Métodos espectrofotométricos em condições estacionária e em fluxo para determinação de Zn(II) foram desenvolvidos com base na formação do complexo de Zn(II) com di-2-piridil cetona saliciloilhidrazona (DPKSH), em meio de etanol-água 50% (V/V) e pH 4,5. Os complexos de Zn(II) com DPKSH foram caracterizados utilizando diferentes técnicas e as respectivas constantes de formação (βl 1,07x105 e β2 1,05x1010) e absortividades molares, em 376 nm, (εl 1,1x104 e ε2 4,5x104) determinadas. Os parâmetros para o método em condição estacionária foram otimizados e, a seguir, construiu-se a respectiva curva analítica. Obteve-se uma faixa de linearidade (lei de Beer) de (0,0293 a 2,01)x10-5mol. L-1, absortividade molar média (em 376 nm, em meio de etanol-água 50% (V/V) e pH 4,5) de 4,83x104 mol-1.L.cm-1 e limite de detecção igual a 1,24x10-7 mol.L-1 (8,11 µg.L-1). Avaliou-se a interferência de 43 íons. Alguns destes que apresentaram interferência positiva são comuns em preparações farmacêuticas e amostras biológicas, porém tal interferência pode ser facilmente eliminada. Aplicou-se o método desenvolvido à preparações farmacêuticas e amostras biológicas. Comparando-se os resultados com a técnica de absorção atômica com chama, obteve-se grande concordância. O método espectrofotométrico em fluxo foi desenvolvido a partir de alguns parâmetros previamente estudados e estabelecidos. Construiu-se a curva analítica e obteve-se uma faixa de linearidade de (0,332 a 7,04)x 10-5 mol.L-1 e limite de detecção 7,46x10-7 mol.L-1 (48,8 µg.L-1). Aplicou-se o método em preparações farmacêuticas e comparando-se os resultados com a técnica de absorção atômica com chama observou-se grande concordância. Com a finalidade de aplicar o método em fluxo a amostras biológicas, foi acoplada ao sistema uma coluna com resina de troca iônica no lugar da alça de amostragem. Após avaliar alguns parâmetros, construiu-se a curva analítica que apresentou uma faixa de linearidade de (0,126 a 3,15)x10-5 mol.L-1 e limite de detecção 2,12x10-7 mol.L-1 (13,9 µg.L-1). / Spectrophotometric methods in stationary and flow conditions were developed for Zn(II) determination based on the complex formation of Zn(II) with di-2-pyridyl ketone salicyloylhydrazona (DPKSH), in ethanol-water 50% (V/V) medium and pH 4.5. The complexes Zn(II)/DPKSH were characterized by several methods and the respective formation constants (β1 1.07x105 and β2 1.05x1010) and molar absorptivities, in 376 nm, (ε1 1.1x104 and ε2 4.5x104) were determinated. Parameters for the stationary method were optimized and, the analytical curve was obtained. A linear behavior (Beer\'s law) was verified in the range (0.0293 and 2.01)x10-5mol.L-1, the medium molar absorptivity (in 376 nm, ethanol-water 50% (V/V) medium and pH 4.5) and the detection limit are 4.83x104 mol-1.L.cm-1 and 1.24x10-7 mol.L-1 (8.11 ppb), respectively. The interference of 43 ions was evaluated. Some of them that showed present a positive interference are commom in pharmaceutical formulations and biological samples, but such interference could be easily eliminated. The developed method was applied to pharmaceutical formulations and biological samples. The results complied well with atomic absorption (flame atomization) technique. A spectrophotometric method in flow was developed upon some previously established parameters. The analytical curve was determined and a linear range was verified between (0.332 to 7.04)x10-5 mol.L-1, and the detection limit 7.46x10-7 mol.L-1 ( 48.8 µg.L-1). This method was applied to pharmaceutical formulations and the results showed a good agreement with atomic absorption (flame atomization) technique. Aiming at employing the flow method to biological samples an ion-exchange resin column was adapted substituting the sampling handle. After evaluating some parameters the analytical curve was determined and it displayed a linear range of (0.126 to 3.15)x10-5 mol.L-1, and detection limit 2.12x10-7 mol.L-1 (13.9 µg.L-1).

