminishing set of materials and rapidly identify the favorable candidates for hydrogen separation. We screened 1059 intermetallics at this high level of theory, which is the largest set of materials studied for this application. We divided the intermetallics into Pd-based and non-Pd based materials using additional screening algorithms to reduce the number of calculations required to identify potential candidate materials. 8 intermetallics were identified that had permeabilities that was comparable or higher to that of pure Pd. MgZn₂ and MnTi were found to have the highest H permeabilities among all the intermetallics studied. In addition to ground state structures, metastable structures were also found to be stabilized in the presence of hydrogen. Our work demonstrates the ability of these computational methods to identify potential novel materials for specific applications from large sets of materials that would not be possible experimentally. In the models for hydrogen permeability developed above, H-induced metal lattice rearrangements were not considered. Experimental evidence suggests that hydrogen heat treated (HHT) Pd-Au alloys undergo lattice rearrangement that results in an ordered structure which has a higher solubility than the non-HHT alloys. Using a combination of cluster expansion methods developed for predicting hydrogen permeability of disordered alloys and Monte Carlo methods, we predicted the extent of H induced lattice rearrangement in Pd₉₆Au₄ and Pd₈₅Au₁₅ alloys. We also predicted the solubility, diffusivity and permeability of these rearranged phases and found that their H permeability is higher than the non-rearranged phases. Our models capture the H-induced lattice rearrangement and provide useful insight of the conditions where this phenomenon is significant. Using the tools developed in this work, similar alloys that have a tendency to undergo lattice rearrangement that results in enhanced H permeability can be identified.

Thesis (MSc)--Stellenbosch University, 2000.
ENGLISH ABSTRACT: The phenolic content of wine is responsible for determining characteristics such as the organoleptic qualities, colour stability, ageing properties and health-beneficial effects associated with wine. The aim ofthis study was to investigate the possibilities offered by capillary electrophoresis (CE) as an alternative separation technique to high performance liquid chromatography (HPLC) for the analysis of polyphenols in wine. The complexity of wine samples was the cause that neither technique was capable of a satisfactory singlestep analysis of wine. Suitable sample preparation techniques such as Sephadex- and Sep- Pak fractionation and ether extraction of wine polyphenols were investigated. These techniques did not, however, prove to be universal. A novel form of sample preparation namely a process analogous to lyophylization used to separate wine volatiles from nonvolatiles was introduced. The versatility of CE was further investigated in an attempt to eliminate the need for sample preparation. The use of polyvinylalcohol (PVA) coated capillaries, micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) were investigated in this regard. Although none of these techniques could offer conclusive results, useful applications were forthcoming and routes for further investigation were outlined. Liquid chromatography coupled to electro spray ionisation mass spectroscopy (LC-ESI-MS) and capillary electrophoresis coupled to electro spray ionisation mass spectroscopy (CE-ESI-MS) were compared for the analysis of polyphenols in wine. While the latter technique could not produce sufficient separation compared to the former, future development ofCE-ESI-MS should make it a powerful technique for these analyses.
AFRIKAANSE OPSOMMING: Die fenoliese komponente in wyn speel 'n bepalende rol by eienskappe soos die organoleptiese karakter, kleur stabiliteit, verouderingspotensiaal en gesondheids-voordele wat met wyn geassosieër word. Die doel van hierdie projek was om ondersoek in te stel na die potensiaal wat kapillêre elektroforese (CE, "capillary electrophoresis") as 'n alternatiewe skeidingstegniek teenoor hoë druk vloeistof chromatografie (HDVC) vir die analise van die polifenole in wyn bied. Die kompleksiteit van wyn monsters is van so 'n aard dat 'n bevredigend enkelstap analise met geeneen van die tegnieke moontlik is nie. Gepaste monster-voorbereidingstappe soos Sephadex- en Sep-Pak fraksionering asook eter ekstraksie van die polifenole in wyn is ondersoek. Geeneen van die tegnieke was egter universeel toepaslik nie. 'n Nuwe metode van monster-voorbereiding, naamlik 'n proses analoog aan liofilisasie wat gebruik word om die wyn te skei in vlugtige en nievlugtige komponente is gedemonstreer. Die veelsydigheid van CE was gevolglik ondersoek in 'n poging om monstervoorbereiding uit te skakel. Die gebruik van polyvinielalkohol-(pVA) bedekte kapillêre, missellêre elektrokinetiese chromatografie (MEKC) en kapillêre gel elektroforese (CGE, "capillary gel electrophoresis) is in hierdie verband ondersoek. Alhoewel geeneen van hierdie tegnieke onweerlegbare resultate gelewer het nie, het bruikbare toepassings hieruit voortgespruit en is die grondslag vir verdere navorsing gelê. Vloeistof chromatografie gekoppel aan eIektrosproei ionisasie massaspektroskopie (LCESI- MS) en kapillêre elektroforese gekoppel aan elektrosproei ionisasie massaspektroskopie (CE-ESI-MS) is vergelyk vir die analise van polifenole in wyn. Alhoewel laasgenoemde tegniek onvoldoende skeiding lewer vergeleke met eersgenoemde, behoort toekomstige ontwikkelinge op die gebied van CE-ESI-MS dit 'n kragtige tegniek vir die analise van hierdie monsters te maak.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: Advances in pharmacology, biochemistry and biotechnology are increasingly dependant upon affinity chromatography as a preferred separation technique for the purification and characterisation of specific biomolecules. In the past few years avidin-biotin technology has been widely and successfully used in the fields of medicine, pharmacy, biology and biochemistry. The avidin-biotin complex (ABC) has been used as a mediator for affinity chromatography, affinity cytochemistry, immunoassay, histopathology, bioaffinity sensors, erosslinking and immobilisation studies. The main reason for the popularity of the ABC and its growing usefulness in biotechnology is the exceptionally high affinity (1015 M-l) and stability of the noncovalent interaction between avidin and biotin. The use of the ABC is broadening as different biotin derivatives and avidin-containing conjugates are becoming commercially available. The aim of this work was to evaluate the usefulness of a plutonic" FI 08 and the ABC conjugate to effect affinity separation. Towards this aim, the adsorption of plutonic" F108 onto hydrophobic polysulphone membrane surfaces was studied. This information was used to determine the theoretical maximum amount of pluronic" FI08 that will adsorb onto a unit surface area of the membrane. It is known that the polypropylene oxide (PPO) centre block ofthe pluronic" F I08 surfactant molecule governs the concentration of pluronic" F I 08 molecules that will adsorb onto a given hydrophobic surface. If the maximum coating concentration of plutonic" FI08 is known, one can assume that the maximum coating concentration of any pluronic derivative, with the same PPO centre block size, will be the same. Adsorption studies were carried out, the Langmuir adsorption isotherm was determined, and subsequently the fractional coating was calculated. The end-groups of plutonic" FI08 were modified as follows and the substituted pluronic was adsorbed onto a membrane that was to act as the solid support matrix in the development of an affinity system: Amino pluronic was synthesised by first tosylating pluronic" FI08, followed by azidation with NaN3 then reduction with LiAI~. The synthesised amino pluronic was then biotinylated using N-hydroxysuccinimide biotin ester. The suitability of this synthetic route was first assessed on a model compound, 2-methoxyethylamine, and validated by NMR (Nuclear Magnetic Resonance) spectroscopy. The synthetic protocol was then used to derivatise the larger pluronic molecule. The affinity system was tested on two different hydrophobic surfaces: polystyrene and polysulphone membranes (PSMs). Avidin-conjugated horseradish peroxidase was obtained and used to interact with the immobilised biotin. The enzymatic reaction of the coupled peroxidase converted the substrate, 2, 2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) to a coloured product. The colour developed is proportional to the amount of biotin that was immobilised on the hydrophobic surfaces studied. Non-covalent immobilisation of the synthesised biotin-pluronic molecule was successfully obtained onto the hydrophobic polystyrene as well as the polysulphone membrane surfaces.
AFRIKAANSE OPSOMMING: Vooruitgang in die farmakologie, biochemie en biotegnologie word al meer afhanklik van affiniteits chromatografie as die verkose tegniek vir die suiwering en karaterisering van spesifieke biomolekules. Oor die afgelope jare het die avidien-biotien tegnologie homself as baie bruikbaar bewys in die mediese, farmakologiese, biologiese en biochemiese velde. Toepassings waar die avidien-biotien kompleks betrokke was sluit in die toepassing as 'n mediator vir affiniteits chromatografie, affiniteits sitologie, immuno bepalings, histopatologie, bioaffiniteits sensors sowel as kruisbinding en immobiliserings studies en vele meer. Die hoofrede vir die gewildheid van die avidien-biotien kompleks en die groeiende bruikbaarheid in die biotegnologie is die buitengewone hoë affiniteit (l015 M-I ) en stabiliteit van die nie-kovalente interaksie tussen avidien en biotien. Die toepassingsveld van die avidien-biotien kompleks word wyer met die verskeidenheid biotien derivate en avidien-bevattende konjugate wat kommersiëel beskikbaar is. Die doel van die werk wat hier gedokumenteer word is om die bruikbaarheid van Plutonic" FI08 en die avidien-biotien kompleks, vir gebruik in 'n affiniteits chromatografie sisteem, te evalueer. Om hierdie doel te bereik is die adsorpsie van Pluronic" FI08 aan hidrofobiese polisulfoon membraan oppervlaktes bestudeer. Die eksperimentele data wat gegenireer is, is gebruik om die teoretiese maksimum hoeveelheid Pluronic wat per eenheids oppervlakte membraan adsorbeer te bepaal. Dit is reeds bekend dat die polipropileen (PPO) middel blok van die Pluronic emulgant die konsentrasie van die geadsorbeerde Pluronic molekules op 'n gegewe hidrofobiese oppervlakte bepaal. Indien die maksimum bedekkingskonsentrasie VIr maksimum oppervlakbedekking van Plutonic" FI08 bekend is, kan teoreties aanvaar word dat die bedekkingskonsentrasie vir enige Pluronic derivaat met dieselfde grootte PPO blok dieselfde sal wees. Adsorpsiestudies was uitgevoer om die Langmuir adsorpsie isoterm te bepaal. Daaropvolgend was die fraksionele bedekking bereken. Amino-pluronic was gesintetiseer deur die eindpunte van Pluronic te derivatiseer. Hierdie Pluronic derivaat was gevolglik geadsorbeer aan 'n membraan wat gedien het as die soliede oppervlakte vir die ontwikkeling van 'n affiniteits chromatografie sisteem. Amino-pluronic was gesintetiseer deur Pluronic eers te tosileer en daarna te asideer met NaN3 en laastens te reduseer met LiAI~. Die produk was gebiotinileer deur gebruik te maak van N-hidroksisuksinimied-biotien-ester. Die bruikbaarheid van hierdie sintetiese roete is eers bepaal deur van 'n model verbinding, 2-metoksiëtielamien, gebruik te maak en dit met behulp van KMR (Kern Magnetiese Resonans) spektroskopie te karakteriseer. Die affiniteits sisteem is getoets op twee verskillende hidrofobiese oppervlaktes naamlik polistireen en polisulfoon membraan oppervlaktes. Avidien gekonjugeerd met 'n peroksiedase ensiem is gebruik om met die geïmmobiliseerde biotien te assosieer. Die ensiematiese reaksie van die gekoppelde peroksiedase het die substraat 2, 2' -azino-di-(3-etiel-benzthiazolien-6-sulfoonsuur) (ABTS) omgesit na 'n gekleurde produk, waar dit teenwoordig is. 'n Reeks wasstappe is gebruik om die gemodifiseerde peroksidase ensiem wat nie aan die hidrofobiese oppervlakte gekoppel nie, weg te spoel. Hierdeur is die mate van binding aan die hirofobiese oppervlakte gekwantifiseer deur die kleur te kwantifiseer wat ontwikkelomdat die kleurontwikkeling direk proporsioneel is aan die hoeveelheid peroksidase wat nog aan die membraan gekoppel is. Nie-kovalente immobilisasie van die gesintetiseerde biotien-pluronic molekule is suksesvolop beide die hidrofobiese polistireen oppervlakte sowel as die polisulfoon membraan verkry.

