Agilent Technologies 1100 Series Reference Manual page 38

2
First Steps with the Fluorescence Detector
These tasks have to be repeated for each compound using either a
fluorescence spectrophotometer or stop-flow conditions in LC. Usually each
compound requires a separate run. As a result, a set of excitation and
emission spectrum is obtained (Figure 10 on page 38) for each compound.
Since this is a tedious procedure, it is applicable only when there is a limited
number of compounds of interest.
The Agilent 1100 Series LC offers three different ways to obtain complete
information on a compound's fluorescence:
Procedure I - Take a fluorescence scan offline for a single compound as
described above for the mobile phase. This is done preferably with a manual
FLD cuvette when pure compounds are available.
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.
Figure 12 on page 42 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
40 1100 Series FD Reference Manual