Chromatography on Florisil in the Quantitative Estimation of Urinary and Other Porphyrins

We describe the use of Florisilchromatography to purify and fractionate porphyrins into uro-, copro-, and proto-porphyrins before their measurement by quantitative flu-orometry. Porphyrinogens in fresh urine may be rapidly oxidized to porphyrins by chloranil, or slowly in weakly alkaline solution, before thesample is applied to the Florisil column. Aliquots of the separate ammoniacal eluates may be lyophilized for additional fractionation by direct thin-layer chromatographic analysis if desired. The behavior of-aminolevulinic acid, porphobilinogen, and other pyrrole compounds in this system is also described. AddItional Keyphrases: fluorometry #{149}-aminolevulinic acid #{149} porphobilinogen #{149} thin-layer chromatographic separation of classes of porphyrins The simple fractionation of porphyrins for quanti-tative analysis is complicated by several factors, in-cluding the multiplicity of porphyrins and their isomers, their excretion mainly as nonfluorescing porphyrino-gens, and the presence in urine of large amounts of in-terfering pigments and of other fluorescing compounds. For several years, we have used chromatography on Florisil as a way of purifying and separating uro-, copro-, and proto- type porphyrins, respectively, and this ap-pars to us to be the most accurate and convenient of numerous methods tested. It also permits easy drying of aliquots of the three NH4OH eluates for subsequent thin-layer chromatography, if desired. We have recently described a simple fluorometric analysis for total porphyrin in urines diluted directly with an acid chloranil solution (1). Because it includes rapid and quantitative oxidation of porphyrinogen to porphyrin, this method can also be used for analysis of freshly passed urines. Results obtained agreed well with the sums of uro- and copro- fractions found by the Florisil method we describe here