Charring, thin layer chromatography

Thin Layer Chromatography Procedures. Silica gel G (Suppelco, Redi-coat, 5 X 20 cm X 0.25 mm) plates were activated by heating in an oven for 1 hour at 110°C. Toxic products were spotted onto duplicate plates at concentrations corresponding to their previously determined LD99 levels. The plates were developed to 14 cm with chloroform, methanol and (6N) ammonium hydroxide (90 9.5 0.5) or chloroform, methanol, and water (60 35 8). Plates were developed and visualized by spraying with 50% aqueous sulfuric acid and charring. Undeveloped plates were scraped, the silica gel fines removed and the extracts concentrated to a residue under a nitrogen gas stream. 

Thin Layer Chromatography. TLC analysis of the WSAP was accomplished By application to silica gel G plates at a concentration of 0.1 mg and development to 14 cm with chloroform-methanol-water (60 35 8). Prior to sulfuric acid-char treatment, five components were visibly resolved. Following such treatment a total of 10 components were visualized. Duplicate plates were divided into six zones, which were scraped, eluted with methanol and assayed at a concentration of 0.2 mg equivalents... 

The reaction may be followed by thin-layer chromatography. The ratio of the Rf values for cyclohexene and c/s-diol 2 is 2 1 (commercial silica gel plates, ethyl acetate). The plates are best visualized by first spraying with 1 % aqueous potassium permanganate, then with methanolic sulfuric acid, followed by charring with heat. The checkers found that, if the procedure is followed exactly, monitoring the reaction by thin-layer chromatography is unnecessary. 

It is best to purify the methyl esters by thin-layer chromatography of the sample on another silica gel G (250 p,m) plate using this solvent system, but without any spray reagent being used. A comparable plate is run and the methyl ester band is detected by the sulfuric acid/char reaction. Then the unsprayed plate is scraped at the methyl ester area and the silica gel is extracted with petroleum-diethyl ether (80 20, v/v) or with chloroform-methanol-water (1 2 0.8, v/v). It is always prudent to spray the latter plate (after removing the desired area by scraping) with sulfuric acid and then charring. This will validate whether the apparent removal of all of the methyl esters has been accomplished. 

Diacetates. The alkenylglycerols are isolated by thin-layer chromatography on silica gel G in a solvent system of hexane-diethyl ether (80 20, v/v). These compounds can be located by use of the TNS spray or by running an additional thin-layer chromatography plate for charring with sulfuric acid. 

Subsequently the alkenylglycerols can be reacted with acetic acid anhydride-pyridine, usually a 1 5 (v/v) mixture, in a sealed tube. The tube is heated at 70-80°C for 45 min and then cooled to room temperature, and the seal is carefully broken. The contents are diluted with an equal volume of water and extracted with n-hexane. Two separate extractions with n-hexane should allow complete recovery of the alkenylglycerol diacetates. The combined hexane extracts are washed with water until neutral and then dried over anhydrous Na2S04. The purity of the preparation can be determined by thin-layer chromatography on silica gel G in a solvent system of petroleum ether-diethyl ether-acetic acid (80 20 1, v/v). Again two separate plates can be run, one for spraying with TNS and the other for charring with sulfonic acid (plus heat). 

Fig. USB. Two-dimensional fractionation of human serum lipids on a layer partly impregnated with silver nitrate [182]. Adsorbents silica gel G and silica gel G-silver nitrate (5%) solvents 1 direction petrol ether (BP 40—60° C)-diethyl ether-acetic acid (85 + 15 + 1), 2 direction petrol ether (BP 40—60° C)-diethyl ether-acetic acid (75 + 25 + 1) visualisation charring by heating with chromic acid/ sulphuric acid amount 300 fxg 26 Thin-Layer Chromatography, 2nd Edition...

Privett, O. S., Dougherty, K. A., and Erdahl, W. L. (1973). Quantitative analysis of the lipid classes by thin layer chromatography via charring and densitometry. In Quantitative Thin Layer Chromatography, J. C. Touchstone (lid.). Wiley, New York, pp. 57-78. 

Spinach chloroplast envelope membranes were prepared according to [5]. Envelope protein samples (30 pg) were phosphorylated in the presence of 33 nM [y PjATP as described in [2]. The envelope protein kinase activities were stopped after incubation at 23°C for 1.5 min by adding one volume of SDS-PAGE sample buffer. The incorporated [ Pjphosphate into proteins was quantified by the Cerenkov procedure. Lipid extraction and thin layer chromatography (TLC) were carried out as described in [2] and [6]. Immediately after charring, the separated lipids on TLC plates were quantified by laser densitometry (Bioimage Millipore). 

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