The fluorometric determination of polycarboxylic acids following chromatography.

Several methods have been presented for the determination of the acids of the tricarboxylic acid cycle by chromatography. The acids may be separated on silica gel (l-3), Dowex 1 (4), or filter paper (5) ; after separation, they may be measured by titration with standard alkali (l-3) or by the usual methods for the individual acids (4, 6). Although the current means of separation are satisfactory, the subsequent methods of determining the acids are laborious and in some instances inaccurate. Titration of the fractions following silica gel chromatography not only is time-consuming because of the large numbers of fractions of non-aqueous material to be titrated, but also may be inaccurate owing to the presence of inorganic acids in some of the fractions. These inorganic acid impurities may arise from elution of the mineral acid with which the column has been impregnated. In ion exchange chromatography, the use of titrimetric methods of determination is even more impractical. The use of specific methods for the individual acids following separation is likewise undesirable because of the multiplicity of analytical procedures required. Furthermore, the presence of a given acid in an individual fraction is not indicated by any gross characteristic such as color; therefore it is difficult to select the exact fractions needed for analysis by specific methods. In the present study, a method was developed for the determination of polycarboxylic acids by a general fluorometric procedure following their chromatographic separation. The method is based on the reaction of organic acids with resorcinol (7) to form fluorescent derivatives. These fluorescent derivatives not only can be used for the quantitative determination of the original acids, but also, because of their solubility in ether, can be extracted for measurements of radioactivity in the parent compound.