Capillary electrophoretic (CE) separations were successfully coupled to time-of-flight mass spectrometric (TOFMS) detection for the analysis of three families of biological compounds that act as mediators and/or indicators of disease, namely, catecholamines (dopamine, epinephrine, norepinephrine) and their O-methoxylated metabolites (3-methoxytyramine, norepinephrine, and normetanephrine), indolamines (serotonin, tryptophan, and 5-hydroxytryptophan), and angiotensin peptides. While electrophoretic separation techniques provided high separation efficiency, mass spectrometric detection afforded specificity unsurpassed by other types of detectors. Both catecholamines and indolamines are present in body fluids at concentrations that make it possible for them to be determined by capillary zone electrophoresis coupled to TOFMS without employing any preconcentration scheme beyond sample work up by solid phase extraction (SPE). Using this hyphenated approach, submicromolar levels of catecholamines and metanephrines in normal human urine and indolamines in human plasma were detected after the removal of the analytes from their biological matrices and after preconcentration by SPE on mixed mode cation-exchange sorbents. The CE-TOFMS and SPE methods were individualized for each group of compounds. While catecholamines and metanephrines in urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard, deuterated isotopes, considered ideal internal standards, were used for the quantitation of indolamines. Because the angiotensin peptides are present in biological fluids at much lower concentrations than the previous two families of analytes, their analysis required the application of additional preconcentration techniques. In this work, the coupling of either of two types of electrophoretic preconcentration methods - field amplified injection (FAI) and isotachophoresis (ITP) - to capillary zone electrophoresis with both UV and MS detection was evaluated. Using FAI-CE-UV, angiotensins were detected at ~1 nM concentrations. Using similar conditions but TOFMS detection, the detection limits were below 10 nM. ITP was evaluated in both single-column and two-column comprehensive arrangements. The detection limits achieved for the ITP-based techniques were approximately one order of magnitude higher than for the FAI-based preconcentration. While the potential usefulness of these techniques was demonstrated using angiotensins standards, substantial additional research would be required to allow these approaches to be applied to plasma as part of clinical assays.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Palladium complexes of bis(di-tert-butylphosphinomethyl)benzene (DTBPMB) show remarkably high activity as alkene methoxycarbonylation catalysts, in addition to numerous other catalytic conversions, and are currently being commercialised by Lucite in ethene methoxycarbonylation to methyl propanoate. Any large-scale exploitation of this catalyst system for heavier products, however, is likely to be hindered by catalyst-product separation problems common to homogeneous catalysts; hence modification of this catalyst system to allow facile product separation was investigated. Tethering of DTBPMB residues onto polystyrene via Suzuki-type coupling of suitable precursors onto bromopolystyrene and boronic acid functionalised polystyrene resins was investigated and the phosphine was successfully immobilised. Phosphination of the resins was not complete however and as such there is concern that other phosphine residues may be present which do not exhibit a bidentate binding motif. The synthesis of a potassium sulfonate derivative of DTBPMB (KBPMBS) was successful and immobilisation of this onto ion exchange resins was also investigated. Some preliminary results from studies into 1-octene methoxycarbonylation using palladium complexes of these resins were obtained. Supporting of this diphosphine onto silica via a sol-gel co-condensation methodology was also investigated; the synthesis of a suitably functionalised precursor containing a sulfonamide linkage was successful via protection of the diphosphine using borane. Although formation of the silica support was successful, attempts to deprotect the phosphine-borane resulted in cleavage of the ligand from the support. An alternative route to this supported ligand was attempted and others discussed. Synthesis of a suitable sol-gel precursor via alkene hydrosilation was also attempted and is discussed. Supporting of the sulfonated phosphine, KBPMBS onto silica functionalised with imidazolium tethered residues was also investigated, although complete leaching of the phosphine from the support by methanol washing was observed. Immobilisation of the synthesised KBPMBS ligand in an ionic liquid (IL) phase was investigated. Complex formation and catalytic activity were demonstrated and a positive effect on conversion was observed upon addition of carbon dioxide to the system; possibly due to the increased CO solubility within the IL phase. Efficient product separation from the IL-immobilised catalyst system was demonstrated, both by organic extraction and using supercritical carbon dioxide flow. However, poor catalyst stability under these conditions appears to present a barrier to recycling this system, with loss of conversion observed on catalyst recycling. Other attempts to immobilise the DTBPMB ligand are discussed and reduction of the sulfide derivative of DTBPMB was demonstrated using hexachlorodisilane, which could be used as a general synthetic strategy for protecting highly electron rich phosphines. It is possible that increasing the bulk of the DTBPMB ligand may increase catalyst stability and result in catalyst systems with higher turnover numbers. Therefore syntheses of bulky ligands based on the DTBPMB backbone were investigated. 1,2,4,5-tetrakis(di(tert-butyl)phosphinomethyl)benzene was successfully synthesised although palladium complexes of this showed no activity in catalytic methoxycarbonylation. Attempts to synthesise a related biphenyl-based tetraphosphine is also discussed, although isolation of this in a pure form was not achieved. Routes toward tetraphenyl and dimethyl-diphenyl functionalised derivatives of DTBPMB have also been explored, although only a monophosphine was isolated due to difficulties in obtaining an intermediate di(chloromethyl) precursor in both synthetic pathways, although this now appears to have been overcome.

