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.

A combinação entre o delapril (DEL), um inibidor da enzima conversora de angiotensina e o manidipino (MAN), um antagonista dos canais de cálcio, produz um efeito anti-hipertensivo sinérgico, podendo ser considerado um ótimo tratamento para pacientes com hipertensão essencial leve e moderada. No presente trabalho foram desenvolvidos e validados métodos cromatográfico e eletroforético para avaliação simultânea de DEL e MAN em produto farmacêutico. As análises por cromatografia líquida em fase reversa (CL-FR) foram executadas utilizando coluna C8 (250 mm x 4,6 mm), mantida a 35 oC. A fase móvel foi constituída por acetonitrila e solução de trietilamina 0,3%, pH 3,0 (55:45; v/v), eluída na vazão de 1,2 mL/min com detecção a 220 nm. Paralelamente, desenvolveu-se método por eletroforese capilar, utilizando modo de separação por cromatografia eletrocinética micelar (MEKC) e ácido salicílico como padrão interno. Foi utilizado capilar de sílica fundida (comprimento efetivo de 72 cm) mantido a 35 °C, com solução eletrolítica composta de tampão borato 50 mM e dodecil sulfato de sódio (SDS) 5 mM, pH 9,0. Voltagem de 25 kV foi aplicada e a injeção foi de 50 mbar durante 5 s, com detecção a 208 nm. A especificidade e a capacidade dos métodos serem indicativos de estabilidade foram demonstradas através de estudos de degradação forçada dos fármacos e pela não interferência dos excipientes nas análises. Além disso, o desenho experimental Plackett-Burman foi utilizado para a avaliação da robustez, observando-se resultados adequados para ambos métodos. Os procedimentos foram validados de acordo com guias aceitos internacionalmente, observando-se resultados em uma faixa aceitável. Os métodos propostos foram aplicados com sucesso na determinação quantitativa simultânea de DEL e MAN em comprimidos, não havendo diferença significativa dos resultados (P>0,05), contribuindo, portanto, para aprimorar o controle da qualidade, assegurando a eficácia terapêutica. / The combination of delapril (DEL), an angiotensin converting enzyme inhibitor and manidipine (MAN), an antagonist of calcium channels, produces a synergic antihypertensive effect and may be regarded as an optimal antihypertensive drug treatment in mild to moderate essential hypertensive patients. The chromatographic and eletrophoretic methods for the simultaneous evaluation of DEL and MAN in pharmaceutical product were developed and validated in the present work. The reversed-phase liquid chromatography (RP-LC) method was carried out on a C8 column (250 mm x 4.6 mm i.d., 5 μm), maintained at 35 ºC. The mobile-phase consisted of acetonitrile and a solution of triethylamine 0.3% pH 3.0 (55:45; v/v), running at a flow rate of 1.2 mL/min, with detection at 220 nm. The capillary electrophoresis method was developed using the micellar electrokinetic chromatography (MEKC) as the separation mode, and salicylic acid as internal standard. The analysis were performed on a fused-silica capillary (effective length of 72 cm) maintained at 35 °C, with 50 mM of borate buffer and 5 mM of sodium dodecyl sulfate (SDS) at pH 9.0 as background electrolyte. The separation was achieved at 25 kV applied voltage and the injection was performed at 50 mbar for 5 s, with detection at 208 nm. The specificity and stability-indicating capability of the methods were demonstrated through forced degradation studies, which also show that there is no interference of the excipients in the analysis. Moreover, the Plackett- Burman experimental design was used for robustness evaluation, giving acceptable results for both methods. The procedures were validated according to Internationals guidelines, giving results within the acceptable range. Therefore, the proposed methods were successfully applied for the simultaneous quantitative analysis of DEL and MAN in the tablet dosage form, showing non-significant difference (P>0.05), contributing to improve the quality control and to assure the therapeutic efficacy.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Interleukin 11 (IL-11) is a multifunctional cytokine in the IL-6 type family of long-chain helical cytokines, which modulates the proliferation, differentiation and maturation of various types of hematopoietic cells. Recombinant human interleukin-11 (rhIL-11) produced by DNA technology in Escherichia coli is currently being used worldwide for the prevention of thrombocytopenia and to reduce the need for platelet transfusions after myelosuppressive chemotherapy in patients with nonmyeloid malignancies. A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11) in biopharmaceutical formulations. The RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), maintained at 25ºC. