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

The applicability of two-photon excitation (TPE) for fluorescence dete ction in now dynamic systems was explored. Emphasis was on conventiona l-size liquid chromatography (LC) and a direct comparison was made wit h one-photon excitation (OPE) by the use of standard laser- and lamp e xcitation. Simultaneous two-photon excitation (STPE) with visible lase r light was used for fluorescence detection of UV-absorbing analytes; in consecutive two-photon excitation (CTPE) fluorescence from higher e xcited states was detected. In both TPE modes fluorescence is measured at the short-wavelength side of the laser light, a spectral region wi th 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 in dole-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 suffic ient to bring the analyte in an excited electronic state so that in th is mode, in principle, both normal and short-wavelength fluorescence c an be measured. CTPE fluorescence has a distinct potential; it was obs erved for aminoanthraquinones, one of them has a very low fluorescence quantum yield in protic eluents. Detection limits were at the nM leve l. Mitoxantrone, a potent anti-tumour drug, could be detected in urine at a similar level by applying excitation at 625 nm and detecting hig her excited state emission in a 290-400 nm window; sample treatment pr ior to LC was very simple.