Electrospray ionisation (ESI) coupled to mass spectrometry (MS) is one of the most important detection techniques for chemical analysis of small drugs as well as large biomolecules in life science today. In this thesis, aspects on improved compatibility between liquid based separation systems and mass spectrometric detection were investigated regarding buffers, sample preparation and analysis of polar compounds as well as peptides and protein digests for enhanced ESI-MS performance.

Capillary electrophoresis (CE) coupled to ESI-MS detection, was evaluated using both a sheath flow interface and a sheathless design. The separation of peptides and small, polar compounds was optimised for both CE-ESI interfaces. The effect of sheath liquid composition was also studied with the aim to improve sensitivity in the ESI-MS detection.

Polar compounds were retained and separated by capillary ion-pair chromatography coupled to ESI-MS detection. Since commonly used ion-pairing reagents are detrimental to the ESI process they were effectively removed before the ionisation by the use of a trapping column after the separation. Alternatively, the ion-pairing reagents were exchanged to volatile constituents.

A method for peptide mapping by liquid chromatography (LC)-ESI-MS was developed for lactate dehydrogenase. The method was further enhanced to involve the proteolysis on-line to the LC-ESI-MS. No manual sample handling was then needed and the total analysis time decreased from 7 to 1.5 hours. The amount of information was also shown to increase in the on-line system.

Finally, the on-line concept was extended to an innovative interface for direct coupling of a pumped liquid flow to an electroosmotically driven flow. This provided a valve-free sample transfer between capillary LC and CE, aiming towards increased peak capacity per unit time for the analysis of complex peptide samples.

In this study, four different chromatographic column chemistries (octadecylsilane/ C18, pentafluorophenyl/ PFP, octadecylated polystyrene-divinylbenzene/ PRP and underivatized silica/ HILIC) were compared under optimal conditions to evaluate the relative strengths and weaknesses of the phases for use in the determination of pain management drugs by LC-MS. Furthermore, different column scaffoldings, traditional silica, porous shell, and porous polymer, were also compared. The drugs included in this study included buprenorphine, fentanyl, methadone, naloxone, oxycodone, and tramadol. Factors such as peak area, peak resolution, theoretical plates, and reproducibility were compared among the columns and analytes using a 2-way analysis of variance (ANOVA). Because of the lipophilic nature of these drugs, the C18 columns tended to offer the best performance; however, PFP and PRP columns were viable alternatives. Finally, HILIC separation was also suitable for most of the compounds under study; often providing higher peak areas (sensitivity) likely associated with higher organic (% B) conditions, thus favoring mass spectrometric detection. To our knowledge, this is the first study to explore viability of other non-C18 stationary phases such as PRP and HILIC for this drug class.

