Multi-spots in Paper Chromatograms

Abstract

During investigations on the synthesis of nucleotides in this laboratory, extensive use has been made of the paper chromatography of phosphorus-containing compounds. In the early experiments it was observed that in a descending pyridine – ethyl acetate – water system, disodium hydrogen phosphate gave two clear spots of approximately equal intensity with R_F 0.17 and 0.55 on spraying with molybdate reagent¹. Identical spots were obtained with sodium or potassium mono- or di-hydrogen phosphates, whereas ammonium phosphate and free orthophosphoric acid gave only the faster-running spot. Trisodium phosphate showed largely the slower spot with some trailing. (These R_F values are quoted to give an indication of the positions of the spots. The temperature was not strictly controlled.) Other experiments showed the effect was not due to complexes with pyridine as the same double-spot phenomenon occurred in a butanol – water system. By buffering the phosphates with acid and alkali, it was found that the number of spots depended on the pH of the solution in which the phosphate was applied to the paper. Potassium dihydrogen phosphate below pH 3 gave only the faster running spot; between pH 3 and pH 9, both spots at R_F 0.17 and 0.55 were in evidence, but by pH 10 (caustic soda added) only the slower spot appeared. Above pH 12 only a spot at R_F 0.05 was obtained with a horseshoe appearance (compare Paterson and Reincke²).