O presente trabalho teve como objetivo a purificação da enzima glicose-6-fosfato desidrogenase pela tecnologia de extração líquido-líquido em Sistemas Micelares de Duas Fases Aquosas (SMDFA). Estes sistemas são constituídos por soluções de tensoativos contendo micelas e oferecem ambientes hidrofóbico e hidrofílico, o que possibilita seletividade na partição da enzima de acordo com sua hidrofobicidade e proporciona um ambiente ameno às biomoléculas. Foram estudados alguns dos fatores que influenciam a partição da G6PD, como: tipo e concentração de diferentes agentes tensoativos não-iônicos (C10E4 e Triton X-114), temperatura e adição de ligantes de afinidade (cibacron blue e procion red) e o efeito da adição dos sais sulfato de amônio ((NH4)2SO4) e sulfato de sódio (Na2SO4). Estudou-se ainda a síntese do tensoativo de afinidade TX-114-Blue. Em todos os ensaios a enzima foi recuperada preferencialmente na fase diluída, pobre em micelas, tanto em sistema Triton X-114/tampão como para C10E4/tampão, no qual existe maior volume disponível, resultando em valores de KG6PD inferiores a 1. A utilização dos ligantes de afinidade na partição da G6PD nos sistemas descritos proporcionou um pequeno aumento nos valores de KG6PD da enzima, porém com valores inferiores a 1. Os sistemas Triton X-114/Sal não influenciaram a partição da enzima para a fase micelar, apesar da existência da diferença de potencial eletrostático entre as fases destes sistemas. O efeito desempenhado pelo volume de exclusão foi dominante em todos os sistemas estudados e, portanto, a enzima foi predominantemente excluída para a fase aquosa, pobre em micelas. A tecnologia por SMDFA para a purificação do homogeneizado celular de Saccharomyces cerevisiae demonstrou ser eficiente em recuperar a biomolécula alvo na fase aquosa, pobre em micelas, permitindo separar da presença de biomoléculas ou mesmo de contaminantes com caráter hidrofóbico. Dessa forma, o SMDFA pode ser empregado como uma possível etapa de purificação num processo biotecnológico. / In this work, the use of two-phase micellar system was studied aiming at the purification of the enzyme glucose-6-phosphate dehydrogenase. Usually, these systems are constituted of micellar surfactants solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to the enzyme partitioning according to its hydrophobicity. Some of the factors influencing the G6PD partition in micellar systems were studied, such as: type and concentration of nonionic surfactant agents (C10E4 and Triton X-114), temperature, addition of affinity ligands (Cibacron Blue and Procion Red) and the addition of the salts ammonium sulfate ((NH4)2SO4) and sodium sulfate (Na2SO4). The synthesis of the affinity surfactant TX-114-Blue was also studied. In all the assays the experiments, G6PD partitioned preferentialy to dilute, micelle-poor phase, in which there is a higher volume available for the enzyme to be, resulting in KG6PD values lower than 1. The use of affinity ligands in G6PD partitioning in both C10E4 and Triton X-114 systems provided some increase in the KG6PD, however with values still lower than 1. Employing a methodology previously described in the literature with some alterations, it was not possible to obtain the affinity surfactant TX-114-Blue. The systems Triton X-114/salt have not shown a significant influence on the enzyme partition to the micelle-rich phase, in spite of the existence of an electrostatic potential difference between the phases of the systems. The excluded-volume effect was dominant in all the systems studied and, therefore, the enzyme predominantly excluded to the dilute, micelle-poor phase. The use of Triton X-114 two-phase aqueous micellar systems to the purification of the Saccharomyces cerevisiae cell homogenate was found to be efficient in the recovery of G6PD in the dilute, micelle-poor phase, partially separating this target molecule from other proteins and contaminants of hydrophobic character. Therefore, aqueous two-phase micellar systems can be considered useful as a possible purification stage in a biotechnology process.

