Step 2: Optimize Limits Of Detection And Selectivity - Agilent Technologies 1260 Infinity User Manual

4
Using the Fluorescence Detector
Method Development

Step 2: Optimize Limits of Detection and Selectivity

To achieve optimum limits of detection and selectivity, analysts must find out
about the fluorescent properties of the compounds of interest. Excitation and
emission wavelengths can be selected for optimum limits of detection and best
selectivity. In general, fluorescence spectra obtained with different
instruments may show significant differences depending on the hardware and
software used.
The traditional approach is to extract an appropriate excitation wavelength
from the UV spectrum that is similar to the fluorescence excitation spectrum
(see
optimum emission wavelength determined, the excitation spectrum is
acquired.
Excitation spectrum
with emission at
440 nm, emission
spectrum with
excitation at 250 nm
of 1 µg/ml quinidine.
Detector settings:
Step size 5 nm, PMT
12 Response time 4 s.
Figure 31
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
78
Figure 31 on page 78) and to record the emission spectrum. Then with an
Excitation and emission spectra of quinidine
(Figure 30 on page 76) for each compound.
Agilent 1260 FLD User Manual