Hitachi CHROMASTER 5160 Instruction Manual page 453

If emission occurs only in the entrance area for excitation
beam, such a sample needs to be diluted with an
appropriate factor before measurement.
The second factor is called "concentration quenching"
where activity is disturbed by interaction of molecules.
Another potential error that may occur when fluorescence
detection is used is the re-absorption of fluorescence (self-
absorption of fluorescence). An example of this
phenomenon is shown in Fig. 3-9. Re-absorption of
fluorescence occurs when the tail of the shorter-wavelength
side of fluorescence spectrum overlaps the tail of the long-
wavelength side of the excitation spectrum. When this
phenomenon occurs, the fluorescence spectrum appears to
be somewhat shifted toward the longer-wavelength.
This phenomenon will very rarely cause a substantial error
in quantitation when fluorescence detection is used, and
should not be a cause for concern.
Excitation
spectrum
Fig. 2-9 Re-absorption of Fluorescence
Anyway if a high concentration may have caused a
measurement error, dilution with an appropriate factor is
necessary.
2 - 13
Fluorescence
spectrum
Fluorescence re-absorbed
Wavelength