O presente trabalho teve como objetivo a purificação da enzima glicose-6-fosfato desidrogenase pela tecnologia de extração líquido-líquido em Sistemas Micelares de Duas Fases Aquosas (SMDFA). Estes sistemas são constituídos por soluções de tensoativos contendo micelas e oferecem ambientes hidrofóbico e hidrofílico, o que possibilita seletividade na partição da enzima de acordo com sua hidrofobicidade e proporciona um ambiente ameno às biomoléculas. Foram estudados alguns dos fatores que influenciam a partição da G6PD, como: tipo e concentração de diferentes agentes tensoativos não-iônicos (C10E4 e Triton X-114), temperatura e adição de ligantes de afinidade (cibacron blue e procion red) e o efeito da adição dos sais sulfato de amônio ((NH4)2SO4) e sulfato de sódio (Na2SO4). Estudou-se ainda a síntese do tensoativo de afinidade TX-114-Blue. Em todos os ensaios a enzima foi recuperada preferencialmente na fase diluída, pobre em micelas, tanto em sistema Triton X-114/tampão como para C10E4/tampão, no qual existe maior volume disponível, resultando em valores de KG6PD inferiores a 1. A utilização dos ligantes de afinidade na partição da G6PD nos sistemas descritos proporcionou um pequeno aumento nos valores de KG6PD da enzima, porém com valores inferiores a 1. Os sistemas Triton X-114/Sal não influenciaram a partição da enzima para a fase micelar, apesar da existência da diferença de potencial eletrostático entre as fases destes sistemas. O efeito desempenhado pelo volume de exclusão foi dominante em todos os sistemas estudados e, portanto, a enzima foi predominantemente excluída para a fase aquosa, pobre em micelas. A tecnologia por SMDFA para a purificação do homogeneizado celular de Saccharomyces cerevisiae demonstrou ser eficiente em recuperar a biomolécula alvo na fase aquosa, pobre em micelas, permitindo separar da presença de biomoléculas ou mesmo de contaminantes com caráter hidrofóbico. Dessa forma, o SMDFA pode ser empregado como uma possível etapa de purificação num processo biotecnológico. / In this work, the use of two-phase micellar system was studied aiming at the purification of the enzyme glucose-6-phosphate dehydrogenase. Usually, these systems are constituted of micellar surfactants solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to the enzyme partitioning according to its hydrophobicity. Some of the factors influencing the G6PD partition in micellar systems were studied, such as: type and concentration of nonionic surfactant agents (C10E4 and Triton X-114), temperature, addition of affinity ligands (Cibacron Blue and Procion Red) and the addition of the salts ammonium sulfate ((NH4)2SO4) and sodium sulfate (Na2SO4). The synthesis of the affinity surfactant TX-114-Blue was also studied. In all the assays the experiments, G6PD partitioned preferentialy to dilute, micelle-poor phase, in which there is a higher volume available for the enzyme to be, resulting in KG6PD values lower than 1. The use of affinity ligands in G6PD partitioning in both C10E4 and Triton X-114 systems provided some increase in the KG6PD, however with values still lower than 1. Employing a methodology previously described in the literature with some alterations, it was not possible to obtain the affinity surfactant TX-114-Blue. The systems Triton X-114/salt have not shown a significant influence on the enzyme partition to the micelle-rich phase, in spite of the existence of an electrostatic potential difference between the phases of the systems. The excluded-volume effect was dominant in all the systems studied and, therefore, the enzyme predominantly excluded to the dilute, micelle-poor phase. The use of Triton X-114 two-phase aqueous micellar systems to the purification of the Saccharomyces cerevisiae cell homogenate was found to be efficient in the recovery of G6PD in the dilute, micelle-poor phase, partially separating this target molecule from other proteins and contaminants of hydrophobic character. Therefore, aqueous two-phase micellar systems can be considered useful as a possible purification stage in a biotechnology process.

Nanoparticles have important applications in ceramics, metal catalysts, semiconductors etc. They are normally required to be of small size (~ nm) and monodisperse. The aim of the present work is to model the formation of nanoparticles, obtained by precipitation in reverse micellar microreactors. These are dispersions of tiny water drops in a surfactant laden oil medium. Two systems were investigated: (i) Reverse micelles, having nanometer sized spherical water droplets in the micellar core and (ii) Water-in-oil emulsions, having micron-sized aqueous drops. Two modes of precipitation, namely, gas-liquid (g-1) and liquid-liquid (1-1) were studied. In each case, the models could predict the number, average size and size distribution of the particles reported in literature. Two groups have obtained widely divergent number and size of CaCO3 nanoparticles, formed by g-1 precipitation in reverse micelles. These particles are used as a fine suspension in lube-oil additives, where they serve to neutralize acid produced during combustion in engines. Kandori et al. (J. Colloid Interface Sci, 122,1988, 78) obtained particles of about 100 nm size, by passing CO2 through a reverse micellar solution, containing dissolved Ca(OH)2 in the micellar core. Roman et al. (J. Colloid Interface Sci., 144,1991, 324), instead of using lime solution; added micron-sized solid lime particles in the oil and generated the reverse micelles by in situ reaction. This is a commercial process known as overbasing. It led to a higher amount of lime in the micelles as well as unreacted lime particles in oil, at the beginning of the experiment Upon passing CO2, they got particles of only 6 nm in size, compared to 100 nm reported by Kandori et al.. Furthermore, while Kandori et al. found that one particle formed from 108 micelles, Roman et al. got one particle out of only ten micelles. We have modelled the two processes in a common framework to explain the reported disparity in particle characteristics. A time scale analysis of CO2 mass