Agilent Technologies InfinityLab LC Series User Manual page 96

5
Optimizing the Detector
Method Development
The Agilent 1200 Infinity 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 1260 Infinity Fluorescence
Detector to separate the compound mix under known conditions and acquire
emission and excitation spectra separately.
Procedure III - Use an Agilent 1200 Infinty 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 1260 Infinity 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 31
with the Agilent 1260 Infinity Fluorescence Detector 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
96
on page 97 shows the complete information for quinidine as obtained
Figure 31 on page 97:
Agilent InfinityLab LC Series 1260 Infinity II FLD User Manual