The antibody immunoglobulin G (IgG) can be tryptically digested into smaller peptides. This study attempted to develop a separation method for those peptides using RP-HPLC with a C18 column at room temperature. Optimizing separation of trypsin cleaved cytochrome C was used as a guideline before analyzing IgG. The optimized analysis of Cytochrome C was performed at wavelength 280nm (UV) and methanol was used as an organic solvent in mobile phase (B). A fast gradient to 100% mobile phase B with low flow rate gave favorable results for cytochrome C. A slow gradient to 100% mobile phase B was suited for IgG separation. The optimized gradient elution of cytochrome C and IgG was performed at 0.3 and 0.8 ml/min, respectively. / Antikroppen Immunoglobulin G (IgG) kan klyvas till peptider med enzymet trypsin. Under denna studie ska utvecklades en separationsmetod för dessa peptider med RP-HPLC. Separationen utfördes med en C18 kolonn i rumstemperatur. Först optimerades en separation av trypsin-klyvt cytokrom C vars optimerade parametrar sedan användes som grund för IgG-separation. Optimeringen utfördes vid våglängden 280 nm(UV) och metanol användes som organiskt lösningsmedel i mobil fas (B). En snabb gradient upp till 100% B med låg flödeshastighet gav mest gynnsamt resultat för cytokrom C. Separationen av IgG gynnades av ett högt flöde och en långsam gradient till 100% B. Den optimerade gradientelueringen för cytokrom C och IgG gjordes vid flödet 0.3 respektive 0.8 ml/min.

1ABSTRACTCarbohydrate-deficient transferrin (CDT) is a biomarker for excessive and long-running intake of alcohol. It is a form of transferrin called disialotransferrin that under normal circumstances is <2 % of total transferrin in human blood. An increase is seen when alcohol consumption exceeds450 g per week. CDT is analyzed in serum usinghigh performance liquid chromatography (HPLC) and UV/VIS detection. The purpose of this study was to investigate if an “in routine” method could be improved by switching columns.With ion exchange chromatography transferrin glycoforms are separated and quantified. The carbohydrate-deficient transferrin glycoforms have an isoelectric point between 5,7-5,9 that depends on the number of sialic acids on the molecule. With the use of a salt gradient and pH above the isoelectric point the glycoforms can beseparated depending on their affinity to the stationary phase. Batches with patient and control serum was first analyzed on the routine column Source 15Q PE and then on the alternative column Reprospher 200 SAX 5μm.Student’s t-test showed that the two methods’results correlated but were significantly different. A Bland-Altman plot illustrated differences between the two columns. High and low control serum values from Reprospher were lower than the accepted interval. In this study Reprospher’s stationary phase seemed to be affected to such an extent that stabile retention time, better resolution, and stabile values could not be achieved and because the information about the column was lacking an attempt to regeneratethe columnwas not conducted.

No description available.

