Capillary electrochromatography of methylated benzo[a]pyrene isomers: I. Effect of mobile phase tuning

It is of increasing importance that chromatographic methods be developed for the separation and identification of biological and environmentally harmful compounds such as methylated polycyclic aromatic hydrocarbons (PAH). Capillary electrochromatography (CEC) is fast becoming a useful technique for analysis of PAH, as it offers both high efficiency and superior resolution. The separation of 12 methylated benzo[a]pyrene (MBAP) isomers is a challenge due to the extreme hydrophobicity and structural similarity of these compounds. In this work, we present Part I of our ongoing study, a method for the systematic mobile phase tuning for CEC separation of the 12 MBAP isomers. The CEC experiments were conducted utilizing a CEC-octadecylsilica (ODS) stationary phase and fused-silica capillary [(75 μm I.D., 363 μm O.D.) 36.5 cm total length, 25.0 cm effective length] which was slurry pressure packed in our laboratory. Several mobile phase parameters were manipulated to provide optimum separation. These included acetonitrile (ACN) concentration, tris(hydroxymethyl) aminomethane (Tris) concentration, pH, and addition of a tertiary buffer constituent such as tetrahydrofuran (THF) and isopropanol (IPA) to ACN–aqueous buffer mixtures. Optimum CEC separation conditions were achieved using 75% (v/v) ACN–25% (v/v) 12.5 mM Tris, pH 8.0, and 30 kV at 25 °C. These mobile phase conditions were then utilized for Part II of our study, the CEC stationary phase optimization for the separation of 12 MBAP isomers.