No description available.

Este estudo apresenta os resultados obtidos na aplicação do método de padronização k0-INAA no LANIPEN, para a análise de amostras biológicas, utilizando o programa k0-IAEA, fornecido pela Agência Internacional de Energia Atômica (IAEA). Os parâmetros de fluxo f e α do reator IEA-R1 do IPEN foram determinados na estação pneumática de irradiação e na posição de irradiação 24B/prateleira2, para irradiações curtas e longas, respectivamente. Para obter esses fatores, foi utilizado o chamado método bare triple-monitor com 197Au-96Zr- 94Zr. Para a verificação da precisão e exatidão do método, foram analisados os materiais biológicos de referência Peach Leaves (NIST SRM 1547), Mixed Polish Herbs (INCT-MPH-2) e Tomato Leaves (NIST SRM 1573a). Os critérios estatísticos Erro Relativo (bias, %), Coeficiente de Variação (CV) e U-score foram aplicados para avaliação dos resultados obtidos (média de seis replicatas). Os erros relativos (bias, %) ficaram, para maioria dos elementos, entre 0 e 30%, em relação aos valores certificados. Os Coeficientes de Variação foram inferiores a 20%, mostrando boa reprodutibilidade nos resultados obtidos experimentalmente. Os valores de U-score mostraram que todos os resultados, com exceção do valor para Na no Peach Leaves e no Tomato Leaves, estão dentro de um intervalo de confiança de 95%. Estes resultados apontam para uma promissora utilização do método k0-INAA no LAN-IPEN para a análise de amostras biológicas. / The results obtained in the application of the k0-standardization method at LAN-IPEN for biological matrices analysis, by using the k0-IAEA software, provided by the International Atomic Energy Agency (IAEA), are presented. The flux parameters f and α of the IEA-R1 reactor were determined for the pneumatic irradiation facility and for one selected irradiation position, 24B/shelf2, for short and long irradiations, respectively. In order to obtain these parameters, the bare triple-monitor method with 197Au-96Zr-94Zr was used. In order to evaluate the accuracy and precision of the methodology, the biological reference materials Peach Leaves (NIST SRM 1547), Mixed Polish Herbs (INCT-MPH-2) e Tomato Leaves (NIST SRM 1573a) were analyzed. The statistical criteria Relative Errors (bias, %), Coefficient of Variation (CV) and U-score were applied to the obtained results (mean of six replicates). The relative errors (bias, %) in relation to certified values, were, for most elements, in the range of 0 e 30. The Coefficients of Variation were below 20%, showing a good reproducibility of the results. The Uscore test showed that all results, except Na in Peach Leaves and in Tomato Leaves, were within 95% confidence interval. These results point out to a promising use of the k0-INAA method at LAN-IPEN for biological sample analysis.