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA, run as follows: time 0 to 0.1 min 40% of B; from 0.1 to 30 min linear up to 65% of B; from 30.01 to 31 min linear down to 40% of B, maintained up to 40 min. The flow rate was 1 mL/min, and using photodiode array (PDA) detection at 214 nm. Chromatographic separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1 200 μg/mL (r2 = 0.9995). The limits of detection and quantitation were 0.34 and 1.12 μg/mL, respectively. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p>0.05). The proposed method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay, showing a higher mean difference of the estimated content/potencies of 2.60% for the RP-LC method, aiming to establish new alternatives to monitor stability, improve quality control and thereby assure therapeutic efficacy of the biological medicine. / A interleucina 11 (IL-11) é uma citocina multifuncional que pertence a família da interleucina- 6 e estimula a proliferação, diferenciação e maturação de células hematopoiéticas. A interleucina-11 humana recombinante (rhIL-11) é produzida pela tecnologia do DNA em Escherichia coli e é usada clinicamente para a prevenção de trombocitopenia grave e redução da necessidade de transfusão de plaquetas após quimioterapia mielossupressiva em pacientes com neoplasias malignas não mielóides. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação de rhIL-11 em formulações de produtos farmacêuticos. Utilizou-se coluna Júpiter C4 (250 mm x 4,6 mm d.i.), mantida a 25ºC. A fase móvel A foi constituída de TFA 0,1% e a fase móvel B de acetonitrila com 0,1% TFA, eluídas no seguinte gradiente: 0 0,1 min, 40% de B; 0,1 30 min, 40 65% de B; 30,01 a 31 min, 65 40% de B, mantendo-se nesta proporção até 40 min. Utilizou-se vazão de 1 mL/min e detector de arranjo de diodos (DAD) a 214 nm. A eluição cromatográfica foi obtida no tempo de 27,6 min, sendo linear na faixa de concentração de 1 200 μg/mL (r2 = 0,9995). Os limites de detecção e quantificação foram 0,34 e 1,12 μg/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,20%, com bias inferior a 1,25%. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais não apresentaram diferença significativa em relação a forma intacta (p>0,05). O método proposto foi aplicado para a avaliação da potência de rhIL-11 e de proteínas relacionadas em formulações farmacêuticas, e os resultados foram comparados com o bioensaio, observando-se diferenças das médias de conteúdo/potência 2,60% superiores para o método por CL-FR. Contribuíu-se assim para estabelecer procedimentos que aprimoram o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biotecnológico.

Febuxostat is a novel non purine drug indicated for the treatment of hyperuricemia in gout. A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of febuxostat in pharmaceutical dosage forms. The LC method was carried out on a XTerra C18 column (150 mm x 3.9 mm I.D.), maintained at 25 ºC. The mobile phase consisted of water (pH 3.5) acetonitrile (40:60, v/v), run at a flow rate of 0.8 mL/min and using photodiode array (PDA) detection at 316 nm. The chromatographic separation was obtained with retention time of 3.9 min, and was linear over the range of 0.25 - 30 μg/mL (r2=0.9995). The specificity and stability-indicating capability of the method was proven through degradation studies were carried out by LC and MS and showing also, that there was no interference of the excipients and degradation products in the quantification of the drug. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p<0.05). The accuracy was 100.54% with bias lower than 0.65%. The limits of detection and quantitation were 0.08 and 0.28 μg/mL, respectively. The procedure was validated evaluating parameters such as the specificity, linearity, precision, accuracy, limits of detection and quantitation, robustness, and system suitability test, giving results within the acceptable range. The proposed method was applied for dissolution studies and the analysis of tablet dosage forms, contributing to assure the safety and therapeutic efficacy. / Febuxostate é um novo fármaco, não purínico, indicado para o tratamento da hiperuricemia em pacientes com gota. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para determinação de febuxostate em produtos farmacêuticos. No método por CL-FR foi utilizada coluna XTerra C18 (150 mm x 3,9 mm d.i), mantida a 25 oC. A fase móvel foi composta de água ultra-pura (pH 3,5): acetonitrila (40:60, v/v), eluída na vazão de 0,8 mL/ min com detecção no ultravioleta a 316 nm. A separação cromatográfica foi obtida no tempo de 3,9 min, sendo linear na faixa de concentração de 0,25-30 μg/mL (r2=0,9995). A especificidade do método foi comprovada através de estudos de degradação realizados por cromatografia líquida e espectrometria de massas, demonstrando que não houve interferência dos excipientes e dos produtos de degradação na quantificação do fármaco. Além disso, o teste de citotoxicidade in vitro das amostras degradadas, apresentou diferenças significativas (p <0,05) em relação à forma intacta. A precisão foi de 100,54%, com bias menor do que 0,65%. Os limites de detecção e de quantificação foram de 0,08 e 0,28 μg/mL, respectivamente. O procedimento foi validado, avaliando-se os parâmetros de especificidade, linearidade, precisão, exatidão, limite de detecção e quantificação, robustez e teste de adequabilidade do sistema, cujos resultados estão de acordo com os requisitos preconizados. O método proposto foi aplicado no estudo de dissolução e análise de formas farmacêuticas de comprimidos, contribuindo, assim, para aprimorar o controle da qualidade de medicamentos, bem como garantir a segurança e eficácia no uso terapêutico.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The interferon is a cytokine with antiviral, antiproliferative, and immunomodulatory properties. It is a protein synthesized by cells in response to viral infection, producing successive biochemistry alterations. The chromatographic methods for evaluation of recombinant interferon-alfa 2a (rhIFN-α2a) in pharmaceutical products were validated in the present work. The reversed-phase liquid chromatography method (RP-LC) was developed and validated using a Jupiter C4 column (250 mm x 4.6 mm), maintained at ambient temperature (25°C). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and mobile phase B consisted of 0.1% TFA in acetonitrile, run in gradient: 0.01 1 min, 38% of B; 1 5 min, 38 43% of B; 5.01 20 min, 43 45% of B; 20.01 30 min, 45 48% of B; 30.01 40 min linear back to 38% of B and 40 42 min, 38% of B. The flow rate used was 1 mL/min with detection at 214 nm. The chromatographic separation was obtained within 42 min and it was linear in the concentration range of 0.5 50 MIU/mL (r2=0.9999). The size exclusion method was developed and validated using a BioSep-SEC-S 2000 (300 mm x 7.8 mm), maintained at ambient temperature (25°C). The mobile phase consisted of 1mM potassium phosphate monobasic, 8mM sodium phosphate dibasic and 200mM sodium chloride buffer, pH 7.4, run at a gradient flow rate: 0.01 20 min, 0.5 mL/min; 20 25 min, 0.5 1.7 mL/min; 25 35 min, 1.7 mL/min; 35 38 min, 1.7 0.5 mL/min; 38 40 min, 0.5 mL/min. The method was linear in the concentration range of 0.5 - 50 MIU/mL (r2=0.9996). The procedures were validated by the parameters of specificity, linearity, precision, accuracy, robustness, limit of quantitation and limit of detection. The methods were applied for the evaluation of the rhIFN-α2a in pharmaceutical products, contributing for the establishment of alternatives which improve the quality control, assuring the safety and therapeutic efficacy of the biological product. / O interferon é uma citocina que possui ação antiviral, imunomoduladora e antiproliferativa. É uma proteína sintetizada pelas células em resposta a infecção viral, gerando sucessivas alterações bioquímicas. No presente trabalho foram validados métodos cromatográficos para a avaliação de interferon-alfa 2a (rhIFN-α2a) em produtos farmacêuticos. O método por cromatografia líquida em fase reversa (CL-FR) foi desenvolvido e validado empregando coluna Júpiter C4 (250 mm x 4,6 mm), mantida a temperatura ambiente (25°C). A fase móvel A foi composta de ácido trifluoracético 0,1% e a fase móvel B de ácido trifluoracético 0,1% em acetonitrila, eluídas no gradiente: 0,01 1 min, 38% de B; 1 5 min, 38 43% de B; 5,01 20 min, 43 45% de B; 20,01 30 min, 45 48% de B; 30,01 40 min, 48 38% de B, mantendo-se nesta proporção até 42 min. Utilizou-se vazão de 1 mL/min e detecção no ultravioleta a 214 nm. A separação cromatográfica foi obtida no tempo de 42 min, sendo linear