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: In this thesis, the recovery of RhIII from both synthetically prepared and authentic industrial PGM-containing solutions was systematically investigated via organic precipitation methods using several commercially available, N-containing organic receptors including (amongst others) diethylenetriamine (Deta), triethylenetetramine (Teta), tetraethylenepentamine (Tepa) and tris(2-aminoethyl)amine (Tren). These organic receptors act as precipitating agents in the presence of an appropriate protonating agent (HCl) by lowering the solubility of the PGM chlorido-anions through an ion-pairing mechanism. The recovery of RhIII from synthetically prepared PGM (RhIII and PtIV) containing solutions using these precipitants was excellent, while poor Rh recovery from authentic industrial process solutions was achieved. The poor Rh recovery from these process solutions was ascribed to the species distribution of the [RhCln(H2O)6-n]3-n complexes. In order to validate the proposition that RhIII speciation effects are responsible for the poor Rh recovery observed during the precipitation studies, attempt were made to describe the species distribution of the [RhCln(H2O)6-n]3-n (n=3-6) by means of high-resolution 103Rh NMR spectroscopy. A detailed high-resolution 103Rh NMR spectroscopic study of the series of [RhCln(H2O)6-n]3-n (n=3-6) complexes was conducted. During this study, all six RhIII aqua chlorido-complexes have unambiguously been characterized by means of high-resolution 103Rh NMR spectroscopy, proving the powerful analytical capability of this technique. Characterization of these complexes is based on the detailed analysis of the 35Cl/37Cl isotope effects which is observed in the 19.11 MHz 103Rh NMR resonances of the [RhCln(H2O)6-n]3-n (n=3-6) complexes in aqueous HCl solutions at 292 K. These resonances show that the “finestructure” of each of the 103Rh resonances may be understood in terms of its unique isotopologue, and in certain cases, the isotopomer distribution of each complex, which manifests as a result of its statistically expected 35Cl/37Cl isotopologue and isotopomer distributions. As a result, the 103Rh NMR resonance structure serves as a unique “NMRfingerprint”, which allows for the unambiguous assignment of [RhCln(H2O)6-n]3-n (n=3-6) complexes, without the reliance on accurate δ(103Rh) chemical shifts. Furthermore, this study reports the first direct species distribution diagram for the [RhCln(H2O)6-n]3-n (n=3-6) series of complexes (in aqueous HCl solutions at 292 K) as a function of the “free” (unbound) chloride concentration, constructed from 103Rh NMR measurements. The need for a revised speciation diagram of [RhCln(H2O)6-n]3-n (n=3-6) complexes is clearly reflected by the vast differences observed in the literature reported species distribution diagrams, which makes it difficult to decide which set of experimental conditions (if any) is required for the quantitative and “selective” recovery of RhIII from aqueous HCl solutions containing associated PGMs (Pt, Pd, Ir, Ru) as well as other transition metals. The documented species distribution diagrams for RhIII have been generally constructed via data from indirect (kinetic and spectrophotometric) measurements using dilute RhIII solutions at relatively high HCl concentrations, which implies that the RhIII:Clmole ratio is higher than what may be expected in authentic process solutions – an important aspect to consider when optimizing RhIII recovery methods. In addition, RhIII kinetic investigations reported in this study shows that ionic strength and temperature effects are important factors that dramatically influences the rate of RhIII ligand exchange (i.e. RhIII aquation reactions) which, in turn, have contributing effects on the species distribution of [RhCln(H2O)6-n]3-n complexes. Notable differences exist between the speciation diagram reported in this study and those documented in literature, especially at a “free” chloride concentration of 1.0 M. At this “free” chloride concentration, the [RhCl5(H2O)]2- complex anion was found to have an abundance of 34%, while literature reports an abundance of 80%. In order to ascertain its practical relevance, the proposed 103Rh NMR speciation method was extended, for the first time, to authentic industrial Rh feed solutions (Anglo Platinum PLC). Each of the 103Rh resonances was unambiguously assigned, and each species quantified. Moreover, the RhIII species distribution of the industrial Rh feed solution was accurately predicted by the “direct” speciation diagram constructed form 103Rh NMR measurements. After careful optimization of the Heraeus industrial feed solutions (optimal chloride concentration followed by thermal treatment for enhancing RhIII chloride anation reactions), the recovery of Rh via precipitation was repeated. In this instance, Rh recovery improved dramatically, with up to 95% of Rh removed from solution. This improvement is ascribed primarily to the increased “free” (unbound) chloride concentration. The presence of associated PGMs as well as other transition metals would lower the effective “free” chloride concentration, since these metals would act as “chloride binders”. By adjusting the total chloride concentration, RhIII chloride anation reactions is enhanced which leads to the [RhCln(H2O)6-n]3-n (n=5,6) complex anions being the dominant species in solution, therefore leading to improved Rh recovery. Moreover, it was shown that, under carefully controlled conditions, “selective” recovery of Rh is achieved using tris(2-aminoethyl)amine (Tren). Considering the fact that Rh is the last precious metal recovered in all PGM refineries, this can possibly provide a cost-effective route for the “upfront” (early stage) recovery of Rh from industrial PGM feed solutions.
AFRIKAANSE OPSOMMING: In hierdie tesis word die herwinning van RhIII uit laboratorium voorbereide sowel as ware industriële PGM-bevattende oplossings sistematies ondersoek deur middel van organiese neerslag metodes, deur gebruik te maak van verskeie kommersieël beskikbare, Nbevattende organiese reseptore insluitende dietileentriamien (Deta), tri-etileentetramien (Teta), tetra-etileenpentamien (Tepa) en tris(2-aminoetiel)amien (Tren). Hierdie organiese reseptore tree op as neerslag-agente in die teenwoordigheid van 'n geskikte protoneringsagent (in hierdie geval HCl) deur 'n verlaging van die oplosbaarheid van die PGM chloriedanione deur middel van 'n ioon parings meganisme. Die herwinning van RhIII vanuit laboratorium voorbereide PGM (RhIII en PtIV) oplossing met behulp van hierdie organiese neerslag-agente was uitstekend, terwyl Rh herwinning vanuit ware industriële oplossings swak was. Die onvolledige Rh herwinning uit hierdie industriële oplossings word toegeskryf aan die spesie-verspreiding van die [RhCln(H2O)6-n]3-n komplekse. Ten einde die effek van RhIII spesie-verspreiding op die herwinning van Rh te bestudeer, is gepoog om die spesieverspreiding van [RhCln(H2O)6-n]3-n (n=3-6) komplekse, deur middel van hoë resolusie 103Rh KMR spektroskopie, te beskryf. 'n Gedetailleerde hoë resolusie 103Rh KMR spektroskopiese studie van die reeks van [RhCln(H2O)6-n]3-n (n=3-6) komplekse was uitgevoer. Tydens hierdie studie was al ses RhIII aqua chlorido-komplekse ondubbelsinnig gekarakteriseer deur middel van hoë resolusie 103Rh KMR spektroskopie, wat bewys lewer van die kragtige analitiese vermoë van hierdie tegniek. Karakterisering van hierdie komplekse is gebaseer op die gedetailleerde analise van die 35Cl/37Cl isotoop effekte wat waargeneem word in die 19.11 MHz 103Rh KMR resonansies van die [RhCln(H2O)6-n]3-n (n=3-6) komplekse in HCl oplossings by 292 K. Hierdie resonansies toon dat die "fyn struktuur" van elk van die 103Rh resonansies verstaan kan word in terme van die unieke isotopoloog, en in sekere gevalle, die isotopomeer verspreiding van elke kompleks, wat manifesteer as 'n gevolg van die die statisties verwagte 35Cl/37Cl isotopoloog en isotopomeer verspreiding. Die 103Rh KMR resonansie-struktuur kan sodoende dien as 'n unieke "KMR-vingerafdruk", wat voorsiening maak vir die ondubbelsinnige karakterisering van [RhCln(H2O)6-n]3-n (n=3-6) komplekse, sonder om vertroue op akkurate δ(103Rh) chemiese verskuiwings te plaas. Hierdie studie rapporteer verder die eerste direkte spesie-verspreiding-diagram vir die [RhCln(H2O)6-n]3-n (n=3-6) reeks komplekse (in HCl oplossings by 292 K) as 'n funksie van die "vrye"(ongebonde) chloried-konsentrasie, verkry van 103Rh KMR metings. Die behoefte vir 'n aangepasde spesiasie-diagram vir die [RhCln(H2O)6-n]3-n (n=3-6) komplekse word duidelik weerspieël deur die groot verskille waargeneem in die literatuur gerapporteerde verspreidings diagramme, wat dit moeilik maak om te besluit watter stel eksperimentele toestande (indien enige) benodig word vir die kwantitatiewe en “selektiewe” herwinning van RhIII in HCl oplossings in die teenwoordigheid van gepaardgaande PGM (Pt, Pd, Ir, Ru) sowel as ander oorgangsmetale. Die gedokumenteerde spesie-verspreiding-diagramme vir RhIII is oor die algemeen verkry via data vanaf indirekte (kinetiese en spektrofotometriese metings) deur gebruik te maak van verdunde RhIII oplossings in relatiewe hoë HCl konsentrasies, wat impliseer dat die RhIII:Cl mol verhouding hoër is as wat verwag kan word in ware industriële proses oplossings - 'n belangrike aspek om te oorweeg gedurende die optimalisering van RhIII herwinning-metodes. Verder, die RhIII kinetiese ondersoeke gerapporteer in hierdie studie toon dat ioniese sterkte sowel as temperatuur effekte belangrike faktore is wat die tempo van RhIII ligand uitruiling (d.w.s. RhIII “aquation” reaksies), wat ‘n betekenisvolle invloed hê op die spesie-verspreiding van [RhCln(H2O)6-n]3-n komplekse. Aansienlike verskille bestaan tussen die spesiasie-diagram gerapporteer in hierdie studie en dit gedokumenteer in die literatuur, veral by 'n "vrye" chloried-konsentrasie van 1.0 M. By hierdie "vrye" chloried-konsentrasie was die [RhCl5(H2O)]2- komplekse anioon gevind om in 34% teenwoordig te wees (hierdie studie), terwyl die publiseerde verslae 80% rapporteer. Ten einde die praktiese toepaslikheid van die voorgestelde 103Rh KMR spesiasiemetode te bepaal, was (vir die eerste keer) ware industriële Rh oplossings (Anglo Platinum PLC) gebruik. Elk van die 103Rh resonansies was ondubbelsinnig gekarakteriseer, en elke Rh spesie teenwoordig gekwantifiseer. Daarbenewens is die RhIII spesie-verspreiding van die industriële Rh oplossing deur die "direkte" spesiasie-diagram saamgestel vanuit 103Rh KMR metings akkuraat voorspel. Die berekende RhIII spesie-verspreiding van die industriële Rh oplossings was akkuraat voorspel deur die voorgestelde “direkte” spesiasie-diagram soos saamgestel vanuit die 103Rh KMR metings. Na deeglike optimalisering van Heraeus industriële oplossings (optimale chloriedkonsentrasie gevolg deur termiese behandeling vir effektiewe RhIII chloried anasie reaksies), is die herwinning van Rh via neerslag metodes herhaal. In hierdie geval, het die Rh herwinning dramaties verbeter, met tot 95% van die Rh uit oplossing verwyder. Hierdie verbetering is hoofsaaklik toegeskryf aan die verhoogde "vry" (ongebonde) chloriedkonsentrasie. Die teenwoordigheid van geassosieerde PGM's sowel as ander oorgangsmetale sal die effektiewe "vrye" chloried-konsentrasie verlaag, aangesien hierdie metale sou optree as "chloried-binders". Deur die aanpassing van die totale chloried-konsentrasie, word RhIII chloried anasie reaksies verbeter, wat daartoe lei dat [RhCln(H2O)6-n]3-n (n = 5,6) komplekse anione die dominante spesies in oplossing word, en dus lei tot verbeterde Rh herwinning. Daarbenewens word verder aangetoon dat, onder noukeurig gekontroleerde voorwaardes, "selektiewe" herwinning van Rh bereik word deur gebruik te maak van tris(2-aminoetiel)amien (Tren). Met inagneming van die feit dat Rh die laaste edelmetaal is wat verhaal word in alle PGM-raffinaderye, kan dit 'n koste-effektiewe roete word vir die "vooraf" (vroeë-stadium) herwinning van Rh vanuit industriële PGM bevattende oplossings.

