No abstract

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.

A phase-modulation fluorometer was used for on-line detection of benzo(k)fluoranthene and benzo(b)fluoranthene in a mixture of the two that was isocratically eluted from a high-performance liquid chromatographic column. Three different mobile-phase solvent compositions provided three degrees of chromatographic separation, ranging from complete to almost no separation. Fluorescence lifetimes were calculated from phase and modulation data that were collected at three different excitation modulation frequencies. Heterogeneity analysis was then used to calculate the fluorescence lifetimes and fractional intensity contributions of each component at each point along the chromatograms. The fractional intensity contributions were multiplied by the fluorescence intensities in the steady-state chromatogram of the mixture in order to construct the fluorescence intensity chromatograms of the individual components. Retention times and fluorescence lifetimes of the components obtained from the constructed chromatograms were in good agreement with values obtained from steady-state chromatograms of the individual components. Relative errors of the peak intensities were as high as 23%. Batch experiments were also performed to determine the best results that could be expected for the heterogeneity analysis of mixtures of the two components in the solvent systems that were used in the chromatographic experiments.