No description available.

No description available.

The initial part of this study involves developments made on an electrospray ionization (ESI) source installed on a large-scale double-focusing reverse-geometry two-sector mass spectrometer. The practical considerations and some basic theory have been given in this thesis for each of the improvements made to the ESI source13 '1. The process of partial re-designing the ion optical system showed that the ion beam was affected by both gas dynamics and by the field penetration from the lenses of the ion optical system in the region immediately after second skimmer. More than one order magnitude stronger ion signal intensities at the mass spectrometer final detector and easier ESI source operation conditions and stable ion signals has been achieved. Mass-analyzed ion kinetic energy spectroscopy (MIKES) of different charge- states of relatively small peptides (substance P and bradykinin) for collision-induced dissociation (CID) and the metastable dissociation was carried out successfully. The translational energy losses caused shifts in the fragment ion peaks in the CID spectra, compared with fragment ion peaks in the metastable dissociation spectra. A major part of the study concerned effects of ESI source condition on the charge state distributions of mass spectra. It has been shown that the ion optical effect of the potential difference (V HCT-S) between the heated stainless steel capillary (HCT) and the first skimmer in the intermediate-pressure region (capillary-skimmer region or orifice-skimmer region) of the ESI source was the main factor in determining the charge state distribution of the ESI mass spectra. The temperature of the HCT and the potential difference between the first skimmer and the second skimmer has been shown to be also important factors. The collision-induced dissociation (or so-called capillary-skimmer dissociation) effect of V HCT-S has been shown to be relatively low in this sector-mass spectrometer ESI source.

No description available.

Flow chemistry has been increasingly used in the last decade as an alternative method to batch chemistry. This methodology allows for conditions that would be unattainable under batch techniques due to the high temperature control, selectivity and safety that flow chemistry allows. The diazidation of styrenes has been investigated under continuous flow conditions, where the inherent safety of flow chemistry allows the use of azides without the concerns usually associated with these reagents. Secondly, the nitroaldol reaction has been transferred to flow conditions. This highlights the safety of continuous flow procedures, as the use of a highly energetic reagent such as nitromethane is easily possible. The Koch-Haaf carbonylation reaction was investigated to demonstrate the use of gases in flow chemistry and the safe handling of toxic gases such as carbon monoxide. Finally the Ritter reaction was used to further demonstrate the suitability of flow chemistry for highly exothermic reactions using concentrated acids; where the temperature control allows for high selectivities.

The objective of the work described in this thesis was to develop novel experimental and analytical methods to study samples of anisotropically oriented fluorophores. The primary development was the fluorescence detection method for linear dichroism spectroscopy (FDLD). The experimental configuration of the measurement has the detector placed facing the propagation direction of the exciting incident light. Long-pass edge filters, selected so that their cut-off wavelength lies between the excitation and emission maxima of the sample’s fluorophore, were placed between the sample and detector to block transmitted incident light, whilst allowing Stoke’s shifted fluorescence emissions to pass freely. The experiment was designed to be conducted using a commercial circular dichroism (CD) spectropolarimeter, which has been adapted to measure linear dichroism (LD). A theoretical framework to calculate FDLD spectra using the output of such an instrument is presented, and used to generate the FDLD spectra of small molecules oriented on stretched oxidised polyethylene films and biological samples oriented in Couette flow. It was found that much of the information that can be obtained from LD may also be derived from FDLD spectra, however, FDLD possessed two clear advantages: i) fluorescence detection is highly sensitive, which significantly lowered the sample volume requirement of a measurement, and ii) fluorescence detection is more selective than absorption methods, as only chromophores that are also fluorophores are detected. A method for the cloning, expression and purification of the Escherichia coli actin homologue MreB is also presented. The composition of secondary structure elements within the protein obtained was analysed using CD and found to be in good agreement with literature values, taken from the crystal structure of Caulobacter crescentus MreB. Fluorescence spectra of the protein were recorded, which indicated that it may be possible to study its in vitro polymerisation kinetics using FDLD. Unfortunately, we were unable to obtain our Escherichia coli MreB in a sufficient yield to develop this assay. Finally, a novel method for detecting the wall shear stress (WSS) exerted on a specific point of a surface by a fluid flowing over it is reported. This information was derived from the response to a shear flow of a fluorescently labelled particle of M13 bacteriophage, which was tethered to the surface. The focus of this thesis was primarily on the analysis of the fluorescence signals. Using a custom made algorithm, microscopy images of the tethered phage particle were segmented to define the region of each image occupied by the particle, and to calculate its orientation and length at all time points. These data were used to calculate the WSS at the point of the particle’s attachment, and show that it varied when the surface was not uniform — highlighting a potential problem with commonly used methods for determining WSS that average over surface dynamics, such as particle image velocimetry and particle tracking velocimetry.

