Wavelength Calibration Process
NOTE
Prior to a wavelength calibration, a wavelength accuracy verification should be performed, see Wavelength Accuracy Test. If the deviation is more than ±3 nm, the wavelength calibration should be done as described in Wavelength Calibration Procedure.
NOTE
The duration of the wavelength calibration is about 20 minutes plus setup time for the calibration sample and system. Depending on the maximum intensity found during this scan, the PMT gain will be changed automatically and requires an additional 1 minute per scan.
Wavelength Calibration Steps shows the steps performed during the wavelength calibration.
The excitation grating and the emission grating are calibrated using Rayleigh stray light from the flow cell or cuvette measured with the photomultiplier tube.
Step | Description | Duration |
|---|---|---|
|
1 |
Preparation |
max 30 s |
|
2 |
Excitation rotation scan, full circle |
60 s |
|
3 |
Excitation rotation scan, high resolution |
44 s |
|
4 |
Excitation position scan, low resolution |
55 s variable |
|
5 |
Excitation position scan, high resolution |
260 s variable |
|
6.n |
Emission rotation scan, full circle (# of scans depends on the required PMT gain, 1 minute per scan) |
61 s variable |
|
6.n |
Em rotation scan, full circle" (instrument profile) |
9 s |
|
6.n |
Em rotation scan, full circle" (instrument profile) |
9 s |
|
6.n |
Em rotation scan, full circle" (instrument profile) |
9 s |
|
6.n |
Em rotation scan, full circle" (instrument profile) |
9 s |
|
7 |
Emission rotation scan, high resolution, part I |
44 s |
|
8 |
Emission rotation scan, high resolution, part II |
44 s |
|
9 |
Emission position scan, low resolution |
50 s variable |
|
10 |
Emission position scan, high resolution |
250 s variable |
NOTE
Variable times means that they could be a little bit longer.
When the lamp is off, the calibration process will stop within the first two steps with " Wavelength Calibration Failed" , see Wavelength Calibration Failed.
If you encounter calibration problems:
Check for air bubbles in the flow cell.
Flush the flow cell with isopropanol.
Change the water.
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