How do you display a 474 spectrum scan in Empower 3? - WKB16835

ENVIRONMENT

• Empower 3
• 474 Scanning Fluorescence Detector

ANSWER

Follow this procedure for Millennium software. It will also work for Empower:

1. Note that scanning success depends on choosing a suitable excitation, as well as on emission wavelength.
2. Emission scans start at a 10 nm higher wavelength than the excitation.
3. Scanning comprises two operations, storing and then outputting the stored spectrum. To troubleshoot, first scan the mobile phase and verify that it is not interfering. Later, you can also subtract it from the standard sample dissolved in the same mobile phase.
4. Fill the cell with the mobile phase using a priming syringe with an HPLC fitting attached that can screw directly into the flowcell bulkhead fitting. Use only the detector, and disconnect flow from the HPLC.
5. On the keyboard, press Scan.
6. It comes up with the first option:  Em Scan.
7. To set scan parameters, push the End key.
8. Hit edit/enter, set scan to STD, if not already set, and hit edit/enter again.
9. Change speed to the slowest scan rate; in other words, at the flashing prompt, use the up arrow key to select “2”, and hit edit/enter.
10. Hit the Monitor key to return to Emission Scan. (If you want to do another type of scan, use the up and down arrows to page through other functions.)
11. Hit edit/enter, and select the memory location, 1-20.
12. Hit edit/enter, and select the excitation wavelength; use the numeric keys (which do not require the shift key).
13. Hit edit/enter, and then hit next to start the scan, using the Program Run key. Start the detector collecting the scan before going to Millennium. If your excitation wavelength is 300 nm, the scan starts at 310 nm.
14. Enter Quickset and build an instrument and method set as usual, but with no flow.
15. In Quickset, load the method set and hit setup. For the initial scan test, just start up monitor baseline. You may need to adjust the Gain to keep the scan on scale.
16. Go back to the detector to output the now-saved scan. Hit scan, and use the up arrow key to select scan function 3, emission spectrum out, or, as it appears on the 474 EM, D.out.
17. Hit edit/enter, select the storage location, and hit edit/enter again.
18. You can change the stop and start wavelengths, but for testing, keep it to the full range; in the example, 310-900 nm. Hit edit/enter to page through.
19. Use the program run key to start. The data should appear on the Millennium monitor baseline. If it goes off scale, reduce the gain setting, re-collect the spectra, and then output again.
20. When gain is set correctly, replace the mobile phase with your active or standard dissolved in mobile phase, and repeat the process. Then now have a comparison of mobile phase with sample to confirm that the mobile phase is not interfering.
21. Of course, if the result is unsatisfactory, you need to select a new excitation wavelength and start the process again.
22. To store an outputted spectrum, make a zero injection and hit the Run program key after the software starts to acquire data. You can calculate the wavelength; it should be apparent where the scan begins at 1 data point per second.

    Minutes of scan time in Millennium =

    {(900 nm – (Excitation Wavelength + 10 nm))/30 nm/second}/Min’s displayed on Millennium

ADDITIONAL INFORMATION