Os catalisadores eletroquímicos binários de PtSn/C, PdSn/C e PtPd/C foram sintetizados em diferentes proporções pelo método da redução via borohidreto, posteriormente estes foram caracterizados por microscopia eletrônica de transmissão, difração de raios X, espectroscopia no infravermelho por transformada de Fourier (PtSn/C e PdSn/C) e energia dispersiva de raios X. As atividades eletroquímicas dos diferentes materiais preparados foram avaliadas por intermédio de voltametria cíclica, cronoamperometria e curvas de polarização em célula a combustível alimentada diretamente por etileno glicol em eletrólito alcalino. As curvas de densidade de potência indicaram que os catalisadores eletroquímicos contendo Sn e Pd são mais ativos para a reação de oxidação do etileno glicol, especialmente a composição 70%:30% - relação molar entre os metais suportados em carbono - dos catalisadores PtSn/C, PdSn/C e PtPd/C todos superando as medidas de potência do Pt/C. Este resultado indica que a adição de Sn e Pd favorece a oxidação do etileno glicol em meio alcalino. O melhor desempenho observado para os catalisadores eletroquímicos PtSn/C, PdSn/C e PtPd/C (70%:30%) poderia estar associado à sua maior seletividade quanto a formação de oxalato, ou seja , a formação deste produto resulta em um maior número de elétrons, por consequência em maiores valores de corrente. / Binary electrochemical catalysts PtSn/C, PdSn/C and PtPd/C were synthesized in different proportions by the method of reduction via borohydride. These were characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy (PtSn/C and PdSn/C) and energy dispersive X-ray. The electrochemical activities of the different materials prepared were evaluated by cyclic voltammetry, chronoamperometry and polarization curves for fuel cell powered directly by ethylene glycol in an alkaline electrolyte. Power density curves indicated that the electrochemical catalysts Sn-containing or Pd-containing are more active for ethylene glycol oxidation reaction, particularly the (70%:30%) composition of PtSn/C, PdSn/C and PtPd/C, all of them exceeding power measurements of Pt/C. These results indicate that the addition of Sn and Pd promotes the oxidation of ethylene glycol in an alkaline medium and this improved performance may be associated with a higher selectivity for the formation of oxalate that results in a larger number of electrons, consequently enhancing the current values.

A adição de corantes fluorescentes a adesivos odontológicos possibilita a investigação da distribuição espacial desses materiais na interface dente-restauração, utilizando-se a microscopia confocal de varredura a laser (MCVL). A literatura indica falta de padronização na aplicação de agentes fluorescentes com tal finalidade. Esse estudo sistematizou estratégias para a adição de rodamina B (RB) e fluoresceína sódica (FS) a um sistema adesivo convencional de três passos, Adper Scotchbond Multi-Purpose (MP), e um autocondicionante de dois passos, Clearfil SE Bond (SE), considerados padrão-ouro na Odontologia. Os objetivos principais foram (a) determinar a menor faixa de concentrações de RB e FS necessária para produzir imagens satisfatórias da interface dentina-adesivo e (b) avaliar o efeito da adição desses corantes sobre algumas propriedades das resinas. Os adesivos foram marcados com RB ou FS em concentrações decrescentes (0,5, 0,1, 0,02 e 0,004 mg/mL) por meio de um método de dispersão semidireto. O comportamento fotofísico/ fluorescente dos adesivos marcados foi investigado por espectroscopia de fotoluminescência e MCVL. Paralelamente, avaliaram-se os adesivos quanto ao grau de conversão (GC) e ao ângulo de contato (AC). Tanto os resultados de GC como os de AC foram submetidos à análise de variância com dois fatores (adesivo e tratamento) com = 0,05, seguida de teste post-hoc de Tukey. Os máximos comprimentos de onda de emissão e de excitação da RB e da FS foram influenciados pelo meio polimérico e pela concentração de corante de modo geral. A MCVL preliminar de amostras de adesivo polimerizado, realizada sob condições experimentais padronizadas, mostrou que o comportamento fluorescente da RB em MP e SE foi muito semelhante na mesma concentração de corante, mas o mesmo não pôde ser dito do comportamento da FS, que foi notavelmente inferior no adesivo autocondicionante, SE, na