OIRAM FILHO, F. Isolamento em escala preparativa de ácidos anacárdicos provenientes do líquido da casca da castanha do caju (LCC). 2017. 64 f. Dissertação (Mestrado em Engenharia Química)-Centro de Tecnologia, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by pgeq ufc (pgeq@ufc.br) on 2017-10-26T17:27:44Z No. of bitstreams: 1 2017_dis_foiramfilho.pdf: 2261095 bytes, checksum: 44ef008226fa11ae68fc6ea4d6a09d08 (MD5) / Rejected by Marlene Sousa (mmarlene@ufc.br), reason: Prezado Oiram, Existe uma orientação para que normalizemos as dissertações e teses da UFC, em suas paginas pré-textuais e lista de referencias, pelas regras da ABNT. Por esse motivo, sugerimos consultar o modelo de template, para ajudá-lo nesta tarefa, disponível em: http://www.biblioteca.ufc.br/educacao-de-usuarios/templates/ Vamos agora as correções sempre de acordo com o template: 1. 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No. of bitstreams: 1 2017_dis_foiramfilho.pdf: 2261032 bytes, checksum: 269a0f76af7bd7648c411c4a819df137 (MD5) Previous issue date: 2017-08-29 / The shells of cashew nut are raw material for obtaining cashew nut shell liquid (CNSL) , a brownish viscous substance with important applications in the chemical and pharmaceutical industry . Several compounds are found in CNSL, such as cardol, cardanol, methyl - cardol and anacardic acids (AnAc). The A nAc are compounds considered as phenolic lipids, due they have a long carbonic chain with different degrees of instauration. Studies in several sc ientific areas show positive results of A nAc for the treatment and prevention of some diseases and their vectors. Therefore, there is a need for more specific studies to monitor, quantify and isolate these compounds in the CNSL. In this work a chromatograp hic separation method was developed, able to isolate the A nAc present in the CNSL . A fractionation of the CNSL was also performed to obtain a fraction containing only A n A c . Samples of CN SL were solubilized in methanol, injected into a high performance liquid chromatograph coupled a diode array detector (HPLC – DAD), monitored at 280 nm, equipped with C 18 column , using as the mobile phase acetonitrile, H 2 O, acetic acid in isocratic mode . The chromatographic method developed showed adequate selectivity to be able to separate anacardic acids triene (15:3 ) , diene ( 15:2 ) and monoene ( 15:1) efficiently presents in the CNSL at the retention times 7.68, 11.09 and 17.85 min, respectively. For the preparative scale chromatographic conditions was used a HPLC – DAD mon itored at wavelength 280 nm, equipped with a C 18 column , using as the mobile phase methanol, H 2 O, acetic acid in isocratic mode . The method was mathematically adjusted to obtaining a greater similarity with the analytical method. The calibration curve w ith linear interval (50 to 1000 μg.mL - 1 ) and validation of the analytical method were made from the external anacardic acid standard (15: 3). The results obtained for method validation were satisfactory for intra - day (CV = 0.60 %) and inter - day (CV = 0.67 %) precision, linearity (y = 2670.8x - 26949, r 2 > 0.9998), repeatability for the retention time (CV = 1.02 %) and area (CV = 0.24 %), selectivity and limits of detection (19,8 μg.mL - 1 ) and quantification (60.2 μg.mL - 1 ). The recovery results obtained by th e purification of the anacardic acid on a preparative scale were 94.02, 87.63 and 97.35 %, for the triene, dieno and monoene, respectively . The data for purity were 99.11, 95.56 and 92.59 % , for the triene, diene and monoene, respectively. The s olvent consumption was 60.52 and 11.09 mL.mg - 1 for analytical and preparative scale, respectively. The productivity was 0.06 and 1.63 g.h - 1 by each g of adsorbent to analytical and preparative scale, respectively. The chromatographic method developed and i ts respecti ve scale - up were adequate for the quantification, monitoring and isolation of the an acardic acids present in the CNSL. The method was validated according to the required standards and the isolation of the analytical standard s was obtained with a n high degree of purity, recovery, low solvent consumption and good productivity. / A casca da castanha de caju é matéria-prima para obtenção do líquido da casca da castanha (LCC), uma substância viscosa de coloração amarronzada, com importantes aplicações na indústria química e farmacêutica. Diversos compostos são encontrados no LCC, tais como, cardol, cardanol, metil-cardol e os ácidos anacárdicos (AcAn). Os AcAn são compostos considerados lipídeos fenólicos, pois possuem uma cadeia carbônica longa com diferentes graus de instauração. Estudos em diversas áreas da ciência mostram resultados positivos dos AcAn para tratamento e prevenção de algumas doenças e seus vetores. Portanto, há uma necessidade de estudos mais específicos para monitorar, quantificar e isolar esses compostos