Thermo Scientific Dionex UltiMate 3000 Series Operating Instructions Manual page 87

Method using 2 samples of different concentrations with a diode array detector and the
fluorescence detector
If your UltiMate 3000 system includes a diode array detector (DAD-3000 or
DAD-3000RS), you can use the UV spectrum determined with the diode array detector to
find the optimum excitation wavelengths and retention times.
1. Use a sample with a concentration that is suitable for diode array detectors (for
example, 100 ng/µL) and run an analysis with the DAD, recording a 3D field. You can
extract the retention times and absorption maxima, and thus the optimum excitation
wavelengths, of each sample component from the Chromeleon 3D plot (→ Fig. 34,
page 79).
2. Use a sample with a concentration that is suitable for fluorescence detectors (for
example, 100°pg/µL for high-fluorescing substances) and run an emission scan for
each sample component with the FLD, using the retention times and excitation
wavelengths. Note that you may have to add a time offset between the DAD and FLD
when determining the retention times if the two detectors are connected in series. You
will get the optimum emission wavelengths (largest peak) for each sample component.
Tip: Chromeleon 7.1 (or later) supports 3D spectra scanning for the FLD, which
facilitates determining the retention times and absorption maxima without a
diode array detector (→ page 76).
Operating Instructions
Fluorescence Detectors FLD-3100 and FLD-3400RS
Absorption-
maximum
peak 2
Fig. 34: 3D data recorded with a diode array detector
Retention time peak 2
UltiMate 3000 Series:
Page 79