Chromatogram developed plates, drying

Automated multiple development (AMD), providing automatic chromatogram development and drying, is a novel form of the PMD technique. Automated multiple development as an instrumental technique can be used to perform normal-phase chromatography with solvent gradients on HPTLC plates. Most of the AMD applications use typical gradients Starting with a very polar solvent, the polarity is varied by means of base solvent of medium polarity to a... 

Detection and result The developed chromatogram was freed from mobile phase by drying for 10 min at 110°C, allowed to cool and immersed for 1 s in the reagent solution. The plate was evaluated as rapidly as possible while it was moist since the fluorescent background increased in intensity as the plate dried out. Cholesterol appeared as a yellow-green fluorescent zone hR 20—25). 

In this case, the TLC system most commonly employed uses silica gel plates and a mobile phase of ethyl acetate/methanol/25% ammonia (85 10 5, by volume). The plates are prepared and the chromatogram developed in the standard way. After development, the plate is removed from the mobile phase, the solvent front marked, and the plate dried. Visualization of barbiturates is best achieved by the use of a mercuric chloride-diphenylcarbazone reagent. The latter is prepared as two component solutions, i.e. (i) 0.1 g of diphenylcarbazone in 50 ml of methanol, and (ii) 0.1 g of mercuric chloride in 50 ml of ethanol. These solutions should be freshly prepared and mixed just before use. The presence of barbiturates will give rise to blue-violet spots on a pink background when using this reagent system. 

In use, the detection reagent is poured into the dipping vessel, either for 20 x 20 cm or 20 x lO cm plates, and then the developed and dried chromatogram is automatically dipped into the reagent. 

Standard soln. of potassium penicillin G prepared in doubly distilled water at cone, of I and 10 f>g/)tl. Appropriate volumes of the standard soln. and the potassium penicillin G dosage forms are applied with a IO-/1I Hamilton syringe, each application being dried in a nitrogen stream before another is made. Chromatograms developed in 7 X 28 X 22 cm glass-lined tanks lined with Whatman No. I filter paper. All plates developed until the solvent rises to within 5 cm of the top (ca. 50 min)... 

On chromatographic plates, 10 pi of racemic mixtures and solutions of pure enantiomers were applied side by side. Chromatograms were developed to a distance of 13 cm at 22 2°C for 40 min in paper-lined rectangular glass chamber pre-equilibrated with the mobile phase for 10-15 min. The developed plates were dried at 60°C for 15 min. 

The chromatogram is freed from mobile phase and evenly sprayed with the spray solution or immersed for 1 s in the dipping solution. After drying the TLC plate is heated to 85 —120°C normally for 10 to 15 min but in exceptional cases for 60 min. It is advisable to observe the chromatogram during the reaction period, because the temperature and duration of heating strongly affect color development. 

The developed chromatogram is freed from mobile phase by heating to 110°C for 10 min in the drying cupboard. It is allowed to cool and immersed for 1 s in or sprayed homogeneously with the reagent the plate is then examined (while still moist). 

Note The developed chromatogram must be completely freed from nonpolar solvents before derivatization, otherwise an intense fluorescence will be stimulated over the whole plate. The fluorescence intensity of the chromatogram zones remains stable for ca. 40 min it decreases slowly as the layer dries out and can be returned to its original intensity by renewed immersion in the reagent solution or in water. 

Procedure. Pour the developing solvent into the chromatographic tank to a depth of about 0.5 cm and replace the lid. Take a prepared plate and carefully spot 5 pL of each indicator on the origin line (see Section 8.6, under Sample application) using a micropipette. Allow to dry, slide the plate into the tank and develop the chromatogram by the ascending solvent for about 1 h. Remove the plate, mark the solvent front and dry the plate in an oven at 60 °C for about 15 min. Evaluate the R value for each of the indicators using the equation... 

In the simplest case the developed chromatograms are heated to the required temperature on a hot plate (Fig. 19) or in a drying cupboard. More rarely infrared heaters are used to heat the system [2]. Gas chromatograph ovens can be used if exact adjustment of the temperature is required [3]. 

---