The water Raman test is a good measure of relative sensitivity between different instruments, provided the experimental conditions used to compare the systems are the same.
Unfortunately, there are a number of different ways of handling the data, all of which are valid but which will give quite different numbers. Therefore, it is important not only to know how the water Raman S/N values is measured, but also how the data were treated.
In general, the water Raman S/N test method combines a value for system sensitivity (in the presence of a signal) with a value for system noise (in the absence of signal) to show the overall performance of the instrument.
At HORIBA Scientific we define the S/N as the difference of peak and background signal, divided by the square root of the Background signal. The peak signal is measured at the water Raman peak (397 nm for 350 nm excitation) and the noise in a region (450 nm) where no Raman signal is present, and an "ideal" system would give a signal value of zero.
Another commonly used method is to divide the difference (Peak signal – Background signal) by the RMS value of the noise on the background signal. This second method is used by a few other manufacturers.
Some actual data from our FluoroLog FL3-22 system (a typical system) will serve to show the difference between the two methods.
The experimental conditions were as follows:
Excitation 350 nm with 5 nm bandpass
Emission 365–450 nm with 5 nm bandpass
Interval 0.5 nm
Integration 1 s
No smoothing of data points
Standard room temperature, red sensitive, detector (Note: make sure the test is carried out with the actual detector you will be using. All HORIBA Scientific systems are specified with a R928P PMT at room temperature).
The measurements provided the following data:
Peak signal (at 397 nm) = 1,017,740 cps
Background (at 450 nm) = 724 cps
Peak to peak noise of background (at 450 nm) = 31.9 c (measured with a separate kinetic scan), which gives an RMS noise of the background signal of 31.9/5 = 6.38.
Therefore, the HORIBA Scientific method gives a water Raman S/N of (1017740–724)/(724)½ = 37,797.
The second method similarly gives a water Raman S/N of (1017740–724)/6.38 = 159,407.
HORIBA Scientific feels that the first method is correct although it gives a lower number. The second method only takes into account the detector noise and the shot noise of the electronics.
On the other hand, by using the background total intensity as a measure of noise, the HORIBA Scientific method is more representative of a real "live" experiment where noise is also influenced by factors like the quality of the optics and scattered light in the system. These additional factors will influence the ability to measure a very low signal from a sample and should not be left out.
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