Agilent Technologies 1100 Series Reference Manual page 40

2 First Steps with the Fluorescence Detector
Procedure II - Use two LC runs with the Agilent 1100 Series FLD to separate the
compound mix under known conditions and acquire emission and excitation spectra
separately.
Procedure III - Use an Agilent 1100 Series FLD/DAD combination and acquire
UV/Visible spectra (equivalent to excitation spectra) with the DAD and emission spectra
with the FLD-both in a single run.
Procedure I - Take a fluorescence scan
Because fluorescence spectra traditionally have not been easily available with previous
LC fluorescence detectors, standard fluorescence spectrophotometers have been used in
the past to acquire spectral information for unknown compounds. Unfortunately this
approach limits optimization, as there are differences expected in optical design
between an LC detector and a dedicated fluorescence spectrophotometer, or even
between detectors. These differences can lead to variations for the optimum excitation
and emission wavelengths.
The Agilent 1100 Series fluorescence detector offers a fluorescence scan that delivers
all spectral information previously obtained with a standard fluorescence
spectrophotometer, independent of the LC fluorescence detector.
shows the complete information for quinidine as obtained with the Agilent 1100 Series
FLD and a manual cuvette in a single offline measurement. The optima for excitation
and emission wavelengths can be extracted as coordinates of the maxima in the three
dimensional plot. One of the three maxima in the center of the plot can be chosen to
define the excitation wavelength. The selection depends on the additional compounds
that are going to be analyzed in the chromatographic run and the background noise that
may be different upon excitation at 250 nm, 315 nm or 350 nm. The maximum of
emission is observed at 440 nm.
Details for
All excitation and emission spectra of Quinidine (1 µg/ml) are shown in graphic.
Fluorescence intensity is plotted vs excitation and emission wavelengths.
Detector settings: step size 5 nm, PMT 12, Response time 4 s
40 1100 Series FD Reference Manual
Figure 12 on page 44:
Figure 12 on page 44