In general, the sensitivity of the fluorometer is expressed in a signal-to-noise ratio (SNR) that is a comparison of a signal value in the presence of a signal, with a value for system noise, in the absence of signal. Typically it is acquired with the excitation wavelength selected at 350 nm, with an emission scan from 365 to 450 nm. The water Raman test is also preferable because ultrapure water is readily available around the world, the sample is stable, the signal is relatively weak, and the Raman band of water can be measured over the entire wavelength range of the instrument allowing for more robust comparisons, than is possible with a single fluorescent probe.This sensitivity specification is derived from an emission spectrum of the Raman vibrational band for pure water. As such, the water Raman test has become an industry standard as a good measure of the relative sensitivity between different instruments. However, today, the highest sensitivity fluorometers are able to detect at such low concentrations of fluorophores, that the ability to accurately perform a serial dilution down to these detection limits becomes questionable. In the past, some commercial manufacturers of fluorometers used detection limits for specific fluorescent molecules, such as quinine sulfate or fluorescein to demonstrate sensitivity. Given that there are so many variables that go into making a sensitive fluorometer, what is needed is a standard reference test that can be performed by any user, to properly compare one fluorometer with another. For example, a system that has a higher wattage lamp, does not guarantee that it offers better overall fluorescence sensitivity. The overall sensitivity of a spectrofluorometer is determined by many factors including, but certainly not limited to, the optical design, coupling optics, intensity of delivered excitation light, efficiency of fluorescence collection, spectrometer design, detector technology and much more. Fluorescence is a highly sensitive analytical technique, so one of the primary specifications to consider when selecting a spectrofluorometer is its sensitivity.
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