WE have previously reported' that hydrolysis of vitamin B,, with 20 per cent. hydrochloric acid at lOO"C., followed by examination of the hydrolysates by unidimensional paper-strip chromatography using the technique described by Consden, Gordon and Martin2, revealed the presence of one " ninhydrin-reacting " substance which could not be identified with any of the known amino-acids. Collateral studies by Brink et aL3 in America, although confirming the absence of amino-acids in the hydrolysates, did not substantiate the existence of the " ninhydrinteacting " substance. We were therefore led to re-examine and extend our observations on this fragment of the B,, complex and now report further data which fully supports our earlier conclusions.Rigorous purification of crystalline samples of vitamin B,, failed to alter the pattern of our results. Paper chromatograms of the hydrolysates, as before, invariably revealed one spot on treatment with ninhydrin, the intensity of which, however, depended markedly on the nature of the irrigation solvent employed. Thus, pronounced purple spots were obtained with solvents consisting of, or containing, the aliphatic acids. Faint or hardly visible spots, in contrast, resulted when n-butyl alcohol or phenol were employed. Brink et aL3, it should be added, used phenol as their irrigation solvent, a fact no doubt explaining the difference between the two sets of results.Again, in another series of experiments the coloured moiety' produced by hydrolysis of vitamin BIZ was quantitatively extracted from the diluted hydrolysate with n-butyl alcohol, and the colourless cobalt-free aqueous phase examined spectroscopically. Selective absorption in the ultra-violet was observed with bands and inflections at 2850, 2768, 2690, 2585 and 2500 A (for convenience this colourless cobalt-free hydrolytic material will subsequently be referred to as " the 285 component "), similar to certain fine structure bands present in the ultra-violet obsorption spectrum of vitamin B,, itself. A paper chromatogram prepared from the aqueous phase, and irrigated with iso-butyric acid, when developed with ninhydrin, gave the typical purple spot obtained using the total hydrolysate. A second chromatogram, run parallel with the first, when examined under a low pressure mercury resonance lamp fitted with a Corning 9863 glass filter', showed a clearly visible localised pale-blue fluorescent area occupying a position corresponding to the " ninhydrin spot." The ultra-violet absorption of the eluate from this fluorescent area, moreover, showed selective absorption substantially the same as that of the aqueous solution from which the chromatogram was prepared.