no LCC. Nesse trabalho foi desenvolvido um método de separação cromatográfica, capaz de isolar os AcAn presentes no LCC. Também foi executado um fracionamento do LCC para obter uma fração contendo apenas AcAn. Amostras de LCC natural foram solubilizadas em metanol, injetadas em um cromatógrafo líquido de alta eficiência acoplado a um detector de arranjo de diodo (CLAE-DAD) monitorado a um comprimento de onda de 280 nm, equipado com uma coluna C18, utilizando como fase móvel acetonitríla, H2O e ácido acético, em modo isocrático. O método cromatográfico desenvolvido apresentou seletividade adequada, sendo capaz de separar os ácidos anacárdicos trieno (15:3), dieno (15:2) e monoeno (15:1) de maneira eficaz, presentes no LCC nos tempos de retenção de 7,68, 11,09 e 17,85 min, respectivamente. Para as condições cromatográficas em escala preparativa, foi usado um CLAE-UV/VIS monitorado à um comprimento de onda de 280 nm, equipado com uma coluna C18 em escala preparativa, utilizando como fase móvel metanol, H2O e ácido acético, em modo isocrático. O método em escala preparativa foi ajustado matematicamente para obter maior similaridade com o método analítico. A curva de calibração com intervalo linear (50 a 1000 µg.mL-1) e validação do método analítico, foram feitos a partir do padrão externo do ácido anacárdico (15:3). Os resultados obtidos para validação do método foram satisfatórios, para precisão intra-dia (CV = 0,60 %) e inter-dia (CV = 0,67 %), linearidade (y = 2670,8x - 26949, r2 > 0,9998), repetibilidade para o tempo de retenção (CV = 1,02 %) e área (CV = 0,24 %), seletividade e limites de detecção (19,8 µg.mL-1) e quantificação (60,2 µg.mL-1). Os resultados da recuperação do isolamento em escala preparativa dos AcAn foram 94,02, 87,63 e 97,35 %, para os AcAn trieno, dieno e monoeno, respectivamente. Os valores de pureza foram 99,11, 95,56 e 92,59 %, para treino, dieno e monoeno, respectivamente. O consumo de solvente foi de 60,52 e 11,09 mL.mg-1, para escala analítica e preparativa, respectivamente. A produtividade foi de 0,06 e 1,63 g.h-1 por cada g de adsorvente para escala analítica e preparativa, respectivamente. O método cromatográfico desenvolvido e sua respectiva ampliação de escala foram adequados para a quantificação, monitoramento e isolamento dos ácidos anacárdicos presentes no LCC. O método foi validado de acordo com as normas exigidas e o isolamento dos padrões analíticos foi obtido com elevado grau de pureza, recuperação, baixo consumo de solvente e boa produtividade.

Analytical chemistry is a field with a vast variety of applications. A robust companion in the field is liquid chromatography, the method used in this thesis, which is an established workhorse and a versatile tool in many different disciplines. It can be used for identification and quantification of interesting compounds generally present in low concentrations, called analytical scale chromatography. It can also be used for isolation and purification of high value compounds, called preparative chromatography. The latter is usually conducted in large scale with high concentrations. With high concentrations it is also possible to determine something called adsorption isotherms. Determination of adsorption isotherms is a useful tool for quite a wide variety of reasons. It can be used for characterisation of chromatographic separation systems, and then gives information on the retention mechanism as well as provides the possibility to study column-column and batch-batch reproducibility. If a protein is immobilised on a solid support, adsorption isotherms can be used for pharmacological characterisation of drug-protein interactions. Moreover, they can be used for the study of unexpected chromatographic phenomena. If the adsorption isotherm is known it is also possible to simulate chromatograms, and subsequently optimise the separation process numerically. The gain of a numerically optimised separation process is higher purity or yield of valuable compounds such as pharmaceuticals or antioxidants, as well as reducing the solvent usage. Taken all together, it saves time, money and the environment. However, the process of the adsorption isotherm determination requires a number of careful experimental considerations and preparations, and these are the main focus of the thesis. Important steps along the way include the choice of separation system and of suitable analytes, preparation of mobile phases and sample solutions, calibration, determination of injection profiles and column void, and of course the adsorption isotherm determination method itself. It is also important to keep track of parameters such as temperature and pH. These issues are discussed in this thesis. At the end, a description of useful methods for processing of the raw adsorption isotherm data is presented, as well as a brief passage on methods for numerical optimisation.