The metabolism of amino acids produces important chemical signaling molecules called neurotransmitters, which are responsible for carrying out important actions within the human body. There are approximately one hundred identified neurotransmitters. Neurotransmitter study is important due to their involvement in biological, physiological, pharmacological, and pathological functions. Commonly employed methods for neurotransmitter detection are mainly based upon microdialysis. However, the methods suffer from disadvantages. Microdialysis fails to determine the absolute concentration of analytes and therefore requires it to be tied in with an analytical technique such as high performance liquid chromatography or capillary electrophoresis. Although high performance liquid chromatography is the most powerful analytical technique to date, it necessitates high maintenance and suffers from poor temporal resolution. While capillary electrophoresis affords more rapid separations than high performance liquid chromatography, it suffers from poor concentration limits of detection and requires large sample dilutions of highly conductive samples, such as biological fluids. Consequently, research is focused on detection of various amino acids and neurotransmitters employing novel analytical techniques along with traditional capillary electrophoresis. First, a method was developed using traditional capillary electrophoresis with laser induced fluorescence detection to detect two major excitatory neurotransmitters, glutamate and aspartate in planaria. The method was later applied to detect several biogenic amines using micellar electrokinetic chromatography with laser induced fluorescence detection in planaria to study the effect of feeding on the levels of biogenic amines within individual planaria homogenates. The concentration sensitivity issue of capillary electrophoresis led to the use of a new method for sensitive neurotransmitter measurements, gradient elution isotachophoresis. Gradient elution isotachophoresis is an efficient capillary-based enrichment and separation technique based on balancing hydrodynamic counter-flow against electrophoresis. Enrichment is achieved with the aid of high concentrations of leading electrolyte in the counter-flow solution that creates an ionic interface near the capillary inlet. Discrete electrolyte spacers or carrier ampholyte mixtures are used to separate analyte zones. The method was applied to the enrichment and separation of physiologically relevant concentrations of aspartate and glutamate labeled with dansyl chloride, phenyl isothiocyanate, or carboxyfluorescein, succinimidyl ester in artificial cerebrospinal fluid using ultraviolet absorbance detection. Finally, gradient elution isotachophoresis was combined with capillary zone electrophoresis to eliminate the use of spacers and provide rapid separations and enrichment. The technique was applied for the detection of biogenic amines in a glass microfluidic device. / Chemistry

The steviolglycosides are the natural, sweet substances from Stevia rebaudiana Bertoni. It affect human health positively and its sweetness is 300x stronger than the sweetness of sucrose. That's why it's used to sweetening the commercial products. Because of its potential properties it's good to have an appropriate method to isolate it. High performance liquid chromatography ( HPLC) is based on the separation of analyte between two immiscible phases with high pressure pump and appropriately chosen stationary phase in the column. Than the analytes come out from the column in different retention times. This master´s thesis follows up selection of the best HPLC system for isolation of the main steviolglycosides and its analysis in commercial products. In the theoretical part of the thesis is described the origin, basic characteristics, botanical description, cultivation and affect to the human health of Stevia rebaudiana Bertoni and its use in food industry. There are also concisely characterized the sweet substances contained in the plant, so called steviolglycosides. Than there are given the theoretical basics of high performance liquid chromatography, HPLC instrumentation and its specific applications at sleviolglycosides with the basic chromatographic parameters. The object of the first experimental part was to research the optimum conditions for time and separation effective chromatographic analysis and select the best chromatographic system for isolation of steviolglycosides. In the second experimental part, I have compared and defined the main steviolglycosides (stevioside, rebaudioside A) in nine selected products, commercially available in Czech republic, with the best chromatographic method. In these products was the contain of the stevioside or rebaudioside A confirmed or refused.