concentração mais alta. Em dentina, os adesivos preparados com RB nas concentrações-alvo de 0,1 e 0,02 mg/mL apresentaram fluorescência ótima; já aqueles preparados com 0,004 mg/mL produziram fraco sinal. Adesivos preparados com FS a 0,5 mg/mL apresentaram ótima fluorescência na interface de adesão, enquanto que concentração menor desse corante não produziu sinal suficiente. Padrões morfológicos aparentemente atípicos foram observados na interface de adesão, quando da associação do adesivo SE com o corante FS. A adição de RB e FS nas quatro concentrações indicadas aos adesivos MP e SE não afetou o GC nem o AC em comparação com os grupos de controle correspondentes. Em suma, a RB mostra-se um corante mais versátil que a FS na avaliação morfológica das interfaces dentina-MP e dentina-SE via MCVL. A menor faixa de concentrações de RB nos adesivos MP e SE, na qual é possível produzir imagens satisfatórias das interfaces, situa-se entre 0,10,02 mg/mL. Já o corante FS deve ser adicionado a esses adesivos a pelo menos 0,5 mg/mL para produzir níveis de fluorescência satisfatórios na interface de adesão. A não ocorrência de efeitos deletérios sobre a polimerização e a molhabilidade das resinas estabelece uma margem de segurança para a incorporação desses agentes fluorescentes (em concentração 0,5 mg/mL) nesses sistemas monoméricos. / The addition of fluorescent dyes to dental adhesives makes it possible to investigate the spatial distribution of such resin-based materials in the tooth-restoration interface, using confocal laser scanning microscopy (CLSM). Literature indicates a lack of standardization on the application of fluorescent agents for this purpose. This work presents strategies for adding rhodamine B (RB) and fluorescein sodium salt (FS) to a three-step etch-and-rinse adhesive system, Adper Scotchbond Multi-Purpose (MP), and a two-step self-etching one, Clearfil SE Bond (SE), both regarded as \"gold standard\" in restorative dentistry. The main objectives were (a) to determine the lowest range of RB and FS concentrations required to produce suitable images of the dentin-adhesive interface via CLSM and (b) to investigate potential effects of addition of these dyes on some resin properties. The adhesives were labeled with RB or FS at decreasing concentrations (0.5, 0.1, 0.02 and 0.004 mg/mL) by means of a semi-direct dispersion method. The photophysical/fluorescent behavior of the labeled resins was investigated by photoluminescence spectroscopy and by CLSM. The adhesives were also investigated with regards to the degree of conversion (DC) and contact angle (CA). A two-way ANOVA of adhesive and treatment was conducted on DC and CA separately, followed by Tukeys test. The maximum emission and excitation wavelengths of RB and FS were influenced by the host polymer and the dye concentration in general. The preliminary CLSM of cured adhesive samples, performed with standardized settings, showed that the fluorescent behavior of RB in MP and SE was very similar in the same dye concentration, unlike the behavior of FS, which was lower in the self-etching adhesive for the highest dye concentration. In dentin, the adhesives prepared with RB at the target concentrations of 0.1 and 0.02 mg/mL presented optimal fluorescence; those with 0.004 mg/mL produced poor signal. Adhesives prepared with FS at 0.5 mg/mL presented optimal fluorescence at the bonding interface, whereas lower concentrations of FS did not produce sufficient signal. Atypical morphological features were observed at the bonding interface, when adhesive SE was used with FS. The addition of RB and FS at the four decreasing concentrations to adhesives MP and SE did not affect DC or CA compared to the corresponding controls. In short, RB is more versatile than FS for the morphological characterization of dentin-MP and dentin-SE interfaces via MCVL. The lowest range of RB concentrations in adhesives MP and SE that can produce suitable images of the bonding interface lies between 0.10.02 mg/mL. The dye FS should be added to these adhesives at 0.5 mg/mL at least to produce satisfactory fluorescence levels at the bonding interface. Since negative effects on polymerization and wettability of the resins were not observed, the use of RB and FS (in concentration 0.5 mg/mL) together with MP and SE should be reliable in terms of resin properties.

