Paper chromatography of pyridine derivatives

WE wish to describe briefly a convenient paper chromatographic separation and identification of pyridine derivatives in use in this laboratory for some time which employs a modification of the wellknown Konig colour-reaction1. Leifer et al.2. have recently described the paper chromatography of nicotinic acid and nicotinamide, but the more involved microbiological method was used for defining the spots. In this laboratory the solvent used for the development of the compounds listed in the accompanying table is n-butyl alcohol saturated with 1.5 N ammonia. After evaporation of the solvent at room temperature, the dry paper is placed for fifteen minutes in a closed glass cylinder containing a few crystals of cyanogen bromide. The vapour pressure of this substance is high enough to provide a sufficient concentration for the first phase of the Konig reaction. The paper is then sprayed with a 0.25 per cent solution of benzidine in 1 : 1 ethanolwater. Amounts down to 5γ can be detected easily. It should be pointed out that although nicotinic acid and iso-nicotinic acid cannot be distinguished on the basis of RF values, they can be distinguished by distinctive differences in colour. This technique should be generally applicable to all non-volatile pyridine derivatives giving a positive Konig colour-reaction.