Use of Phenol in Application of Prebluda-McCollum Reagent for Determining Vitamin B 1

Recently Prebluda and McCollum 1 reported that Vitamin B¹ reacts in alkaline solution with diazotized p-amino acetophenone to yield a water-insoluble, purple-red compound. Subsequently, we found 2 that xylene quantitatively extracts this pigment and that the xylene layer lends itself to colorimetric evaluation. This procedure, coupled with our adsorption and subsequent elution technics using synthetic zeolite,† appears highly specific for the determination of the vitamin.

Ten cc of a thiamin hydrochloride solution at a pH of 7.0 are pipetted into a 50 cc centrifuge bottle. This is followed by the addition of 20 cc of the Prebluda-McCollum reagent. After 24 hours at room temperature 2 cc of xylene are added and the mixture shaken vigorously for 1 1/2 minutes. After centrifugation the color in the xylene layer is compared in a micro-colorimeter with a standard similarly treated. Subsequently, in studies involving the use of phenol for extracting the vitamin from saturated salt solutions, recoveries greater than 100% were consistently obtained due to the presence of trace quantities of phenol in the final concentrates, which escaped ether extraction. Other organic hydroxy compounds, such as ethyl alcohol and pH indicators, also increase the sensitivity of the reaction.

In order to investigate the beihavior of phenol in this respect, a series of determinations were made in which varying amounts of phenol were added to the vitamin solutions, each 10 cc in volume and containing a total of 100 gamma of thiamin hydrochloride. No phenol was added to the standard solution. Although phenol, like some other organic compounds, reacts with the reagent to yield a colored solution, only the red pigment due to the coupled vitamin has been found to be extracted by the organic solvent. The results of these determinations are presented in Fig. 1.