Liu, Haijing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 112-116). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of contents --- p.vi / List of figures --- p.xii / List of tables --- p.xv / List of abbreviations --- p.xvi / Chapter / Chapter 1 --- Introduction / Chapter 1.1 --- Fermentation --- p.1 / Chapter 1.1.1 --- Biopharmaceutical production --- p.1 / Chapter 1.1.2 --- History of fermentation --- p.1 / Chapter 1.1.3 --- Fermentation Technology --- p.2 / Chapter 1.1.3.1 --- Fermentation process --- p.2 / Chapter 1.1.3.2 --- Fermenter --- p.4 / Chapter "1,1.3.3" --- Mode of fermentation process --- p.5 / Chapter 1.1.3.4 --- Bacterial growth --- p.6 / Chapter 1.1.4 --- High cell density fermentation for plasmid DNA production --- p.7 / Chapter 1.1.4.1 --- Porcine growth hormone releasing factor (pGRF) --- p.8 / Chapter 1.1.4.2 --- H5N1 avian influenza viruses DNA vaccine --- p.9 / Chapter 1.1.4.3 --- Fed-batch high-cell-density fermentation --- p.9 / Chapter 1.1.5 --- Fermentation process monitoring --- p.11 / Chapter 1.2 --- Near-infrared spectroscopy (NIRS) --- p.12 / Chapter 1.2.1 --- Basic near-infrared spectroscopy --- p.13 / Chapter 1.2.1.1 --- Rationale of near-infrared spectroscopy --- p.13 / Chapter 1.2.1.2 --- NIR spectra acquisition --- p.16 / Chapter 1.2.1.3 --- Interpretation of NIR spectra --- p.18 / Chapter 1.2.2 --- Multivariate calibration --- p.20 / Chapter 1.2.2.1 --- Why multivariate calibration --- p.22 / Chapter 1.2.2.2 --- The problem of collinearity --- p.25 / Chapter 1.2.2.3 --- Spectral range selection --- p.26 / Chapter 1.2.2.4 --- Signal optimization --- p.26 / Chapter 1.2.2.5 --- Spectral pretreatment --- p.27 / Chapter 1.2.2.6 --- Parameter selection --- p.28 / Chapter 1.2.3 --- Applications of NIRS in high cell density E. coli fermentation --- p.31 / Chapter 1.2.4 --- Adaptive calibration strategy --- p.33 / Chapter 1.3 --- Aims of study --- p.34 / Chapter 2 --- Materials and methods / Chapter 2.1 --- High cell density fermentation of plasmid DNA --- p.35 / Chapter 2.1.1 --- Fermentation system --- p.35 / Chapter 2.1.2 --- pGRF fermentation --- p.38 / Chapter 2.1.2.1 --- Prepare seed flask --- p.38 / Chapter 2.1.2.2 --- Reagents for bacterial culture by fermenter --- p.38 / Chapter 2.1.2.2.1 --- LB based complex fermentation medium --- p.38 / Chapter 2.1.2.2.2 --- Batch fermentation medium --- p.39 / Chapter 2.1.2.2.3 --- Feeding medium-1 --- p.41 / Chapter 2.1.2.2.4 --- Base feed --- p.41 / Chapter 2.1.2.3 --- Fermentation methods --- p.42 / Chapter 2.1.2.3.1 --- Fermenter set-up --- p.42 / Chapter 2.1.2.3.2 --- Inoculate seed culture into the fermenter --- p.43 / Chapter 2.1.2.3.3 --- Transfer the feeding medium --- p.44 / Chapter 2.1.2.3.4 --- Heat induction --- p.44 / Chapter 2.1.2.3.5 --- Fermentation harvest --- p.44 / Chapter 2.1.2.4 --- Scale-up fermentation and large scale production 45 / Chapter 2.1.3 --- H5N1 fermentation --- p.45 / Chapter 2.1.4 --- Reference analytical testing --- p.45 / Chapter 2.1.4.1 --- Optical density --- p.45 / Chapter 2.1.4.2 --- Cell dry weight --- p.45 / Chapter 2.1.4.3 --- Specific plasmid DNA yield --- p.46 / Chapter 2.1.4.3.1 --- Plasmid DNA isolation --- p.46 / Chapter 2.1.4.3.2 --- Plasmid DNA measurement --- p.46 / Chapter 2.1.4.3.3 --- Plasmid DNA identification --- p.46 / Chapter 2.1.4.4 --- Analysis of glycerol and acetate --- p.46 / Chapter 2.1.4.4.1 --- Standard samples --- p.46 / Chapter 2.1.4.4.2 --- Enzymatic test kits --- p.47 / Chapter 2.1.4.4.3 --- Automatic biochemistry analyzer --- p.47 / Chapter 2.1.5 --- Summary of all fermentation batches --- p.48 / Chapter 2.2 --- Full factorial design and semi-synthetic samples --- p.49 / Chapter 2.2.1 --- Adaptive calibration samples --- p.49 / Chapter 2.2.1.1 --- Matrix 1 calibration samples --- p.49 / Chapter 2.2.1.2 --- Matrix 2 calibration samples --- p.50 / Chapter 2.2.1.3 --- Matrix 3 calibration samples --- p.50 / Chapter 2.2.2 --- Summary of all samples --- p.51 / Chapter 2.3 --- NIR sample presentation