Optimizing the Fluorescence Detector 3
1100 Series FD Reference Manual 71
Reducing Stray Light
Cut-off filters are used to remove stray light and 2
nd
order or higher stray light
by allowing complete transmission above the cut-off and little or no
transmission below the cut-off point. They are used between excitation and
emission gratings, to prevent any stray excitation light from reaching the
photomultiplier tube, when it is measuring emission.
When the emission and excitation wavelengths are close together, the
distortion due to scattering severely limits the sensitivity. When the emission
wavelength is twice the excitation wavelength the 2
nd
order light is the
limiting factor. To explain the effect of such higher order light, assume the
detector is on, but no sample is eluting through the flow cell.
The lamp sends 1 million photons into the flow cell at, for example 280 nm.
Scattering on the surface of the flow cell and scattering from the molecules of
solvent allow 0.1% of this light to leave the cell through the window at right
angles to the incident light. Without a cut-off filter, these remaining 1000
photons will reach the emission grating. 90% will be reflected totally without
dispersion onto the photomultiplier. The other 10% disperses at 280 nm
(1
st
order) and at 560 nm (2
nd
order). To remove this stray light, you need a
cut-off filter around 280 nm.
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