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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

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2009

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Vol. 21, no. 1

29-38

EN

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.

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Comparison of spraying, dipping, and the derivapress for postchromatic derivatization with phosphomolybdic acid in the detection and quantification of neutral lipids by high-performance thin-layer chromatography

Vasta J. D., Sherma J.

Acta Chromatographica

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2008

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Vol. 20, no. 1

15-23

EN

Neutral lipids were measured by scanning densitometry after postchromatographic derivatization. Neutral lipids are often derivatized with phosphomolybdic acid reagent, which reacts to form highly visible, blue zones. Uniform application of this reagent to HPTLC plates is critical if quantification of neutral lipid bands is desired. In this paper we compare the quantitative and qualitative results observed by use of three methods of application of derivatization reagent-manual spraying, manual dipping, and use of the Derivapress. Results showed that dipping is the best method of reagent application for achieving quantitative data, and spraying is the best method for qualitative purposes. The Derivapress proved to be a simple and economical device for application of phosphomolybdic acid reagent to HPTLC plates. It enabled qualitative and quantitative analysis of neutral lipids.

3

Analysis of lycopene in nutritional supplements by silica gel high-performance thin-layer chromatography with visible-mode densitometry

Vasta J. D., Sherma J.

Acta Chromatographica

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2008

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Vol. 20, no. 4

673-683

EN

A quantitative method using silica gel 60 HPTLC plates, automated bandwise sample application, petroleum ether-dichloromethane 9:1 as mobile phase, and automated slit-scanning densitometry has been developed for the analysis of lycopene in nutritional supplements consumed to reduce the risk of prostate cancer and other forms of cancer and cardiovascular disease. Four products containing 300 µg, 3 mg, 5 mg, or 10 mg lycopene plus other ingredients were analyzed using a ChromaDex lycopene standard. The amount of lycopene in the tested samples ranged from 77.7 to 98.1% relative to the stated label values. Accuracy was validated by spiked blank analysis, and precision by performing replicate analysis. Accuracy was found to be within 1.90% of theoretical values for the 3 mg softgels and 1.10% of theoretical values for the 10 mg softgels, and precision was 1.44% relative standard deviation (RSD) for the 10 mg softgels and 2.39% RSD for the spiked blank for the 3 mg softgels. Lycopene standards available from two other companies were analyzed and found to contain 55.6, 57.6, and 20.0% of the minimum amount expected from the stated label values.