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Made available in DSpace on 2014-10-09T12:33:04Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:06Z (GMT). No. of bitstreams: 0 / O carvão ativado é um tipo comum de rejeito radioativo que contém elevada concentração de produtos de ativação e fissão. O gerenciamento deste rejeito inclui a sua caracterização, visando à determinação e quantificação dos radionuclídeos específicos, incluindo aqueles conhecidos como Radionuclídeos de Difícil Medição (RDM). A análise dos RDMs geralmente envolve análises radioquímicas complexas para purificação e separação dos radionuclídeos, as quais são caras e demandam muito tempo. O objetivo deste trabalho foi definir uma metodologia de análise sequencial de isótopos de urânio, netúnio, plutônio, amerício e cúrio, presentes em um tipo de rejeito radioativo, avaliando-se rendimento químico, tempo de análise, quantidade de rejeito secundário gerado e custo. Foram comparadas e validadas três metodologias que empregam a troca iônica (TI + EC), extração cromatográfica (EC) e extração com polímeros (ECP). O rejeito estudado foi o carvão ativado, proveniente do sistema de purificação de água do circuito primário de refrigeração do reator IEA-R1. As amostras de carvão foram dissolvidas por digestão ácida, seguida de purificação e separação dos isótopos com resinas de troca iônica, extração cromatográfica e extração com polímeros. Os isótopos foram analisados em um espectrômetro alfa, equipado com detectores de barreira de superfície. O rendimento químico de todos os elementos foi satisfatório para os métodos TI + EC e EC. Para o método ECP, apenas o rendimento químico do U foi comparável aos outros métodos. As análises estatísticas dos resultados bem como a análise de custo e volume de rejeito secundário gerado demonstraram que o método EC é o mais adequado para a identificação e quantificação dos isótopos estudados em carvão ativado. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Oligonucleotides (ONs) are widely used in different applications in life science, forensic, in i.e. family tree DNA test for humans and in diagnostic applications in several fields. The use of ONs in biopharmaceutical therapeutic areas also generates new challenges handling more complex molecules which results in the need of further developed analytical techniques. Anion exchange chromatography (AEX) is a common separation technique for biomolecules and is based on charge attraction between the analyte and the stationary phase. The chromatographic system is complex and often high pH and a high salt concentration is needed for the elution to occur, which in some systems can be corrosive for both the column and the instrument. The aim of this study was to evaluate new mobile phase compositions with lower salt concentrations, organic modifier, and usage of a buffer to increase the control of the pH. This was done by evaluation of three columns developed for AEX and uses different chemical and methodical modification of the mobile phase to control the retention to the stationary phase. The influence of pH, temperature, and methanol (MeOH) content in the buffer were studied by evaluation of resolution, asymmetry, and efficiency responses. Three oligonucleotides with 16, 18 and 19 T-bases in the chain were used in the study of three AIE columns. High pH, elevated temperature and the addition of an organic modifier were used for unfolding of the oligonucleotide chain and generating more efficient separations. Other parameters such as gradient slope and initial concentration of the eluting buffer were also studied, and the findings clearly show that the chromatographic conditions influence the resolution, asymmetry, and efficiency. / Oligonukleotider används i flera olika branscher som forensik och inom life sience till diagnostiska och medicinska applikationer. Eftersom applikationsområdena ökar och forskningen går framåt så blir molekylerna mer komplexa, vilket i sin tur kräver att dom analytiska teknikerna också behöver utvecklas. En vanlig separationsteknik för biomolekyler är anjonbyteskromatografi, då den bygger på laddningsattraktion mellan analyten och den stationära fasen. Oligonukleotider har en negativ nettoladdning på grund av den negativt laddade fosfatgruppen i kedjan. Genom att modifiera de kemiska egenskaperna i den mobila fasen är det därför möjligt att i viss grad kontrollera retentionen till den stationära fasen.       Då det kromatografiska systemet är komplext och det ofta behövs högt pH och/eller en hög saltkoncentration för eluering av analyten kan det i vissa system verka korrosivt för både kolonnen och instrumentet. Det initiala målet med detta arbete var att ta fram olika mobila faser med lägre saltkoncentration, organiskt lösningsmedel och användande av buffer för att kontrollera pH. Detta gjordes genom att utvärdera den kromatografiska kapaciteten hos tre olika anjonbyteskolonner samt använda olika kemiska- och metodiska modifieringar av den mobila fasen för att kontrollera analytens retention. Mer specifikt så kommer påverkan av pH, temperatur och innehåll av metanol i den mobila fasen att studeras genom att utvärdera upplösning, asymmetri och effektivitet i de erhållna kromatogrammen. Analytmixen innehåller tre olika oligonukleotider vilka består av 16, 18 eller 19 T-baser i sekvensen. Genom att höja pH, temperatur och innehåll av metanol i den mobila fasen påvisas mer effektiva separationer. Andra faktorer som gradientlutning och initialkoncentration av den eluerande fasen studeras också med goda resultat när det gäller dess påverkan på upplösning, asymmetri och effektivitet.