MSc (Chemistry) / Department of Chemistry

Extensive research on amino acids, and even other biochemical assays usually present in low concentration and volume face challenges using known analytical techniques for analysis of traces amounts. Some limiting factors are the achievable efficiency, sensitivity (resulting from instrument limit of detection and/or experimental methods), volume requirement, and total analysis time. Counterflow electrofocusing techniques combining forces of electrophoresis and bulk flow (pressure driven flow and/or electroosmotic flow) provides a basis for the development of alternative detection techniques geared towards improving peak efficiency, sensitivity and time. The work presented gives a vivid description of recently developed capillary counterflow techniques: gradient elution isotachophoresis (GEITP) using UV detection, GEITP coupled to Capillary Zonal Electrophoresis (GEITP-CZE), temperature gradient focusing (TGF), and temperature gradient denaturing focusing (TGDF). A first demonstration of GEITP using UV detection was applied to enrichment and separation of tyrosine and tryptophan under optimized conditions. Primarily, separation is achieved as the result of the difference in electrophoretic velocity of analytes in a discontinuous buffer system. First, a plug of sample is allowed to preconcentrate (or enrich) between high mobility leading electrolyte (LE) and low mobility trailing electrolyte (TE) under controlled hydrodynamic pressure and continuous injection. This preconcentration is initiated outside the capillary in a conductivity bubble. Although analyte focus according to their electrophoretic velocity, the inclusion of spacer molecule in sample matrix was instrumental in achieving separation with tradeoff between analyte resolution and enrichment. Gradient produced results from reduction in pressure as sample is loaded on column. Separation using this technique is a one step process. A hybrid method marking the first successful coupling of GEITP to CZE with laser induced fluorescence detection was used for separation of six fluorescently labeled amino acids (which formulates the Mars-7). An eleven minute separation was achieved under optimized conditions. A proof-of-concept demonstration of TGF with LIF detection showed focusing and separation of fluorescein and carboxyfluorescein dye molecules, and carboxyfluorescein-labeled glutamate and aspartate. The generation of null focusing points along the thermal separation column (set between 80-20oC) was produced in collaboration with continuous sample injection, discontinuous buffer system and balancing of counterflows (electrophoresis and bulk flow). Preliminary results showed stability in instrument. The TGDF method carried out on a TGF apparatus is a modification to the temperature gradient gel electrophoresis and denaturing gradient gel electrophoresis methods. In principle, TGDF primarily achieves focusing and separation on a thermal separation column (set between 20 to 80 oC) as a result of conformational changes. It is currently being developed for the detection and simultaneous separation of single and double stranded DNA. Preliminary results show enrichment of wildtype and mutant synthetic DNA strands (containing twenty-four base pairs in sequence) in different buffer matrices. / Chemistry

Novel sol-gel titania-poly(dimethylsiloxane) (TiO2-PDMS) and titania-silica-N-(triethoxysilylpropyl)-O-polyethylene oxide urethane (TiO2-SiO2-TESP-PEO) coatings were developed for capillary microextraction (CME) to perform on-line preconcentration and HPLC analysis of trace impurities in aqueous samples. Due to chemical inertness of titania, effective covalent binding of a suitable organic ligand to its surface is difficult via conventional surface modification methods. In this research, sol-gel chemistry was employed to chemically bind hydroxy-terminated poly(dimethylsiloxane) (PDMS) and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane (TESP-PEO) to sol-gel titania and sol-gel titania-silica network, respectively. A method is presented describing in situ preparation of the titania-based sol-gel PDMS and TESP-PEO coatings and their immobilization on the inner surface of a fused-silica microextraction capillary. To perform on-line CME-HPLC, the sol-gel TiO2-PDMS or TiO2-SiO2-TESP-PEO capillarywas installed in the HPLC injection port as an external sampling loop, and a conventionalHPLC separation column was used for the liquid chromatographic separation. The sol-gel TiO2-PDMS-coated microextraction capillary was used for on-line CME-HPLC analysis of non-polar and moderately polar analytes, and the sol-gel coatings showed excellent pH (1-13), and solvent (acetonitrile and methanol) stabilities under elevated temperatures (150 C) over analogous non-sol-gel silica-based coatings. Extraction of highly polar analytes, especially from aqueous phases is not an easy task. However, the sol-gel TiO2-SiO2-TESP-PEO-coated capillaries showed excellent capability of extracting underivatized highly polar analytes from aqueous samples. This opens the possibility to employ sol-gel titania-based polar coatings for solvent-free extraction and trace analysis of target analytes in environmental and biomedical matrices. To our knowledge, this is the first research on the use of sol-gel titania (or titania-silica)-based organic-inorganic materials as a sorbent in capillary microextraction. The newly developed sol-gel titania (or titania-silica)-based organic-inorganic hybrid extraction media provides an effective solution to coupling CME with HPLC (CME-HPLC), and this can be expected to become a powerful analytical tool in environmental investigations, proteomic research, early disease diagnosis and biomarker research. Being a combination of a highly efficient solvent free sample preconcentration technique (CME) and a powerful separation method (HPLC), CME-HPLC poses to become a key analytical tool in solving complex chemical, environmental, and biomedical problems involving complex matrices.

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