Rj. Vandenesse et al., SIMULTANEOUS AND CONSECUTIVE 2-PHOTON-EXCITED FLUORESCENCE DETECTION IN CONVENTIONAL-SIZE LIQUID-CHROMATOGRAPHY, Analytica chimica acta, 309(1-3), 1995, pp. 135-144
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.