Synthetic oligonucleotides, which are short strings of DNA or RNA, are a grooving area of importance for the pharmaceutical industry and for companies that manufacture diagnostic components. The manufacturing process of synthetic oligonucleotides involves many complex processes that use separation and purification techniques like ion-exchange chromatography, ion-pair reversed phase chromatography and ultra-performance liquid chromatography. In this study, the focus lies on the purification process, where the main aim is to develop a separation and purification method for modified oligonucleotides that can be applied on different scales, from an analytical to a preparative scale. Three modified oligonucleotides, and one unmodified with 44 bases, provided by Scandinavian Gene Synthesis (Västerås, Sweden), were analysed and purified on an ultra-performance liquid chromatography and on a preparative-system. Several parameters were investigated, e.g. mobile phase composition, gradients and concentration. Practical analysis and purification were made in two scales; analytical and semi-preparative.  The results showed that the samples contained impurities that were hard to separate from the main sample. The scaling-up tests showed that, with increasing concentration, the impurities become more aggregated with the main product. Fraction analysis showed that several pure fractions were collected from the semi-preparative purification, and therefore some amount of pure sample were collected from the semi-preparative run. In conclusion, the method developed in this master thesis worked well as a significant amount of samples were purified in the semi-preparative purification, and the method worked on modified and unmodified oligonucleotides, containing different amount of modifications. / Syntetiska oligonukleotider, vilket är korta strängar av DNA eller RNA, är ett framväxande område i läkemedelsindustrin och för företag som tillverkar diagnostiska komponenter. Tillverkningsprocessen för syntetiska oligonukleotider involverar många komplexa processer som använder separation- och reningstekniker som jonbyteskromatografi, jonparskromatografi och ultra-performance kromatografi. I denna studie ligger fokus på reningsprocessen där det huvudsakliga syftet är att utveckla en separation- och renings metod för modifierade oligonukleotider som kan appliceras på olika skalor – från analytisk till preparativ skala.  Tre modifierade oligonukleotider, samt en omodifierad med 44 baser, tillhandahållet av Scandinavian Gene Synthesis (Västerås, Sverige), analyserades och renades på ett ultra-performance kromatografi system och ett preparativt reningssystem. Flertal parametrar undersöktes, bland annat mobilfasens komposition, gradienter och koncentration. Analys och rening utfördes i två skalor; analytisk och semi-preparativ skala.  Resultatet visade att proverna innehöll föroreningar som var svåra att separera från huvudkomponenten. Uppskalningstesterna visade att föroreningarna blandade sig mer med huvudkomponenten då koncentrationen ökade. Fraktionsanalyser visade att flera rena fraktioner blev ihopsamlade från den semi-preparativa reningen, som därav visade att en betydelsefull mängd rent prov blev renat i den semi-preparativa reningen. Sammanfattningsvis, den metod som utvecklats i denna uppsats fungerade bra då betydelsefulla mängder oligonukleotider kunde renas till olika grad vid den semi-preparativa reningen, samt att metoden fungerade för både modifierade och icke-modifierade oligonukleotider som innehöll olika mängder modifikationer.

