Flavonoid dyes were analysed by negative ion mode electrospray ionisation quadrupole ion trap mass spectrometry (ESI QIT MS). New structure-dependent breakdown pathways were identified and the mechanisms were supported by deuterium labelling experiments. The results were extended to provide important structural information on unidentified flavonoids in natural yellow dye extracts. Samples of alum mordanted wool dyed with pure flavonoids or with natural dyes were exposed to accelerated light ageing. Under these conditions photooxidation was found to be the main degradation process. The photodegradation products were analysed by photodiode array high performance liquid chromatography (PDA HPLC) and by liquid chromatography electrospray ionisation quadrupole ion trap mass spectrometry (LC-ESIMS). In general the photodegradation products of flavonols retain information on the nature of substituents of the B-ring and therefore provide partial structural information related to the original dye molecule. The nature of the light source was found not to influence the nature of the photodegradation products detected. No photodegradation products could be identified from flavone or flavonol 3-<i>O</i>-glycosides dyed textiles under the light ageing conditions used. The acid hydrolysis dye extraction process was found to influence the chemical profile of the dye extracts. The identification of the degradation produces allowed their quantification and hence the kinetics of the photooxidation process of flavonoid dyes on wool fibres could be studied. It was found that experimental data did not fit a consecutive first order reaction model. A rapid initial rate of decay of the flavonol was followed by a slower process. The results suggest that a model where the dye population is divided into two groups with different decay rates fits the experimental data adequately. The rates of decay were found to be influenced by the nature of the light source.

Conductive polymers nanowires have been prepared using DNA-templating methods from monomer units designed in modular form. The monomer units comprise a polymerisable, pyrrolyl group, and a flexible alkyl linker attached to bipyridine groups in order to provide a metal-binding functionality within the polymers. The possibility for these DNA/polymer nanowires to act as templates for deposition of metal with enhanced electrical conductivity was also explored. Pyrrole with a flexible alkyl linker was combined with; pyridine (mono-I) as a control experiment, 2,2` bipyridyl (mono-II) and 4,4` bipyridyl (mono-III) with a metal ion binding site (nitrogen atom). This was in order to provide the metal-binding functionality for metal deposition to improve the conductivity as well as the morphology of the aimed hybrid templated nanowires. This series of pyrrole-pyridine derivatives were characterised using a range of techniques such as Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy, Mass Spectroscopy (MS) and Elemental Analysis. Prior to the nanowires fabrication, pyrrole, as a control, and the prepared monomer units (mono-I, mono-II and mono-III) were chemically polymerised in a bulk scale using FeCl3 as an oxidant, then spectroscopic data and electrical conductivity of the resulting polymers were measured. A significant decrease in conductivity of poly-I, poly-II and poly-III compared to PPy, but was especially observed for the bipyridinium derivatives. This was suggested to be due to the steric hindrance of the alkyl side chain in the polymer backbone, in addition, the involvement of the non-quaternised pyridyl nitrogen in poly-II and poly-III by the nucleophilic attack on pyrrolyl groups in the polymerisation reaction. DNA-templated poly(N-substituted pyrrole)bipyridinium nanowires were synthesised at room temperature using the chemical oxidation method. The resulting CPs/DNA hybrids have been characterised using electronic and vibrational spectroscopic methods especially Ultraviolet-Visible (UV-Vis) spectroscopy and FTIR spectroscpy. The nanowires morphology was characterised using Atomic Force Microscopy (AFM). The electrical properties of the prepared nanowires were characterised using Electrostatic Force Microscopy (EFM), and measured using conductive AFM (c-AFM) and two iii terminal I/V technique, where the temperature dependence of the conductivity was probed. The conductivities of the prepared CPs/DNA nanowires are generally lower than PPy/DNA nanowires showing the large effect on N-alkylation in decreasing the conductivity of the polymer, but these are higher than the conductivity of their corresponding bulk films. This enhancement in conductivity could be attributed to the ordering of the polymer chains on DNA during the templating process. Finally, the prepared CPs/DNA nanowires were used as templates for the growth of copper nanowires at room temperature using aqueous solution of Cu(NO3)2 as a source of Cu2+ and ascorbic acid as reducing agent. AFM images showed that these nanowires were uniform and continuous compared to copper nanowires prepared using the templating method directly onto DNA. Electrical characterization of the nanowires by c-AFM revealed slight improvement in conductivity of these nanowires (Cu-CPs/DNA) compared to CPs/DNA nanowires before metallisation. Using similar preparation method, Poly-II/DNA nanostructures were also used as templates to direct the formation of Pd nanowires. An aqueous solution of PdCl2 was used as a source of Pd2+ ions and NaBH4 solution was used as reducing agent. AFM studies show that the resulting Pd-poly-II/DNA nanowires exhibit continuous and smooth morphology. Electrostatic Force Microscopy showed that these nanowires are electrically conductive.