na faixa de concentração de 0,5 - 50 MUI/mL (r2=0,9999). Paralelamente, desenvolveu-se e validou-se método cromatográfico por exclusão molecular (CL-EM) empregando coluna BioSep-SECS 2000 (300 mm x 7,8 mm), mantida a temperatura ambiente (25°C). A fase móvel foi composta de tampão fosfato de potássio monobásico 1mM, fosfato de sódio dibásico 8mM e cloreto de sódio 200mM, pH 7,4, eluída no gradiente de fluxo: 0,01 20 min, 0,5 mL/min; 20 25 min, 0,5 1,7 mL/min; 25 35 min, 1,7 mL/min; 35 38 min, 1,7 0,5 mL/min; 38 40 min, 0,5 mL/min. O método foi linear na faixa de concentração de 0,5 - 50 MUI/mL (r2=0,9996). Ambos os procedimentos foram validados com base nos parâmetros de especificidade, linearidade, precisão, exatidão, robustez, limite de quantificação e detecção. Os métodos foram aplicados para avaliação de rhIFN-α2a em produtos farmacêuticos, contribuindo para o estabelecimento de alternativas que aprimoram o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biológico.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that regulates the proliferation and differentiation of hematopoietic progenitor cells and activates mature granulocytes and macrophages. Clinically is used in enhancing hematopoietic recovery after cancer chemotherapy and bone marrow transplantation. A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of the non-glycosylated recombinant rhGM-CSF (Molgramostim) in biopharmaceutical formulations. The RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), maintained at 45 ºC. The mobile phase A consisted of 0.1% TFA and the mobile phase B consisted of 0.1% TFA in acetonitrile, run at a gradient from: 0.01 34 min, 37% 50% of B; 34 35 min linear back to 37% of B and 35 40 min, 37% of B. The flow rate was 1 mL/min, and using photodiode array (PDA) detection at 214 nm. The chromatographic separation was obtained with the retention time of 29.2 min, and was linear over the concentration range of 2-300 μg/mL (r2 = 0.9992). The procedure was validated evaluating parameters such as the specificity, linearity, precision, accuracy, robustness, limit of detection and limit of quantitation. The specificity was proven through degradation studies, showing that also there was no interference of the excipients. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p<0.05). The proposed method was applied for the analysis of molgramostim and their related proteins, and the results were correlated to the bioassay, showing mean differences of the potency 1.02% higher for the RP-LC method, contributing to establish alternatives to characterize and monitoring its instability, improving the quality control and assuring the therapeutic efficacy. / O fator estimulador da colônia de granulócitos e macrófagos (GM-CSF) é uma citocina que regula a proliferação e diferenciação de células progenitoras hematopoiéticas e ativa granulócitos e macrófagos maduros. É clinicamente indicado após quimioterapia para o câncer e após transplante de medula óssea. No presente trabalho foi validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação da molgramostima, forma recombinante não-glicosilada do GM-CSF, em formulação biofarmacêutica. Utilizou-se coluna Júpiter C4 (250 mm x 4,6 mm d.i.), mantida a 45 °C. A fase móvel A foi composta de ácido trifluoracético 0,1% e a fase móvel B de ácido trifluoracético 0,1% em acetonitrila, eluídas no gradiente: 0,01 34 min, 37 50% de B; 34 35 min, 50 37% de B, mantendo-se nesta proporção até 40 min. Utilizou-se vazão de 1 mL/min, usando detector de arranjo de diodos (DAD) a 214 nm. A separação cromatográfica foi obtida no tempo de 29,2 min, sendo linear na faixa de concentração de 2 300 μg/mL (r2 = 0,9992). O procedimento foi validado, avaliando-se os parâmetros de especificidade, linearidade, precisão, exatidão, robustez, limite de detecção e quantificação. A especificidade foi comprovada através de estudos de degradação, demonstrando também que não houve interferência dos excipientes. Além disso, realizou-se o teste de citotoxicidade in vitro dos produtos degradados que apresentaram diferença significativa (p<0,05). O método foi aplicado para avaliação de potência de molgramostima e de proteínas relacionadas, e os resultados foram correlacionados com o bioensaio, observando-se diferenças médias de potência 1,02% superiores por CL-FR. Contribuiu-se assim para o estabelecimento de alternativas que aprimoram o controle da qualidade, garantindo a segurança e eficácia terapêutica.

