Determining the Optimum Emission Wavelength (RF2000)Detector Control
Determining the Optimum Emission Wavelength (RF2000)
Fluorescence detectors not only allow you to automatically change the wavelength from time to time (see 
Switching the Wavelength), but also to scan the entire range of the spectrum. Thus, spectra acquisition is possible for determining the optimum emission and excitation wavelengths. For more information, refer to Recording Fluorescence Spectra (RF2000).
The PGM File below assists you in finding the optimum values for the emission wavelength (that is, the EmWavelength parameter). Start the chromatographic analysis using the same conditions as before. Decrease the pump flow to 0.000 ml/min as soon as you reach the peak for which to optimize the emission wavelength (in the example below: 5.370 min) and then start scanning.
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-0.1 |
Pressure.LowerLimit = |
0 |
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Pressure.UpperLimit = |
400 |
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%A.Equate = |
"%A" |
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%A.Type = |
Automatic |
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%B.Equate = |
"%B" |
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%B.Type = |
Automatic |
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%C.Equate = |
"H2O/MeOH 80/20" |
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%C.Type = |
Automatic |
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%D.Equate = |
"%D" |
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%D.Type = |
Automatic |
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Flow = |
1.000 |
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%B = |
0.0 |
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%C = |
100.0 |
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%D = |
0.0 |
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Emission.ExWavelength = |
275 |
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Emission.EmWavelength = |
350 |
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Emission.Gain = |
1.0 |
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Emission.Response = |
0.5 |
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Emission.Sensitivity = |
Low |
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Emission.Step = |
Auto |
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Emission.Average = |
On |
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0.000 |
Emission.Autozero |
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Inject |
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Emission.AcqOn |
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5.370 |
; stop flow |
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Flow = |
1.000 |
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Flow = |
0.000 |
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; start scanning |
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Emission.ScanEmission |
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; wait for start of scan |
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Wait |
Emission.Scanning |
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; wait for end of scan |
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Wait |
Not emission.Scanning |
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; continue flow |
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Flow = |
0.000 |
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Flow = |
1.000 |
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10.000 |
Emission.AcqOff |
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End |
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You receive the usual chromatogram until 5.370 min. From 5.370 min on, the emission spectrum of the substance currently being in the flow cell is recorded. The excitation wavelength of 275 nm is kept.
Stop the pump flow at the corresponding retention time to optimize the emission wavelength for all other peaks as well. Determine the optimum excitation wavelength (ExWavelength) in the same way.