This work has been conducted to investigate the chemical processes involved in the remediation of acidic mine drainages (AMD) using electroanalytical methods to study the chemistry of Fe(III) in pure laboratory solutions and samples obtained from Shilbottle Colliery (Northumberland, UK), which are heavily contaminated with soluble Fe leached from the spoil heap. One method for remediation is to raise the pH of the waters and precipitate Fe(III) in an artificial wetland; however microelectrode voltammetry shows significant soluble Fe(III) remains. The characterization of hydrolysed species of Fe(III) in pure laboratory solutions and Shilbottle samples has been studied using voltammetry, Raman spectroscopy, electrospray ionization - mass spectrometry (ESI-MS) and X-ray diffraction (XRD). The voltammetric and Raman studies show that, there is some similarity in square wave voltammograms and Raman spectra between Shilbottle solutions and aqueous laboratory Fe(III)-sulfate solutions. ESI-MS of laboratory Fe(III) solutions with non-coordinating, or weak coordinating anions such as perchlorate shows the presence of monomers, dimers, trimers and tetramers of soluble Fe(III) complexes. However no Fe-related peaks were observed in sulfate-containing media and the Shilbottle samples due to the presence of neutral species such as FeSO4OH. XRD data shows formation of basic sulfate complexes in the case of Fe(III)-sulfate and no XRD peaks can be assigned to Fe(III) in perchlorate media. Determination of soluble fractions of [Fe(III)/Fe(II)] and [SO42-] in Shilbottle samples have been performed using developed voltammetric methods. The total soluble [Fe] varied between 281 and 446 ppm and soluble [SO42-] between 3460 and 7400 ppm. A standard geochemical model (PHREEQC) has been used to study chemical speciation in AMD; a comparison of the model and our measurements indicates that the Shilbottle samples are clearly out-of-equilibrium. Kinetic studies of hydrolysis of Fe(III) in pure laboratory solutions and Shilbottle samples have been performed using potentiometric, voltammetric and Raman spectroscopy techniques. The obtained data show that the hydrolysis of Fe(III) upon raising the pH by addition of NaOH is second order in [Fe]; the rate constants were measured and a mechanism which can rationalize the rate constants is proposed. Also, ii effects of sulfate ions on the hydrolysis of Fe(III) in aqueous solutions have been investigated. The study shows that the SO42- ions catalyse formation of soluble dimers, oligomers and basic sulfate complexes. Furthermore, FeSO4+ stays dissolved in the solution longer than simple Fe(III) species such as Fe3+, Fe(OH)2+ and Fe(OH)2+.

Background and aims Lead exposure has irreversible health effects in children who are susceptible even at very low levels of exposure. The usual test for lead exposure is blood lead level (BLL), but this indicates only recent exposure. This study aimed to ascertain the suitability of milk teeth as biomarker of the history of lead exposure and to develop a methodology for this novel biomarker. Methods My study comprised three stages: Firstly, I explored potential determinants of dentine lead levels (DLLs) in children living in Newcastle upon Tyne (the Tooth Fairy Study). Secondly, I developed a methodology for assessing the history of early life lead exposure using dentine, and thirdly I applied my methodology to newly extracted teeth from children in Teesside. The Tooth Fairy study cohort consisted of 69 children aged 5-8 years. DLLs were measured in primary dentine using Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). To identify determinants of early life exposure, a questionnaire was used. I assessed associations between lifestyle characteristics and DLLs. As teeth develop chronologically they offer the opportunity to study histories of exposure in detail. I collected two deciduous molars each from 15 children aged 6-8 years living in Northeast England. By combining high spatial resolution LA-ICP-MS with dental histology, I acquired information on the age specific concentrations of lead in dentine from in utero to several years after birth. Results Dentine lead levels in the Tooth Fairy cohort ranged from 0.06 to 0.77μg/g, median 0.21μg/g. Unlike other studies, I did not find significant associations between socio-economic status or other possible determinants and lead exposure. In developing the biomarker I found that the 100 micron ablation pit represented 42 days of dentine growth, enabling me to assign an age interval to each ablation pit. I found lead ratios in primary dentine to be consistent between teeth from the same child, and at the same age within each tooth. This indicated that the history of exposure can be determined using a single, multi-point ablation transect on high quality longitudinal sections of individual teeth. Conclusions  I found no association between socio-economic status and dentine lead levels in a cohort of children from Newcastle upon Tyne.  I developed a novel technique to date ablation points in dentine in milk teeth, and, using this technique have demonstrated that primary dentine is a potential biomarker for characterising the early life history of lead exposure in children.

