Dans le but d'étudier l'impact de la taille et de la morphologie de cristaux zéolitiques sur les propriétés texturales, chimiques et diffusionnelles, une série de zéolithe beta a été étudiée.L'augmentation logique des surfaces et des volumes poreux avec la diminution de la taille des cristaux est constatée. En outre, cela implique également une baisse de l'acidité globale ainsi que de la force des sites. Toutefois, la compréhension de l'acidité complexe de ces matériaux nécessite des techniques poussées de caractérisation.L'adsorption de molécules sondes (pyridine, CO, éthylène) suivie par infrarouge ainsi que la thermodésorption d'ammoniac ont été corrélées à des réactions modèles de transformation de l'éthanol et de craquage du n-hexane. Lors de celui-ci, la morphologie des cristaux a peu d'influence et les performances sont dictées par les sites de Brønsted. La présence d'aluminiums extra-réseaux permet d'exalter la force acide des sites protoniques. Avec la transformation de l'éthanol, de meilleures activités et stabilités sont observées dans le cas de zéolithes hiérarchisées.L'étude de la diffusion par chromatographie en phase gaz à flux inversées (RF-IGC) a permis de montrer que, si les coefficients de diffusion augmentent à l'opposé de la taille des cristaux, la porosité secondaire réduit le chemin diffusionnel. De plus, les sites faibles majoritairement présents sur les matériaux hiérarchisés facilitent la désorption des molécules et sont impliqués dans les interactions latérales mesurées par RF-IGC.Ce travail apporte donc des éléments de réponse à la stabilité et aux comportements catalytiques complexes observés sur la zéolithe beta. / In order to study the impact of the size and morphology of zeolite crystals on the textural, chemical and diffusion properties, a series of beta zeolites was studied.The logical increase of the porous surfaces and volumes with the decrease in crystal size is observed. In addition, this also implies a decrease in the overall acidity as well as strength of the adsorption sites. However, understanding the complex acidity of these materials requires advanced characterization techniques.The adsorption of probe molecules (pyridine, CO, ethylene) followed by infrared as well as ammonia thermodesorption were correlated to model reactions of ethanol conversion and n-hexane cracking. In this case, the morphology of the crystals has little influence and performance is mainly dictated by the Brønsted sites. The presence of extra-framework aluminums makes it possible to exalt the acid strength of the protonic sites. With the transformation of ethanol, better activities and stabilities are observed in the case of hierarchical zeolites.The study of diffusion by the reversed-flow gas chromatography (RF-IGC) showed that if the diffusion coefficients increase as opposed to crystal size, the secondary porosity reduces the diffusion path. Moreover, the weak sites predominantly present on the hierarchized materials facilitate the desorption of the molecules and are involved in the lateral interactions measured by RF-IGC.This work thus provides elements of response to the stability and the complex catalytic behaviors observed on the beta zeolite.

This project relates to the non-classical growth of inorganic crystals with interesting morphologies that are highly desirable in industry. All crystals were synthesized via hydrothermal or solvothermal methods and their growth was studied by stopping each reaction at a range of different times, extracting the particles and analysing them using a variety of characterisation techniques. The main techniques used were scanning electron microscopy and transmission electron microscopy but other techniques, such as powder X-ray diffraction and thermal gravimetric analysis, were also employed. Decorated ZnO microstadiums were studied where ZnO nanocones coat the inner and outer columnar walls of ZnO microstadiums. It was revealed that the polymer in the synthetic solution enhanced the aggregation of nanocrystallites of precursor ions on the microstadium surfaces, which then underwent recrystallization, forming ZnO nanocones. The presence of organic agents was also found to be crucial in the non-classical growth mechanisms of CaCO₃ and RHO-ZIF crystals as the presence of charged groups on the organic molecules led to the aggregation of precursor molecules/ions, preventing classical growth. The disordered aggregates underwent surface recrystallization, forming ‘core-shell' structures where a thin layer of single crystal encased a disordered core. Over time the crystallisation extended from the surface inwards, towards the core, until true single crystals were formed. Organic molecules were also shown to play a role in the non-classical growth of 8-branched Cu₂O structures. In this case, however, studies of the electronic configuration of the main terminating facets of Cu₂O crystals revealed another key factor in their non-classical growth. Terminating hydroxyl groups on the Cu₂O surfaces could have different charges depending on the number of Cu⁺ ions they were coordinated to. The terminating {111} faces were the only ones to be coated with negatively charged hydroxyl groups, which explained the rapid growth on these surfaces as they were able to attract the positively charged metal/polymer precursor clusters. This new phenomenon was also found to be the main driving force in the rapid growth of branches in snowflake-like Fe₂O₃ crystals despite no organic agent being used. In this case, the {11-20} faces of the seed crystals had positively charged hydroxyl groups that were able to rapidly attract the negatively charged [Fe(CN) ₆]³⁻ ions in the aqueous solution.

