Determination of Thiamine by Thiochrome Method Effects of Temperature and Dissolved Oxygen on Fluorescence of Quinine Standard and of Thiochrome

Abstract

The effect of dissolved oxygen and changes in temperature upon the quinine standard used in the thiochrome reaction and upon thiochrome solutions is large enough to warrant an attempt to control these variables. Temperature effects can be minimized by the use of a water bath to keep the quinine at a standard temperature. The effect probably does not alter the thiochrome fluorescence very much if the room temperature does not vary greatly. Oxygen effects can be minimized by controlling the temperature of the quinine so that no dissolved air is lost, or b y the use of glass standards. Because of the shaking operation the oxygen content of thiochrome solutions is probably a constant factor. Since several types of instruments used to measure fluorescence will gradually heat up the cuvette chamber, the quinine standard should not be left in the instruments.T HAS long been apparent to many analysts that the thio-I chrome method of assaying the thiamine content of biological and other products is occasionally subject to unexplained sources or error that appear and disappear in an erratic manner. Usually these errors limit the accuracy of the method to from *5% to 110% (6), but they may be considerably larger, m has occasionally been observed in this laboratory. While engaged in an attempt to run down some of these sources of error the authors became suspicious of the accuracy of their quinine standard. The order in which samples of thiamine were oxidized and read against the quinine standards appeared to affect the results. If a sample of material was assayed twice on, any given day, and if several hours elapsed between the two oxidations, the last result was the higher if the same quinine standard was used for both oxidations, This phenomenon made it appear as if the quinine exhibited less fluorescence the longer it was used on any given day. The authors had been using fresh daily aliquots of the standard, kept at about 6" C. when not in use.According to Vavilov's equations (6) the fluorescence of a substance in solution is a function of the absolute temperature, other variables being constant. Vavilov also demonstrates the quenching of fluorescence by foreign molecules in the fluorescing solution. He divides quenching into two types (13): quenching by redistribution of the absorbed radiant energy among the degrees of freedom of the fluorescence molecule itself and collisions of the second type, which may or may not involve a chemical reaction. A consideration of Vavilov's work, together with the observations mentioned above, led to the conclusion that the possible effects of temperature and quenchers on the fluorescence of quinine and thiochrome should be investigated.Quenching by redistribution of energy among the molecules of the fluorophor, if it occurs in the quinine standards and in concentrations of thiochrome usually used in the thiochrome method, would appear to be a constant factor, at least for quinine. The only possibilities for quenching by foreign molecules lie in the presence of dissolved atmospheric gas...

Studies on lipolytic molds. Comparative study of lipases obtained from molds grown on oil seeds

“…The vitamin contents of the lipolytie molds were determined just to see whether there is any relation between the vitamins present and the lipolytic activity of the molds. The thiamine content of the molds was found according to the method of Clausen and Brown (13) and the ascorbic acid content of the molds according to the method of Ga~vron and Berg (14). The results are given in Table XIII.…”

The Determination of Thiamine in White Bread by the Thiochrome Method

“…quinine sulphate. The standard quinine solution was renewed after every measurement (Clausen and Brown, 1944). The development and measurement of thiochrome in aliquots of the effluents were always carried out in duplicate or triplicate.…”

Stability-Indicating Photochemical Method for the Assay of Thiamine by Spectrophotometry

“…iamine undergoes chemical [1,8,9,11,14,16,17,19,[51][52][53][54] and photochemical oxidation [55,56] to form thiochrome (TC) which exhibits a blue fluorescence (440 nm). is has been made as the basis of fluorimetric determination of TH [8,9,11,14,16,17,19,[54][55][56][57][58][59][60].…”