Este estudo apresenta os resultados obtidos na aplicação do método de padronização k0-INAA no LANIPEN, para a análise de amostras biológicas, utilizando o programa k0-IAEA, fornecido pela Agência Internacional de Energia Atômica (IAEA). Os parâmetros de fluxo f e α do reator IEA-R1 do IPEN foram determinados na estação pneumática de irradiação e na posição de irradiação 24B/prateleira2, para irradiações curtas e longas, respectivamente. Para obter esses fatores, foi utilizado o chamado método bare triple-monitor com 197Au-96Zr- 94Zr. Para a verificação da precisão e exatidão do método, foram analisados os materiais biológicos de referência Peach Leaves (NIST SRM 1547), Mixed Polish Herbs (INCT-MPH-2) e Tomato Leaves (NIST SRM 1573a). Os critérios estatísticos Erro Relativo (bias, %), Coeficiente de Variação (CV) e U-score foram aplicados para avaliação dos resultados obtidos (média de seis replicatas). Os erros relativos (bias, %) ficaram, para maioria dos elementos, entre 0 e 30%, em relação aos valores certificados. Os Coeficientes de Variação foram inferiores a 20%, mostrando boa reprodutibilidade nos resultados obtidos experimentalmente. Os valores de U-score mostraram que todos os resultados, com exceção do valor para Na no Peach Leaves e no Tomato Leaves, estão dentro de um intervalo de confiança de 95%. Estes resultados apontam para uma promissora utilização do método k0-INAA no LAN-IPEN para a análise de amostras biológicas. / The results obtained in the application of the k0-standardization method at LAN-IPEN for biological matrices analysis, by using the k0-IAEA software, provided by the International Atomic Energy Agency (IAEA), are presented. The flux parameters f and α of the IEA-R1 reactor were determined for the pneumatic irradiation facility and for one selected irradiation position, 24B/shelf2, for short and long irradiations, respectively. In order to obtain these parameters, the bare triple-monitor method with 197Au-96Zr-94Zr was used. In order to evaluate the accuracy and precision of the methodology, the biological reference materials Peach Leaves (NIST SRM 1547), Mixed Polish Herbs (INCT-MPH-2) e Tomato Leaves (NIST SRM 1573a) were analyzed. The statistical criteria Relative Errors (bias, %), Coefficient of Variation (CV) and U-score were applied to the obtained results (mean of six replicates). The relative errors (bias, %) in relation to certified values, were, for most elements, in the range of 0 e 30. The Coefficients of Variation were below 20%, showing a good reproducibility of the results. The Uscore test showed that all results, except Na in Peach Leaves and in Tomato Leaves, were within 95% confidence interval. These results point out to a promising use of the k0-INAA method at LAN-IPEN for biological sample analysis.

L’approche analytique d’interrogation sur puce par spectrométrie de masse est une techniqueparticulièrement bien adaptée à l’analyse multiplexée requise pour la recherche de biomarqueurs dansle diagnostic moderne. L’objectif a été de contribuer aux développements technologiques etméthodologiques d’une plateforme d’analyse baptisée SUPRA-MS (Imagerie par Résonance desPlasmons de Surface en array combinée à la Spectrométrie de Masse). Des puces d’or compatiblesavec la SPRi et la MS ont été conçues et réalisées à l’aide des techniques de dépôt sous vide. Uneétude originale couplant la SPRi avec l’AFM a permis d’établir une relation entre le signal SPRmesuré et la quantité réelle de protéines fixées sur des puces nanostructurées. Nous avons ensuitedéveloppé une procédure d’immobilisation en format array (16 à λ6 spots) par liaison covalente enmonocouche des anticorps spécifiques, dirigés contre un biomarqueur du cancer du sein (LAG3) pourune analyse multiplexée d’échantillons biologiques. Du plasma humain contenant 300 ng/mL deLAGγ est injecté à la surface de la puce. L’injection est suivie en temps réel par SPRi et conduit à unecapture du biomarqueur à l’échelle de la femtomole par spots. Un traitement collectif des spots parspray pour la digestion in situ des protéines et le dépôt de matrice en vue d’une interrogation MS enMALDI-TOF a été mis au point par l’équipe du Dr Patrick Ducoroy (CLIPP-CHU Dijon). Lesrésultats MS obtenues ont permis 100 % d’identification du biomarqueur. Cette technologie sansmarquages spécifiques est particulièrement bien adaptée à la caractérisation fine des biomarqueurs et àla discrimination de variants protéiques. / The analytic approach of interrogation on chip by mass spectrometry is a suitable technique tomultiplexed analysis required for biomarker research in modern diagnosis. The aim was to contributeto the technological and methodological developments of analysis platform called SUPRA-MS(Surface Plasmon Resonance in Array coupled to Mass Spectrometry) whose goal is to provideadditional data to assay on the target protein by mass spectrometry. At first, gold chips compatiblewith SPRi and MS were designed and fabricated using vacuum deposition techniques. An originalstudy coupling SPRi with AFM has established a relationship between the SPR signal measured andthe real amount of proteins bound to nanostructured chips. We developed an immobilization procedurein array format (spots 16-96) by covalent monolayer of specific antibodies directed against the proteinLAG3, a biomarker of breast cancer for multiplex analysis of human biological samples (plasma).Human plasma containing 300 ng/mL of LAG3 is injected to the chip surface. The injection ismonitored in real time by SPRi and leads to the biomarker capture at the femtomole scale. After thebiosensing step, a collective treatment of spots by spray for in situ protein digestion and matrixdeposition in view of a MS analysis was developed by Dr. Patrick Ducoroy's team (CLIPP-CHUDijon). The MS and MS-MS analysis by MALDI-TOF was developed to analyze all spotsautomatically and determine their peptide mapping leading to 100% of the biomarker identification.This technology does not require the use of specific markers, is suitable to the biomarkerscharacterization and discrimination of protein variants.