transfer, reaction, collision-fusion of micelles, nucleation, and growth of particles was carried out It showed that, in the experiments of Kandori et al., the rate limiting steps are nucleation and fusion. The analysis also indicates that the contents of a particular micelle are well mixed and reaction of lime and incoming CO2 can be treated as instantaneous. In the process of Kandori et al., the amount of lime taken initially being very small, the average number of product molecules in a micelle is well below one. Rapid Brownian coalescence and exchange of micellar contents leads to Poisson distribution of CaCO3(l) molecules formed by reaction. The low occupancy therefore suggests that most of the micelles are empty. Nucleation in a particular micelle is much slow and occurs when it has a critical number of molecules. Thus only very few micelles can nucleate. Comparison of nucleation and growth time scales - both intrinsic growth in a micelle and growth during fusion of nucleated and non-nucleated micelles - show that growth is much faster than both nucleation and collision. Hence a micelle can have only one nucleus, with subsequent growth during collisions. A population balance equation (PBE) is written involving the above steps. Solution of the moments of the distribution yields the number of CaCO3 particles, its size, coefficient of variance (COV) etc. The model not only predicts the ratio of number of micelles to particles, obtained experimentally as 108, but also captures the maxima in this quantity with increasing micellar size. The increase in average particle size with micellar size is also predicted well. The process of of Roman et ai, in addition, involves the time scale of solubilization of solid lime into micelles. Its comparison with other time scales demarcates their experiments into two distinct phases. Phase I consists of reaction of lime initially present in micelles. Time scale analysis also suggests that, as the lime content in the micelles is large, a high degree of supersaturation is rapidly generated. This results in a burst of nuclei. The other conclusions, like, well-mixed micelle, Poisson distribution of CaCO3(l) molecules, instantaneous growth and mono-nucleated micelles are found to hold good. Once the pre-existing lime is finished, relative time scales indicate that, further precipitation is controlled entirely by fresh solubilization of lime. This marks the beginning of phase II. However, solubilization being the slowest step, CaCO3(l) in micelles never builds up for any further nucleation. Phase II thus consists of pure growth of the particles formed in phase I. On developing more general PBEs and with solution of resulting moment equations - written separately for the two phases - the experimental data on number of particles and temporal evolution to the final particle size of 6 nm could be predicted very well. The model also captures the qualitative trend in COV of particle radius with time. Thus within the same framework we could successfully predict both the results, differing by seven orders of magnitude. The above analysis indicates that relative rates of nucleation, fusion-growth and mass transfer of gas controls the carbonation process. We further simplify the process and obtain an analytical solution in the limit of instantaneous mass transfer. The solution gives close first estimates for both the experiments and also indicates the smallest panicle size that could be obtained for a given experimental condition. In contrast to g-1 mode, precipitation in 1-1 mode - using two reverse micellar solutions having two reactants- occurs only on coalescence of two micelles. To obviate the solution of multivariate PBEs, we have developed a general Monte Carlo (MC) simulation scheme for nanoparticle formation, using the interval of quiescence technique (IQ). Starting with a fixed number of micelles, we conduct each coalescence-redispersion and nucleation events in this population, in the ratio of their relative frequencies. Our simulation code is much more general and realistic than the scheme of Li and Park (Langmuir, 15,1999, 952). Poisson distribution with realistic micellar occupancies of reactants, binomial redispersion of solutes after fission, a nucleation rate with critical number of molecules and Brownian collision-fusion rates were used. These considerations are based on our earlier findings in g-1 precipitation and those known in the literature too. The simulation of Li and Park then becomes a special case of our code. Our simulation code was then used to predict experimental data on two systems. The results of Lianos and Thomas (Chem. Phys. Lett. 125, 1986, 299 and /. Colloid Interface 5c/., 117, 1987, 505), on number of molecules per CdS particle, as a function of micelle size and reactant concentrations have been predicted very well. For the Fe(OH)3 nanoparticles, our simulation provides a better prediction of the experimental particle size range, than that of Li and Park. Finally, 1-1 precipitation on mixing two emulsions, having respectively the two reactants, has been simulated. Here, large reactant amount leads to multiple nucleation in a single drop and renders growth rate to be finite. This requires solving a PBE for particle population in each drop. Moreover, emulsions have a drop size distribution due to independent coalescence and breakage. The IQ technique was used for handling these events. Thus a composite model of PBE and MC for a drop population was developed. Simulation of particle size distribution in MgCO3 precipitation shows that nearly monodisperse nanoparticles can be produced in emulsions. Furthermore, average particle size can be controlled by changing reactant concentration in a drop. The findings of the thesis have provided new issues to be addressed in modelling nanoparticle formation. It points out the importance of finding models for coalescence efficiency and critical nuclear size in micelles. Extension of our model and simulation to precipitation in other organized surfactant assemblies can be done by starting from appropriate time scale analysis.

