Waters 2475, Operator'S Manual, Page: 30 / 228

1-16 Theory of Operation
wavelength pairs and plots the maximum fluorescence signal value for each
sample component. The MaxPlot outputs the greater of the fluorescence
values on the selected channels.
Difference plot
The detector allows you to obtain a difference plot in multichannel mode. The
difference plot function monitors fluorescence at user-selected
excitation/emission wavelength pairs and plots the difference in signal value
between them.
Spectrum scanning
You can use the detector as a fluorometer to acquire spectra and store them as
a file. The major difference between this detector and a double-beam
spectrophotometer is that this detector employs only one flow cell rather than
a simultaneous sample and reference flow cell pair. The detector obtains a
fluorescence spectrum by performing the following types of scan on the flow
cell:
• Zero scan – Characterizes the baseline spectrum of a solvent.
• Excitation sample scan – Subtracts the zero scan, so the displayed or
charted results are of the sample’s excitation spectra only.
• Emission sample scan – Subtracts the zero scan, so the displayed or
charted results are of the sample’s emission scan only.
To obtain an excitation or emission spectrum of a sample, run a zero scan
followed by the appropriate sample scan. Typically, a zero scan is run with
pure solvent. A sample scan is usually run with the analyte dissolved in the
same solvent.
Lamp energy and performance
In conventional designs of fluorescence detectors, the signal-to-noise
performance of the instrument is directly proportional to the lamp energy
input to the instrument. Lamp energy input to the detector can be affected by
• age and efficiency of the lamp.
• improperly maintained optics and/or flow cell.
• normal degradation of optical components (including the PMT).