Derivatized $ beta$-cyclodextrin was used as mobile phase additive for the high performance liquid chromatographic resolution of amino acid derivative enantiomers while using an achiral C$ sb{18}$ stationary phase. Dimethyl-$ beta$-cyclodextrin gave improved enantioselectivity over the underivatized $ beta$-cyclodextrin. The effects of cyclodextrin concentration, pH, and methanol and buffer concentration on enantioselectivity and capacity factor were examined.
$ beta$-Cyclodextrin and two derivatized cyclodextrins were utilized as mobile phase additives for the liquid chromatographic resolution of equilin and estrone as well as estrone from 2-, 4-, and 16$ alpha$-hydroxyestrone. $ beta$-cyclodextrin proved to be suitable in these separations but the modified $ beta$-cyclodextrins provided better resolution. Apparent inclusion complex strengths were calculated for estrone and its derivatives were calculated for each cyclodextrin.
Micellar electrokinetic chromatography using sodium dodecyl sulphate with $ beta$-cyclodextrins was found to give comparable or improved separation of a series of fat-soluble vitamins over reversed-phase high performance liquid chromatographic techniques with cyclodextrins in the mobile phase.
Charged carboxymethyl-$ beta$-cyclodextrin was used in the capillary electrophoretic separation of a series of nine tricyclic antidepressants. Cyclodextrin alone was successful in separating some of the compounds under investigation while complete separation required the addition of a micellar pseudophase. A variety of coatings were investigated to reduce or eliminate the electroosmotic flow.

