Browsing by Subject "capillary electrophoresis"
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Item Fluorescent Labeling Reagents Optimized for Capillary Electrophoretic Separations(2012-02-14) Estrada, Roy Tonacao, IIIFluorescent labeling can improve the detection sensitivity in capillary electrophoretic (CE) separations down to attomolar concentrations. However, most fluorescent labels are not compatible with CE because their fluorescence properties and charge states are pH-dependent, they are often hydrophobic and they have a tendency to significantly change the properties of the analytes after labeling. A group of fluorescent labeling reagents have been prepared whose fluorophores have properties that are optimized for CE separations. These fluorophores have fluorescence properties and charge states that are independent of pH in the 2 < pH < 11 range. Their excitation maxima are also compatible with the 488 nm line of the Argon ion laser. A mono-cationic acridine-based fluorescent label was prepared and was found to not shift the pI of a labeled model protein in capillary isoelectric focusing separation (cIEF). Lower loading, due to increased sensitivity, led to better resolution of closely spaced isoform peaks having a pI = 0.05. A tri-anionic pyrene-based fluorescent labeling reagent was also synthesized and was used in the sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) separation of proteins. The fluorophore led to an LOQ in the nM range, and did not alter the migration behavior of proteins in the sieving matrix. A third fluorescent labeling reagent was developed as a solid phase reagent (SPR) where the fluorophore was immobilized on a solid surface through a cleavable anchor. The fluorophore is di-anionic and is based on pyrene. The SPR was designed to allow the simultaneous capture and labeling of an analyte and the efficient release of the label-analyte conjugate under mild acidic conditions. The use of the SPR allowed the labeling of a diamine whose concentration was in the low nanomolar range. The SPR opens up the possibility for mono-labeling and proportional multiple labeling of proteins.Item Spatial and temporal controls on biogeochemical indicators at the small-scale interface between a contaminated aquifer and wetland surface water(2009-05-15) Baez-Cazull, Susan EnidThis high-resolution biogeochemical study investigated spatial and temporal variability in the mixing interface zones within a wetland-aquifer system near a municipal landfill in the city of Norman, Oklahoma. Steep biogeochemical gradients indicating zones of enhanced microbial activity (e.g. iron/sulfate reduction and fermentation) were found at centimeter-scale hydrological and lithological interfaces. The small resolution study was achieved by combining passive diffusion samplers with capillary electrophoresis for chemical analysis. The spatial and temporal variability of biogeochemical processes found at the interfaces was evaluated in a depth profile over a period of three years. Correlations between geochemical parameters were determined using Principal Component Analysis (PCA) and the principal factors obtained were interpreted as a dominant biogeochemical process. Factors scores were mapped by date and depth to determine the spatial-temporal associations of the dominant processes. Fermentation was the process controlling the greatest variability in the dataset followed by iron/sulfate reduction, and methanogenesis. The effect of seasonal and hydrologic changes on biogeochemistry was evaluated from samples collected in a wet/dry period from three locations exhibiting upward, downward, and negligent hydrologic flow between aquifer and wetland. PCA was used to identify the principal biogeochemical processes and to obtain factor scores for evaluating significant seasonal and hydrological differences via analysis of variance. Iron and sulfate reduction were dominated by changes in water table levels and water flow paths, whereas methanogenesis and bacterial barite utilization were dominated by season and associated with a site with negligible flow. A preliminary study on microbial response to changes in geochemical nutrients (e.g. electron acceptors and electron donors) was conducted using in situ microcosms with the purpose of quantifying iron and sulfate reduction rates. Problems encountered in the experiment such as leaks in the microcosms did not allow the determination of respiration rates, therefore the experiments will be repeated in the future. The results suggest that iron and sulfate reduction were stimulated with the addition of sulfate and ferrihydrite (electron acceptors) and acetate and lactate (electron donors). This research demonstrates the importance of assessing biogeochemical processes at interface zones at appropriate scales and reveals the seasonal and hydrological controls on system processes.Item Synthesis, characterization and capillary electrophoretic use of new, single-isomer hexasulfated alpha-cyclodextrins(Texas A&M University, 2005-08-29) Li, ShulanThe first three, pure, single-isomer, 6-O-sulfo a-cyclodextrins, the sodium salts of hexakis(6-O-sulfo)-a-CD (HxS), hexakis(2,3-di-O-methyl-6-O-sulfo)-a-cyclodextrin (HxDMS) and hexakis(2,3-di-O-acetyl-6-O-sulfo)-a-cyclodextrin (HxDAS) have been synthesized, analytically characterized and utilized as chiral resolving agents in capillary electrophoresis. The purity of each synthetic intermediate and of the final product was determined by HPLC-ELSD and indirect UV-detection capillary electrophoresis. The structural identity of each intermediate and final product was verified by 1D and 2D NMR, and mass spectrometry.HxS, HxDMS and HxDAS have been used to separate a series of neutral, basic, ampholytic and acidic enantiomers in pH 2.5 and pH 9.5 aqueous and acidic methanol background electrolytes using capillary electrophoresis. Rapid separations with satisfactory peak resolution values were obtained for most of the analytes, indicating that HxS, HxDAS and HxDMS can serve as chiral resolving agent for a wide range of analytes. The observed separation patterns follow the predictions of the CHArged Resolving agent Migration (CHARM) model. The separation patterns observed with HxS, HxDAS and HxDMS as chiral resolving agent were compared with those of (1) b-cyclodextrin analogues, such as, heptakis(6-O-sulfo)-b-cyclodextrin (HS), heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-cyclodextrin (HDAS) and heptakis(2,3-di-O-methyl-6-O-sulfo)-b-cyclodextrin (HDMS); (2) g-cyclodextrin analogues, such as, octakis(6-O-sulfo)-g-cyclodextrin (OS), octakis(2,3-di-O-acetyl-6-Osulfo)- g-cyclodextrin (ODAS) and octakis(2,3-di-O-methyl-6-O-sulfo)-g-cyclodextrin (ODMS). The effects of the structure of the analytes, and those of the pH and the solvent of the background electrolyte were also studied.Item Two new, single-isomer, sulfated β-cyclodextrins for use as chiral resolving agents for enantiomer separations in capillary electrophoresis(Texas A&M University, 2006-08-16) Busby, Michael BrentTwo novel, single-isomer, sulfated cyclodextrins, the sodium salts of heptakis(2- O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose (HMAS) and heptakis(2-O-methyl- 6-O-sulfo)cyclomaltoheptaose (HMS) were used as chiral resolving agents in both aqueous and non-aqueous electrophoretic separation of a set of pharmaceutically active weak base enantiomers. Enantiomers of twenty one of the twenty four weak bases were baseline resolved in one or more of the background electrolytes (BGE’s) used. An eight-step synthetic method was used to produce, on a large scale, the title compounds in greater than 97% purity. The purity of the synthetic intermediates and the final products were characterized by HPLC-ELSD and indirect UV-detection capillary electrophoresis (CE), respectively. X-ray crystallography, MALDI-TOF mass spectrometry and 1H as well as 13C NMR spectroscopy allowed for unambiguous characterization of the structure of each intermediate and the final product.