PerkinElmer 200A Series, User Manual

Page: 46 / 172

46.
Series 200a Fi User’s Guide
In this discussion, we will consider the detection of a sample that has a transmittance of 99% (relative to
the blank). If we assume that the inaccuracy of the % transmittance measurement is 0.1%, the reliability
of the observed data is as follows:
Percent transmittance of blank
Percent transmittance of sample
100.0 ± 0.1%
99.0
±
0.1%
Difference (proportional to concentration of sample) 1.0 ± 0.2%
In this example, the uncertainty in the concentration measurement is ± 20%. In contrast, in fluorometry,
a difference in the signal is directly proportional to the concentration of sample.
An analysis of the error in the measurement is as follows:
Output signal level at measurement of sample
Value corresponding to blank
100
±
0.1
0
± 0.1
Difference(proportional to concentration of sample) 100 ± 0.2
The error in the measurement using fluorescence is considerably smaller than that obtained using
absorbance, thus fluorometry is very advantageous for analyzing samples with a low concentration of the
compound(s) of interest. If a compound exhibits a strong fluorescence signal, it is likely that fluorescence
can detect concentrations that are two orders of magnitude lower than absorbance.
A graphical description of why fluorescence can provide better sensitivity than absorbance is presented in
Fig. 3-6. 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.
(a) Absorptiometry (b) Fluorometry
Fig. 3-6 Comparison of Absorptiometry and Fluorometry
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