ON-THE-FLY FLUORESCENCE LIFETIME DETECTION IN HPLC USING A MULTIHARMONIC FOURIER-TRANSFORM PHASE-MODULATION SPECTROFLUOROMETER

Frequency-domain fluorescence lifetime detection has been demonstrated recently as a technique for detecting and resolving overlapping peaks on-the-fly in reversed-phase high-performance liquid chromatography (RP-HPLC). However, instrument limitations necessitate multiple injections of the same sample for peak resolution. The introduction of a commercially available multiharmonic Fourier transform spectrofluorometer (MHF) eliminates this problem and offers other advantages as well. The MHF data acquired on-the-fly during a single chromatographic run contain all of the multifrequency information needed to determine lifetimes and to indicate and resolve overlapping peaks at intervals as short as several milliseconds. This allows essentially continuous monitoring of both lifetime and intensity during chromatographic elution. This approach to the addition of lifetime in fluorescence detection increases the information content of chromatographic-based analysis without increasing the time per analysis. This paper presents the first results obtained using MHF detection, including measurements of single components and simple mixtures. Optimization of instrumental and experimental conditions is discussed, including the nature of the raw data and the procedures that were developed to extract and process the necessary lifetime and intensity information.