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We present a planar chromatographic separation method for the phytoestrogenic active compound equol, separated on RP-18 W (Merck, 1.14296) phase. It could be shown that an ethanolic cattle manure extract contains this phytoestrogenic active compound to a larger amount. As solvents for the mobile phase, hexane, ethyl acetate, and acetone (45:15:10, v/v); acetone and water (15:10, v/v); and n-hexane, CH2Cl2, ethyl acetate, methanol, and formic acid (40:40:20:5:1, v/v) have been used. After separation, a modified yeast estrogen screen (YES) test was applied, using the yeast strain Saccharomyces cerevisiae BJ3505 containing an estrogen receptor. Its activation by equol induces the reporter gene lacZ which encodes the enzyme β-galactosidase. The enzyme activity is measured directly on the TLC plate by using the substrate MUG (4-methylumbelliferyl-β-d-galactopyranoside) or the substrate X-β-Gal (5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside). β-Galactosidase cleaves MUG into a fluorescing compound. X-β- Gal is also hydrolyzed and then oxidized by oxygen forming the deep-blue dye 5,5′-dibromo-4,4′-dichloro-indigo. Both reactions in combination with a thin-layer chromatography (TLC) separation allow very specific detecting of equol in cattle manure, although that is a very challenging matrix. Preliminary results show that the average content of equol in liquid manure is roughly 60 μg g−1. The value for urine is 50 μg mL−1.
We present a video-densitometric quantification method in combination with diode-array quantification for the methyl-, ethyl-, propyl-, and butylparaben in cosmetics. These parabens were separated on cyanopropyl bonded plates using water-acetonitrile-dioxane-ethanol-NH3 (25%) (8:2:1:1:0.05, v/v) as mobile phase. The quantification is based on UV-measurements at 255 nm and a bioeffectively-linked analysis using Vibrio fischeri bacteria. Within 5 min, a Tidas S 700 diode-array scanner (J&M, Aalen, Germany) scans 8 tracks and thus measures in total 5600 spectra in the wavelengths range from 190 to 1000 nm. The quantification range for all these parabens is from 20 to 400 ng per band, measured at 255 nm. In the V. fischeri assay a CCD-camera registers the white light of the light-emitting bacteria within 10 min. All parabens effectively suppress the bacterial light emission which can be used for quantifying within a linear range from 100 to 400 ng. Measurements were carried out using a 16-bit MicroChemi chemiluminescence system (biostep GmbH, Jahnsdorf, Germany), using a CCD camera with 4.19 megapixels. The range of linearity is achieved because the extended Kubelka-Munk expression was used for data transformation. The separation method is inexpensive, fast, and reliable.
Die Weltwirtschaftskrise 2008 hat mit ihrer zeitweisen Verknappung von Acetonitril eindringlich gezeigt, dass man nicht nur auf eine einzige chromatographische Methode setzten sollte. Genau dies wird aber im Augenblick getan, denn Industrie und Forschung setzen mehrheitlich auf die High Performance Liquid Chromatography (HPLC) als die Trennmethode ihrer Wahl. Für viele Anwendungen in der Pharmazie, in der Umweltanalytik, der Lebensmittelanalytik, aber auch in der Inprozesskontrolle gibt es mit der Dünnschichtchromatografie eine Alternative.
Die Weltwirtschaftskrise 2008 hat mit ihrer zeitweisen Verknappung von Acetonitril eindringlich gezeigt, dass man nicht nur auf eine einzige chromatographische Methode setzten sollte. Genau dies wird aber im Augenblick getan, denn Industrie und Forschung setzen mehrheitlich auf die High Performance Liquid Chromatography (HPLC) als die Trennmethode ihrer Wahl. Für viele Anwendungen in der Pharmazie, in der Umweltanalytik, der Lebensmittelanalytik, aber auch in der Inprozesskontrolle gibt es mit der Dünnschichtchromatografie eine Alternative.
We present a two-dimensional (2D) planar chromatographic separation of estrogenic active compounds on RP-18 W (Merck, 1.14296) phase. A mixture of 8 substances was separated using a solvent mix consisting of hexane, ethyl acetate, acetone (55:15:10, v/v) in the first direction and of acetone and water (15:10, v/v) in the second direction. Separation was performed on an RP-18 W plate over a distance of 70 mm. This 2D-separation method can be used to quantify 17α-ethinylestradiol (EE2) in an effect-directed analysis, using the yeast strain Saccharomyces cerevisiae BJ3505. The test strain (according to McDonnell) contains the estrogen receptor. Its activation by estrogen active compounds is measured by inducing the reporter gene lacZ which encodes the enzyme β-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylumbelliferyl-β-d-galactopyranoside).