horiba FluoroMax-3, Operation Manual

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FluoroMax-3 v. 3.1 (3 Mar 2006) Optimizing Data
5-12
Selecting the appropriate bandpass
The bandpass (wavelength spread) affects the resolution of your spectra. If the band-
pass is too broad, narrow peaks separated by a small change in wavelength may be un-
resolved. For example, for two 2-nm peaks 5 nm apart, and a bandpass of 10 nm, one
broad peak, instead of two well-defined ones, is visible.
By adjusting the slit widths, the intensity and bandpass of the light is controlled. The
slits of the excitation spectrometer determine the amount of light that passes through
the excitation spectrometer to the sample. The emission spectrometer slits control the
amount of fluorescence recorded by the signal detector. Signal level is proportional to
the square of the slit width, that is,
signal
level
∝
(slit width)
2
Bandpass is calculated using the following formula:
bandpass (nm) = slit width (mm) × dispersion (nm/mm)
A FluoroMax
®
-3, which has a single-grating monochromator and 1200 grooves/mm
gratings, has a dispersion of 4.25 nm/mm.
For steady-state fluorescence measurements, set the entrance and exit slits the same for
a monochromator. (This occurs automatically when using bandpass units in FluorEs-
sence™.) For biological samples that may photobleach, try narrowing the excitation
slits and opening the emission slits wider.