La chromatographie bidimensionnelle préparative suscite de plus en plus d'intérêt dans l'élucidation d'échantillons complexes car elle permet de collecter un grand nombre de molécules à haute pureté et quantitée récupérée. Bien que la chromatographie liquide (LC) soit souvent choisie en deuxième dimension, la chromatographie de partage centrifuge (CPC) à de multiples avantages qui en font une technique de choix pour la première dimension. Dans le but de purifier plusieurs molécules d’intérêt dans les matrices végétales, le couplage «comprehensive» CPCxLC représente une technique à fort potentiel. Après avoir expliqué son intérêt et les enjeux liés à la séparation préparative en mode «comprehensive», le développement d’une telle séparation est étudiée selon trois axes. Tout d’abord, une purification de deux molécules d’intérêt dans la plante Edelweiss est réalisée à l’échelle industrielle grâce à la réalisation de cartographies 2D au laboratoire. Cette application permet de montrer l’intérêt du couplage et de mettre en évidence les verrous liés aux conditions de transfert total des fractions en deuxième dimension. Dans une deuxième partie, la séparation CPCxLC en mode « comprehensive » est développée avec le transfert total de l’échantillon en deuxième dimension pour la purification de cinq composés cibles présents dans la plante Edelweiss. Les points clés de la séparation CPCxLC, à savoir le temps d’échantillonnage et le transfert en deuxième dimension, sont étudiés au regard du couplage LCxLC afin de garantir une qualité de séparation permettant la récupération totale des composés. Enfin, la troisième partie consiste à la mise en place d’une méthodologie de sélection des systèmes CPCxLC basée sur l’évaluation quantitative du potentiel des systèmes bidimensionnels à apporter de la distance entre les pics. Cette procédure de sélection est développée sur l’échantillon Cyclopia genistoides avec l’objectif d’isoler huit composés cibles / Preparative two-dimensional chromatography is gaining interest in the elucidation of complex samples as it allows the collection of a large number of molecules with high recovered purity and quantity. While the second dimension is often selected to be liquid chromatography (LC), centrifugal partition chromatography (CPC) is a technique with multiple advantages representing a suitable first dimension. In order to purify several molecules of interest in plant matrices, the comprehensive CPCxLC represents a technique with high potential. After explaining its interest and the issues related to the preparative separation in comprehensive mode, the development of such a separation is studied according to three axes. Firstly, a purification of two targeted molecules in Edelweiss plant is carried out at industrial scale thanks to the realization of 2D-contour plot. This application allows to expose the interest of the separation and to highlight the locks related to the conditions of total transfer of the fractions in second dimension. In a second part, the comprehensive CPCxLC separation is developed with the total transfer of the sample in second dimension applied to the purification of five target compounds from Edelweiss plant. The key points of the CPCxLC separation, namely the sampling time and the second dimension transfer, are studied with regard to the LCxLC separation in order to ensure a separation quality allowing the total recovery of the compounds. Finally, the third part consists in the implementation of a CPCxLC system selection methodology based on the quantitative evaluation of the potential of two-dimensional systems to generate distance between peaks. This selection procedure is developed on the sample Cyclopia genistoides with the objective of isolating eight target compounds

This thesis concerns the development and validation of methods for the industrially important area of adsorption isotherm parameter estimation in preparative, nonlinear high performance liquid chromatography (HPLC). Preparative chromatography is a powerful separation method to get pure compounds from more or less complex liquid mixtures, e.g., mixtures of mirror-image molecules. Computer simulations can be used to optimize preparative chromatography, but then competitive adsorption isotherm parameters are usually required. Here two methods to estimate adsorption isotherm parameters are treated: (i) the perturbation peak (PP) method and (ii) the inverse method (IM). A new theory for the PP method was derived and led to a new injection technique which was validated experimentally. This injection technique solved the severe problem with vanishing peaks and enabled us to use the PP method to estimate binary competitive adsorption isotherms valid over a broad concentration range. Also, the injection technique made it possible to estimate competitive adsorption isotherms for a quaternary mixture for the first time. Finally, an interesting perturbation peak phenomenon, known as the “Helfferich Paradox”, was experimentally verified for the first time. The IM is a relatively new method to determine adsorption isotherm parameters. It has the advantage of requiring very small samples, but also requires an advanced computer algorithm. An improved implementation of this computer algorithm was developed and tested experimentally. Also, a variant of the IM called “the inverse method on plateaus” was tested experimentally and the estimated adsorption isotherm parameters were shown to be valid over a broader concentration range than those estimated with the standard IM.