The aim of this project was to assess the feasibility of using UV spectroscopy for a simple detection system for formaldehyde gas in the indoor environment. Formaldehyde gas is hazardous to human health causing irritation of the eyes, nose and throat, headaches, limited pulmonary function and is a potential carcinogen. Formaldehyde derivatives are used in plywood and fibre board, carpeting, fabrics and some paints. The gas can be emitted from these materials and can build up in the indoor environment. Current methods for detecting formaldehyde gas that are simple, reliable and inexpensive are limited. A literature study of chemicals common to the indoor environment was carried out, and their UV absorption spectra compared to that of formaldehyde. 85 substances and substance groups were considered, 11 of which had absorption spectra that overlapped with the formaldehyde UV absorption band. A region was found between 320 and 360nm with very little spectral interference. Given the number of gases considered, this was a surprising result. Formaldehyde has several strong absorption peaks between regions of very low absorption, allowing for low resolution detection using a single LED source. Two prototype detection systems were developed. The first used a UV LED light source and used a beam splitter to provide one detection channel and one reference channel. The channels used narrow band (laser-line) optical filters. It was thoroughly optimised for noise performance, giving a best case limit of detection of 4.2ppm, limited by source fluctuation and shot noise, and LED thermal drift. Future developments could include a temperature controller inside the casing of the LED, or a multi- pass gas cell to increase sensitivity. With only two channels, the two filter system was susceptible to spectral interference from nitrogen dioxide and nitric acid. The second prototype system was developed using a novel method of passing un-collimated light through a laser-line filter to produce multiple wavelength channels with an angular spread. The principle was validated using two-channel detection with a limit of detection of 6ppm.

Pyrrolocytosine (Pc) and 2-aminopurine (2-Ap) are modified fluorescent nucleobases that can respectively replace the non-fluorescent natural nucleobases cytosine (C) and adenine (A) and therefore, can be used to probe the structure and dynamics of nucleic acids. Changes in fluorescence emission intensity of these modified bases as a function of polarity (general solvent effect) were measured. Lippert plots were generated and the difference between the excitation and emission maxima (Stokes shift) wavelengths of Pc and 2-Ap in water and acetonitrile mixtures indicate that the measured Stokes shifts are mainly due to hydrogen bonding (specific solvent effect) rather than polarity. The data of the quantum yield measurements show that the fluorescence intensity of 2-Ap decreases in organic solvents as opposed to that of Pc. The big difference in dipole moment of the bases in the excited and ground state experimentally found may be due to intramolecular charge transfer. Computational studies performed at DFT (Density Functional Theory) level by using the B3LYP functional revealed that the most stable 2-Ap tautomer is the N9H amino tautomer in both gas and liquid phase. In the case of Pc, our results show that the most stable tautomer in the gas phase is the N9H enol tautomer whereas the N1H N9H keto one is the most stable tautomer in solution. The theoretical absorption and emission maxima are in excellent agreement with the experimental data. The optimized geometry of 2-Ap A in the ground state was found to be non-planar, i.e., with an out of plane pyramidal amino group with respect to the purine ring whereas the optimized molecule appears to be planar in the first excited state. When 2-Ap A is explicitly bound to water molecules, the energy gap between the dark state n-π* and the bright state π-π* is bigger than the electronic energy gap between the two states previously predicted in implicit solvent. The quantum yield of 2-Ap in phosphate buffers is lower than the one measured in water. This decrease in fluorescence emission is almost certainly due to dynamic quenching. The fluorescence emission of both 2-Ap and Pc is drastically reduced when the bases are incorporated into single and double-stranded oligonucleotides but, the degree of fluorescence depression is more marked in water than in buffer solutions. Oligonucleotides may form secondary structures in buffers because of the presence of salts; in particular, G-quadruplexes. The spectroscopic data acquired by using UV, fluorescence, CD (circular dichroism), and anisotropy steady-state techniques seem to rule out the presence of G-quadruplex secondary structure in selected fluorescent nucleobase containing oligonucleotides.