Stereochemistry plays an important role in nature because biologically important molecules such as amino acids, nucleotides and sugars, only exist in enantiomerically pure forms. Semiochemicals carry messages, between the same species (pheromones) and between different species (allelochemicals). Both pheromones and allelochemicals can be used as environmentally friendly pest management. Many semiochemicals, i.e. behaviour modifying chemicals, consist of pure or well-defined mixtures of stereoisomers, where some of the other stereoisomers can be repellent. It is therefore important to be able to separate them to produce a synthetic pheromone in a mixture that is attractive. Pine sawflies are a family of insects that in some cases can be severe defoliators of conifer trees. Diprion pini, Diprion similis and Neodiprion sertifer are severe pests for these trees and have got the most attention in pine sawfly pheromone studies. The pheromone precursors are stored in the female body as long-chain secondary alcohols, which, when released, are esterified to acetates or propionates. The alcohols are chiral, and normally one of the stereoisomer is the main pheromone component, sometimes possible together with other stereoisomers as essential minor components. Bicyclus is a genus of African butterflies, and especially Bicyclus anynana has become a popular model for the study of life history evolution, morphology, mating choice and genetics. The wing pattern of Bicyclus differs depending on the season, with large eyespots during the rain-season and small or absent spots during the dry season.  Euglossa is one of the genera among the orchid bees in the Neotropics that does not produce its own pheromone. Instead, the males collect fragrances from orchids and other sources and store them in a pocket in their hind legs. Both Bicyclus and Euglossa use semiochemicals similar to pine sawflies, and thus can be analysed by the same methods. Pheromones and other semiochemicals in insects are often present in low amounts in a complex matrix, and purification of the sample before chemical analysis is often required. A common method is gradient elution on a solid phase silica column. Separation of stereoisomers can be achieved either by using a column with a chiral stationary phase (CSP) or with pre-column derivatisation using a column with an achiral stationary phase (ASP) or a combination of both, with mass detection as the dominant detection method. The purpose of this work has been to improve the purification method, find suitable methods to separate the stereoisomers of secondary alcohols, and to apply this on extracts of insects. By selecting the right fractions to collect during gradient elution the purification method was optimised. To reduce plasticizer contamination from ordinary columns, solid phase columns of Teflon or glass were used. For pre-column derivatisation of different chiral alcohols various acid chlorides were tested. For the pine sawfly pheromone precursors enantiopure (2S)-2-acetoxypropionyl chloride was the best choice. To separate some of the stereoisomers achiral 2-naphthoyl chloride was used. For derivatisation of 6,10,14-trimethylpentadecan-2-ol (R)-trans-chrysanthemoyl chloride was the best choice. The derivatised alcohols were separated on different columns, both chiral and non-chiral. Varian FactorFour VF-23ms was chosen as a general-purpose column, the Agilent HP-88 column was the best column with an ASP of those tested, and the Chiraldex B-PA column (CSP) was the only one that could separate all eight stereoisomers of derivatised 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, and 3,7-dimethyltridecan-2-ol. To determine the stereoisomeric purity of standard solutions used in field experiments and extracts of different species of insects the optimised methods were applied. For extracts from B. anynana, Euglossa and Neodiprion lecontei this work describe the first determination of the stereochemistry of some of their semiochemicals. For the determination of the stereochemistry of chiral semiochemicals the methods for purification and separation presented herein have shown to be of great value. The results will hopefully contribute to a better understanding of the communication among insects, and ultimately to a more environmentally friendly pest control.
Många naturligt förekommande kemiska ämnen finns som två spegelbilder av varandra, ungefär som höger och vänster hand. Dessa kan ha helt olika egenskaper och det är därför viktigt att kunna separera dem. Insekter och andra djur använder olika doftämnen för att kommunicera med varandra, om det är inom samma art kallas de för feromoner. De kan bestå av ett ämne eller en blandning av flera. Dessa doftämnen kan man även använda för att på ett miljövänligt sätt bekämpa skadeinsekter. En fälla med syntetiskt feromon för en viss insekt lockar endast till sig den arten, medan alla andra är opåverkade. Eftersom dessa ämnen ofta finns som spegelbilder där kanske bara den ena är aktiv och den andra rent av frånstötande, måste man kunna separera dem för att framställa ett syntetiskt feromon som är attraktivt. Målet med detta arbete har varit att bestämma feromonet hos olika arter av tallsteklar som kan vara svåra skadedjur på tallskog. De metoder som tagits fram har även tillämpats på några arter av afrikanska fjärilar samt orkidébin från Centralamerika eftersom de använder snarlika doftämnen. Att få fram feromonet från en insekt är lite som att leta efter in nål i en höstack eftersom de ofta bara innehåller några miljarddels gram per individ. Provet behöver först renas, och en del av arbetet i det här projektet har gått ut på att ta fram en lämplig reningsmetod. Huvudfokus har dock varit på att ta fram metoder som kan separera och identifiera det eller de ämnen, och spegelbilder av dessa, som doftämnena består av. När lämpliga metoder tagits fram har extrakt av olika insektsarter analyserats. I några fall är det första gången som deras feromon bestämts i detalj. Resultaten kan förhoppningsvis bidra till en ökad kunskap om insekters sätt att kommunicera, och i slutändan till miljövänligare bekämpning av skadeinsekter.

A successful supercritical fluid extraction method includes removal of the analyte from the matrix into the bulk fluid as well as trapping or concentration of the analyte prior to analysis. In the first phase of this research, the trapping capacities of three solid-phase traps (glass beads, 50/50 (w/w) glass beads/octadecylsilica), 50/50 (w/w) Porapak Q®/glass beads) were determined as a function of trap composition for a mixture of components varying in polarity and volatility. The Porapak Q®/glass beads mixture was found to be the most successful solidphase investigated exhibiting the highest trapping capacity. The use of the Porapak Q®/glass beads as a solid-phase trap was investigated in later extraction studies in this dissertation. The extraction of highly polar, multifunctional analytes may not be completely successful with modified carbon dioxide, therefore, a secondary modifier (i.e. additive) may be added directly to the extraction fluid in hopes of improving the recoveries. In the second phase of this research, the effect of secondary modifiers in the subcritical fluid extraction of lovastatin from in-house prepared tablet powder mixtures and MEVACOR® tablets was investigated. The effect of in-line methanol-modifier percentage, additive type (acidic, basic, neutral) to the in-line methanol, and additive concentration on the extraction efficiency were examined. The extraction recoveries of lovastatin from MEVACOR® tablets were shown to be highly dependent on methanol concentration and additive type. Isopropylamine was shown to be the most successful additive investigated. An optimized and reproducible extraction method was developed. The extraction of ionic compounds with carbon dioxide may be difficult due to the high polarity of the compounds. In the third phase of this research, the addition of ion-pairing additives to the matrix in hopes of forming an ion-pair complex of reduced analyte polarity was investigated. Therefore, a screening study consisting of a fractional-factorial design was performed in order to identify the factors which contribute most to the recovery of an anionic species, triphenylphosphinetrisulfonate (TPPTS), from a spiked-sand surface employing supercritical fluid extraction with carbon dioxide. The experimental parameters investigated were: type of ion-pairing additive (i.e. tetralkylammonium hydrogen sulfates) and its concentration, carbon dioxide density, extraction temperature, static extraction time, CO₂ mass used, liquid CO₂ flow rate, and the volume of methanol spiked into the matrix prior to extraction. Of the eight factors investigated, four factors were identified as significantly affecting the recovery of the anionic species. They were: 1) ion-pairing reagent added to the spiked sand surface and its concentration; 2) static extraction time; and 3) volume of methanol present in the extraction vessel. The experimental parameters and settings identified as influential by the statistical approach were later shown in concert to yield 100% recovery of TPPTS from the spiked-sand. In the fourth phase, the extraction of a cationic species, pseudoephedrine hydrochloride, from spiked-sand and Suphedrine tablets, with pure and methanol-modified CO₂ was examined. Once the extraction was shown to feasible, several strategies were compared in terms of their effectiveness in enhancing the analyte's extractability. The first strategy involved the addition of ion-pairing additives. Several sodium salts of alkylsulfonic acids varying in lipophilicity and concentration were investigated. The addition of 1-heptanesulfonic acid, sodium salt, in methanol, in a 5:1 mole ratio of reagent to analyte was shown to be the most useful in recovering the drug from the spiked-sand. The second strategy considered the influence of acids and bases and other modifier compositions such as a methanol/water mixture with or without 1-heptanesulfonic acid, sodium salt, on the pseudoephedrine recovery. The recoveries obtained from the drug spiked-sand were shown to comparable in the presence of a methanol/water solution, a tetrabutylammonium hydroxide in methanol solution, and a methanol solution with 1-heptanesulfonic acid, sodium salt. Next the extraction of pseudoephedrine hydrochloride from Suphedrine tablets was performed with pure and modified CO₂. Similar to the sand-spike studies, the effect of the addition of the ion-pairing reagent and other in-cell modifiers were examined. Once again, the recoveries obtained when the matrix was in the presence of a methanol/water mixture and a methanol solution containing 1-heptanesulfonic acid, sodium salt were similar. Finally, the identity of the extracted analyte was determined via IR analyses, and it was shown that pseudoephedrine hydrochloride was indeed extractable from the tablets with in-line modified CO₂ in the absence of any in-cell modifier. In the last phase of this research, a supercritical fluid chromatographic separation with evaporative light scattering detection was developed for the separation of five phospholipids varying in polarity and ionic characteristics. Several parameters were investigated and shown to be influential in the separation. They were: 1) stationary phase composition, 2) addition of an acidic additive and its concentration, 3) mobile phase ramp rate, and 4) column outlet pressure.
Ph. D.

