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Silica-based monolithic capillary columns modified to zwitterionic stationary phase for hydrophilic interaction liquid chromatography
Moravcová, Dana ; Planeta, Josef ; Kahle, Vladislav ; Horká, Marie ; Roth, Michal
Zwitterionic monolithic capillary columns intended for isocratic gradient hydrophilic interaction chromatography (HILIC) separations are introduced. Silica-based capillary columns (150 mm x 0.1 mm) were prepared by acidic hydrolysis of tetramethoxysilane in the presence of polyethylene glycol and urea. The modification by a 3-(trimethoxysilyl)propyl methacrylate and then by a zwitterionic [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide to HILIC stationary phase bearing sulfoalkylbetaine groups on its surface followed. Prepared columns were characterized in HILIC separation mode employing mobile phase containing 10% (v/v) of 5 mM ammonium acetate pH = 4.5 in acetonitrile. Comparison with the commercially available ZIC-HILIC® column (Merck SeQuant®) under the same separation conditions using a mixture of aromatic carboxylic acids as a sample was done on the basis of separation efficiency of tested columns as well as retention factors and peak asymmetry of individual solutes.
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Filtration microcartridge and capillary isoelectric focusing for the analysis low number of microorganisms in real samples
Kubesová, Anna ; Horká, Marie ; Šalplachta, Jiří ; Horký, J.
At present, detection and identification of microorganisms in real biological samples needs very sensitive methods for correct diagnosis and therapy in medical practice, biotechnology, agriculture, food safety and quality etc. Accordingly, it is essential to develop new cheap techniques for pathogen identification, particularly for pathogen in complex biological samples. Pre-concentration, separation, and sensitive detection of whole cells in one step are great potential and advantage for detection of pathogens in low concentration. Combination of filtration microcartridge and capillary isoelectric focusing was developed for pre-concentration and pre-separation of microorganisms from real suspensions and the possibility its application to real samples was verified.
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Dynamic modification of proteins for fluorometric detection in CZE
Horká, Marie ; Šlais, Karel
The separation techniques employing fluorescence detection are sensitive and selective so they have been often applied for the trace analysis of biological samples. The commonly used derivatization of proteins can lower the detection limits; however, they can change the acido-basic properties and mobilities when compared to the native species. Recently, we have used the colored tenside as a buffer additive for the photometric detection of proteins in the UV region. The selectivity, efficiency and resolution of CZE separation using this dye were found to be similar to the CZE with SDS as the additive. In this study, the ampiphilic fluorescent compound is suggested as a buffer additive in CZE for dynamic modification of the sample of several proteins. Using the deuterium lamp for the excitation in the UV region for the on column fluorometric detection, the amol minimum detectable amounts of the proteins sampled on the CZE capillary were achieved.
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