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- Dünnschichtchromatographie (8)
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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.
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 (Merck, 1.05559) and silica gel (Merck, 1.05721) phase. A mixture of 13 substances was separated using a solvent mix consisting of methanol–acetonitrile–water (2 + 2 + 1, v/v/v) on RP-18 phase in the first direction and cyclohexane–butylacetate–methanol (8 + 6 + 1, v/v/v) in the second direction on silica gel plate. Both developments were carried out over a distance of 70 mm. We used the grafted method to combine both plates in a 2D-separation. 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 inducting the reporter gene lacZ that encodes the enzyme ß-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylumbelliferyl ß-D-galactopyranoside).
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).
A Validated Quantification of Sudan Red Dyes in Spicery using TLC and a 16-bit Flatbed Scanner
(2018)
We present a video-densitometric quantification method for Sudan red dyes in spices and spice mixtures, separated by TLC. Application was done band-wise in small dots using a 5 μL glass pipette. For separation, the RP-18 plates (20 × 20 cm with fluorescent dye; Merck, Germany, 1.05559) were developed in a vertical developing chamber without vapor saturation from the starting point to a distance of 70 mm by using acetonitrile, methanol, and aqueous ammonia solution (25%; 8 + 1.8 + 0.2, v/v) as mobile phase. The quantification is based on direct measurements using an inexpensive 16-bit flatbed scanner for color measurements (in red, green, and blue). Evaluation of only the green channel makes the measurements very specific. For linearization, an extended Kubelka-Munk expression for data transformation was used. The range of linearity covers more than two magnitudes and lies between 20 and 500 ng. The extraction from a 2 g sample with acetonitrile, evaporation, and reconstitution to 200 μL with methanol and the band-wise application (7 mm) of a 10 μL sample allows a statistically defined LOD of less than 500 ppb of Sudan red dyes. To perform the analysis, a separation chamber, RP-18 plates, 5 μL glass pipettes, and a 16-bit flatbed scanner for 105 € are needed; therefore, the separation method is inexpensive, fast, and reliable.
Limits of quantification of some neonicotinoid insecticides measured by thin-layer chromatography
(2012)
A simple method to quantify the neonicotinoid insecticides nitenpyram, thiamethoxam, acetamiprid, imidacloprid, thiacloprid and clothianidin directly on an HPTLC-plate is presented. As stationary phase silica gel 60 RP-18WF254 s plates were used and a mixture of methyl-t-butyl ether, 2-butanone, NH3 (25%) (5 + 2+0.1, v/v) was used as solvent. All neonicotinoid insecticides show light absorptions below 300 nm. The calculated limits of quantification (LOQ) by UV-detection are in the range from 12 ng to 26 ng on plate depending on the different insecticides.Nitenpyram can be stained using fast blue salt B, forming red zones. The observed LOQ is 25 ng on plate. Acetamiprid can be specifically stained using phenylglyoxylic acid forming a yellow/green fluorescent compound. The LOQ is 52 ng per spot.The compounds thiamethoxam, acetamiprid, thiacloprid and clothianidin can be transformed into blue fluorescing zones, using a relatively new staining solution. This consists of tetraphenylborate and HCl. This is the first publication mentioning that neonicotinoids undergo this reaction. The calculated limits of quantification are in the range from 10 ng to 27 ng on plate.A simple pre-treatment procedure using an acetonitrile extraction and a Chromabond SiOH clean up procedure leads to overall LOQs for bee samples of 48 to 108 µg/Kg. The method can be used to measure neonicotinoid contaminations of bees.