120 Agilent InfinityLab LC Series 1260 Infinity II FLD User Manual
5
Optimizing the Detector
Optimization Overview
3 Finding the optimum wavelength
Most fluorescent active molecules absorb at 230 nm (see “Finding the Best
Wavelengths” on page 122). Set the excitation wavelength to 230 nm and
on-line scan the emission spectra (multi-emission mode). Then set the
determined emission wavelength and perform a multi-excitation scan
(multi-excitation mode) to find the best excitation wavelength.
4 Evaluating fluorescence spectra
In contrast to diode array based UV detectors where UV spectra are
evaluated by taking a spectrum at the peak maximum and selecting a
reference spectrum at the baseline, correct fluorescence spectra are
obtained by selecting a peak maximum spectrum and a reference around
the inflection points. Selecting reference spectra at the baseline is not
useful because the spectrum on the baseline is very noisy (no light!).
5 Switching lamp ON only for analysis
Unless maximum sensitivity is needed, the lamp lifetime can significantly
be increased by switching it on just for analysis. In contrast to other LC
detectors the fluorescence detector equilibrates within seconds after the
lamp is switched ON.
6 Do not overpressurize the detector flow cell
Be aware to not exceed a 20 bar pressure drop after the flow cell when
hooking up additional devices like other detectors or a fraction collector.
It's better to place a UV detector before the fluorescence detector.
For highest reproducibility and linearity change the lamp setting to always ON (default is
on only during run).
One hour of initial warm-up of the instrument is recommended.
When comparing fluorescence excitation spectra directly with DAD spectra or literature
based absorbance spectra, you should consider large differences in the used optical
bandwidth (FLD = 20 nm) which cause a systematic wavelength maximum shift depending
on the absorbance spectrum of the compound under evaluation.
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