Use of narrow-bore high-performance liquid chromatography-diode array detection for the analysis of intermediates of the biological degradation of 2,4,6-trinitrotoluene.

A single method was developed for the separation and quantitation of hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene (TNT), and most of the known and suspected biodegradation intermediates of TNT by RP-HPLC and diode array detection. The known biodegradation intermediates of TNT analyzed were 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,6-diamino-4-nitrotoluene, 2,4-diamino-6-nitrotoluene, 2,4,6-triaminotoluene, 2,2',6,6'-tetranitro-4,4'-azoxytoluene, and 4,4',6,6'-tetranitro-2,2'-azoxytoluene. The suspected biodegradation intermediates of TNT included 1,2,3-benzenetriol (pyrogallol), 1,3,5-benzenetriol (phloroglucinol), 2-methyl-1,3,5-benzenetriol (methyl phloroglucinol) and 4-methylphenol (p-cresol). Mobile phases consisting of aqueous buffers adjusted to three different pH values in a gradient with acetonitrile were examined for their efficiency in separating the intermediate compounds and for the minimization of speciation of the ionizable intermediates (e.g. 2,4,6-triaminotoluene). A final aqueous buffer pH of 3.2 was selected to minimize the interference to the separation caused by 2,4,6-triaminotoluene speciation. Solvent consumption was minimized by the use of a narrow-bore column. All of the known reduction products as well as p-cresol and methyl phloroglucinol were identified in culture supernatants from TNT-degrading cultures while pyrogallol and phloroglucinol were not.