PerkinElmer 200A Series, Руководство пользователя

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50.
Series 200a Fi User’s Guide
3. Second-order and Third-order Radiation
A diffraction grating that is set to transmit radiation of a specific wavelength (e.g. 250 nm) will also
transmit multiples of the radiation (e.g. 500 nm). When a fluorescence spectrum is collected, the
analyst should be aware of the second order (and third order) phenomena and take care that peaks
are not misidentified.
For instance, if the excitation wavelength is 240 nm, the second-order and third-order excitation occur
at 480 and
720 nm, respectively. To eliminate second and third order radiation, it is simply necessary to place a
short-wavelength cutoff filter in the path of fluorescing radiation (before the fluorescence
monochromator).
4. An Example of a Fluorescence Spectrum
Figure 3-11 provides an example of a fluorescence spectrum, which contains the various types of
peaks indicated above. In most cases, the fluorescence of the compound of interest lies closer to the
excitation than shown in Fig. 3-10, and overlapping of peak of interest and the second order radiation
is rarely a problem. Typically, the peak for the compound of interest might correspond to peak *D,
and the analyst might have to correct for the Raman peak (e.g. use a shorter excitation wavelength).
*A Scattering of excitation beam
*B Raman scattering due to eluent
*C Fluorescence of impurity
*D Fluorescence of compound of interest
*E Second-order scattering of excitation beam
Fig. 3-11 Measurement Example of Fluorescence Spectrum
5. Temperature Dependency of Fluorescence Intensity
In most cases, the fluorescence intensity decreases by 1 to 2% as the temperature of sample
increases by 1
° C. It should be noted, however, that in some biochemical samples, the
fluorescence intensity decreases by approx. 10% as the temperature of sample increases by 1 ° C
(especially if the fluorescence is due to the indole ring system).
Relative intensity
Wavelength EX
×
2 EX
*A
*B
*C
*D
*E