Detection of nitro-polycyclic aromatic hydrocarbons in liquid chromatography by zinc reduction and peroxyoxalate...

Detection limits in the range 0.25-8.5 pg have been obtained for nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) in high-performance liquid chromatography (HPLC). The nitro-PAHs are reduced on-line to the corresponding amino-PAHs and detected by peroxyoxalate chemiluminescence. Quantitative reduction is achieved on a short (3.5 X 0.32 cm) column packed with a 1:1 mixture of glass beads (ca. 40 micron) and zinc particles (40-80 micron). The mobile phase consists of 70-80% acetonitrile, the balance being a 50-mM tris(hydroxymethyl)aminomethane hydrochloride (pH 6.5) buffer, which is necessary both for the reduction and for catalysis of the chemiluminescent reaction. The amino-PAHs are excited by energy transfer from the decomposition products of the reaction between hydrogen peroxide and bis(2,4,6-trichlorophenyl)oxalate. These reagents are introduced by post-column mixing, and the emission is detected by means of a conventional fluorescence detector with its light source turned off. We reported the high sensitivity and selectivity of peroxyoxalate chemiluminescence toward amino-PAHs in earlier work. The method yields a linear response over at least three orders of magnitude, and in most cases the detection limits are better than those obtained by fluorescence detection with the same fluorometer. The reduction column may be placed either before or after the analytical column, so that the analytes are eluted either as the nitro-PAHs or the corresponding amino-PAHs. This feature provides a second characteristic retention time and is useful in identifying the detected compounds. The technique has been applied to the selective detection of nitro-PAHs in carbon black. The carbon black samples were Soxhlet extracted with toluene, evaporated to dryness, and the extracts redissolved in methylene chloride for direct injection into the HPLC column. The principal compounds found in the carbon black samples were poly-substituted nitro-PAHs and unsubstituted PAHs. This method provides a sensitive and selective method for the analysis of complex samples for these environmentally important mutagens.