Hitachi CHROMASTER 5160 Instruction Manual page 449

High
concentration
I
s
I
o
I
t
(a) Absorptiometry
Fig. 2-5 Comparison of Absorptiometry and Fluorometry
A graphical description of why fluorescence can provide better
sensitivity than absorbance is presented in Fig. 3-5. In this
figure, the signal I
the intensity of the incident beam I
transmitted beam I
point where the difference between I
noise level. In contrast, when fluorometry is used, the observed
signal I is directly proportional to the concentration and the
F
background has a fluorescence of zero. When a small signal is
observed it is compared to a very small signal (since the blank
does not fluoresce) and is readily amplified for detection.
In addition, since the fluorescence emission wavelength is
different from the excitation wavelength (incident beam
wavelength), scattering due to the excitation radiation is
negligible.
Low
concentration
I
o
I
t
In addition to providing high sensitivity, fluorescence detection
can provide a fluorescence spectrum and an excitation spectrum
(which is very similar to the absorbance spectrum).
If the sample contains two compounds, selection of the
appropriate excitation and emission wavelengths may be used to
provide qualitative and quantitative information about the
components in the mixture. This point is described in Fig. 3-6.
An attempt to quantitate compound B in a mixture of A and B
using absorbance will not be successful because the absorbance
spectra of the two compounds overlap at all wavelengths (Fig.
3-6 (a)).
2 - 9
is used to represent the difference between
S
and the intensity of the
o
in absorptiometry. The detection limit is the
t
t
High
concentration
I
s
I
F
and I is equivalent to the
o
Low
concentration
I
F
Amplification
I
F
(b) Fluorometry