La molécule d'ammoniac est une des molécules les plus présentes dans les environnements astrophysiques en particulier dans l'atmosphère des planètes solaires comme Jupiter et Saturne où la mesure du rapport isotopique 14N/15N est une donnée importante. Les informations disponibles pour l'isotopologue 14NH3 dans la gamme 6300-7000 cm-1 [Sung et al JQSRT, vol. 113, 1066 2012] ont été incluses dans la base de données HITRAN 2012. Pour l'isotopologue 15NH3, Lees et al [JMS, 251, 241–251, 2008] ont identifié un certain nombre de transitions (états vibrationnels ν1+ν3, ν1+2ν4 et ν3+2ν4). Lins et al [App.Phy. B,102,293–301, 2011] ont cherché des transitions susceptibles de convenir à la mesure du rapport isotopique.Dans notre travail, nous avons étendu la connaissance de la spectroscopie dans la gamme 6367-6578 cm-1, à l'aide d'un système diode laser à cavité étendue et de mesures à quatre températures (150 K, 180 K, 220 K et 296 K). Les spectres ont été calibrés et chaque transition ajustée sur un profil de Voigt. L'évolution du spectre en fonction de la température a permis de déterminer l'énergie de l'état initial de chaque transition. Un traitement spécifique considérant le rapport d'intensité de paires de transitions a permis de s'affranchir de la mesure précise de la pression. Une liste des transitions (2206) a été construite pour 14NH3 comportant la position, la force de raie et l'énergie de l'état initial en étendant les précédents travaux (1661 transitions). Pour l'isotopologue 15NH3, une liste position, force de raie à T= 296 K a été proposée pour la première fois dans la gamme 6367-6578 cm-1 où elle porte le nombre de transitions mesurées de 240 à 2604 transitions. / The ammonia molecule is one of the most abundant species in the atmosphere of solar planets and isotopic ratio 14N/15N is an important parameter to measure. Spectroscopy of ammonia was recently updated for 14NH3 by Sung et al [JQSRT, vol. 113, 11,1066 2012] in the range 6300-7000 cm−1 and included in HITRAN 2012 database.For the 15NH3 isotopologue, Lees et al [JMS, 251, no. 1-2, 241–251, 2008] assigned some transitions belonging to ν1 + ν3, ν1 + 2ν4 et ν3 + 2ν4 in the range 6412 to 6819 cm−1.Furthermore few line positions and strengths of transitions have been measured by Linsand al [App.Phy. B,102, 293–301, 2011.] with the goal of isotopic ratio measurement. In our work the spectroscopy of 14NH3 and 15NH3 have been extended with the help of an External Cavity Diode Tunable Laser spectrometer (ECDTLS) in the range 6369 to 6578 cm−1 and recordings at four temperatures (150K, 180K, 220K,296K).The spectra has been calibrated and each transition fitted to Voigt profile. Lines positions and strengths have been measured. The intensity ratio temperature dependence have been used to derive the energy of lower state for each transition. A specific treatment has been developed considering pairs of transitions intensities removing pressure dependence.For 14NH3 a transition list has been proposed including line position, strength and lower stateenergy. Compared with Sung et al 's work, the knowledge is increased by one third. For 15NH3 compared to the Lees et al 's work (240 lines), a line list including position and strength for 2604 transitions at room temperature has been proposed for the first time.

In the following thesis the calculation of the NMR shieldings of the nuclei within a variety of molecules in differing situations is presented. A detailed introduction to modern quantum chemical methodology is initially laid down including descriptions of the Hartree-Fock method as well as electron correlation methods such as Configuration Interaction and Many Body Perturbation Theory. This is followed by a description of the common methods used in the calculation of NMR shielding values including Gauge Invariant Atomic Orbitals (GIAO), Individual Gauge for Local Orbitals (IGLO) and Localized Orbitals/Localized Origins (LORG), as well as the existing methods which account for electron correlation effects. The theoretical background is then completed with a discussion of the methods used in optimizing molecular geometries, both to minimum energy structures or first order saddle points (i.e. equilibrium and transition structures), as well as reaction path following and a brief description of frequency calculations. The remaining chapters are then devoted to the three studies of this thesis. The first study presented is that of the investigation of the effects of hydrogen bonding upon the 19F shielding in monofluorobenzene. It is found that the 19F shielding is sensitive to hydrogen bonding, which has implications for the use of fluoroaromatic compounds as probes into large biological molecules such as proteins. The second study is into the performance of a variety of basis sets with respect to the calculation of the 19F shielding in three fluorobenzenes, as well as an investigation into the convergence of the NMR shielding toward the Hartree-Fock limit. This study shows that the moderately sized Karlsruhe-TZF and 6-311G** basis sets offer a good compromise between size and accuracy, but the investigation into the shielding convergence is less conclusive. In the final study the effect of molecular inversion and Berry pseudorotation upon both the shielding and temperature dependent shielding of five small molecules is examined. It is found that in the case of four of the five molecules studied (NH3, NF3, PF3 and PF5) the contribution to the temperature dependent shielding is low, but in the case of one of the molecules (PH3) the contribution is large.