A combinação entre o delapril (DEL), um inibidor da enzima conversora de angiotensina e o manidipino (MAN), um antagonista dos canais de cálcio, produz um efeito anti-hipertensivo sinérgico, podendo ser considerado um ótimo tratamento para pacientes com hipertensão essencial leve e moderada. No presente trabalho foram desenvolvidos e validados métodos cromatográfico e eletroforético para avaliação simultânea de DEL e MAN em produto farmacêutico. As análises por cromatografia líquida em fase reversa (CL-FR) foram executadas utilizando coluna C8 (250 mm x 4,6 mm), mantida a 35 oC. A fase móvel foi constituída por acetonitrila e solução de trietilamina 0,3%, pH 3,0 (55:45; v/v), eluída na vazão de 1,2 mL/min com detecção a 220 nm. Paralelamente, desenvolveu-se método por eletroforese capilar, utilizando modo de separação por cromatografia eletrocinética micelar (MEKC) e ácido salicílico como padrão interno. Foi utilizado capilar de sílica fundida (comprimento efetivo de 72 cm) mantido a 35 °C, com solução eletrolítica composta de tampão borato 50 mM e dodecil sulfato de sódio (SDS) 5 mM, pH 9,0. Voltagem de 25 kV foi aplicada e a injeção foi de 50 mbar durante 5 s, com detecção a 208 nm. A especificidade e a capacidade dos métodos serem indicativos de estabilidade foram demonstradas através de estudos de degradação forçada dos fármacos e pela não interferência dos excipientes nas análises. Além disso, o desenho experimental Plackett-Burman foi utilizado para a avaliação da robustez, observando-se resultados adequados para ambos métodos. Os procedimentos foram validados de acordo com guias aceitos internacionalmente, observando-se resultados em uma faixa aceitável. Os métodos propostos foram aplicados com sucesso na determinação quantitativa simultânea de DEL e MAN em comprimidos, não havendo diferença significativa dos resultados (P>0,05), contribuindo, portanto, para aprimorar o controle da qualidade, assegurando a eficácia terapêutica. / The combination of delapril (DEL), an angiotensin converting enzyme inhibitor and manidipine (MAN), an antagonist of calcium channels, produces a synergic antihypertensive effect and may be regarded as an optimal antihypertensive drug treatment in mild to moderate essential hypertensive patients. The chromatographic and eletrophoretic methods for the simultaneous evaluation of DEL and MAN in pharmaceutical product were developed and validated in the present work. The reversed-phase liquid chromatography (RP-LC) method was carried out on a C8 column (250 mm x 4.6 mm i.d., 5 μm), maintained at 35 ºC. The mobile-phase consisted of acetonitrile and a solution of triethylamine 0.3% pH 3.0 (55:45; v/v), running at a flow rate of 1.2 mL/min, with detection at 220 nm. The capillary electrophoresis method was developed using the micellar electrokinetic chromatography (MEKC) as the separation mode, and salicylic acid as internal standard. The analysis were performed on a fused-silica capillary (effective length of 72 cm) maintained at 35 °C, with 50 mM of borate buffer and 5 mM of sodium dodecyl sulfate (SDS) at pH 9.0 as background electrolyte. The separation was achieved at 25 kV applied voltage and the injection was performed at 50 mbar for 5 s, with detection at 208 nm. The specificity and stability-indicating capability of the methods were demonstrated through forced degradation studies, which also show that there is no interference of the excipients in the analysis. Moreover, the Plackett- Burman experimental design was used for robustness evaluation, giving acceptable results for both methods. The procedures were validated according to Internationals guidelines, giving results within the acceptable range. Therefore, the proposed methods were successfully applied for the simultaneous quantitative analysis of DEL and MAN in the tablet dosage form, showing non-significant difference (P>0.05), contributing to improve the quality control and to assure the therapeutic efficacy.