and spectral acquisition --- p.52 / Chapter 2.3.1 --- NIR spectrophotometers --- p.52 / Chapter 2.3.2 --- Cuvettes for transmittance spectral acquisition --- p.53 / Chapter 2.3.3 --- Bottles for reflectance spectral acquisition --- p.54 / Chapter 2.3.4 --- Spectral acquisition --- p.55 / Chapter 2.3.4.1 --- Transmittance --- p.55 / Chapter 2.3.4.2 --- Reflectance --- p.55 / Chapter 2.4 --- Multivariate calibration and validation --- p.56 / Chapter 2.4.1 --- Spectral preprocessing --- p.56 / Chapter 2.4.2 --- Multivariate calibration --- p.57 / Chapter 2.4.3 --- Model validation --- p.57 / Chapter 3 --- Results and discussion / Chapter 3.1 --- Sample presentation and NIR spectrum --- p.59 / Chapter 3.1.1 --- Transmission measurement --- p.60 / Chapter 3.1.2 --- Reflectance measurement --- p.63 / Chapter 3.1.3 --- Spectral responses and pre-treatment method --- p.64 / Chapter 3.1.4 --- Design of experiments for calibration sample preparation --- p.67 / Chapter 3.1.5 --- Summary --- p.68 / Chapter 3.2 --- Adaptive calibration --- p.69 / Chapter 3.2.1 --- Selection of Multivariate calibration model --- p.70 / Chapter 3.2.1.1 --- Matrix 1 calibration models --- p.70 / Chapter 3.2.1.2 --- Matrix 2 calibration models --- p.74 / Chapter 3.2.1.3 --- Matrix 3 calibration models --- p.76 / Chapter 3.2.1.4 --- Summary --- p.78 / Chapter 3.2.2 --- Model validation --- p.79 / Chapter 3.2.2.1 --- Performance of Reference analytical methods --- p.79 / Chapter 3.2.2.1.1 --- Enzymatic test kits --- p.79 / Chapter 3.2.2.1.2 --- Automatic biochemistry anaylyzer (Bioprofile) --- p.81 / Chapter 3.2.2.1.3 --- Summary --- p.83 / Chapter 3.2.2.2 --- Model validation using external test samples --- p.83 / Chapter 3.2.2.2.1 --- Matrix 1 models --- p.83 / Chapter 3.2.2.2.2 --- Matrix 2 models --- p.87 / Chapter 3.2.2.2.3 --- Matrix 3 models --- p.89 / Chapter 3.2.2.2.4 --- Overall NIR measurement errors --- p.90 / Chapter 3.2.2.2.5 --- Summary --- p.91 / Chapter 3.3 --- Use of calibrated NIRS in at-line monitoring and control of fermentation --- p.93 / Chapter 3.3.1 --- Analysis of small-scale fermentation --- p.94 / Chapter 3.3.1.1 --- pGRF plasmid DNA production --- p.94 / Chapter 3.3.1.2 --- H5N1 plasmid DNA production --- p.95 / Chapter 3.3.1.3 --- Summary --- p.97 / Chapter 3.3.2 --- Analysis of large scale fermentation --- p.97 / Chapter 3.3.2.1 --- 30 L clinical production of H5N1 plasmid DNA --- p.97 / Chapter 3.3.2.2 --- 80 L scale-up production of H5N1 plasmid DNA --- p.99 / Chapter 3.3.2.3 --- Summary --- p.100 / Chapter 3.3.3 --- Effective control of fermentation production using at-line NIR analysis --- p.101 / Chapter 3.3.3.1 --- At-line monitoring of Batch 11 --- p.101 / Chapter 3.3.3.2 --- At-line monitoring of Batch 12 X --- p.102 / Chapter 3.3.3.3 --- Summary --- p.104 / Chapter 3.4 --- General discussion and conclusion --- p.105 / Chapter 3.5 --- Future prospects --- p.108 / References --- p.112 / Appendix 1 --- p.117 / Appendix 2 --- p.118 / Appendix 3 --- p.123 / Appendix 4 --- p.131 / Appendix 5 --- p.134

A classificação e caracterização do petróleo cru em tempo real é um dos mais importantes desafios atuais da indústria petroquímica e agencias ambientais. Um método rápido e econômico antes da destilação do petróleo iria a fornecer informações químicas de grande importância para o ajuste em tempo real dos parâmetros críticos de uma refinaria, permitindo a otimização do processo, resultando em benefícios econômicos e ambientais. Uma variedade de técnicas espectroscópicas está sendo utilizadas nas ultima décadas para as analises, classificação e caracterização de petróleo cru nos campos de perfuração, para as análises dos produtos petrolíferos e para detecção de derrames. As vantagens do uso desta técnica incluem uma rápida resposta, requerimento mínimo de preparação da amostra e equipamentos relativamente econômicos. As técnicas espectroscópicas envolvidas na tese (NIR) mostraram o maior potencial no campo. No entanto, os estudos realizados com todas estas