Identification of Phosphates on TLC Plates

01% fluorescein sodium (w/v) in ethanol B Hanes reagent 0.5 g ammonium molybdate are dissolved in 5 ml H2O and supplemented with 2.5 ml of 25% HCl followed by 2.5 ml of 70% perchloric acid. After cooling acetone is filled up to 50 ml. The reagent is ready for use the next day and it is stable for 2-3 weeks in the dark. 

Phosphorescence is observed when the dry sheet is dipped in a container with liquid nitrogen and illuminated by a UV lamp. Purine derivatives phosphoresce light blue pyrimidines are not visible. 

To detect the spots on the TLC plate spray it with Soln. A and dry it. Illumination with a UV lamp (254 nm) shows the spots dark in a greenish fluorescent surrounding. Indicate the spots with a pencil. 

Spray the dry plate with Soln. B and dry with air and heat in a drying oven to 110 °C until blue spots of phosphoric acid esters, e.g., phospholipids, appear. 

Touchstone JC (1992) Practice of thin layer chromatography. 3rd. ed. Wiley, New York 

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