Botulinum toxin type A (BTTA) is a polypeptide with 1296 amino acids and its used in some pathologies like hyperhidrosis and migraine, but the cosmetic area is the most important application. BTTA is produced from broth-culture synthesized by the Clostridium botulinum as a single peptide with 150 kDa. Reversed-phase liquid chromatography (RP-LC) method was developed and validated for the assessment of botulinum toxin type A in biopharmaceutical formulations. A RP-LC method was carried out on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d.), maintained at 45 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.8, and the mobile phase B was acetonitrile, run isocratically at flow rate 0.3 mL/min. Cromatographic separation was obtained with retention time of 11.4 min, and was linear over the concentration range of 0.2-100 IU/mL (r2 = 0.9999) with photodiode array (PDA) detection at 214 nm. The limits of detection and quantitation were 0.04 and 0.16 IU/mL, respectively. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.31% with bias lower than 0.80%. The method was correlated with in vivo and in vitro bioassays. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p <0.05) compared to intact molecule. The validated methods were applied for the determination of BTTA in biopharmaceutical-derived products, giving mean values of the estimated content/potencies 1.16% lower compared to the in vivo bioassay. It is concluded that represents a contribution to establish new alternatives to monitor stability, quality control and thereby assure efficacy of the biotechnology-derived product. / A toxina botulínica tipo A (BTTA) é um polipeptídio constituído de 1296 aminoácidos e é recomendada para patologias, como hiperhidrose e enxaqueca, mas é especialmente usada como cosmético. A toxina botulínica é produzida através do processo de fermentação bacteriana e após sucessivas etapas de purificação resulta em uma proteína com 150 kDa responsável pelo seu efeito biológico. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação de potência de BTTA em formulações de produtos biofarmacêuticos. No método foi utilizada coluna Zorbax 300 SB C18 (150 mm x 4,6 mm d.i.), mantida a 45 ºC. A fase móvel A foi constituída por tampão sulfato de sódio 0,05 M, pH 2,8, e a fase móvel B por acetonitrila, eluídas em vazão isocrática de 0,3 mL/min. O método utilizou detector de arranjo de diodos (DAD) a 214 nm. A separação cromatográfica foi obtida em 11,4 min, sendo linear na faixa de concentração de 0,2-100 UI/mL (r2 = 0,9999). Os limites de detecção e quantificação foram 0,04 e 0,16 UI/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,31 com bias inferior a 0,80. O método validado foi correlacionado com bioensaios in vivo e in vitro. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais apresentaram diferença significativa em relação a forma intacta (p <0,05). O método proposto foi aplicado para avaliação da potência de BTTA em formulações biofarmacêuticas, e os resultados foram comparados com o bioensaio in vivo, observando-se diferença das médias de teor/potência de 1,16% inferior para o método cromatográfico. Contribuíu-se assim para estabelecer procedimentos que aprimoram a caracterização ou o controle da qualidade, garantindo a segurança e eficácia do produto biotecnológico.