Cette thèse vise à comprendre l’effet des dynamiques des micelles de copolymères sur leurs propriétés rhéologiques. En effet les copolymères amphiphiles peuvent s'auto-assembler dans l’eau pour former diverses microstructures micellaires sphériques, cylindriques ou lamellaires. Leur dynamique est un paramètre crucial pour définir leur spectre d’applications, en particulier dans le transport de médicaments et dans la synthèse de matériaux méso-structurés. Les micelles se structurent en continu: elles peuvent se former et se dissocier en monomères, se fusionner ou se fragmenter. Nous avons développé une méthode basée sur la fluorescence pour sonder directement les dynamiques micellaires collectives de fusion et de fission dans un bon nombre de copolymères triblocs de la famille PEO-PPO-PEO. Cette thèse se concentre sur l'étude des systèmes micellaires formés par des copolymères triblocs (tensioactifs non ioniques) et les tensioactifs cationiques à des concentrations et des températures où micelles sphériques et micelles cylindriques sont formées. De telles structures peuvent être formées spontanément lorsque le système est soumis à des conditions appropriées de concentration et de température ou à l'induction entre elles en ajoutant des sels inorganiques ou organiques, ou en appliquant des contraintes de cisaillement au système tensioactif/eau.La technique de diffusion de la lumière dynamique (DLS) a été utilisée pour évaluer la transition de la structure dans des solutions micellaires en changeant la température du système et de la structure du système correspondant à des conditions de concentration - température.Des expériences de relaxation de contraintes ont été effectuées où l’assouplissement temporaire de contrainte est mesuré après une déformation instantanée dans des solutions aqueuses de P103. Les temps de relaxation (ou taux) sont comparés avec ceux obtenus par diffusion de la lumière et de leur rapport à la taille des micelles est montré. La relaxation des contraintes présente le comportement de Maxwell. Les temps de relaxation ont montré la dépendance de la température avec des comportements caractéristiques des régimes de micelles sphériques et allongées respectivement.Dans ce travail, nous étudions également le système micellaire CTAnFB avec n la substitution de fluor en positions ortho (F: 2), meta (F: 3) et para (F: 4), à savoir les systèmes CTAortoFB/, CTAmetaFB/eau et CTAparaFB/eau à des concentrations diluées. Nous présentons aussi une étude de la dynamique de la simulation moléculaire de l'hydratation de tensioactifs ortho-, méta- et para-fluorobenzoate et son effet sur la formation de micelles.Nous avons étudié l'effet des contre-ions organiques hydrotropes, 2-, 3- et 4-fluorobenzoïque (2FB-, 3FB- et 4FB-, respectivement) sur le comportement d'épaississement par cisaillement de solutions aqueuses micellaires de micelles en forme de tige de leurs sels avec des cations de cétyltriméthylammonium (CTA2FB, CTA3FB et CTA4FB).Ce travail a contribué à la compréhension de la synergie entre la structure, la dynamique micellaire et la rhéologie dans cette famille de copolymère. / This thesis aims to understand the effect of the dynamics of copolymer micelles on their rheological properties. Indeed amphiphilic copolymers can self-assemble in water to form various micellar microstructures spherical, cylindrical or lamellar. Their dynamic is a critical parameter to define their range of applications, particularly in the transport of drugs and in the synthesis of mesostructured materials. Micelles are structured continuous: they can form and dissociate into monomers, merge or fragment. We have developed a method based on fluorescence to probe directly collective micellar dynamics of fusion and fission in a number of triblock copolymers of PEO -PPO -PEO family.This thesis focuses on the study of micellar systems formed by triblock copolymers (nonionic surfactants) and cationic surfactants at concentrations and temperatures where spherical micelles and rod like micelles are formed. Such structures may be formed spontaneously when the system is subjected to appropriate conditions of concentration and temperature or to the induction of these by adding inorganic or organic salts, or by applying shear stresses to the surfactant/water system.The technique of dynamic light scattering (DLS) was used to evaluate the transition of the structure in micellar solutions by changing the temperature and the structure of the system itself at given concentration - temperature conditions.Stress relaxation experiments were performed where temporary relaxation of stress is measured after a momentary deflection in aqueous solutions of P103. The relaxation times (or rate) are compared with those obtained by light scattering and their relation to the size of the micelles is shown. The stress relaxation presented a Maxwelian behavior. The relaxation times showed temperature dependence with characteristic behavior schemes for elongated and spherical micelles respectively.In this work, we also study the micellar system CTAnFB where “n” is substitution of fluorine in the ortho position (F:2) meta ( F:3) and para (F:4), namely CTAortoFB/water CTAmetaFB/water and CTAparaFB/water systems in diluted concentrations. We presented a study of molecular dynamics simulation of the hydration of ortho-, meta-, para- Fluorobenzoate surfactants and its effect on the formation of micelles.The effect of hydrotropes organic counterions , 2- , 3- and 4- fluorobenzoic acid (2FB-, 3FB- and 4FB-, respectively), the shear thickening behavior of aqueous micellar solutions of rod like micelles thereof salts with cations of cetyltrimethylammonium (CTA2FB, CTA3FB and CTA4FB ) were studied.This work has contributed to the understanding of the synergy between the structure, dynamics and rheology of micellar in this family of copolymer.