Les procédés multi-colonnes de chromatographie ont connu depuis quelques années un développement tel qu'ils sont devenus des standards industriels à toutes échelles, depuis celle des produits pharmaceutiques à haute valeur ajoutée jusqu'à celle des grands intermédiaires chimiques. La spécificité du présent travail consiste à étudier, pour ces procédés, l'influence d'un gradient d'élution. Il s'agit de faire varier au cours du temps la force éluante de la phase mobile. L'objectif est d'augmenter la productivité et le taux de récupération d'un produit à haute valeur ajoutée, tout en répondant à des contraintes de pureté. L'utilisation d'un gradient de solvant, courante en chromatographie analytique, fait l'objet d'un intérêt plus récent en chromatographie préparative. Les applications visées concernent des séparations de mélanges complexes où l'espèce cible a une affinité intermédiaire pour le support solide par rapport à celle des autres espèces, ce qui est souvent le cas lors de la purification de biomolécules issues de matières premières naturelles ou issues des biotechnologies. Dans ce cas, la séparation conduit à trois fractions, des impuretés faiblement retenues, la fraction intermédiaire et des impuretés fortement retenues. Pour notre étude, un mélange modèle, peu coûteux et non toxique, de cinq acides aminés a été choisi. Ces acides aminés ont été choisis en tenant compte de leur caractère apolaire et hydrophobe. Les séparations ont été réalisées par chromatographie en phase inverse. Dans un premier temps, une étude expérimentale, réalisée à l'aide d'une chaîne HPLC, a permis de déterminer les paramètres des isothermes d'adsorption de chaque acide aminé pour différentes teneurs en solvant organique de l'éluant. Une loi empirique a permis de relier le facteur de rétention k à la composition de la phase mobile (K = f (xméthanol)). Un travail de modélisation/simulation, reposant sur l'approche d'une cascade de mélangeurs, a ensuite permis de simuler les séparations obtenues dans le cas d'une seule colonne, puis dans le cas d'un système multi-colonnes. L'utilisation des lois reliant les facteurs de rétention k à la concentration en modifieur a alors permis de réaliser des simulations pour différents gradients de solvants. Dans le cas d'une seule colonne, le gradient a été optimisé en minimisant la durée de la séparation et en respectant une contrainte sur la résolution des pics des 2 espèces les plus difficiles à séparer. Une bonne adéquation a été observée entre les simulations et les résultats expérimentaux obtenus avec un gradient sur une seule colonne. Des expérimentations numériques ont alors été réalisées dans le cas du système multi-colonnes. Les paramètres opératoires optimaux ont été déterminés dans le cas du mélange étudié. Ces réglages seront ainsi utilisés lors de la validation expérimentale qui sera réalisée sur l'unité pilote. Cette unité comporte trois colonnes. Il s'agit d'un procédé séquentiel cyclique. Pour le mode opératoire retenu, chaque cycle comporte 8 étapes. A chaque étape les alimentations et soutirages des différentes colonnes sont modifiées. Pour le soutirage qui correspond à la fraction de l'espèce cible, les critères étudiés seront la pureté et le taux de récupération / Multi-column chromatographic processes have known, for a few years, a development on all scales, from high added value pharmaceutical products to major chemical intermediates. The specificity of the present work is to study the influence of a gradient elution for these processes. It consists in varying the eluent strength of the mobile phase over the time. The aim is to increase the productivity and the recovery ratio of a high added value product, while satisfying the constraints of purity. Solvent gradient is currently used in analytical chromatography and presents a recent interest in preparative chromatography. The applications concern separations of complex mixtures where the target species has an intermediate affinity for the solid phase compared to other species, which is often the case during the purification of biomolecules extracted from natural raw materials or resulting from biotechnologies. In this case, separation leads to three fractions, impurities weakly retained, an intermediate fraction and impurities strongly retained. For our study, a model mixture, inexpensive and nontoxic, of five amino acids was selected considering their nonpolar and hydrophobic character. The separations were carried out by reversed phase chromatography. An experimental study using a HPLC system was first carried out with single-element solution of each amino acid in isocratic mode. This enabled to determine adsorption isotherm parameters. An empirical law giving the retention factor as a function of eluent composition was determined (K = f (xmethanol)). A work of modeling / simulation, assuming linear isotherm and based on the mixed cells approach, permitted to simulate the separations obtained in the case of a one-column process, then in the case of a multi-column system. The use of retention factors laws allowed to carry out simulations for different solvent gradients. In the case of a single column, a simple methodology was developed to calculate the optimal solvent gradient. The gradient was optimized by minimizing the separation time and by respecting a constraint on the peaks resolution of the two species which are the most difficult to separate. A really good adequacy was observed between simulations and the experimental results. Numerical experimentations, executed in the case of the multi-columns process, made it possible, yet, to find the optimal operating parameters in the case of the studied mixture. These settings will be applied in the experimental validation which will be realized on the pilot unit. This unit has three columns. It is a cyclic sequential process. For the selected operating mode, each cycle contains eight steps. At each step, inlets ant outlets streams of different columns are switched. The criteria for the target species fraction are purity and recovery

