On-the-Fly Fluorescence Lifetime Determination with Total Emission Detection in HPLC.

The entire fluorescence decay profile during HPLC elution has been directly measured on-the-fly in HPLC at higher sensitivity than in previous literature reports. The fluorescence is excited with the fourth harmonic (266 nm) of a pulsed Nd:YAG laser system and detected broadband with a photomultiplier tube and a digital storage oscilloscope. Detection limits in the range 1-10 ppb are found for several individual polycyclic aromatic hydrocarbons (PAHs) when the total time-integrated fluorescence is analyzed. The chromatograms of PAH mixtures containing 8-10 species were lifetime analyzed with a simple phase plane analysis, in which a single lifetime is determined from the fluorescence decay profile for each point on the chromatogram. The determination of lifetimes under coelution conditions is also illustrated and discussed.