O ácido clavulânico é um potente inibidor de β-lactamases utilizado como terapêutico em associação à amoxicilina. O processo industrial de obtenção envolve o cultivo bacteriano em processo descontínuo, enquanto que a sua purificação é realizada, principalmente, por processos de extração líquido-líquido com solventes orgânicos e etapas cromatográficas. Assim, métodos alternativos como a purificação empregando sistemas micelares de duas fases aquosas (SMDFA), os quais oferecem um ambiente hidrofóbico e hidrofílico que possibilita seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, são de grande interesse. Neste trabalho estudou-se a viabilidade da utilização de sistema micelar misto (não-iônico/aniônico) de duas fases aquosas formados pelos tensoativos não-iônico Triton X-114 e aniônico AOT na extração do ácido clavulânico proveniente de cultivo submerso de Streptomyces clavuligerus em processo descontínuo. A estabilidade do ácido clavulânico em relação ao pH, sais e suas concentrações, temperatura e aos tensoativos foi investigada visando limitar as regiões a serem estudadas nas extrações. Determinou-se ainda o ponto névoa dos sistemas Triton X-114/Tampão e Triton X-114/AOT/Tampão em diferentes condições, bem como, estudos de migração da biomolécula para as fases e do tempo necessário para o equilíbrio do sistema. A partir destas investigações iniciais, foram definidos os níveis das variáveis: concentração de AOT (0,50, 0,75 e 1,00 mM), Triton X-114 (1, 3 e 5% m/m), NaCl (0, 0,05 e 0,10 M) e temperatura (24, 26 e 28°C), cujas influências sobre o coeficiente de partição (KAC) e recuperação da biomolécula na fase diluída (nAC) foram investigadas através de um planejamento fatorial. A análise estatística e o modelo matemático obtido a partir dos resultados para KAC revelou ser possível obterem-se KAC≈ 1,65 e nAC≈ 71,58%. No entanto, a análise estatística empregando nAC como resposta apontou condições experimentais desprovidas de significado físico-químico. Em experimentos seriados realizados com Triton X-114 3% m/m, Tampão Mcllavine pH 6,5 e adição gradual de AOT, um aumento da concentração deste tensoativo levou a maiores KAC e Balanço de massa (BMAC)≈ 85%. Desta forma é possível concluir que o sistema micelar misto de duas fases aquosas proporciona baixa perda da biomolécula e partição para a fase diluída, que é uma vantagem devido a menores problemas em uma etapa subsequente de purificação. / Clavulanic acid is a potent inhibitor of β-lactamases that is used as a therapeutic in conjunction with amoxicillin. Its industrial process of production involves bacterial growing in a batch process, while its purification is carried out mainly by process of liquid-liquid extraction with organic solvents and chromatographic steps. Thus, alternative methods like the purification employing two-phase aqueous micellar systems, which offer both hydrophobic and hydrophilic environments to solute species and allow selectivity in partitioning depending on the hydrophobicity of biomolecules, are of great concern. This work studied the feasibility of using two-phase aqueous mixed (nonionic/anionic) micellar systems formed by nonionic surfactant Triton X-114 and anionic surfactant AOT to extract clavulanic acid from broths fermented by Streptomyces clavuligerus in batch process. The stability of clavulanic acid at different values of pH, salts and their concentration, temperature and in the presence of different surfactants was investigated so as to limit the areas to be submitted to further studies. The cloud-point of the systems Triton X-114/Buffer and Triton X-114/AOT/Buffer was also determined under different conditions, and studies were performed on partitioning the biomolecule between the phases and the time needed to balance of the system. From these initial investigations the following levels of the variables were defined: concentration of AOT (0.50, 0.75, and 1.00 mM), Triton X-114 (1, 3 and 5% w/w), NaCl (0, 0.05 and 0.10 M) and temperature (24, 26 and 28°C), whose influences on the partition coefficient (KCA) and yield in the top phase (nCA) were investigated using factorial design. Statistical analysis and the mathematical model derived from the results allowed obtaining KCA≈ 1.65 e nCA≈ 71.58%. However, the statistical analysis using the response nCA indicated experimental conditions devoid of any physico-chemical meaning. In serial experiments performed with Triton X-114 3% w/w, Mcllvaine buffer at 6.5 pH and gradual addition of AOT, an increase in the concentration of anionic surfactant led to higher KCA and mass balance (BMCA≈ 85%). Thus, is possible conclude that the two-phase aqueous mixed micellar system provides low loss and partition to dilute phase, which is an advantage because it implies less problems in a subsequent step of purification.

O ácido clavulânico é um potente inibidor de β-lactamases utilizado como terapêutico em associação à amoxicilina. O processo industrial de obtenção envolve o cultivo bacteriano em processo descontínuo, enquanto que a sua purificação é realizada, principalmente, por processos de extração líquido-líquido com solventes orgânicos e etapas cromatográficas. Assim, métodos alternativos como a purificação empregando sistemas micelares de duas fases aquosas (SMDFA), os quais oferecem um ambiente hidrofóbico e hidrofílico que possibilita seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, são de grande interesse. Neste trabalho estudou-se a viabilidade da utilização de sistema micelar misto (não-iônico/aniônico) de duas fases aquosas formados pelos tensoativos não-iônico Triton X-114 e aniônico AOT na extração do ácido clavulânico proveniente de cultivo submerso de Streptomyces clavuligerus em processo descontínuo. A estabilidade do ácido clavulânico em relação ao pH, sais e suas concentrações, temperatura e aos tensoativos foi investigada visando limitar as regiões a serem estudadas nas extrações. Determinou-se ainda o ponto névoa dos sistemas Triton X-114/Tampão e Triton X-114/AOT/Tampão em diferentes condições, bem como, estudos de migração da biomolécula para as fases e do tempo necessário para o equilíbrio do sistema. A partir destas investigações iniciais, foram definidos os níveis das variáveis: concentração de AOT (0,50, 0,75 e 1,00 mM), Triton X-114 (1, 3 e 5% m/m), NaCl (0, 0,05 e 0,10 M) e temperatura (24, 26 e 28°C), cujas influências sobre o coeficiente de partição (KAC) e recuperação da biomolécula na fase diluída (nAC) foram investigadas através de um planejamento fatorial. A análise estatística e o