Fluorimetric and Microbiological Assays of Riboflavine in Malted Preparations

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Abstract
Summary: Using a more sensitive fluorimeter than has previously been described in this country, a method is given for estimating riboflavine in malted preparations which gives much better agreement with microbiological assays than had previously been obtained. These preparations when oxidised with permanganate during the purification procedure exhibit a non-specific blue fluorescence which interferes with the fluorimetric assay. In old samples of malt extract this blue fluorescence is quite marked before permanganate treatment. The behaviour of the fluorescent substance with Florisil and its low solubility in chloroform show it not to be lumichrome. It is most effectively separated from riboflavine by adsorption on Florisil and careful elution with 1 per cent. pyridine, the process being observed continuously under the ultra-violet lamp. Spectroscopic studies indicated Wratten 47 as a satisfactory primary filter. Comparison of fluorimetric and microbiological results confirmed this. When calculating the results of riboflavine fluorimetric assays a method based on the assumption that the net fluorescence is proportional to the concentration, and using solvent blanks to determine the net fluorescence, is preferable to the method more usual in this country of using calibration curves obtained by plotting gross fluorescence against concentration. We are indebted to Mr. E. J. Bowen, F.R.S., for advice, to Miss Janet Horsford and Mr. R. Evans for technical assistance, to Mr. G. Slaughter for the fluorescence spectrum of riboflavine, to Messrs.*** Hilger and Watts, Ltd., and Messrs. Kodak, Ltd., for data on the transmission of filters.