Development of a Liquid Chromatography Method to Separate and Fractionate EPA’s 16 Priority Polycyclic Aromatic Hydrocarbons (PAHs)

Abstract: Polycyclic Aromatic Hydrocarbons (PAHs) are persistent, toxic and carcinogenic environmental pollutants consisting of two or more aromatic rings. The estimation of the toxicity of PAHs can be challenging and time-consuming due to the complexity of PAH-mixtures found in the environment, for example at contaminated sites. To solve the problem of a high complexity, PAH-mixtures can be divided into fractions. In the present study, Normal Phase (NP) and Reverse Phase (RP) Liquid Chromatography (LC) methods were evaluated and developed to separate and fractionate 16 U.S. Environmental Protection Agency’s (EPA) priority PAHs. Two stationary and six mobile phases, different column temperatures, flow rates, injection volumes, gradient and isocratic elutions were evaluated in order to achieve the best separation and fractionation. Best separation of PAHs was achieved with the RP-LC method using a phenyl stationary phase and gradient elution with mixture of methanol, acetonitrile and water. Fractionation was performed based on PAHs’ retention time windows, as well as peaks’ threshold and slope values. The elution order of fractions was verified by identification and quantification of the PAHs by GC-MS. Fractionation of a soil extract with the developed RP-LC method resulted in successful separation and fractionation of five EPA’s priority PAHs naphthalene, fluorene, phenanthrene, anthracene and chrysene, as well as two non-priority PAHs carbazole and 4H-Cyclopenta[d,e,f]phenanthrene. The study shows that developed RP-LC method can be further optimized to fractionate complex mixtures of alkyl substituted, oxygenated and other polycyclic aromatic compounds (PACs).