modelo matemático obtido a partir dos resultados para KAC revelou ser possível obterem-se KAC≈ 1,65 e nAC≈ 71,58%. No entanto, a análise estatística empregando nAC como resposta apontou condições experimentais desprovidas de significado físico-químico. Em experimentos seriados realizados com Triton X-114 3% m/m, Tampão Mcllavine pH 6,5 e adição gradual de AOT, um aumento da concentração deste tensoativo levou a maiores KAC e Balanço de massa (BMAC)≈ 85%. Desta forma é possível concluir que o sistema micelar misto de duas fases aquosas proporciona baixa perda da biomolécula e partição para a fase diluída, que é uma vantagem devido a menores problemas em uma etapa subsequente de purificação. / Clavulanic acid is a potent inhibitor of β-lactamases that is used as a therapeutic in conjunction with amoxicillin. Its industrial process of production involves bacterial growing in a batch process, while its purification is carried out mainly by process of liquid-liquid extraction with organic solvents and chromatographic steps. Thus, alternative methods like the purification employing two-phase aqueous micellar systems, which offer both hydrophobic and hydrophilic environments to solute species and allow selectivity in partitioning depending on the hydrophobicity of biomolecules, are of great concern. This work studied the feasibility of using two-phase aqueous mixed (nonionic/anionic) micellar systems formed by nonionic surfactant Triton X-114 and anionic surfactant AOT to extract clavulanic acid from broths fermented by Streptomyces clavuligerus in batch process. The stability of clavulanic acid at different values of pH, salts and their concentration, temperature and in the presence of different surfactants was investigated so as to limit the areas to be submitted to further studies. The cloud-point of the systems Triton X-114/Buffer and Triton X-114/AOT/Buffer was also determined under different conditions, and studies were performed on partitioning the biomolecule between the phases and the time needed to balance of the system. From these initial investigations the following levels of the variables were defined: concentration of AOT (0.50, 0.75, and 1.00 mM), Triton X-114 (1, 3 and 5% w/w), NaCl (0, 0.05 and 0.10 M) and temperature (24, 26 and 28°C), whose influences on the partition coefficient (KCA) and yield in the top phase (nCA) were investigated using factorial design. Statistical analysis and the mathematical model derived from the results allowed obtaining KCA≈ 1.65 e nCA≈ 71.58%. However, the statistical analysis using the response nCA indicated experimental conditions devoid of any physico-chemical meaning. In serial experiments performed with Triton X-114 3% w/w, Mcllvaine buffer at 6.5 pH and gradual addition of AOT, an increase in the concentration of anionic surfactant led to higher KCA and mass balance (BMCA≈ 85%). Thus, is possible conclude that the two-phase aqueous mixed micellar system provides low loss and partition to dilute phase, which is an advantage because it implies less problems in a subsequent step of purification.

This thesis reports the results of light-scattering measurements and visual investigations of critical phenomena in the complex mixture 1-propanol (1P) + water (W) + potassium chloride (KCl) which has a special critical point (or a special thermodynamic state) known as the double critical point (DCP). The main theme of the thesis is the critical behavior on approaching a special critical point (i.e., the DCP) in a complex or associating mixture in contrast with that in simple, nonassociating mixtures. The asymptotic critical behavior in complex or associating fluids, such as polymer solutions and blends, ionic and nonionic micellar solutions, microemulsions, aqueous and nonaqueous electrolyte solutions, protein solutions, etc., is now commonly accepted to belong to the 3D-Ising universality class. However, the temperature range of the asymptotic regime in these fluids, with universal behavior, has a nonuniversal width and is, in general, smaller than that in simple or nonassociating fluids. In complex mixtures, which are made up of relatively large molecules or particle clusters of mesoscopic range, the coupling between the conventional correlation length of the critical fluctuations ( ξ) and an additional length scale associated with the mesoscale structures (ξD) is known to modify the approach towards the universal nonclassical critical behavior near their critical points. Nevertheless, the generality of this approach needs to be confirmed. There are also instances of a pure classical or close to classical behavior being observed in the critical domain of complex mixtures, although recent experimental results contradict the earlier observations. Therefore, further experimental evidences than that presently available are necessary before one can say how far the analogy between simple and complex fluids can be pushed. Variations in the effective dielectric constant of a mixture have been known to affect the critical behavior. Furthermore, we anticipate the presence of special critical points in complex mixtures to cause nontrivial modifications in the approach towards the universal asymptotic critical behavior. Special thermodynamic states are characterized by critical fluctuations with exceptionally large correlation length, and are displayed by multicomponent liquid mixtures, in which there are a multitude of thermodynamic paths by which a critical point can be approached, and offers rich information about the critical phenomena. These issues are being addressed in this research work. This thesis is organized into 7 Chapters. Chapter 1 begins with an account of the historical development of the field of critical point phenomena with a brief introduction to critical phenomena in simple fluids. Critical phenomena observed in various complex systems such as aqueous and nonaqueous ionic fluids, polymer solutions and blends, micellar and microemulsion systems, etc., are discussed, with particular attention to investigations into crossover from Ising to mean-field critical behavior observed in these systems, which are relevant to the present work. Theoretical attempts at modeling ionic criticality are cited and summarized. This is followed by a discussion of re-entrant phase transitions in multicomponent liquid systems. An account of the various types of special critical points, such as double critical point, critical double point, critical inflection point, quadruple critical point, etc., highlighting the critical