Foram desenvolvidas metodologias de cromatografia a líquido de fase reversa baseadas em injeção sequencial (SIC) e em cromatografia a líquido de alta eficiência (HPLC) para determinação de triclosan em amostras de produtos de higiene pessoal e em estudos de adsorção em argilominerais naturais e modificados, visando determinar parâmetros de adsorção de triclosan frente a alguns de seus metabólitos. A determinação de triclosan em enxaguadores bucais foi realizada por SIC com eluição isocrática usando fase móvel constituída por acetonitrila: tampão fosfato de trietilamina 70 mM pH 3,5 na proporção 70:30 (v v-1), obtendo-se limites de detecção e de quantificação de 0,22 e 0,72 mg L-1, respectivamente. Taxas de recuperação entre 96 e 98% foram obtidas da aplicação a amostras reais, sendo que os resultados obtidos pelo método proposto não apresentaram evidências de diferenças estatisticamente significativas em comparação a uma metodologia de referência baseada em HPLC com coluna empacotada. A separação de triclosan (TCS), 2-clorofenol (2-CP), 2,4-diclorofenol (2,4-DCP), 2,4,6-triclorofenol (2,4,6-TCP), 2,3,4-triclorofenol (2,3,4-TCP) e metiltriclosan (MTCS) foi estudada por SIC, obtendo-se a separação de TCS, 2-CP, 2,4-DCP e 2,4,6-TCP com duas etapas de eluição isocrática, a primeira delas com fase móvel 60:40 (v v-1) metanol: tampão acetato de amônio 20 mM (pH 5,5) seguida de eluição com fase móvel 70:30 (v v-1) metanol : tampão acetato de amônio 20 mM (pH 5,5). Nesse caso, os isômeros 2,4,6-TCP e 2,3,4-TCP coeluem. Metiltriclosan, o menos polar desses compostos, pode ser separado de TCS com etapas subseqüentes de eluição. Os métodos foram aplicados para estudar a adsorção de triclosan e seus metabólitos 2,4-DCP, 2,4,6-TCP e metiltriclosan em montmorilonita homoiônica (K+) e modificada com sal de hexadeciltrimetilamônio (HDTMA), observando-se forte adsorção de triclosan e metiltriclosan em comparação a 2-CP, 2,4-DCP e 2,4,6-TCP. A incorporação de HDTMA no argilomineral causou significativo aumento da capacidade de adsorção desses metabólitos, determinada a partir do ajuste dos dados experimentais à equação linearizada de Langmuir, observando-se que a ordem de adsorção é 2,4,6-TCP > 2,4-DCP > 2-CP / Reversed-phase liquid chromatography methodologies based on sequential injection (SIC) and high performance liquid chromatography (HPLC) have been developed for determination of triclosan in samples of personal hygiene products and in studies of adsorption on natural and modified clay minerals aiming to determine kinetic and thermodynamic parameters of adsorption of triclosan in comparison with some of its metabolites. The determination of triclosan in oral rinses with SIC was performed by isocratic elution using a mobile phase of acetonitrile : 70 mM triethylamine phosphate buffer pH 3.5 at the ratio 70:30 (v v-1), obtaining limits of detection and quantification of 0.22 and 0.72 mg L-1, respectively. Recovery rates between 96 and 98 % were obtained from the application to commercial samples, and the results obtained by the proposed method showed no evidence of statistically significant differences compared to the reference methodology based on HPLC with packed column. The separation of triclosan (TCS), 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,3,4 trichlorophenol (2,3,4-TCP) and methyltriclosan (MTCS) was studied by SIC, resulting in the separation of TCS, 2-CP, 2,4-DCP and 2,4,6-TCP with two isocratic elution steps, the first of them with a mobile phase 60:40 (v v-1) methanol: 20 mM ammonium acetate buffer (pH 5.5) followed by elution with 70:30 (v v-1) mobile phase of methanol : 20 mM ammonium acetate buffer (pH 5.5). In this case, the isomers 2,4,6-TCP and 2,3,4-TCP coeluted. Methyltriclosan, the less polar of these compounds, can be separated from TCS with subsequent elution steps. The methods were applied to study the adsorption of triclosan and its metabolites 2-CP, 2,4-DCP, 2,4,6-TCP and methyltriclosan on homoionic montmorillonite (K+) as well as in hexadecyltrimethylammonium salt (HDTMA) modified montmorillonite, noticing a stronger adsorption of triclosan and methyltriclosan compared with 2-CP, 2,4-DCP and 2,4,6-TCP. Incorporation of HDTMA in the clay mineral caused significant increase in adsorption capacity of these metabolites. This capacity was determined by fitting the experimental data to the linearized Langmuir equation. The adsorption order was 2,4,6-TCP > 2,4-DCP > 2-CP.