Orientador: Cesar Costapinto Santana / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-09-05T13:20:08Z (GMT). No. of bitstreams: 1 Veredas_ViniciusDe_D.pdf: 13205142 bytes, checksum: 97c5009c255088bef6fcdc1fd92294c3 (MD5) Previous issue date: 2005 / Doutorado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Les travaux de cette thèse portent sur le développement de nouvelles méthodologies de purification des peptides pharmaceutiques par chromatographie de partage centrifuge (CPC) dans le but de l'introduction de cette technique comme outil de R&D mais surtout de production en milieu industriel. Le caractère original de ces travaux porte essentiellement sur l'introduction de nouveaux systèmes de solvants et la mise au point de nouveaux procédés de purification en mode CPC co-courant. Les différents aspects liés à l'industrialisation des différents procédés de purification ont également été étudiés.La première partie des travaux a consisté en l'étude de quelques nouveaux aspects de l'intérêt de l'application du mode co-courant en chromatographie de partage centrifuge. Une méthodologie originale de purification des peptides tensioactifs non ioniques en mode CPC co-courant a été mise au point. Cette méthodologie a permis de résoudre les problèmes de perturbations hydrodynamiques et de perte de phase stationnaire engendrés par le caractère tensioactif de ces molécules et a été appliquée avec succès à la purification d'une cyclosporine modifiée douée d'une activité anti-virale et faiblement soluble dans les solvants usuellement utilisés en CLHP. Une étude fondamentale de l'effet du peptide sur le comportement hydrodynamique des deux phases lors de la séparation et la visualisation des modèles d'écoulement au sein de la colonne CPC a permis la mise en évidence du role de la ciclosporine modifiée dans la perturbation de la composition des phases du système chromatographique. D'autres aspects de l'intérêt du mode co-courant en CPC ont été étudiés lors de cette étude, notamment l'amélioration de la robustesse et de la résolution de la séparation.La seconde partie des travaux a porté sur le développement de nouveaux systèmes biphasiques de solvants particulièrement adaptés à la purification des peptides hydrophobes non-ioniques, notamment les intermédiaires de synthèse protégés, qui sont très faiblement solubles dans la plupart des solvants communs utilisés en chromatographie. Deux gammes quaternaire et quinaire de systèmes biphasiques de solvants, ainsi qu'un système biphasique ternaire ont été introduits. L'originalité de ces systèmes porte sur l'usage de solvants verts à fort caractère solvatant tel que le Methyl-THF et le cyclopentyl methyl ether (CPME). Les systèmes développés ont été efficacement utilisés pour la purification en CPC d'une exénatide protégée de 39 acides aminés et d'un peptide protégé de 8 acides aminés intermédiaire de la synthèse de la bivalirudine. Ces systèmes devraient être utiles pour une utilisation générale en CPC pour la séparation des peptides synthétiques hydrophobes libres ou protégés. / The work presented in this thesis deals with the development of new methodologies for the purification of pharmaceutical peptides by centrifugal partition chromatography (CPC) in order to introduce this technique as a tool for R & D but also in industrial production. The original character of this work relies on the introduction of new solvent systems and the development of new purification processes based on the co-current CPC mode. The different aspects of the process intensification and industrialization have also been studied.In the first part of the work, a study of some new aspects of the interest of the application of the stationary phase co-current mode in CPC is described. An original method for the purification of non-ionic tensioactive peptides in the co-current CPC mode was developed. This method has been successfully applied to the purification of a modified cyclosporine showing a therapeutic interest. This particular elution mode, taking advantage of the liquid nature of the stationary phase, appears to be an efficient solution to get round some hydrodynamic instabilities that sometimes appears during a purification intensification by CPC. A fundamental study of the effect of the peptide on the hydrodynamic behavior of the two phases in the separation and visualization of flow patterns within the CPC column allowed highlighting the role of the peptide in the disruption of phases composition of the chromatographic system. Other aspects of the interest of the co-current mode in CPC were investigated in this study, including the improvement of the efficiency and the resolution of the separation.The second part of the work focused on the development of new biphasic solvent systems particularly suitable for the purification of hydrophobic non-ionic peptides, including protected intermediates, which are very poorly soluble in the most common solvents used in chromatography. Two new scales of biphasic solvent systems showing a wide range of polarity and a ternary biphasic system were introduced to overcome solubility problems often encountered with synthetic hydrophobic protected peptides. The originality of these systems relies on the use of green solvents with high solvating character such as Methyl-THF and cyclopentyl methyl ether (CPME). The developed systems have been effectively used for the purification in CPC of a 39mer protected exenatide and and a 8mer protected peptide intermediate of bivalirudin synthesis.

