TLC Silica Gel Silanised Plate

Support of glass, metal or plastic, coated with a layer of silanised silica gel of a suitable thickness and particle size (usually 2 to 10 µm for fine particle size (high performance thin layer chromatography, HPTLC, plates) and 5 to 40 µm for normal plates). If necessary, the particle size is indicated after the name of the reagent in the tests where it is used. An organic binder may be included.

Chromatographic separation Introduce 0.1 g each of methyl laurate, methyl myristate, methyl palmitate and methyl stearate into a 250 mL conical flask, add 40 mL of alcoholic potassium hydroxide solution and heat under a reflux condenser on a water bath for 1 hour. Allow to cool, transfer the solution to a separating funnel with the aid of 100 mL of water, acidify to pH 2 to 3 with 2m hydrochloric acid and extract with three 10-mL quantities of dichloromethane. Dry the combined extracts over anhydrous sodium sulfate, filter and evaporate to dryness on a water bath. Dissolve the residue in 50 mL of dichloromethane. Examine by thin-layer chromatography, Appendix III A, using a TLC silica gel silanised plate and a mixture of 10 volumes of glacial acetic acid, 25 volumes of water and 65 volumes of 1,4-dioxan as the mobile phase but allowing the solvent front to ascend two thirds of the height of the plate. Apply separately to the plate an appropriate quantity (about 10 µL of normal plates and about 1 µL to 2 µL for fine particle size plates) of the dichloromethane solution at each of three separate points. After removal of the plate, dry it at 120° for 30 minutes, allow to cool, spray with a 3.5% w/v solution of phosphomolybdic acid in propan-2-ol and heat at 150° until the spots become visible. Treat the plate with ammonia vapour until the background is white. The chromatograms show four clearly separated, well-defined principal spots.