behavior on approaching these special critical points, and some of the models of reentrant miscibility are briefly given. The Chapter ends with a statement on the goals of the present research work. Chapter 2 describes the instrumentation developed and the data acquisition procedures adopted for the study. Details of the thermostats and precision temperature controllers used for visual and light-scattering measurements are provided. The important design considerations relating to the achievement of a high degree of temperature stability (~ ±1 mK in the range 293-383 K) are elucidated clearly. The temperature sensors used in the present experiments and their calibration procedures are discussed. The light-scattering instrumentation is discussed in depth. The problems associated with the light-scattering techniques when it is used to study critical point phenomena, and the strategies adopted to overcome them are discussed. The sample cells used for visual investigations and light- scattering experiments, along with the procedure adopted for cleaning and filling of sample cells are also described. Chapter 3 essentially deals with the characterization of the system 1P + W + KCl. It begins with a brief introduction to the critical behavior in complex mixtures, and the motivation behind choosing the present system. The phase behavior in the present mixture, the generation of the coexistence curves and the line of critical points in the mixture, and the method used for preparation of the samples are described. The criticality of the samples is judged by the equal volume phase separation criterion through visual investigations. Addition of a small amount of salt (i.e., KCl) to the 1P + W solution induces phase separation in the mixture as a result of a salting-out process. Decreasing the salt concentration has the same effect as that of increasing pressure on the liquid-liquid demixing of this mixture. Therefore, KCl may be considered as an appropriate field variable analogous to pressure in this mixture. The mixture 1P + W + KCl exhibits reentrant phase transitions and has an array of lower (TL) and upper (TU) critical solution temperatures. It is found that the line of TL’s and TU’s, known as the line of critical points, merge (TU - TL = ΔT → 0) to form a special thermodynamic state known as the DCP. The DCP is approached as close as 509 mK (i.e., ΔT ~ 509 mK) in this work. An analysis of the critical line shows that it is roughly parabolic in shape, which is in consonance with the predictions of the lattice models and the Landau-Ginzburg theory of phase transition. In addition to the presence of a special critical point, various structure probing techniques like small angle X-ray scattering (SAXS), small angle neutron scattering (SANS), etc., indicate the presence of large-scale density inhomogeneities or clusters in 1P + W solution and its augmentation on adding small amount of KCl. Therefore, the present mixture provides a unique possibility to investigate the combined effects of molecular structuring as well as a special critical point on the critical behavior. Only a section of the coexistence surface of the mixture could be generated, owing to various experimental limitations and other problems inherent to the system. This limited further studies on the coexistence curves in the mixture. Chapter 4 reports the critical behavior of osmotic susceptibility in the present mixture. The behavior of the susceptibility exponent is deduced from static light-scattering measurements, on approaching the lower critical solution temperatures (TL’s) along different experimental paths by varying t [ =| (T - T TL)/ TL|] from the lower one-phase region. The light-scattering data analysis emphasizes the need for correction-to-scaling terms for a proper description of the data over the investigated t range. Renormalization of the critical exponents is observed as the critical line is approached along certain special paths. Experimental evidence for the doubling of the extended scaling exponent Δ1 near the DCP is shown. There is no signature of Fisher renormalization in the values of the critical exponents. The data analysis yields very large magnitudes for the correction amplitudes A1 and A2, with the first-correction amplitude A1 being negative, signifying a nonmonotonic crossover behavior of the susceptibility exponent in the mixture. The magnitudes of the correction amplitudes are observed to increase gradually as TL approaches the DCP. The increasing need for extended scaling in the neighborhood of special critical points has been noted earlier in several aqueous electrolyte solutions, in polymer-solvent systems, etc. However, the magnitudes of the correction amplitudes were not as large as that in the present case. Analysis of the effective susceptibility exponent γeff in terms of t indicate that, for the TL far away from the DCP, γeff displays a nonmonotonic crossover from its single limit 3D Ising value (~ 1.24) towards its mean-field value with increase in t. While for that closest to the DCP, γeff displays a sharp, nonmonotonic crossover from its nearly doubled 3D-Ising value (~ 2.39) towards its nearly doubled mean-field value (~ 1.84) with increase in t. For the in-between TL’s, the limiting value of γeff in the asymptotic as well as nonasymptotic regimes gradually increases towards the DCP. The renormalized Ising regime extends over a relatively larger t range for the TL closest to the DCP, and a trend towards shrinkage in the renormalized Ising regime is observed as TL shifts away from the DCP. Nevertheless, the crossover behavior to the mean-field limit extends well beyond t > 10¯2 for the TL’s studied. The crossover behavior is discussed in terms of the emergence of a new lengthscale ξD associated with the enhanced ion-induced clustering seen in the mixture, as revealed by various structure probing techniques, while the observed unique trend in the crossover is discussed in terms of the varying influence of the DCP on the critical behavior along the TL line. The discussion is extended to explain the observed critical behavior in various re-entrant systems having other special critical points. The extended renormalized Ising regime towards the DCP is also reflected in a decrease in the correlation length amplitude (ξ0) as TL approaches the DCP. It is observed that the first-correction amplitude A1 corresponding to fit using two correction terms becomes more negative as TL approaches the DCP, implying an increase in the value of the parameter ū of the crossover