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THIs paper embodies further observations made upon and in relation to the formaldehyde azo reaction for vitamin B1 [Kinnersley & Peters, 1934] and its' possible application to semi-quantitative estimation of vitamin B1. The development of the more sensitive thiochrome method of estimation [Jansen, 1936; Karrer & Kubli, 1937; Pyke, 1938] during the progress of this work, may ultimately make the method here described obsolete. Nevertheless, we think that some independent check may prove useful, and further, that the method may prove of value, when enough vitamin B1 is available, owing to the stability of the standards, and to the fact that a dark room and a source of ultra-violet light are not required. Unfortunately, neither method is successful with phosphoric esters of vitamin B1; these do not give the formaldehyde azo test (as stated by Lohmann & Schuster [1937]); they form a blue fluorescent compound with alkaline ferricyanide, but this does not pass into a butyl alcohol layer, which is a basis of separation from interfering fluorescent substances [Kinnersley & Peters, 1937]. We have found that phosphatase can be used inthe pre-treatment of the extracts to liberate the vitamin B1. In our previous communication we stated that the condition of alkalinity chosen and the presence of formaldehyde largely reduced interference by other substances; a particularly troublesome impurity giving a pseudo-vitamin B1 colour reaction could be distinguished by its relative instability in acid solution. We have realized for some time that butyl alcohol would extract the bulk of the pink substance formed in the reaction. By an error the colour was compared in our previous paper [1934] with phenol red in phosphate buffer pH 5; it should have been "approximately pH 7-3 ". A considerable improvement is again made by shaking the butyl alcohol with dilute acid, when the pink substance passes back into the acid aqueous phase; the whole test now compares favourably as regards specificity with other methods and can be readily applied to vitamin B1 concentrates from the International Standard Clay (1936) and also the cruder "charcoal" concentrates. We have found certain conditions which interfere with this and other azo tests. The work falls into two parts: I. The method and some conditions modifying it; II. Application to crude vitamin B1 extracts.

Improvements in the use of the formaldehyde azo reaction for vitamin B1