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Evaluation of sample application techniques, stationary and mobile phases, and detection reagents for HPTLC — Densitometry analysis of glucose in fecal samples of mice infected with Echinostoma caproni (Trematoda)

Cicchi M., O’sullivan C. O, Fried B., Sherma J.

Acta Chromatographica

2011

Vol. 23, no. 2

295--305

Seven different thin-layer chromatography stationary phases, one additional stationary layer pretreatment, eight mobile phases, two spotting techniques, and three detection reagents were evaluated for the determination of glucose in mouse fecal samples. Quantitative analysis was performed by slit-scanning densitometry. The optimal system was found to be Merck silica gel HPTLC plates with a concentrating zone developed with 1-butanol-glacial acetic acid-diethyl ether-deionized water 27:18:5:3. α-Naphthol-sulphuric acid detection reagent was found to give the best quantitative results, while the naphthoresorcinol reagent was the most useful for qualitative analysis. Semiautomatic application of samples with a CAMAG Linomat was found to give more compact bands and better separations than manual application. Using this system, quantification of glucose was achieved in mouse fecal samples. The amounts of glucose in the fecal samples of BALB/c mice infected with the intestinal trematode E. caproni were compared to control samples of uninfected mice. On the third and tenth days of postinfection, it was determined that the amount of glucose in the infected fecal samples was significantly greater than in the control samples. This indicates that metabolic profiling of glucose using TLC is possible in the mouse model and that TLC may potentially be used to test for the presence of E. caproni in humans.

Evaluation of thin-layer chromatography systems for analysis of amino acids in complex mixtures

Vasta J. D., Cicchi M., Sherma J., Fried B.

Acta Chromatographica

2009

Vol. 21, no. 1

29-38

Different thin-layer chromatography (TLC) systems were evaluated for analysis of 21 biologically important essential and nonessential amino acids in complex mixtures such as biological tissues and fluids. Amino acids were visualized on the layers by derivatization with ninhydrin reagent, and R F values were determined by slit-scanning densitometry. The five systems found to be most useful for analysis of amino acids were cellulose and silica gel high-performance TLC (HPTLC) plates developed with either 2-butanol-pyridine-glacial acetic acid-deionized water, 39:34:10:26, or 2-butanol-pyridine-25% ammonia-deionized water, 39:34:10:26, and ion exchange TLC plates developed with citrate buffer, pH 3.3. Using these five systems with ninhydrin detection, identification of all amino acids except for leucine and isoleucine in complex mixtures is possible, and quantification can be achieved if the amino acid to be quantified is well separated from adjacent components of the mixture. Example chromatograms are illustrated for separation and identification of amino acids in a snail tissue sample on a cellulose HPTLC plate.