técnicas não foram enfocados em aplicações em linha. O NIR é tradicionalmente a técnica espectroscópica mais utilizada para a análise de petróleo cru. Mas os problemas típicos que esta técnica apresenta são sobreposição das diferentes bandas de absorção e saturação do sinal devido à alta absorbância característico do petróleo cru. A Fluorescência é um fenômeno mais complexo do que a absorção (NIR) e efeitos como quenchinge transferência de energia têm que ser consideradas. No entanto, a espectroscopia de fluorescência pode fornecer sinais bidimensionais com muito mais informação do que uma emissão unidimensional do espectro de absorção. Enquanto a espectrometria de massa, a técnica MALDI TOF faz possível a ionização de macromoléculas que são difíceis de serem ionizadas por não ser facilmente decompostas. Neste trabalho, as vantagens e limitações das técnicas disponíveis são analisadas e o potencial do analises remoto em linha do petróleo cru é avaliado. A pesar da boa analise e classificação obtida com algumas técnicas, nem sempre é possível integrá-los em um sistema em linha quando uma preparação previa da amostra é necessária ou quando o sistema é demasiado complexo. Para a extração da informação química contida nos espectros foram utilizados métodos matemáticos e estatísticos (quimiométricos) nos espectros: Mínimos Quadrados Parciais (PLS). / Online remote characterization and real-time classification of crude petroleum is one of the most important current challenges faced by the petrochemical industry and environmental agencies. A rapid and inexpensive method for the remote analysis and classification of petroleum prior to distillation of the crude would provide chemical information of great importance for real time adjustment of the critical parameters of a refinery, permitting an optimization of the process and resulting in economic and environmental benefits. A variety of spectroscopic techniques have been used over the last few decades for the analysis, characterization and classification of crude oil in drilling fields, for the analysis of petroleum products and for the detection of oil-spills. The advantages of using these techniques include rapid response, the requirement of minimal sample preparation and relatively inexpensive equipment costs. The spectroscopic techniques involved in the thesis (NIR, fluorescence and mass spectrometry) showed the greatest potential in the field. Nevertheless, the studies carried out with these techniques typically do not focus on online applications. NIR is traditionally the spectroscopic techniques most used for crude petroleum analysis. Overlap of the different absorption bands and saturation of the signal due to the high absorbance of crude petroleum are typical problems encountered with this technique. Fluorescence is a more complex phenomenon than absorption (NIR) and effects such as quenching and energy transfer have to be considered. However, fluorescence spectroscopy can provide two-dimensional signals with much more information than a one-dimensional emission or absorption spectrum. While mass spectrometry, MALDI TOF technique makes possible the ionization of macromolecules that are difficult to be ionized not be easily broken. In this work, the advantages and limitations of the available techniques are analyzed and the potential for remote on-line analysis of crude petroleum is evaluated. In spite of the good analysis and classification obtained with some of the techniques, it is not always possible to integrate them into a remote on-line analytical system when some prior sample preparation is required or when the system is too complex. For the extraction of chemical information contained in the spectra were used mathematical and statistical (chemometric) in the spectra: Partial Least Squares (PLS).