This work concerns a proton transfer reaction time-of-flight mass spectrometer, PTR-TOF-MS, a bespoke model manufactured by Kore Technology Ltd. for Smiths Detection. This instrument achieves 'soft' ionization of volatile 'organic compounds (VOCs) by proton transfer from protonated water vapour in a reaction chamber at 1 mbar (= 100 Pa). The resulting ions are separated by mass in a field-free time-of-flight tube prior to detection by a multi-channel plate. The instrument was modified to facilitate direct determination of the electric field in the reaction chamber. Sensitivity measurements determined a value of 4-6 counts per second per parts per billion by volume (ncps ppbv·1 ) normalised to 106 H30+. The calibration gas mixture used in this investigation consisted of 14 compounds, (alkylbenzenes and chlorobenzenes) spanning an mlz range of 78 to 180. Each of these was separately investigated over EIN = 90 to 245 Td to establish fragmentation behaviour and possible interfering contributions. For example, several of the alkylbenzenes fragmented to product ions occurring at mlz 79, the same value as that of protonated benzene. Most of this occurred at the higher EIN values with ethylbenzene a notable exception. The isobaric compounds ethyl benzene and the xylenes exhibit very different fragmentation patterns so enabling differentiation of these two compounds. However, it is not possible to distinguish the individual xylene isomers using this method. Benchmarking was continued using the hexenol compounds cis-3-, cis-2-, trans-3- and trans-2- hexen-I-ols. This work demonstrated that the same four product ions are seen for all of the hexenol isomers at mlz 39 (C3H/), 41 (C3H/), 55 (C4H/) and 83 (C6Hl1+) when reacted with H30+ in a PTR-TOF-MS. A characteristic peak at mlz 99 was seen in trans-2-hexen-1-01 and cis- 2-hexen-1-01 at low EINvalues « 140 Td) when the protonated parent ion, mlz 101, is absent. In trans-3-hexen-l-ol and cis-3-hexen-1-01 the MW ion at mlz 101 is seen at these lower EIN values but there is no product ion at mlz 99. This suggests a possible method for distinguishing between the 2- and 3-hexenols. It may also be possible to further identify the individual isomers from the differences in the percentage yield of these product ions.

There are several approaches to reducing mobile phase viscosity in order to overcome high pressures related to stationary phase permeability. This can be achieved either by elevating column temperature and or by employing organic rich eluents. To begin with, a standard application of UHPLC-MS was developed for the high throughput quantification of quinine and its major metabolite. In attempting to expand the performance capabilities of ultra-pressure systems a high temperature column oven was evaluated. It was found to be mostly unsuitable for incorporating micro-bore columns due to excessive system volume necessary for installation, and due to the effects of frictional heating associated with operation at ultra-high pressures. An attempt to reduce the effect of frictional heating by modifying the column hardware was evaluated, however only slight improvements were observed. This system has benefits to operate at elevated temperatures, however reduced column performance was always observed as shown by the separation of anabolic steroids and neutral test substances. An evaluation of HILIC column efficiency was undertaken by assessing the performance merits of several commercially available packings using the Knox/van Deemter and kinetic plots approaches. This work centred on evaluation using realistic test substances on underivatised silica, highlighting that very high efficiencies could be achieved using these phases, otherwise not achievable with reversed-phase systems due to peak shape improvements. The justification of which was further addressed by comparing the performance merits of ephedrine-substances separated by both reversed-phase and HILIC noting that the latter was far superior. Arguably the biggest disadvantage with HILIC is the reliance on acetonitrile based mobile phases. An in-depth evaluation of acetone as an alternative, aprotic organic modifier for HILIC-ESI-MS was undertaken, showing that this may not be suitable for the analysis of small polar molecules in positive ion electrospray.

