Waters 2475, Operator'S Manual, Page: 32 / 228

1-18 Theory of Operation
electronics. Too low a gain reduces sensitivity to emission signals, degrading
signal-to-noise ratios. The detector therefore requires you to specify a gain
setting for the PMT before you inject a sample. However, before the injection,
you cannot know the magnitude of your fluorescence signal. Users
traditionally resolve this difficulty by running several injections to determine
a suitable gain setting, a tedious process, especially when they run timed
event changes in gain and/or wavelength.
The Auto-Optimize Gain diagnostic function runs a trial chromatogram and
displays the ideal gain values. The reported values are based on an algorithm
that ensures a 2× margin against overloading the PMT and its associated
electronics with variations in fluorescence intensity for concentrated samples.
In the case of timed event changes in gain and/or wavelength, the report
reflects adjusted values representing the ideal gain setting for each critical
timed event region. You should incorporate the reported gain values in the
method, including its timed event table, to optimize the method’s
performance.
The detector also monitors the maximum fluorescence signal level throughout
the run. When you use the analog outputs during data collection, it displays a
minimum EUFS value that applies to the entire chromatogram. Like the ideal
gain value, the EUFS value assumes a 2× margin to account for any
variations in fluorescence intensity. Based on this report, you must adjust the
gain values in the method, including its timed event table, to optimize the
performance of the method.
The detector also monitors the maximum fluorescence signal level throughout
the entire run. It recommends a minimum EUFS value, which applies to the
entire chromatogram and appears when you use the analog outputs during
data collection. This value is also computed assuming a 2× margin for error.
Method optimization
You can download a method that includes timed event changes. The timed
event changes that alter gain, excitation wavelength, or emission wavelength
are critical “light condition” changes, the points at which the signal peak
maximum search is renewed. You must therefore enter any timed event gain
changes at strategic points before peaks to improve the detector’s sensitivity
to peaks. The goal is to provide a retention time demarcation point at which a
gain change could be tolerated without disrupting the integration of peaks in
the chromatogram. Before you run the Auto-Optimize Gain diagnostic
function, you must set the initial conditions. Timed events are not absolutely
necessary, but this causes the detector to recommend only one gain value