A zidovudina, conhecida como AZT, é um inibidor da transcriptase reversa, enquanto que a lamivudina é um fármaco antirretroviral que atua na inibição da síntese de ácidos nucléicos. Estes são dois dos 21 fármacos componentes dos medicamentos distribuídas pelo Ministério da Saúde Brasileiro em programas de combate a Síndrome da imunodeficiência Adquirida (Acquired Immunodeficiency Syndrome - AIDS), configurando-se assim uma grande demanda de produção de medicamentos com estes fármacos. Programas de Tecnologia Analítica em Processo (Process Analytical techology - PAT), embasadas por avanços nos guias internacionais da Conferência Internacional sobre a Harmonização dos Requerimentos Técnicos para o Registro de Produtos Farmacêuticos para o uso Humano (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use - ICH) e pela agência norte-americana para a Administração de Alimentos e Medicamentos (Food and Drugs Administration - FDA), estão ganhando força como alternativas para aumentar a eficiência e a segurança na produção de medicamentos, tanto para aqueles já em processo produtivo como também para medicamentos em fase de desenvolvimento. Estes últimos são denominados desenvolvimento em programas de Qualidade por Design (QbD). Métodos de quantificação por espectroscopia (NIR, MID, RAMAM, entre outras) são reconhecidos como ferramentas para a PAT. Neste contexto propôs-se comparar objetivamente o método tradicional de quantificação destes dois fármacos frente a um método de quantificação desenvolvido utilizando-se a espectroscopia no infravermelho médio (MID). Prepararam-se assim 41 amostras de calibração e 23 amostras de validação, compostas por misturas de zidovudina, lamivudina e placebo (qs) em escala laboratorial, na faixa de 80 a 120% da concentração nominal de uma associação comercial dos dois fármacos. As concentrações de referência de todas as preparações foram determinadas empregando-se o método de referência por Cromatografia Líquida de Alta Eficiência (CLAE) da Farmacopeia Americana (United States Pharmacopeia - USP). Subsequentemente, obtiveram-se cinco espectros no infravermelho de cada uma das preparações, na faixa de 450 a 4000 cm-1. Os espectros foram então pré-processados e utilizados para a construção de um modelo de calibração multivariado por PLS (mínimo quadrados parciais), de acordo com a ASTM E1655-05. Adicionalmente, o método de CLAE foi transferido para um método de UPLC de acordo com o Capitulo Geral descrito no volume 37(3) do Fórum da USP (United States Pharmacopeia). O desempenho do método MID foi então comparado com o método tradicional, bem como com o novo método de quantificação por UPLC. Foram definidaLs assim regiões de confiança para embasar a utilização dos métodos desenvolvidos. O método de quantificação por MID apresentou uma grande variabilidade enquanto que o método por UPLC foi totalmente comparável com o método tradicional, reduzindo o tempo de corrida de 60 minutos para 12.55 minutos. / Zidovudine, also known as AZT is a reverse transcriptase inhibitor, whereas lamivudine is an antiretroviral drug that acts on the inhibition of nucleic acid synthesis. These are two of the 21 active ingredients components of medicines distributed by Brazilian Health Ministry in programs against the Acquired Immunodeficiency Syndrome (AIDS), becoming thus a great demand for production of these two drugs. Process Analytical Technology (PAT) programs, supported by advances in international guides from the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and by the FDA (Food and Drugs Administration), are gaining momentum as alternatives to increase efficiency and safety in the production of medicines, both for those medicines already in the production process as well as to those medicines under development. The latter are called Quality by Design (QbD) programs. Spectroscopy quantification methodologies methodologies (NIR, MID, Ramam, among others) are recognized as PAT tools. In this context it was proposed to compare objectively the traditional method for quantification of these two drugs against a quantification method developed using the MID (middle infrared spectroscopy). Thus 41 calibration and 23 validation samples, comprising