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Development of a new reversed-phase liquid chromatography and fluorescence method with multichannel selective wavelength detection for the analysis of benzo[a]pyrene and six of its isomers



Walter B. Wilson, Stephen A. Wise, Lane C. Sander


The baseline separation of benzo[a]pyrene in complex samples via reversed-phase liquid chromatography (RPLC) is particularly challenging due to the potential for interferences from other molecular mass 252 (MM, g/mol) polycyclic aromatic hydrocarbons (PAHs). The work presented here explores the use of different types of RPLC stationary phases and different fluorescence detection conditions for application to method development. Six different stationary phases were investigated with SRM 869b and SRM 1647f for potential use in the development of PAH methods: polymeric C18, monomeric C18, narrow pore C30, wide pore C30, phenylhexyl, and pentafluorophenyl phases. Although the best chromatographic separation was obtained with the polymeric C18 stationary phase, seven MM 252 PAH isomers were not fully resolved and selective detection was utilized to eliminate interferences. Stop-flow fluorescence (excitation and emission) spectra were recorded for the seven PAHs; these spectra differed significantly among the isomers. Based on these spectra, the appropriate excitation/emission wavelengths were determined to be 406/440 nm, 243/509 nm, and 290/411 nm. These conditions were used in the new RPLC/FL method and the seven MM 252 PAH isomers were baseline resolved in their respective chromatograms. The new RPLC/FL method was used to identify the MM 252 PAHs in a diesel particulate extract (SRM 1975).


Reversed-Phase Liquid Chromatography, Standard Reference Materials, Stationary Phases, Polycyclic Aromatic Hydrocarbons, Fluorescence
Created September 28, 2018, Updated January 29, 2019