<p>This thesis concerns the development and validation of high performance liquid chromatography (HPLC) methods aimed for two industrially important areas: (i) analysis of biotechnological synthesis and (ii) determination of adsorption isotherm parameters. There is today a lack of detailed recommendations for analytical procedures in the field of biotechnological production of drugs. Therefore, guidelines were given for analytical development and validation in this field; the production of 9α-hydroxyprogesterone was used as model. In addition, a rapid method using HPLC coupled with diode-array-detection (DAD) and mass spectrometry (MS), was developed for the preliminary identification and quantification of the product. In addition, requirements and recommendations were developed for the selection of the internal standard and for its inclusion in the process liquid. By using this approach the precision and accuracy of the quantitative method were considerably improved. </p><p>Preparative chromatography is a powerful separation method for the purification of pure compounds from more or less complex sample mixtures. One such mixture can be the process liquid from a fermentation, another example can be a racemic mixture of compounds whose enantiomeric constituents must be isolated. Computer-assisted modeling can be used to optimize preparative chromatography. However, competitive adsorption isotherm parameters are required as input data for the computer simulations. In this thesis, a new injection technique, based on a firm theoretical basis, was developed for the peak perturbation (PP) method allowing the determination of binary competitive adsorption isotherm parameters from a broad concentration range. With the new method the determination of adsorption isotherm parameters from a quaternary mixture could be done for the first time. The profiles simulated with these parameters showed excellent agreement with the corresponding experimental profiles, validating the accuracy of the adsorption isotherm parameters derived by the new method.</p>

This thesis concerns the development and validation of high performance liquid chromatography (HPLC) methods aimed for two industrially important areas: (i) analysis of biotechnological synthesis and (ii) determination of adsorption isotherm parameters. There is today a lack of detailed recommendations for analytical procedures in the field of biotechnological production of drugs. Therefore, guidelines were given for analytical development and validation in this field; the production of 9α-hydroxyprogesterone was used as model. In addition, a rapid method using HPLC coupled with diode-array-detection (DAD) and mass spectrometry (MS), was developed for the preliminary identification and quantification of the product. In addition, requirements and recommendations were developed for the selection of the internal standard and for its inclusion in the process liquid. By using this approach the precision and accuracy of the quantitative method were considerably improved. Preparative chromatography is a powerful separation method for the purification of pure compounds from more or less complex sample mixtures. One such mixture can be the process liquid from a fermentation, another example can be a racemic mixture of compounds whose enantiomeric constituents must be isolated. Computer-assisted modeling can be used to optimize preparative chromatography. However, competitive adsorption isotherm parameters are required as input data for the computer simulations. In this thesis, a new injection technique, based on a firm theoretical basis, was developed for the peak perturbation (PP) method allowing the determination of binary competitive adsorption isotherm parameters from a broad concentration range. With the new method the determination of adsorption isotherm parameters from a quaternary mixture could be done for the first time. The profiles simulated with these parameters showed excellent agreement with the corresponding experimental profiles, validating the accuracy of the adsorption isotherm parameters derived by the new method.