Hitachi PRIMAIDE 1440, Инструкция по эксплуатации

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2 - 9
A graphical description of why fluorescence can provide better
sensitivity than absorbance is presented in Fig. 3-5. In this
figure, the signal I
S
is used to represent the difference between
the intensity of the incident beam I
o
and the intensity of the
transmitted beam I
t
in absorptiometry. The detection limit is the
point where the difference between I
t
and I
o
is equivalent to the
noise level. In contrast, when fluorometry is used, the observed
signal I
F
is directly proportional to the concentration and the
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.
Fig. 2-5 Comparison of Absorptiometry and Fluorometry
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)).
Low
concentration High
concentration High
concentration Low
concentration
I
o
I
F
I
F
I
F
I
s
I
t
I
o
I
s
I
t
Amplification
(a) Absorptiometry (b) Fluorometry