Ion exchange chromatography (IC) is an analytical technique used to separate charged molecules including ions, proteins, small nucleotides, and amino acids. It can function in anion or cation mode. In this dissertation, anion exchange chromatography was used, and column materials were made in our lab with resorcinarene-based compounds called cavitands. Cavitands create cavities to bind to molecules because of their three-dimensional structure. Two new gradient IC methods were established to identify and quantify perrhenate and perfluoroalkyl substances (PFAS) by customized resorcinarene-based column, zinc cyclen resoecinarene (ZCR) and arginine methyl ester (RUE) columns. The ZCR column accomplished outstanding separation of perrhenate from other anions such as chloride and sulfate by using a gradient elution of 2-60 mM NaOH. There was a logarithmic relationship between the perrhenate concentration and its retention time. In addition to separating anions, the ZCR column was able to preconcentrate perrhenate with over 90% recovery in different conditions. RUE was successfully synthesized and attached to polystyrene resin and used in IC to separate the PFAS, perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorobutanesulfonic acid (PFBS), perfluorohexanoic acid (PFHxA), perfluorohexanesulphonic acid (PFHxS), and perfluorooctanoic acid (PFOA). The sample preparation for the PFAS was simple and only needed filtration. A gradient method starting with 70 mM NaOH and going to pure water was necessary to separate the PFAS. There was no detectable PFAS in Provo tap water and Utah Lake water by our method. Although the LOD and LOQ of PFAS were not as low as the existing methods, the IC method does not require complicated sample preparation steps to separate and quantify PFAS. Binding studies of RUE and RUA were done with organic acids, including citric, malic, and succinic acid, and PFAS including PFBA, and PFHxA. The strongest binding was for L-malic acid followed by succinic acid, D-malic acid, pentanoic acid, citric acid, and dimethyl L-malate. RUE displayed some chiral recognition between L-malic acid and D-malic acid. Unfortunately, it did not show significant differences in binding between the different PFAS even though RUE had been able to separate them by IC.

The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli. A positively charged purification tag, Zbasic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Zbasic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Zbasic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Zbasic, was effected by adsorption to a second cation-exchanger. Using a similar strategy, a purification tag with a negatively charged surface, denoted Zacid, was constructed and thoroughly characterised. Contrary to Zbasic, the negatively charged Zacid was highly unstructured in a low conductivity environment. Despite this, all Zacid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer. The positively charged domain, Zbasic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin. / QC 20101020

<p>The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.</p><p>A positively charged purification tag, Z_basic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Z_basic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified <i>Escherichia coli</i> homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Z_basic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Z_basic, was effected by adsorption to a second cation-exchanger. </p><p>Using a similar strategy, a purification tag with a negatively charged surface, denoted Z_acid, was constructed and thoroughly characterised. Contrary to Z_basic, the negatively charged Z_acid was highly unstructured in a low conductivity environment. Despite this, all Z_acid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography</p><p>Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed <i>in vivo</i> by the use of a flow cytometer. </p><p>The positively charged domain, Z_basic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Z_basic as a reversible linker to the cation-exchanger resin.</p>

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122 f. / Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2013-03-22T17:20:17Z No. of bitstreams: 1 Tese corrigida final.pdf: 1322795 bytes, checksum: a277616fb5634d49040e3b569eae8dd2 (MD5) / Approved for entry into archive by Ana Hilda Fonseca(anahilda@ufba.br) on 2013-06-05T17:38:45Z (GMT) No. of bitstreams: 1 Tese corrigida final.pdf: 1322795 bytes, checksum: a277616fb5634d49040e3b569eae8dd2 (MD5) / Made available in DSpace on 2013-06-05T17:38:45Z (GMT). No. of bitstreams: 1 Tese corrigida final.pdf: 1322795 bytes, checksum: a277616fb5634d49040e3b569eae8dd2 (MD5) Previous issue date: 2012 / CAPES / No presente trabalho, desenvolveu-se um método alternativo para determinação de cobre, ferro, níquel e zinco, usando a espectrometria de fluorescência de raios-X com energia dispersiva (EDXRF), em amostras de etanol combustível e cachaça, após procedimento de pré-concentração. O procedimento consistiu na retenção dos analitos em papel cromatográfico de troca iônica, suporte sólido que se mostrou apropriado para medições diretas por EDXRF. Algumas variáveis foram otimizadas e os melhores resultados obtidos foram: vazão de amostragem de 2,0 mL min-1, pH entre 5,0 e 7,5 e volume de amostragem de 10 mL. No procedimento, com auxílio de uma bomba peristáltica, alíquotas da solução padrão ou amostra foram passadas em papel cromatográfico de troca catiônica, que foi cortado em círculos com 13 mm de diâmetro e adaptados em suporte para filtração. Após a secagem dos papéis cromatográficos, foram realizadas medições diretas no papel empregando espectrômetro de fluorescência de raios-x de energia dispersiva. As curvas de calibração apresentaram resposta linear no intervalo de 0,050 a 1,50 μg mL-1 dos analitos. As repetibilidades, expressas em termos de RSD das soluções-padrão contendo 0,25 μg mL-1 de Cu, Fe, Ni e Zn, calculadas a partir de 15 determinações consecutivas, foram de 2,5, 2,8, 3,0 e 2,7%; e 3,1, 2,9, 2,8 e 2,7%, para matrizes com 96% e 50% de etanol, respectivamente. Obteve-se limites de quantificação (LQ) iguais a 43, 50, 50 e 40; e 47, 54, 59 e 45 μg mL-1, para matrizes com 96% e 50% de etanol, respectivamente. O procedimento foi verificado através de testes de recuperação, adicionando-se 100 μg L-1 de cada metal nas diferentes amostras, com resultados entre 92 e 99%. Os resultados obtidos pelo método proposto foram comparados com os resultados obtidos por um método comparativo usando ICP OES, após digestão das amostras, não apresentando diferenças significativas, com um nível de confiança de 95%. O método proposto é simples e não exigiu que as amostras fossem submetidas a qualquer pré-tratamento drástico ou demorado. / Salvador

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