model [by Anisimov et al., Phys. Rev. Lett. 75, 3146 (1995)] as the DCP is approached. This increase in reflected in a trend towards a relatively sharp crossover behavior of γeff as TL shifts towards the DCP, i.e., towards the high temperature critical points. The significance of the field variable tUL in understanding different aspects of reentrant phase transitions is manifested in the present system as well. Analysis of the data in terms of tUL led to the retrieval of universal values of the exponents for all TL’s. The effective susceptibility exponent as a function of tUL displays a nonmonotonic crossover from its asymptotic 3D-Ising value towards a value slightly lower than its nonasymptotic mean-field value of 1. The limited (TL _ T) range restricted such a behavior of the effective exponent (in terms of t as well as tUL) for the lowest TL. This feature of the effective susceptibility exponent is interpreted in terms of the possibility of a nonmonotonic crossover to the mean-field value from lower values in the nonasymptotic, high tUL region, as foreseen earlier in micellar systems. The effective susceptibility exponent in terms of tUL also indicates an increase in the sharpness of crossover towards the high temperature TL’s. An increase in the sharpness of crossover with polymer chain length has been observed in polymer solutions. Therefore, our results suggest the need for further composition and temperature-dependent study of molecular structuring in the present mixture. There is also a large decrease in the dielectric constant of the mixture towards the high temperature TL’s. In Chapter 5 the light-scattering measurements are performed on approaching the DCP along the line of the upper critical solution temperatures (i.e., TU’s), by varying t [ = (T - TU )/ TU ] from the high temperature one-phase region in the mixture. A trend towards shrinkage in the simple scaling region is observed as TU shifts away from the DCP. Such a trend was not visible in the data analysis of the TL’s using the correction terms, due to the varying (TL - T) ranges. The light-scattering data analysis substantiates the existence of a nonmonotonic crossover behavior of the susceptibility exponent in the mixture. As with the TL’s, for the TU closest to the DCP, γeff displays a nonmonotonic crossover from its 3D-Ising value towards its nearly doubled mean-field value with increase in t. While for that far away from the DCP, γeff displays a nonmonotonic crossover from its single limit Ising value towards a value slightly lower than its mean-field value of 1 with increase in t. The limited (TL – T) range restricted such a behavior of γeff for the TL far away from the DCP, This feature of γeff in the nonasymptotic, high t region is yet again interpreted in terms of the possibility of a nonmonotonic crossover to the mean-field value from below. Unlike TL’s, the crossover behavior in the present case is pronounced and more sharp for all TU’s. However, the variation in the width of the renormalized Ising regime on approaching the DCP along the TU line is quite similar to that observed along the TL line. The crossover behavior is attributed to the strong ion-induced structuring seen in the mixture, while the observed trend in the crossover as TU shifts towards/away from the DCP is attributed to the varying influence of the DCP. The influence of the DCP on the critical behavior along the TU (or TL) line decreases as TU (or TL) shifts away from the DCP. Our observations indicate an increase in the sharpness of crossover as the critical temperature shifts from TL towards TU, or in other words, as the critical point shifts towards higher temperatures. SANS measurements on the present mixture indicate no difference in the growth of mesoscale clusters in the lower and upper one-phase regions in the mixture. Hence, the observed increase in the sharpness of crossover towards the TU’s is very puzzling. The dielectric constant of the major constituent (i.e., water, ~ 62 %) of the present mixture decreases from around 80 to 63 as the critical temperature shifts from TL towards TU. Therefore, our results suggest the need to look at the crossover phenomena probably from two perspectives, namely, the solvent or dielectric effect and the clustering effect. The increase in the sharpness of the crossover behavior on approaching the high temperature critical points is probably related to the macroscopic property of the mixture, i.e., to the decrease in the dielectric constant of the mixture, while the actual nonmonotonic character of the crossover behavior is related to the microscopic property of the mixture, i.e., to the clustering effects, the extent of which determines the width of the asymptotic critical domain. However, this conclusion is somewhat subtle and calls for rigorous theoretical and experimental efforts to unravel the exact dependence of the crossover behavior on the dielectric constant. Analysis using the field variable tUL in lieu of the conventional variable t led to the retrieval of unique, universal exponents for all TU’s irrespective of the ΔT value. For all TU’s, the effective susceptibility exponent in terms of tUL displays a nonmonotonic crossover from its asymptotic 3D-Ising value towards a value slightly lower than its nonasymptotic mean-field value of 1, as that observed in the t analysis of the effective exponent for the TU far away from the DCP. Like with the TL’s, the crossover behavior extends over nearly the same tUL range for the TU’s studied. However, the crossover is again sharper when compared to the TL’s. Chapter 6 reports light-scattering measurements (by heating as well as cooling) on a non phase-separating 1P + W + KCl mixture in the vicinity of the DCP. The results indicate that despite the lack of phase-separation or critical points, critical-phenomena-like fluctuations can still occur in homogeneous mixtures if they reside in some other direction than temperature or composition (like, pressure or salt concentration) of the phase diagram. Unlike earlier studies on non phase-separating mixtures, our results indicate a crossover behavior of the effective susceptibility exponent, in addition to the power-law behavior. Chapter 7 sums up the major findings of the work reported in this thesis. It also presents a range of open problems that need to be explored further in order to fully understand the results that are reported in this thesis, especially, regarding the exact dependence of dielectric constant of the mixture on the character of the crossover behavior.