La conversion de la biomasse lignocellulosique en biocarburants et molécules biosourcées produit des matrices liquides complexes thermosensibles qui couvrent une large gamme de polarités et de masses moléculaires. Les outils analytiques développés dans la littérature donnent une description partielle de ces matrices oxygénées. Pour en comprendre la réactivité et mieux guider le développement des procédés de conversion, une meilleure caractérisation est nécessaire. L’objectif de cette thèse est de démontrer l’apport d’une dimension de fractionnement pertinente en amont de techniques séparatives pour accéder à la caractérisation à l’échelle moléculaire d’échantillons ex-biomasse. Une déformulation complète et structurée par familles chimiques est visée, sans perte ni modification des composés. Deux voies de fractionnement ont été investiguées : (1) fractionnement par solubilité à l’aide de l’extraction liquide-liquide (LLE) et de la chromatographie de partage centrifuge (CPC) et (2) fractionnement par taille avec la chromatographie d’exclusion stérique (SEC). Ces techniques se veulent complémentaires à une analyse par chromatographie liquide à polarité de phase inversée avec détection par spectroscopie ultraviolet-visible et spectrométrie de masse haute résolution (RPLC-UV/HRMS). Des méthodes de fractionnement LLE, CPC et SEC ont été développées sur molécules modèles afin d’identifier les mécanismes et la sélectivité chimique mis en jeu. Des cartographies 2D inédites ont ainsi été obtenues, assurant un gain important en pouvoir résolutif et une structuration nouvelle des chromatogrammes en comparaison à l’approche RPLC-UV/HRMS. Dans un second temps, le potentiel des couplages SECxRPLC-UV/HRMS et CPCxRPLC-UV/HRMS pour la description de matrices complexes a été illustré via l’étude de deux échantillons issus d’expérimentations en unités pilotes et de compositions chimiques très différentes, représentant deux voies possibles de transformation (biochimique et thermochimique) de biomasse lignocellulosique. La complémentarité entre les approches de séparation mises au point a ainsi permis de doubler le nombre de pics détectés tout en bénéficiant de l’organisation chimique des composés. Cette aide précieuse à l’identification a été renforcée par les informations structurales délivrées via les différents modes de détection, en particulier l’HRMS. La compréhension de la structuration des cartographies 2D a été présentée et discutée afin de proposer la stratégie la plus adaptée pour déformuler complètement un échantillon en s’appuyant sur la mesure de descripteurs pertinents. Enfin, l’une des approches développée dans cette thèse a été mise en œuvre pour l’isolement sélectif et l’élucidation structurale de molécules clefs au sein d’une matrice complexe à l’aide d’expériences en fragmentation MS et spectroscopie de résonance magnétique nucléaire (RMN) / The conversion of lignocellulosic biomass into biofuels and biosourced molecules produces complex thermosensitive liquid matrices which cover a wide range of polarity and molecular weight. Analytical tools developed in the literature only give a partial description of these oxygenated matrices. To understand the reactivity of these samples and optimize the development of conversion processes, a better characterization is required. The objective of this thesis is to demonstrate the interest of a relevant fractionation step prior to separation techniques to help the molecular characterization of biomass samples. The reverse engineering proposed for the sample is desired complete and chemically controlled (without loss or sample modification). Two fractionation pathways were investigated: (1) solubility fractionation with liquid-liquid extraction (LLE) and centrifugal partition chromatography (CPC) and (2) size fractionation with size exclusion chromatography (SEC). These techniques intend to be complementary to reversed-phase liquid chromatography hyphenated to ultraviolet-visible spectroscopy detection and high resolution mass spectrometry (RPLC-UV/HRMS). LLE, CPC and SEC methods were developed on model molecules to understand mechanisms involved and control the chemical selectivity. 