The thesis presents experimental study on application of various polypropylene membranes (with different thicknesses, porosity and pore size) for direct coupling of membrane microextractions with capillary electrophoresis. No comprehensive study, which describes effect of these membrane parameters on extraction recoveries, was published in the past. Previous scientific works prefer application of 100 µm polypropylene membranes for their easy handling and satisfactory extraction efficiencies. Experimental part includes examination of selected polypropylene membranes as supported liquid membranes in-line coupled to capillary electrophoresis for analysis of basic drugs from complex samples. Membranes with three thicknesses (25, 100 and 170 µm) were tested. The highest extraction recoveries were achieved for the 25µm thick polypropylene membrane. Various pH conditions of donor and acceptor operational solutions were examined for extractions from real complex matrices (urine, plasma). The optimal extraction conditions were 10 mM NaOH as donor phase and 10 mM HCl as acceptor phase. 25µm membranes offer higher extraction recoveries, reduced consumption of organic solvents for membrane impregnation, similar mechanical stability and similar clean-up ability compared to thicker polypropylene membranes.

<p>In this thesis, porous graphitic carbon (PGC) has been used as packing material in packed capillary liquid chromatography. The unique chromatographic properties of PGC has been studied in some detail and applied to different analytical challenges using both electrospray ionization-mass spectrometry (ESI-MS) and ultra violet (UV) absorbance detection. </p><p>The crucial importance of disengaging the conductive PGC chromatographic separation media from the high voltage mass spectrometric interface has been shown. In the absence of a grounded point between the column and ESI emitter, a current through the column was present, and changed retention behaviors for 3-O-methyl-DOPA and tyrosine were observed. An alteration of the chromatographic properties was also seen when PGC was chemically oxidized with permanganate, possibly due to an oxidation of the few surface groups present on the PGC material. </p><p>The dynamic adsorption of the chiral selector lasalocid onto the PGC support resulted in a useful and stable chiral stationary phase. Extraordinary enantioselectivity was observed for 1-(1-naphthyl)ethylamine, and enantioseparation was also achieved for other amines, amino acids, acids and alcohols. </p><p>Finally, a new strategy for separation of small biologically active compounds in plasma and brain tissue has been developed. With PGC as stationary phase it was possible to utilize a mobile phase of high content of organic modifier, without the addition of ion-pairing agents, and still selectively separate the analytes. </p>

In this thesis, porous graphitic carbon (PGC) has been used as packing material in packed capillary liquid chromatography. The unique chromatographic properties of PGC has been studied in some detail and applied to different analytical challenges using both electrospray ionization-mass spectrometry (ESI-MS) and ultra violet (UV) absorbance detection. The crucial importance of disengaging the conductive PGC chromatographic separation media from the high voltage mass spectrometric interface has been shown. In the absence of a grounded point between the column and ESI emitter, a current through the column was present, and changed retention behaviors for 3-O-methyl-DOPA and tyrosine were observed. An alteration of the chromatographic properties was also seen when PGC was chemically oxidized with permanganate, possibly due to an oxidation of the few surface groups present on the PGC material. The dynamic adsorption of the chiral selector lasalocid onto the PGC support resulted in a useful and stable chiral stationary phase. Extraordinary enantioselectivity was observed for 1-(1-naphthyl)ethylamine, and enantioseparation was also achieved for other amines, amino acids, acids and alcohols. Finally, a new strategy for separation of small biologically active compounds in plasma and brain tissue has been developed. With PGC as stationary phase it was possible to utilize a mobile phase of high content of organic modifier, without the addition of ion-pairing agents, and still selectively separate the analytes.

