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Agilent InfinityLab LC Series 1260 Infinity II FLD User Manual 95
Optimizing the Detector
5
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
Figure 30 on page 95) and to record the emission spectrum. Then with an
optimum emission wavelength determined, the excitation spectrum is acquired.
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 29 on page 94) for each compound.
Since this is a tedious procedure, it is applicable only when there is a limited
number of compounds of interest.
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 30 Excitation and emission spectra of quinidine
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