The work presented here demonstrates two unique approaches in the application of LC/MS for the analysis of drugs and endogenous compounds in biofluids. First, the use of basic aqueous mobile phases in conjunction with both methanol and acetonitrile by LC/MS operating in electrospray positive ionization mode was investigated. Second, the design and development of a prototype ceramic micro-fluidic device (CMFD) and optimized MS source was carried out. The CMFD was packed with sub 2 μm bridged ethyl hybrid (BEH) chromatographic particles that could withstand operating pressures of up to 12,000 psi. The MS source was built to operate in both positive and negative electrospray ionization mode with the operating flow rates corresponding to the 300 um i.d. of the CMFD. The results generated from studies utilizing the addition of a base, such as ammonium hydroxide to modify the aqueous mobile phase, showed significant benefits for LC/MS/MS based bioanalytical assays when analyzed with electrospray positive ionization mode. Increases in the signal-to-noise values were observed for twenty-two out of twenty-four of the probe pharmaceuticals tested. Increase in the chromatographic retention of poorly retained compounds was also observed however, this increase in analyte retention did not occur for many highly polar compounds that eluted in the column void when chromatographed with the basic mobile phase conditions. The effect of the pH of the mobile phase further showed that the phospholipid fraction, present in protein precipitated plasma, was only slightly affected by the change in mobile phase pH. It provided complementary data to that obtained with traditional acidic based mobile phases and results in an increased number of ions detected overall for metabonomic studies. Further it was observed that by changing the pH to a basic system it was possible to resolve compounds that had previously co-eluted on traditional acidic reversed-phase LC/MS. A 0.3 x 100 mm i.d. CMFD and compatible MS source was successfully designed for the analysis of biological samples. The device showed average chromatographic efficiencies of 9038 plates compared to 10219 plates for standard silica capillary columns. Gradient performance utilizing a diverse mix of compounds yielded a peak capacity of 55 as the average peak widths for all analytes was 0.11 minutes for a 6 minute separation. Resolution of the probe pharmaceutical alprazolam and associated hydroxylated metabolite was maintained between 1.2 and 1.5 for four different devices. Testing of the device with plasma samples prepared by protein precipitation resulted in over 1000 injections being carried out over approximately a one week period. The system was however unable to withstand the high pH (10.5) utilized in the previous study but could operate with a mobile phase pH of 10. The resulting MS source built to operate under these conditions and with the flow rates required to operate the 0.3 x 100 mm CMFD yielded signal to noise increase in the range of 8.3 to 38.6 for the molecules tested. The CMFD/MS system was successful in the analysis of biofluid samples in both ESI + and ESI – ionization modes and was shown to allow for the analysis of small sample injection volumes from plasma prepared by protein precipitation, and dried blood spots. This device was successfully utilized for the profiling of metabolites from the beta blocker drug, propranolol. Further separation of complex biofluid samples derived from axenic rats and bile from dogs again illustrated the separation and sensitivity capabilities offered by the CMFD/MS system.

This thesis comprises studies of ultra high performance liquid chromatography (UHPLC) performance with a particular focus on the application of liquid chromatography-mass spectrometry (LC-MS) for the analysis of basic drugs. UHPLC-MS technologies are investigated to facilitate fast, sensitive and robust analysis of drugs in biological matrices, particularly those related to doping in human sport. Mobile phase solvent, pH and temperature significantly affect the chromatographic performance of hydrophilic, basic analytes. Manipulation of mobile phase pH suppresses the protonation of basic analytes for improved retention, peak shape and resolution. This is exemplified by the ephedrines, which are inherently difficult to separate by reversed-phase liquid chromatography (RPLC). A high pH RPLC separation is coupled with MS detection and validated for the identification and quantification of ephedrines in doping control analysis. The effects of mobile phase composition and pH on efficient and stable ionisation are studied for robust and sensitive analysis of basic analytes. Different mobile phase conditions are evaluated with electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI), with high pH eluents generating greater signal intensities for the basic analytes studied with ESI. Hydrophilic interaction liquid chromatography (HILIC) is evaluated as an alternative approach for accurate and robust quantification. A HILIC approach to separate the ephedrines is evaluated for robustness, and the validated method is compared with the high pH RPLC method in terms of linearity, accuracy, precision, matrix effects and sensitivity. Finally, a switching system comprising two different, complementary stationary phase materials is designed and evaluated to widen the elution window, allowing for the simultaneous analysis of both polar and non-polar analytes. A combination of Hypercarb and Cig stationary phases are used with UHPLC column switching, and the suitability of the approach is illustrated by the analysis of selected doping agents covering a wide polarity range in a single injection.