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Multifrequency Phase-Modulation Fluorescence Lifetime Determinations On-the-Fly in HPLC

Volume 43, Number 8 (Dec. 1989) Page 1363-1367

Cobb, W. Tyler; McGown, Linda B.

The first use of a multifrequency, phase-modulation spectrofluorometer for fluorescence lifetime determinations on-the-fly in HPLC is described. Direct, simultaneous measurements of fluorescence intensity, phase-shift, and demodulation are made at one-second intervals for polycylic aromatic hydrocarbons as they are eluted from the chromatograph. Fluorescence lifetime is calculated from both the phase-shift and the demodulation; the two independent values can be used to indicate the absence or presence of more than one component at any point in the chromatogram. Special considerations regarding data correction and calibration for phase-modulation lifetime determinations under continuous-flow conditions are discussed, along with effects of flow rate and mobile phase composition.