Related: Principle and Working of HPLC
Preparation of platesUnless otherwise specified in the monograph, the plates are prepared in the following manner. Prepare a suspension of the coating substance in accordance with the instructions of the supplier and, using the spreading device designed for the purpose, spread a uniform layer of the suspension, 0.25 to 0.30 mm thick, on a flat glass plate 20 cm long. Allow the coated plates to dry in air, heat at 100° to 105° for at least 1 hour (except in the case of plates prepared with cellulose when heating for 10 minutes is normally sufficient) and allow to cool, protected from moisture. Store the plates protected from moisture and use within 3 days of preparation. At the time of use, dry the plates again, if necessary, as prescribed in the monograph.
Adjustment of chromatographic conditions
ApparatusThe apparatus for direct measurement consist of: a device for exact positioning and reproducible application of the amount of solutions onto the plate, a mechanical device for moving the plate or the measuring device along the x-axis or the y-axis, a recorder and a suitable integrator or a computer, and a photometer with a source of light, an optical device for generating monochromatic light and a photo cell of adequate sensitivity; for measurement of fluorescence, a suitable filter to prevent light used for excitation from reaching the detector while permitting emitted light or a specific portion thereof to pass.
MethodPrepare the test solution and reference solutions as prescribed in the individual monograph. Use the same solvent for all the solutions and apply the same volume of each and develop the plate. Prepare and apply not fewer than 3 reference solutions of the substance under examination, the concentrations of which span the expected value in the test solution (about 80 per cent, 100 per cent and 120 per cent).
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