SIMULTANEOUS AND CONSECUTIVE 2-PHOTON-EXCITED FLUORESCENCE DETECTION IN CONVENTIONAL-SIZE LIQUID-CHROMATOGRAPHY

The applicability of two-photon excitation (TPE) for fluorescence detection in now dynamic systems was explored. Emphasis was on conventional-size liquid chromatography (LC) and a direct comparison was made with one-photon excitation (OPE) by the use of standard laser- and lamp excitation. Simultaneous two-photon excitation (STPE) with visible laser light was used for fluorescence detection of UV-absorbing analytes; in consecutive two-photon excitation (CTPE) fluorescence from higher excited states was detected. In both TPE modes fluorescence is measured at the short-wavelength side of the laser light, a spectral region with relatively low background signals. STPE fluorescence will only have potential if the background measured in real samples is sufficiently low; this was investigated for the LC analysis of urine spiked with indole-3-acetic acid. The relative intensities of signals from the urine matrix and indole-3-acetic acid were not improved; that is, no extra selectivity was gained by applying STPE. In CTPE, one photon is sufficient to bring the analyte in an excited electronic state so that in this mode, in principle, both normal and short-wavelength fluorescence can be measured. CTPE fluorescence has a distinct potential; it was observed for aminoanthraquinones, one of them has a very low fluorescence quantum yield in protic eluents. Detection limits were at the nM level. Mitoxantrone, a potent anti-tumour drug, could be detected in urine at a similar level by applying excitation at 625 nm and detecting higher excited state emission in a 290-400 nm window; sample treatment prior to LC was very simple.