First, I'd like to ask is there a threshold for the amplitude of 2nd order scattering. I noticed i get offscale peak with increasing emission slit width, which means the scattering is more significant than sample emission.
Second, why background (Blank) scattering is stronger than from sample scattering.
finally, any advice to overcome the 2nd order rayleigh scattering in both emission and excitation spectra?
Its an issue with the monochromator. To solve it use the monochromator with a combination of optical cut off filters. Usually there are slots on fluorimeters to accommodate these although some newer instruments will have an order sorting filter wheel.
The steady-state section of my PhD thesis gives probably one of the most through overviews of using a fluorimeter, I've left a link to it.
Second order and Rayleigh scattering can be examined by measuring an Excitation Emission Matrix of water. Figure 59.
So for instance if you are exciting at 300 nm and measuring at 320-800 nm you will get a second order Rayleigh peak at 600 nm. Therefore placing a LongPass 330 nm filter when measuring say at 400-800 nm on the emission side will get rid of the 2nd order.
Likewise if you are measuring excitation at say 600 nm you can place a LP590 nm on the emission side to get rid of any 2nd order.
You can measure the transmission spectra of any long pass filters used using a spectrophotometer and use these curves in conjunction with the wavelength correction factors. See Figure 21 for examples.
The upper limit of your fluorimeter depends on the model of fluorimeter (how the data is processed) and also what detector you have. Likely you have a PMT detector. These have a linear regime. Outwith the linear regime the PMT's quantum efficiency decreases. More details about the linear regime of a PMT and CCD are in Figure 47 and Figure 51 respectively. In extreme cases more photons reaching the PMT leads to less counts as the quantum efficiency decreases.
Thesis Nanometrology using Time-Resolved Fluorescence
Thanks Philip.
your detailed simple explanation is much appreciated.
There is an alternative solution.
Collect emission in between excitation wavelength and the second order scatter, if your sample permits. For most of the samples this range should be fine. For example, if the excitation wavelength is at 400nm, collect emission from 420nm to 780nm. Narrower slits will help you go close to 400nm and 800nm.
To add further. Using cut-off filters causes loss in fluorescence intensity which in turn may lead to decreased sensitivity.
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