The mixing of the hydrocarbon surfactant cetyltrimethyl ammonium bromide (CTAB) and the fluorocarbon surfactant, Zonyl-FSN-100 with the average chemical structure of C8F17C2H4 (OC2H4)9OH, is quantified. The critical micelle concentration (CMC), the size and shape of the micelles and their composition have been investigated by surface tension, fluorescence, small-angle neutron scattering (SANS), electron paramagnetic resonance (EPR), pulsed-gradient spin-eco NMR spectroscopy (PGSE-NMR), 1H-NMR and 19F-NMR. The pure surfactant aqueous solutions and the mixtures have also been studied in the presence of hydrocarbon oil (hexane) and fluorocarbon oil (perfluorohexane, PFH) in order to investigate the swollen micelle shape and structure. The surfactants mix nonideally except for a degree of ideality at some CTAB mole fraction (0.5 > αCTAB > 0.7).

Protein molecules perform a vast array of functions in living organisms and the characterisation of their structures and dynamics is a key step towards a full understanding of many biological processes. Magic angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy has emerged as a uniquely powerful technique for the extraction of such information at atomic resolution, with mounting successes founded on continual developments in methodology and technology. In this thesis, a number of new approaches for probing the structures and dynamics of proteins are presented, towards the aim of overcoming current challenges regarding sensitivity, spectral resolution and a shortage of quantitative experimental observables. A streamlined method for simultaneously obtaining long-distance homonuclear (13C-13C) and heteronuclear (15N-13C) contacts is introduced that relies on the third spinassisted recoupling (TSAR) mechanism. The experiment, dubbed "time-shared TSAR" (TSTSAR), effectively doubles the information content of spectra and reduces the required experimental time to that needed for just one of the equivalent PAR or PAINCP experiments. An approach for the quantitative study of large proteins and complexes is presented, relying on a combination of proton detection at "ultrafast" (≥55 kHz) MAS frequencies, sample deuteration and optional paramagnetic doping. This is successfully employed for the characterisation of a >300 kDa precipitated complex of the protein GB1 with full length human immunoglobulin (IgG), with only a few nanomoles of sample. Recent advances in MAS technology have enabled spinning frequencies of 100 kHz and above to be obtained. Using the dipeptide β-Asp-Ala, it is found that under such conditions, protons lines are narrowed to an extent similar to that achievable using contemporary homonuclear decoupling methods, leading to a time-efficient method for obtaining resolved spectra of small, natural-abundance molecules. Similar experiments with a GB1-IgG complex sample confirm the technology’s applicability to non-model biological systems, despite the tiny rotor volume of 0.7 μL (≤3 nanomoles of complex). 15N R1ρ relaxation rates are measured for the same complex and compared with identical measurements in crystalline GB1, allowing for a direct comparison between the slow (ns-ms) dynamics of the protein in different molecular environments. Motions on this time scale are found to be more prevalent in the complex, possibly evidence of an overall collective molecular motion. An approach for the measurement of aliphatic 13C relaxation rates in fully protonated samples is presented, based on a combination of ultrafast MAS rates and alternately labelled samples. Sample spinning at ≥80 kHz enables resolved 13Cα-1H correlations, forming a base for 13Cα relaxation experiments that are subsequently performed on crystalline [1,3-13C,15N]GB1 and analysed using a simple model-free (SMF) treatment. It is noted that without further data, this analysis is likely inadequate for an accurate description of the dynamics of the protein. The measurement of 13C’ R1ρ relaxation rates at ultrafast MAS rates is introduced as a probe of backbone protein dynamics in fully protonated samples. 13C and 15N R1 and R1ρ relaxation rates are measured in crystalline [U-13C,15N]GB1 and analysed using the SMF formalism. An examination of simulated spectral densities rationalises the apparent inconsistencies that arise from this and reveals that motions in GB1 occur on at least two time scales. A combined 15N/13C extended model-free (EMF) analysis is conducted for peptide plane motions in GB1, whereupon it is found that the addition of 13C data helps to remove fitting artefacts present in a 15N-only analysis.