Determining The Optimum Excitation And Emission Wavelength - Thermo Scientific Dionex UltiMate 3000 Series Operating Instructions Manual

UltiMate 3000 Series:
Fluorescence Detectors FLD-3100 and FLD-3400RS

5.7.1 Determining the Optimum Excitation and Emission Wavelength

The most important parameters that need to be optimized are the excitation and emission
wavelength. Note the following key criteria for determining the wavelength for an
analysis:
• Preferably, select an excitation wavelength on the absorption maximum of the sample
components.
• The mobile phase should be "transparent", showing little or no absorption, at the selected
wavelength. Thus, the excitation wavelength should always be above the UV cutoff of
the solvent (→ table page 123).
• Select an emission wavelength that is at least 20 nm above the excitation wavelength.
The following section outlines two methods to determine the optimum wavelengths. After
you have determined the optimum wavelengths and the retention times, you should perform
another analysis run using these parameters to determine a suitable sensitivity (→ page 81).
Method using 1 sample with the fluorescence detector
1. Set the excitation wavelength to a wavelength at which most sample components
absorb. Many fluorescent molecules absorb at 250 nm. Set the filter wheel setting to a
wavelength that is at least 30 nm above the excitation wavelength (in the example
280 nm). Run a sample analysis in Zero Order Mode. To activate the Zero Order
Mode in Chromeleon, set the EmWavelength property to ZeroOrder. You will get a
peak for most sample components, provided that the emission wavelength is not
(significantly) smaller than the selected filter wavelength.
2. From step 1 you have obtained the retention times of the sample components. Perform
an emission scan for each sample component at its retention time. The excitation
wavelength remains 250 nm. You will get the optimum emission wavelengths (largest
peak) for each sample component. (Even if the excitation wavelength is not selected at
the excitation maximum, the resulting emission spectrum is identical in most cases.)
3. Now perform an excitation scan for each sample component using the optimum
emission wavelengths to determine the optimum excitation wavelengths (largest peak).
Page 78 Operating Instructions