Orientador: Isabel Cristina Sales Fontes Jardim / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T02:08:02Z (GMT). No. of bitstreams: 1 Begnini_FernandaRibeiro_M.pdf: 1951363 bytes, checksum: fe0989f49b67e9b16db7e81f2c1544f5 (MD5) Previous issue date: 2011 / Resumo: Novas fases estacionárias (FE) para Cromatografia Líquida de Alta Eficiência em fase reversa (CLAE-FR) foram preparadas a partir da sorção e imobilização por micro-ondas do copolímero poli(2- fenilpropil)metilsiloxano (PFPMS) sobre suporte de sílica Kromasil (tipo B, esférica, 5 mm). A FE preparada com carga do copolímero de 50%, imobilizada a 540 W por 80 minutos e extraída com clorofórmio (3 horas) e metanol (2 horas) após 8 dias de imobilização, apresentou desempenho cromatográfico satisfatório, com eficiências acima de 60000 pratos/m e parâmetros cromatográficos na faixa aceita pela literatura. Através da caracterização físico-química da FE preparada, pelas técnicas de RMN Si e C, e espectroscopia de absorção no infravermelho, entre outras, foi possível sugerir que o PFPMS encontra-se fisicamente sorvido e quimicamente ligado à sílica. A caracterização cromatográfica mostrou que a FE possui seletividade hidrofóbica, seletividade estérica, pouca interação com analitos polares e grupos silanóis residuais, sendo características apropriadas para uma FR. A FE preparada apresenta grande potencial na separação de agrotóxicos, fármacos e hidrocarbonetos policíclicos aromáticos, além de possuir estabilidade em meio ácido / Abstract: A new stationary phase for reversed-phase high-performance liquid chromatography (RP-HPLC) was prepared by sorption and microwave immobilization of the copolymer poly(2- phenylpropyl)methylsiloxane (PPPMS) onto a silica support (Kromasil®, type B, 5 mm). The stationary phase prepared with copolymeric content of 50%, immobilized at 540 W for 80 min and extracted with chloroform (3 hours) and methanol (2 hours) eight days after immobilization, showed satisfactory chromatographic performance, with efficiency above 60000 plates/m and symmetrical peaks. The physical¿chemical characterization of the new microwave immobilized stationary phase using solid-state NMR spectroscopy and infrared spectroscopy suggested that the copolymer is physically sorbed and chemically bonded onto the silica support. The chromatographic characterization showed that the stationary phase has hydrophobic selectivity, steric selectivity, low interaction with polar analytes and residual silanols, suggesting that the stationary phase prepared is promising to be used in RP-HPLC. The microwave immobilized stationary phase presented chemical stability and potential applicability to separation of pesticides, pharmaceutical drugs and polycyclic aromatic hydrocarbons / Mestrado / Quimica Analitica / Mestre em Química

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

Introduction Oligonucleotides are short nucleic acid chains, usually 19-27mer long. They bind to their corresponding chain, making a specific inhibition possible. In pharmaceuticals, this can be used to inhibit the expression of a gene or protein of interest. Oligonucleotides are usually analyzed based on separation using both hydrophobic and ion-exchange properties. In this project, the possibility to use a mixed-mode column to separate these oligonucleotides and their impurities were explored. Method Liquid chromatography is used as the separation method and the method of detection is both mass spectrometry and UV. Three different columns are evaluated; C18, DNAPac RP, and mixed-mode RP/WAX. Results and discussion Different compositions of mobile phases and gradients are evaluated based on a literature study. Triethylamine, triethylammonium acetate, ammonium formate, hexafluoroisopropanol is used along with both methanol and acetonitrile. Phosphate buffer is evaluated on LC-UV. The results from the C18 column displays a good separation of the oligonucleotides, whilst the DNAPac RP is not as sufficient using the same mobile phases. The mixed-mode column provides good separation and selectivity using phosphate buffer and UV detection. Conclusion Mixed-mode column has the potential to be used for separation of oligonucleotides and one future focus would be to make the mobile phase compatible with mass spectrometry. Phosphate buffer and UV detection seems to be the go-to mobile phase using mixed-mode column even though MS is a more powerful tool for the characterization and identification of oligonucleotides. This provides a hint about the challenge in making the mobile phase MS compatible.