Phloroglucinol"
has been determined by a wide variety of methods, most of w*hich rely on the phenolic nature of the compound, and none of which is specific for phloroglucinol itself (l-9). Many of the available procedures suffer from the disadvantages of being rather cumbersome and not very sensitive. During the course of studies on an enzyme which cleaves phloretin hydrolytically to yield phloroglucinol and phloretic acid, and which will be referred to as phloretin hydrolase (Chatterjee and Gibbins, in preparation), it became necessary to develop a method for the determination of either the substrate for, or the end-product of, the enzyme reaction in order to formulate a suitable procedure for the assay of the activity. There being no method available for the specific assay of either phloretin or phloretic acid, attention was focused on the vanillin-HCl reagent as being, potentially, the most useful from the points of view of sensitivity, reliability, relative specificity, and convenience in technique.
In preliminary tests with the vanillin-HCI reagent (VHR) (1) it was observed that phloretin gave too high a response to the reagent (Fig. l), for the reagent to be suitable for the assay of phloretin hydrolase, very erratic assay values being obtained. It was found that, by separating the vanillin and concentrated HCI components of the reagent (a procedure carried out when using vanillin to detect phenols on paper chromatograms (10) ) , and by decreasing the vanillin concentration, a considerable reduction in the response of phloretin was obtained. The phloroglucinol response, while being reduced, was only marginally so, and an investiga-' To whom requests