During the course of our study on certain aspects of metabolism of nitrobenzene it became necessary to separate and estimate isomeric nitrophenols. Earlier work with these phenols relied on separation of o-nitrophenol from its isomers by means of steam distillation; the meta and para iso,mers were then approximately estimated after their partial chromatographic separation (1, 2). Park (3) determined a#ccurately the nitrophenols arising as metabolites of 14C-nitrobenzene, by carrying out isotopic dilutions, preparing derivatives of the nitrophenols, and recrystallizing these to constant count. Parihar et al. ( 4) performed an extensive study of 25 chromatographic systems and found that ptoluenesulfonates of mononitrophenols could be separated on acidic alumina with petroleum ether/ether solvent. Free nitrophenols separated from each other only partially in the chromatographic systems studied by Graham (5). Gasparic (6) achieved varying degrees of separation of the mononitrophenols by chromatography on formamidetreated paper; the best separation between the meta and para isomers was obtained in acetic acid/benzene solvent system (95:5) where approximate Rf's were 0.89, 0.23, and 0.15 for ortho, meta, and para, respectively.
Other investigators, while looking for metabolites of the insecticide parathion, addressed themselves to the problem of p-nitrophenol detection and estimation in biological materials (mainly urine). Thus, Gardocki and Hazelton (7) assayed p-nitrophenol after its reduction to p-aminophenol; the precedure resulted in approximately 57% recovery of the p-nitrophenol added to urine. Hladka and Hla,dky (8) pointed out the problem of interference by urine pigments in the determination of p-nitrophenol in urine extracts. They separated these interfering materials by chromatography on silica gel thin layer in acetone/n-hexane (2O:SO) solvent. In an earlier work, Lawford and Harvey (9) tried to minimize the quantities of interfering pigments