Mestrado em Química - Química Analítica e Qualidade
In the last decades, the effects of the air pollution have been increasing, especially in the case of the human health diseases. In order to overcome this problem, scientists have been studying the components of the air. As a part of water-soluble organic compounds, amino acids are present in the atmospheric environment as components of diverse living organisms which can be responsible for spreading diseases through the air. Liquid chromatography is one technique capable of distinguish the different amino acids from each other. In this work, aiming at separating the amino acids found in the aerosols samples collected in Aveiro, the ability of four columns (Mixed-Mode WAX-1, Mixed-Mode HILIC-1, Luna HILIC and Luna C18) to separate four amino acids (aspartic acid, lysine, glycine and tryptophan) and the way the interaction of the stationary phases of the columns with the analytes is influenced by organic solvent concentration and presence/concentration of the buffer, are being assessed. In the Mixed-Mode WAX-1 column, the chromatograms of the distinct amino acids revealed the separation was not efficient, since the retention times were very similar. In the case of lysine, in the elution with 80% (V/V) MeOH, the peaks appeared during the volume void. In the Mixed-Mode HILIC-1 column, the variation of the organic solvent concentration did not affect the elution of the four studied amino acids. Considering the Luna HILIC column, the retention times of the amino acids were too close to each other to ensure a separation among each other. Lastly, the Luna C18 column revealed to be useful to separate amino acids in a gradient mode, being the variation of the mobile phase composition in the organic solvent concentration (ACN). Luna C18 was the column used to separate the amino acids in the real samples and the mobile phase had acidified water and ACN. The gradient consisted in the following program: 0 – 2 min: 5% (V/V) ACN, 2 – 8 min: 5 – 2 % (V/V) ACN, 8 – 16 min: 2% (V/V) ACN, 16 – 20 min: 2 – 20 % (V/V) ACN, 20 – 35 min: 20 – 35 % (V/V) ACN. The aerosols samples were collected by using three passive samplers placed in two different locations in Aveiro and each sampler had two filters - one faced up and the other faced down. After the sampling, the water-soluble organic compounds was extracted by dissolution in ultra-pure water, sonication bath and filtration. The resulting filtered solutions were diluted in acidified water for the chromatographic separation. The results from liquid chromatography revealed the presence of the amino acids, although it was not possible to identify each one of them individually. The chromatograms and the fluorescence spectra showed the existence of some patterns: the samples that correspond to the up filters had more intense peaks and signals, revealing that the up filters collected more organic matter.
Nas últimas décadas, os efeitos da poluição atmosférica têm aumentado, essencialmente no caso das doenças nos seres humanos. De modo a ultrapassar este problema, a comunidade científica tem-se dedicado ao estudo dos componentes atmosféricos. Enquanto parte dos compostos orgânicos solúveis em água, os aminoácidos estão presentes na atmosfera como componentes dos organismos vivos, seres responsáveis por dispersarem doenças através do ar. A cromatografia líquida consiste numa das técnicas capazes de separar os diferentes aminoácidos entre si. Neste trabalho, com o intuito de separar os aminoácidos presentes em amostras de aerossóis recolhidas em Aveiro, foi avaliada a capacidade de separação de quatro colunas cromatográficas (Mixed-Mode WAX-1, Mixed-Mode HILIC-1, Luna HILIC e Luna C18) para quatro aminoácidos diferentes (ácido aspártico, lisina, glicina e triptofano) e a forma como a interação da fase estacionária destas colunas com os analitos é influenciada pela percentagem de solvente orgânico e pela presença/concentração do tampão. Na coluna Mixed-Mode WAX-1, os cromatogramas dos diferentes aminoácidos revelaram que a separação não era eficiente, sendo os tempos de retenção bastante similares. No caso da lisina, na eluição a 80% (V/V) MeOH, os picos apareceram durante o volume morto. No caso da coluna Mixed-Mode HILIC-1, a variação da concentração do solvente orgânico não influenciou a eluição dos quatro aminoácidos em estudo. Considerando a coluna Luna HILIC, os tempos de retenção dos vários aminoácidos eram demasiados próximos para garantir uma separação entre os mesmos. Por fim, a coluna Luna C18 mostrou-se útil na separação de aminoácidos no modo gradiente, no qual a variação da constituição da fase móvel ocorreu em termos de volume do solvente orgânico (ACN), tendo sido esta a coluna utilizada para a separação das amostras reais. A fase móvel era constituída por água acidificada e ACN, e o gradiente consistia no seguinte programa: 0 – 2 min: 5% (V/V) ACN, 2 – 8 min: 5 – 2 % (V/V) ACN, 8 – 16 min: 2% (V/V) ACN, 16 – 20 min: 2 – 20 % (V/V) ACN, 20 – 35 min: 20 – 35 % (V/V) ACN. As amostras de aerossóis foram obtidas com recurso a três amostradores passivos colocados em dois locais distintos de Aveiro, cada amostrador contendo dois filtros - um virado para cima e outro para baixo. Após a amostragem, a matéria orgânica solúvel em água foi extraída com recurso a dissolução em água ultra-pura, banho de ultrassons e filtração. Os filtrados recolhidos foram diluídos em água acidificada para a separação cromatográfica. Os resultados da cromatografia líquida evidenciaram a presença de aminoácidos, não tendo sido possível efetuar a sua identificação individual. Os cromatogramas e os espetros de fluorescência indicaram a existência de padrões: as amostras correspondentes aos filtros de cima possuíam sinais mais intensos, indicando que os filtros de cima recolheram maior quantidade de matéria orgânica.

Thesis (MSc)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: In the ongomg search for better and faster ways to characterize complex polymer systems, it is often necessary to couple different analytical techniques in order to obtain information on more than one distributed property. In this study, the coupling of chromatography and spectroscopy to atomic force microscopy (AFM) was attempted for the first time, and thus the term "LC-FTIR-AFM" was coined. This new hyphenated technique combines the separation power of liquid chromatography (LC) and the ability of infrared spectroscopy (IR) to identify almost any organic compound, with the AFM's ability to be used for thermal analysis of individual phases in polymer mixtures. The first two steps of this new technique comprise (i) the separation of compounds in a mixture via gradient polymer elution chromatography (GPEC) and (ii) the identification of each compound by means of LC-FTIR analysis. In the final step, LC-FTIR analysis is coupled to AFM through the use of the LC-FTIR interface. A number of polymer mixtures were analysed by means of the novel technique that was developed, in order to establish its validity and value as a characterization technique of the future. The influence of fllm thickness and molar mass on the thermal parameters of individual components in mixtures, measured by this technique, were also investigated. This technique adds a new dimension to conventional thermal analysis methods, since it allows the thermal transitions of individual polymer phases in multiphase polymers to be resolved directly after separation and identification.
AFRIKAANSE OPSOMMING: In die voortdurende soektog na beter en vinniger maniere om komplekse polimeersisteme te karakteriseer, is dit soms nodig om verskillende analitiese tegnieke met mekaar te koppel ten einde inligting aangaande twee of meer verspreide eienskappe te bekom. Gedurende hierdie studie is daar gepoog om chromatografie en spektroskopie met atoominteraksie-mikroskopie (atomic force microscopy, AFM) te koppel. Gevolglik het die term "LC-FTIR-AFM" ontstaan. Hierdie nuwe koppelingstegniek kombineer die kragtige skeidingspotensiaal van vloeistofchromatografie en die vermoë van infrarooispektroskopie om byna enige organiese verbinding positief te identifiseer, met die atoominteraksie-mikroskoop se potensiaal om as 'n termiese analise metode vir individuele fases in polimeermengsels te dien. Die eerste twee stappe van die tegniek behels (i) die skeiding van verbindings in 'n mengsel deur middel van gradient-hoë-druk-vloeistofchromatografie en (ii) die identifisering van afsonderlike verbindings deur vloeistofchromatografie gekoppel aan infrarooispektroskopie. Gedurende die finale stap word vloeistofchromatografie en infrarooispektroskopie aan die atoominteraksie-mikroskoop gekoppel deur gebruik te maak van die LC-FTIR koppelingsapparaat. 'n Aantal polimeermengsels is geanaliseer deur die nuwe tegniek hierbo beskryf, ten einde die geldigheid en waarde daarvan as 'n analitiese metode vir die toekoms vas te stel. Die invloed van film diktes en molekulêre massa op die termiese oorgange van individuele komponente in mengsels, soos gemeet deur hierdie metode, is ook ondersoek. Hierdie tegniek voeg 'n nuwe dimensie tot konvensionele termiese analise metodes deurdat dit die bepaling van termiese oorgange van individuele polimeerfases III multifase polimere, direk na afloop van skeiding en identifikasie moontlik maak.