of laboratorial scale mixtures of lamivudine, zidovudine and placebo (qs), were prepared in the range equivalent to 80 to 120% of the nominal concentration of the commercial tablets product. The concentrations of all calibration and validation samples were determined using the HPLC reference method of USP (United States Pharmacopeia). Subsequently, there were obtained five infrared spectra of each of the preparations in the range 450-4000 cm-1. The spectra were then pre-processed and used to build a multivariate calibration model for PLS (Partial Least Squares) according to ASTM E1655-05. Additionally, the HPLC method was transferred to a UPLC method according to General Chapter described in volume 37 (3) Forum USP (United States Pharmacopeia). The performance of the method MID was then compared with the traditional method and with the new method of quantification by UPLC. Confidence regions were built to support the use of the methods developed. The MID quantification method presented considerable variability, while the method the UPLC method was fully comparable to the traditional method. Another advantage of the UPLC method was the reduction of running time from 60 minutes to 12:55 minutes.

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.

Chemometrics is a discipline of chemistry which uses mathematical and statistical tools to help in the extraction of chemical information from measured data. With the assistance of chemometric methods, infrared (IR) spectroscopy has become a widely applied quantitative analysis tool. This dissertation explores two challenging applications of IR spectroscopy facilitated by chemometric methods: (1) passive Fourier transform (FT) remote sensing and (2) process monitoring by near-infrared (NIR) spectroscopy. Passive FT-IR remote sensing offers a measurement method to detect gaseous species in the outdoor environment. Two major obstacles limit the application of this method in quantitative analysis: (1) the effect of both temperature and concentration on the measured spectral intensities and (2) the difficulty and cost of collecting reference data for use in calibration. To address these problems, a quantitative analysis protocol was designed based on the use of a radiance model to develop synthetic calibration data. The synthetic data served as the input to partial least-squares (PLS) regression in order to construct models for use in estimating ethanol and methanol concentrations. The methodology was tested with both laboratory and field remote sensing data. Near-infrared spectroscopy has attracted significant interest in process monitoring because of the simplicity in sample preparation and the compatibility with aqueous solutions. For use in process monitoring, the need exists for robust calibrations. A challenge in the NIR region is that weak, broad and highly overlapped spectral bands make it difficult to extract useful chemical information from measured spectra. In this case, signal processing methods can be helpful in removing unwanted signals and thereby uncovering useful information. When applying signal processing as a spectral preprocessing tool and regression analysis for building a quantitative calibration model, optimizing the parameters that specify the details of the methods is crucial. In this research, particle swarm optimization, a population-based optimization method was applied. Digital filtering and wavelet processing methods were evaluated for their utility as spectral preprocessing tools. Both a pump-controlled flowing system and bioreactor runs involving the yeast, Pichia pastoris, were studied in this work. In investigating the bioreactor runs, insufficient reference data resulted in difficulties in employing the PLS calibration method. Instead, the augmented classical least-squares modeling technique was applied since it requires only pure-component or composite spectra of the analyte and background matrix rather than a large set of mixture samples of known analyte concentration.

