PerkinElmer 200A Series, User Manual

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System Description
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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). These may be useful in
identifying the eluent.
If the eluent 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. 3-7. 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. 3-7(a)).
In contrast, if the fluorescence spectra do not overlap (Fig. 3-7(b)), quantitative analysis of each
compound can be made by selecting the appropriate fluorescence emission wavelength (even if the
absorption wavelengths of two components are the same).
(a) Absorptiometry (b) Fluorometry
Fig. 3-7 Spectroscopic Measurement of a Sample Containing Two Components
Table 3-4 compares the analytical information obtained via absorptiometry and fluorometry.
Table 3-4 Comparison of Analytical Information
Attainable with Absorptiometry and Fluorometry
Absorptiometry Fluorometry
Absorption spectra only
(corresponding to excitation spectra)
• Excitation spectra
• Fluorescence spectra
Notes on Fluorescence Analysis
1. Raman Scattering
When fluorescence is measured, two additional peaks may appear in the spectrum. The Rayleigh peak
appears at the excitation wavelength and is due to scattered light, while the Raman peak appears at
longer wavelength than the excitation. The position of the Raman band is dependent on the excitation
wavelength, while the position of the fluorescence is independent of the excitation wavelength.
Absorption spectra
Component A
Component B
Excitation spectra
Component A
Fluorescence spectra
Component B