Step 3: Set Up Routine Methods - Agilent Technologies 1260 Infinity User Manual

4
Using the Fluorescence Detector
Method Development

Step 3: Set up Routine Methods

In routine analysis, sample matrices can have a significant influence on
retention times. For reliable results, sample preparation must be thorough to
avoid interferences or LC methods must be rugged enough. With difficult
matrices, simultaneous multi-wavelength detection offers more reliability than
timetable-controlled wavelength switching. The FLD can, in addition, acquire
fluorescence spectra while it records the detector signals for quantitative
analysis. Therefore qualitative data are available for peak confirmation and
purity checks in routine analysis.
Multi wavelength detection
Time-programmed wavelength switching traditionally is used to achieve low
limits of detection and high selectivity in routine quantitative analysis. Such
switching is difficult if compounds elute closely and require a change in
excitation or emission wavelength. Peaks can be distorted and quantitation
made impossible if wavelength switching occurs during the elution of a
compound. Very often this happens with complex matrices, influencing the
retention of compounds.
In spectral mode, the FLD can acquire up to four different signals
simultaneously. All of them can be used for quantitative analysis. Apart from
complex matrices, this is advantageous when watching for impurities at
additional wavelengths. It is also advantageous for reaching low limits of
detection or increasing selectivity through optimum wavelength settings at
any time. The number of data points acquired per signal is reduced and thus
limits of detection may be higher, depending on the detector settings
compared to the signal mode.
PNA analysis, for example, can be performed with simultaneous multi
wavelength detection instead of wavelength-switching. With four different
wavelengths for emission, all 15 PNAs can be monitored (Table on page 91).
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