Optimization Overview

Setting the right PMT value

For most applications a setting of 10 is adequate (see Finding the Best Signal Amplification). The FLD A/D converter exhibits a large linear range making PMT switching unnecessary for most applications. For example, if at high concentrations a peak is cut off; decrease the PMT setting. Remember that low PMT settings decrease the signal to noise ratio.

The built-in PMT gain test uses the parameters in the detector. When using the PMT gain test, the wavelength setting and lamp energy mode (depending on Multiwavelength-Mode and Lamp-Economy) will affect the PMT gain calculation.

NOTE: If you have changed one or more parameter(s), you have to press 'OK' to write down the new settings into the FLD. Then re-enter 'FLD-Signals' and start the PMT gain test.

Using an appropriate response time

For most applications a setting of 4 seconds is adequate (see Selecting the Best Response Time). Only for high speed analyses (short columns at high flow rates) a lower setting is recommended. Bear in mind that even if the response time is too high fast peaks will appear a little smaller and broader but retention time and peak areas are still correct and reproducible.

Finding the optimum wavelength

Most fluorescent active molecules absorb at 230 nm (see Finding the Best Wavelengths). Set the excitation wavelength to 230 nm and on-line scan the emission spectra (multi-emission mode). Then set the determined emission wavelength and perform a multi-excitation scan (multi-excitation mode) to find the best excitation wavelength.

Evaluating fluorescence spectra

In contrast to diode array based UV detectors where UV spectra are evaluated by taking a spectrum at the peak maximum and selecting a reference spectrum at the baseline, correct fluorescence spectra are obtained by selecting a peak maximum spectrum and a reference around the inflection points. Selecting reference spectra at the baseline is not useful because the spectrum on the baseline is very noisy (no light!).

Switching lamp ON only for analysis

Unless maximum sensitivity is needed, the lamp lifetime can significantly be increased by switching it on just for analysis. In contrast to other LC detectors the fluorescence detector equilibrates within seconds after the lamp is switched ON.

NOTE: For highest reproducibility and linearity change the lamp setting to always ON (default is on only during run).One hour of initial warm-up of the instrument is recommended.

Do not overpressurize the detector flow cell

Be aware to not exceed a 20 bar pressure drop after the flow cell when hooking up additional devices like other detectors or a fraction collector. It's better to place a UV detector before the fluorescence detector.

NOTE: When comparing fluorescence excitation spectra directly with DAD spectra or literature based absorbance spectra, you should consider large differences in the used optical bandwidth (FLD = 20 nm) which cause a systematic wavelength maximum shift depending on the absorbance spectrum of the compound under evaluation.