This thesis describes two different sample preparation strategies for inductively coupled plasma atomic emission spectrometry (ICP-AES), and their ability regarding multi element quantification in complex samples. Sensitivity, repeatability, reproducibility and accuracy were investigated. The aim was to increase the over all efficiency, the speed of analysis, and/or the sensitivity of the analytical method. The intention was to measure analytes with concentrations ranging from ng/g to mg/g simultaneously. The aim was additionally to study chemical and physical processes occurring during the sample preparation, the sample transport to the plasma, and the atomization therein. In the first sample preparation strategy, a hydrophilic highly cross-linked iminodiacetate-agarose adsorbent, IDA-Novarose, was used for preconcentration of metal ions, and matrix elimination in natural water samples. The sorbent was synthesized with different binding capacities. The effect of the capacity on preconcentration, matrix elimination, and uptake capability at high flow rates was studied. For a high capacity IDA-Novarose (≥ 45 µmole/ml) quantitative uptake was seen even at high flow rates (100 ml/min) for Cu2+ with a high affinity to the adsorbent, and for Cd2+ with a moderate affinity. For lower capacities the uptake of Cd2+ was affected by the sample matrix and the flow rate. A method based on the determination of the conditional stability constant of the metal sorbent complex was suggested for the prediction of the sorbent capacity needed to obtain quantitative recovery and optimal matrix elimination. The sorbent was used in a flow system with online buffering for the analysis of a certified riverine water (SLRS-3), tap water and lake water. With few exceptions the results obtained by ICP-AES after preconcentration agreed well with the certified concentrations and results obtained by ICP-MS. The other sample preparation strategy discussed is a method for non digested biological samples from different animal organs for the multi element analysis by ICP-AES. This “mix and measure method” consists of a simple homogenization of the sample with a mixing rod in a small amount of neutral media, followed by dilution and direct measurement with ICP-AES. The total time of analysis is only a few minutes. The ability of this fast method to accurately quantify some elements of toxic, environmental, and/or physiological concern with the lowest possible sample dilution and the highest possible plasma load was evaluated. In 10 % liver slurry Cd, Co, and Sr, at concentration levels around 0.05 µg/g were quantified simultaneously with P and K around 2000 µg/g and with several other elements in between (Al, Ca, Cu, Fe, Mg, Mn, Pb, and Zn). The relative standard deviation of repeated measurements of samples was around 5 - 6 % for regardless of the concentration of the element. The method was also used for fast screening of the elemental distribution in mice organs (brain, heart, kidney, liver, lung and spleen).

This thesis focuses on low-cost microextraction techniques and their application for purification and preconcentration of biological samples, specifically on the experimental study of supported liquid membrane (SLM) extraction. The described microextraction technique uses commercially available filtration plates as the extraction units and allows the extraction of basic drugs from biological samples of urine and blood (in the form of dried blood spots). The experimental part includes the optimization of microextraction conditions of basic drugs from real samples through a SLM coupled in-line to lab-made capillary electrophoresis. The basic optimization of microextraction conditions involved selecting the appropriate organic phase for membrane impregnation (1:1 mixture of ENB and DHE), appropriate agitation speed for sample convection during extraction (1000 rpm), and optimal ratio of donor to acceptor volumes for high preconcentration of the analytes (400:15 µL). After basic optimization, the effect of donor alkalization with NaOH on extraction recovery (ER) was investigated. For all matrices used (saline solution, undiluted human urine samples, human capillary blood eluted from dry blood spots with deionized water), the highest ER values were achieved using a neutral donor and an acidic acceptor. The extraction time (60 minutes) was optimized based on the time profile of the microextraction for 120 minutes. This optimized microextraction method is suitable for the determination of basic drugs in real matrices with sufficient sample clean-up, preconcentration and ER values.