Contents Preparative Layer Chromatography

This reaction is run under the same conditions as described earlier and the products isolated by preparative thin-layer chromatography. The products should be analyzed for P content (which would be primarily monoacylphos-phatidylcholine and unreacted sn-l diacylphosphatidylcholine) and for optical activity. Whereas the starting rac-diacylphosphatidylcholine would exhibit no optical activity, each of the above products of the reaction should have optical activity. The liberated free fatty acid can be converted to a methyl ester form and examined by gas-liquid chromatography coupled with mass spectrometry (GC-MS). 

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). 

Subsequent to recovery of the total lipids of a cellular preparation as a chloroform-soluble fraction, the total phosphorus content can be determined (see Chapter 3) and then, depending on the amount of lipid phosphorus (or whether the preparation is radiolabeled or not, see below), analytical and/or preparative thin-layer chromatography can be undertaken. In either case, if the experimental protocol is centered on a signal-transduction process, then there may be insufficient material for a phosphorus analysis. In the latter instance, the cellular preparation is prelabeled with 32P or [3H]inositol and the labeled products are located by autoradiography. A preferred type of adsorbent (for thin-layer chromatography) is Merck silica gel 60 (oxalate impregnated). An effective solvent for separation of the phosphatidylinosi-tols and other lipids is chloroform-acetone-methanol-acetic acid-water (80 30 26 24 14, v/v). The approximate / values of cellular phospholipids under these conditions are presented as follows ... 

The ethanol-chloroform-water (5 2 1, v/v) eluate is evaporated to dryness in vacuo and the residue is dissolved in chloroform-methanol (2 1, v/v). This fraction can be analyzed for total P, and its contents can be evaluated by thin-layer chromatography on silica gel H (250 pm) plates in a solvent system of chloroform-methanol-ammonium hydroxide (28% 65 35 7, v/v). Two ninhydrin-positive spots will be found one is at Rf 0.50, which is phospha-tidylcthanolamine the other is at Rf 0.20, which is phosphatidylserine. These findings then make it possible to isolate phosphatidylserine by preparative thin-layer chromatography. 

B. Assemble an apparatus for Thin-Layer Chromatography (see Chromatography, Appendix IIA), using chromatographic silica gel as the adsorbent and a 4 1 mixture of cyclohex-ane ether as the solvent system. Prepare a Standard Solution by dissolving the contents of 1 capsule of USP Vitamin A Reference Standard in sufficient chloroform to make 25.0 mL. 

Further comparatively voluminous literature on the detection and identification of narcotine has appeared (68-91) paper chromatography and paper electrophoresis were repeatedly utilized for the detection of narcotine (92-117) as well as for its quantitative determination (65, 80, 84, 118-152) and separation (153-175). Paper chromatography, even in conjunction with electrophoresis, has been advocated for the quantitative separation and estimation of narcotine (116, 176-191). Thin-layer chromatography (154,155,167,192-196) and gas chromatography (197) are more recent techniques. Dry poppy heads with a content of 0.01% narcotine may be utilized for preparative purposes (198-201). 

The content of narcotoline in poppy capsules (0.1-0.2%) is useful for preparative purposes (212, 221-224). In connection with this problem, colorimetric determination (223), paper chromatography (92, 94, 103, 107, 108, 112, 189), potentiometric determination (225), and thin-layer chromatography (161) were utilized. Indirect determination of narcotoline is possible via cotarnoline (149), especially polarographically (226, 227). The dissociation constant of narcotoline has been determined (228). 

Pedersen has examined the pyrrolizidine alkaloid content of 19 Danish species of the Boraginaceae. The isolated alkaloids from ten of these species were purified by preparative thin-layer chromatography and investigated by mass spectrometry. The... 

Use of the cELISA to determine methoprene content of tobacco residues prepared as described above, showed that the extracts contained materials which interfered with the assay (Figure 8). Extracts of tobacco not only contained methoprene, but also plant substances that interfered with the assay. Thin-layer chromatography (TLC) was then used to clean up crude extracts from tobacco samples to reduce and/or eliminate interference. 

Quantitative estimation of the O-acyl content of sialic acid preparations can be made using the hydroxamic acid reaction as described by Ludowieg and Dorfman (1960), modified from the method of Hestrin (1949). Calibration with ethyl acetate or other ester standards provides a simple quantitative assay, measuring the chromophore at 520 nm. Volume reductions in the assay procedure allow quantitation of 0.05(i,mole of sialic acid acyl ester (Schauer 1978). Qualitative identification of different acylhydroxamates can be made on thin-layer chromatography (see section III.3.b)). 

For the polymerization studies, multilamellar vesicular dispersions were prepared by vortexing the hydrated lipid dispersions. These dispersions were gel filtered on a Sephadex G 50-150 column and were polymerized at 5 C by 254 nm irradiation in a Rayonette Photoreactor. The course of the polymerization was monitored by thin layer chromatography on silica gel using a chloroformrmethanol water (65 25 4) solvent system. The monomer participation was measured in polymerized and freeze dried samples by dissolving the monomers in chloroform and spotting on the TLC plate, developing in lipid solvent and measuring the phosphorus content under monomer and polymer spots. 

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