2D contour plots were obtained, improving the resolving power and structuring chromatograms in comparison with RPLC-UV/HRMS. Then, SECxRPLC-UV/MS and CPCxRPLC-UV/MS hyphenations were applied to describe two complex samples from different substrates produced on experimental pilot units from two possible conversion pathways of lignocellulosic biomass (biochemical and thermochemical). The complementarity of separation modes allows to double the number of peaks detected, benefiting from the chemical organization of compounds. This constitute a support to identification also enhanced by multi-detection which provide additional structural information on compound detected, especially HRMS. Chemical organization in 2D contour plots were presented and discussed to propose the most adapted strategy to fully fractionate a sample based on the measurement of relevant descriptors. Finally, one of the fractionation approach developed in this thesis was used to isolate and structurally elucidate key molecules of a complex sample through MS fragmentation experiments and nuclear magnetic resonance spectroscopy (NMR)

(EN) In the first part of this work, analytical methods for determination of impurities of active pharmaceutical ingredients (API) in combined pharmaceutical dosage forms were developed and validated. Development of the methods covered both the optimization of sample preparation procedure and chromatographic conditions. The methods were validated according to International Conference on Harmonization guideline and both of them were confirmed to be able to analyze stability samples. Impurities in paracetamol, codeine phosphate hemihydrate and pitophenone hydrochloride in the presence of fourth API fenpiverinium bromide were separated by using ion-pair reversed phase chromatography with gradient elution. Symmetry C18, 250 x 4,6 mm, 5 µm heated to 35 řC was used as a separation column. A diode array detector was used. The detection wavelengths were set as follows: 220 nm for paracetamol impurity K, 245 nm for paracetamol and its other impurities and 285 nm for codeine, pitophenone and their impurities. Impurities in valsartan, amlodipine besylate and hydrochlorothiazide were separated by reversed phase UHPLC method with gradient elution. Chromatographic column Zorbax Eclipse C8 RRHD, 100 x 3,0 mm, 1,8 µm heated to 30 řC and spectrophotometric detection were used. The detection wavelengths were set as...

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Rigid poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] (Poly(HEA-co-PEGDA) monoliths were synthesized inside 75-µm i.d. capillaries by one-step UV-initiated copolymerization using methanol and ethyl ether as porogens. The optimized monolithic column was evaluated for hydrophobic interaction chromatography (HIC) of standard proteins. Six proteins were separated within 20 min with high resolution using a 20 min elution gradient, resulting in a peak capacity of 54. The performance of this monolithic column for HIC was comparable or superior to the performance of columns packed with small particles. Monoliths synthesized solely from PEGDA were also found to show excellent performance in HIC of proteins. Continuing efforts showed that rigid monoliths could be synthesized from PEGDA or poly(ethylene glycol) dimethacrylates (PEGDMA) containing different ethylene glycol chain lengths for HIC of proteins. Effects of PEG chain length, bi-porogen ratio and reaction temperature on monolith morphology and back pressure were investigated. Monoliths prepared from PEGDA 258 were found to provide the best chromatographic performance with respect to peak capacity and resolution. An optimized PEGDA 258 monolithic column was able to separate proteins using a 20-min elution gradient with a peak capacity of 62. The preparation of these in situ polymerized single-monomer monolithic columns was highly reproducible. The single-monomer synthesis approach clearly improves column-to-column reproducibility.The highly crosslinked monolith networks resulting from single crosslinking monomers were found to enhance the surface area of the monolith and concentrations of mesopores. Thus, monolithic columns were developed from four additional crosslinking monomers, i.e., bisphenol A dimethacrylate (BADMA), bisphenol A ethoxylate diacrylate (BAEDA, EO/phenol = 2 or 4) and pentaerythritol diacrylate monostearate (PDAM) for RPLC of small molecules. Gradient elution of alkyl benzenes and alkyl parabens was achieved with high resolution using all monolithic columns. Porogen selection for the BADMA and PDAM was investigated in detail with the intention of obtaining data that could possiblly lead to a rational method for porogen selection.

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