As espécies patogênicas do gênero Colletotrichum apresentam importância mundial, pois causam danos a várias culturas de interesse agronômico. Diversas medidas de controle são empregadas, mas estas nem sempre são eficazes devido à ocorrência de linhagens resistentes. Desta forma, se faz necessário a busca por novos compostos que possam ser utilizados no manejo integrado desta doença. Os produtos naturais isolados de micro-organismos podem ser uma alternativa para o desenvolvimento de novos defensivos agrícolas. Dentre os micro-organismos, as actinobactérias são conhecidas pela produção de inúmeros compostos antimicrobianos. Neste contexto, o presente estudo teve como objetivo o isolamento e a identificação de compostos antifúngicos produzidos por actinobactérias da rizosfera de guaranazeiros. Para isto, a seleção de actinobactérias foi baseada em dois ensaios. No primeiro, as 65 actinobactérias foram avaliadas em ensaio de cultivo pareado frente ao fitopatógeno Colletotrichum gloeosporioides. Destas, os isolados mais promissores foram AM1 (43,78 % de inibição do crescimento micelial), AM3 (43,98 %), AM18 (37,86 %), AM25 (43,17 %), AM30 (47,12 %), AM61 (40,12 %) e AM68 (47,94 %). No segundo ensaio, estes isolados foram cultivados em meio BD e, após o cultivo, o meio metabólico foi submetido a três métodos de extração: (a) partição líquido-líquido com n-butanol; (b) partição líquido-líquido com acetato de etila e (c) coluna sílica gel C18. As frações obtidas a partir das três metodologias foram avaliadas pelo método de difusão em disco de papel contra C. gloeosporioides. Neste ensaio de difusão em disco foram selecionadas as linhagens AM1(n-butanol), AM3 (acetato de etila) e AM25 (C18) para o estudo de bioprospecção. Estas foram identificadas por técnicas moleculares como pertencentes ao gênero Streptomyces. A partir do extrato bruto da Streptomyces sp. AM1 foi isolado um composto análogo do ácido proclavamínico, o qual apresentou atividade mínima inibitória (MIC) de 1,25 mg mL-1 contra o fitopatógeno C. gloeosporioides. Da linhagem Streptomyces sp. AM3 foi isolado o composto streptimidona que apresentou MIC de 1,25 mg mL-1. Já no estudo de Streptomyces sp. AM25 um composto não identificado apresentou MIC de 2,50 mg mL-1. Estes três compostos apresentaram atividade superior aos fungicidas Captan SC® (Captana) e Dithane NT® (Mancozeb), e inferior ao Score® (Difenoconazol). A atividade antifúngica destes compostos ao C. gloeosporioides está sendo relatada pela primeira vez.
The pathogenic species of the genus Colletotrichum present importance worldwide because they cause damage to numerous crops of agronomic interest. Several control methods are employed, but they are not always effective due to the occurrence of resistant strains. Thus, it is necessary searching for new compounds that can be used in the integrated management of this disease. Natural products isolated from microorganisms can be an alternative for the development of new agricultural pesticides. Among microorganisms, actinobacteria are known to produce numerous antimicrobial compounds. In this context, the present study aimed to isolate and identify antifungal compounds produced by actinobacteria from guarana rhizosphere. For this, the selection of actinobacteria was based on two tests. In the first one, the 65 actinobacteria were evaluated in paired cultivation test against the plant pathogen Colletotrichum gloeosporioides. Among them, the most promising isolates were AM1 (43.78% inhibition), AM3 (43.98%), AM18 (37.86%), AM25 (43.17%), AM30 (47.12%), AM61 (40.12%) and AM68 (47.94%). In the second assay, these isolates were cultured in BD medium and, after culturing, the metabolic medium was subjected to three extraction methods: (a) liquid-liquid partition with n-butanol; (B) liquid-liquid partition with ethyl acetate and (c) silica gel column C18. The fractions obtained from the three methodologies were evaluated by paper disc diffusion method against C. gloeosporioides. In this disk diffusion assay, the strains AM1 (n-butanol), AM3 (ethyl acetate) and AM25 (C18) were selected for the bioprospecting study. These were identified by molecular techniques as belonging to the genus Streptomyces. From the crude extract of Streptomyces sp. AM1 the analogous compound proclavaminic acid was isolated, which presented minimal inhibitory activity (MIC) of 1.25 mg mL -1 against the plant pathogen C. gloeosporioides. From Streptomyces sp. AM3, the compound streptimidone was isolated, which presented MIC of 1.25 mg mL-1. In the study of Streptomyces sp. AM25 an unidentified compound had MIC of 2.50 mg mL-1. These three compounds presented superior activity to the fungicides Captan SC® (Captan) and Dithane NT® (Mancozeb), and inferior to the Score® (Difenoconazole). The antifungal activity of these compounds to C. gloeosporioides is being reported here for the first time.

Synthetic musk compounds (MUSK) or synthetic fragrances are organic substances commonly used as fragrant constituents of perfumes, detergents, cosmetics and personal care products. These compounds are discharged after use via domestic wastewater and sewage treatment plants to the aquatic environment. The result is accumulation in the environment and occurrence in a food chain especially of aquatic and marine ecosystems. Synthetic musk compounds are also found in surface water, fish tissues and also in body fluid (blood, urine, milk) and tissues of human body. Quantitative detection and studying properties of these compounds is an important activity because we have low knowledge about their fate in the environment (persistence, bioaccumulation) and toxicity. This diploma thesis is focused on three commonly used synthetic fragrances – tonalide, musk ketone and musk xylene. The base of this study was to perform a method optimisation for the determination of selected compounds in water and water biota (fish). The aim was determination of selected musk compounds in real samples (water, fish) and evaluation of the results. Pressurized solvent extraction (PSE) for fish samples and solid phase microextraction (SPME) for water samples were used for isolation of analytes from samples. The identification and quantification of analytes was carried out by high resolution gas chromatography – mass spectrometry.