Electron transfer is one of the fundamental process occurring in many chemical reactions. Electron transfer process has been under intensive study for many applications, for example artificial photosynthesis, where electrons from photo-excited chromophore molecules are harnessed to produce solar fuels in various forms. Transition metal complexes, such as ruthenium and rhenium complexes, play an important role in the continuing development of artificial photosynthetic devices. The electron transfer process in chromophores involving transition metal complexes often occurs on an ultrafast time scale from sub-ps to ns. To resolve such dynamics, ultrafast spectroscopic techniques are required. A variety of ultrafast techniques, such as time-resolved infrared spectroscopy and multi-pulse transient absorption spectroscopy, were used in this study to unravel the excited state electron transfer dynamics in a series of Re(I) complexes. Transition metal complexes often feature excited states that involve only partial electron transfer between the electron donating and accepting ligands, even for ligands with strong electron donating and accepting properties. It is often difficult to design a compact complex feature a full electron transfer excited state. Therefore, part of the work presented in this thesis was dedicated to the study of the electron transfer extent in the excited states of a series of [Re(N,N)(CO)3L]+ compounds, where N,N stands for electron accepting and L stands for electron donating ligands. By carefully designing the structure and redox properties of both the electron donor and acceptor, we demonstrated that essentially a full-electron charge transfer excited state can be prepared, while the designed Re(I) complex is still compact. To further extend the understanding of the electron transfer in transition metal complexes, modulation of the electron transfer rate in a compact Re(I) complex was studied. By perturbing the electron transfer process with a femtosecond mid-IR pulse, we showed that a 28% increase of the electron transfer rate was achieved. This study demonstrated the possibility of using a small energy mid-IR quanta to change the energy conversion process in a chromophore. Vibrational energy transfer in molecules is another important process in nature. Detailed understanding of the vibrational energy transfer on a molecule level is fundamentally important and essential for the development of molecular optical devices. It was recently discovered that the transport of vibrational energy in molecules can be fast and efficient due to its ballistic character. To understand the mechanism of the ballistic energy transport, experiments with several series of oligomers were performed using a relaxation-assisted two-dimensional infrared method. The energy transport speed was found to be dependent on transport initiation method and the transport pathways for different cases of initiation were identified. Detailed analysis on the chain band structure, group velocity and vibrational relaxation dynamics is presented. / acase@tulane.edu

The aim of this work was to use Fourier Transform Infrared Spectroscopy to characterize and identify bacteria of particular significance to the food industry. FT-IR spectroscopy is a rapid technique that can be applied to all groups of bacteria. The two objectives were to determine a suitable sampling procedure to record a spectrum and to determine a suitable statistical technique to identify characteristic regions of the spectrum associated with the genus and, potentially, the species. Pure cultures of bacteria were grown in broth, suspended in saline and dried to produce a film on a halide salt crystal. These films were then used to produce FT-IR spectra. In total, 80 spectra were recorded from seven genera, seven species and four strains of bacteria. Some of the spectra were considered to be too low in intensity to be included in statistical analysis. Data points from three specific windows of the remaining spectra were used to determine spectral distances between spectra. These spectral distances were used to perform cluster analysis using Ward's method, the Complete Linkage method and the Centroid method. The statistical analysis created successful clusters for several of the species used but was inconclusive overall in being able to distinguish between spectra at the genus, species and strain level. This may be due to inconsistent growth of bacteria and insufficient manipulation of the data. This study has shown the potential for FT-IR spectroscopy to be used to identify bacteria with significance for food but further development is needed to reproduce the consistent results demonstrated in current literature.

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