When using an ACQUITY UPLC FLR Detector in emission mode, does changing the gain change the sensitivity? - WKB118420

ENVIRONMENT

• ACQUITY UPLC FLR Detector
• 2475 Multi-Wavelength Fluorescence Detector

ANSWER

■ Mode that can acquire with the fluorescence detector
Energy mode
Outputs the direct fluorescence intensity. The intensity changes depending on the gain. Detection limit: 10000
Emission mode
In this mode, the fluorescence intensity is corrected and calculated inside the detector and output.
The intensity is calculated using the relative value of water Raman.
The emission intensity is output by correcting the fluorescence intensity value with the Raman intensity and gain value at the normalization.
For the acquisition mode where the output value is not affected by the gain, the feature is that there is no difference in intensity between individual detectors or due to detector deterioration.

■ For the emission mode
The following calculation is performed inside the detector to output the intensity.
Emission unit = ( Fluorescence intensity at acquisition / Gain at acquisition) × ( Gain at normalization / Raman intensity at normalization ) × 100

Fluorescence intensity at acquisition: Value affected by gain with direct fluorescence intensity
Gain at acquisition: Gain value during acquisition (value set by the instrument method)
Gain at normalization: Gain value determined at normalization
Raman intensity at normalization: Raman intensity obtained at normalization

From the above formula, it is clear that the emission unit is corrected with the value obtained by normalizing thefluorescence intensity per gain .
(In normalization, a gain value that keeps Raman intensity constant is determined.)
Therefore, changing the gain does not change the intensity.

However, if the gain is too high or too low, the intensity may change depending on the gain set even in emission mode.
If Fluorescence intensity at acquisition exceeds the upper detection limit of FLR (Max = 10000 in energy mode), the emission unit may also be off-scale. If this happened, you can get the correct peak by lowering the gain to a gain value where the intensity in energy mode drops below 10000.
Also, if the gain value is extremely low, the intensity may differ depending on the gain even in emission mode.
The considered reason is that extremely low Fluorescence intensity at acquisition value results in a large error in emission intensity calculation.

■ Conclusion
The emission unit does not change in intensity when the gain is changed within the optimum gain range for the compound,
however, if the gain value is inappropriate as too high or too low, the intensity may differ or the gain value may be off-scale.
When calculating the emission intensity, the gain value is used in the calculation, therefore it is incorrect that the emission mode and the gain are completely unrelated.

ADDITIONAL INFORMATION