Some New Reactions for the Detection of Sterols

Abstract

OBSERVATIONS made in relation to the colour reactions in vogue for the de-tection of sterols have indicated that in their general character all or most of these reactions are in essence similar, and have led to the discovery of several new reactions which present certain interesting features including a greater degree of delicacy than that possessed by reactions now employed. In what follows, first three new reactions for sterols and a reaction for sterolins are described, and then some considerations are brought forward concerning the mechanism of the colour reactions shown by sterols. Reaction A. To 2 cc. of a chloroform solution of the sterol (containing preferably 1-2 mg. of the sterol) are added 2 cc. of a reagent prepared by mixing conc. sulphuric acid and formalin in the proportions 50 vols.: 1 vol., and the contents of the test-tube are then shaken. When ther layers have separated, the upper, chloroform layer is seen to be cherry-red in colour, and the lower, sulphuric acid layer to be brownish-red in colour and to show an intense green fluorescence. The upper layer is then poured off into a dry test-tube and treated with two or three drops of acetic anhydride; as a result it assumes a bright blue colour, which lasts for a considerable time, but passes into a green within, say, an hour. The reaction is much more sensitive and striking than the Salkowski [1872] reaction, of which it may be regarded as an elaboration. In the Salkowski reaction, a chloroform solution of a sterol is shaken with an equal volume of conc. sulphuric acid; when the liquids have separated, the chloroform layer is seen to be brownish-yellow, and the sulphuric acid layer to be yellow-brown with a green fluorescence; on allowing the test-tube to stand for several hours, the sulphuric acid layer becomes deeper and redder in colour and more strongly fluorescent, while the chloroform layer assumes, if the sterol was present in sufficient amount, a cherry-red or purple colour. Reaction A de-scribed above has several advantages over thejust-described Salkowski reaction, namely: (a) the cherry-red colour is obtained immediately in the former, whereas in the latter it is necessary to wait several hours for its appearance; (b) the former reaction is given by a much smaller quantity of the sterol than the latter (vide infra); (c) the coloured chloroform solution obtained in the G. S. WHITBY former is converted by acetic anhydride into a comparatively lasting blue, whereas when the ch…