Scan Fluorescence Application

1. Click the Windows Start button, point to Programs, Cary Eclipse and then select Scan. The Scan Application is displayed.

2. Select the Setup button to display the Setup dialog and specify the method parameters for a new method.
        A. Set the Data Mode to Fluorescence

        B. Set the Scan Setup mode to Emission. During the emission scan, the excitation monochromator is set to a fixed wavelength and the emission monochromator is scanned over a wavelength range.

        C. Set the X Mode to Wavelength (nm).

      D. Enter an Excitation (nm) value that is within the region where the fluorescent molecule to be scanned will absorb light. For test purposes, Anthracene will be used as an example and therefore a value of 360 nm should be set.

      E. Enter an Excitation slit (nm) value of 5 and an Emission slit (nm) value of 5. Slits determine the resolution of the spectrum and therefore are used in conjunction with the PMT Detector Voltage (set on the Options page) to determine concentration. If a compound is highly fluorescent and has reasonable signal intensity (as is the case for Anthracene) the slits can be set quite narrow.

      F. Enter a Start (nm) value of 370 nm and enter a Stop (nm) value of 520 nm.The Start (nm) value should be set to the Excitation (nm) value plus the sum of the slits. In this example, as the Excitation value is 360 nm, and the sum of the slits is 10 nm, the Start (nm) value is set to 370. Typically the Stop (nm) value should be set to 150-200 nm greater than the Start (nm) value.

     G. If selected, clear the 3-D Mode check box.

     H. Set the Scan Control to Medium.
3. Select the Status Display check box so that you can view various instrument parameters during the scan to setup visual system monitoring.
4. Set up the scan options
     A. Select the Options tab to display the Options page.
     B. In the Display Options group, select the Overlay traces check box to overlay the results of the traces on one graph.

     C. If selected, clear the CAT or S/N Mode, Cycle Mode and Smoothing check boxes.

     D. Set the Excitation filter to Auto. This will automatically move the filter wheel to the appropriate position for the selected excitation wavelength.

     E. Set the Emission filter to Open in order to minimize steps in the spectra.

     F. Set the PMT Detector Voltage to Medium. This setting can later be adjusted if results are over-range. If the signal is too high, decrease the PMT Detector Voltage. If the signal is too low, increase the PMT Detector Voltage.

5. Ensure that no options are selected on any of the tabs of the Accessories page.

6. Set up reporting and printing requirements

Select the Reports tab to display the Reports page where you can specify your reporting requirements for this method. Enter your name in the Name entry field. If required, enter any comments relating to your experiment in the Comment field. Set up your report style by selecting the appropriate check boxes in the Options group.

For example, select the Auto print check box to obtain a printout of your report automatically.Select the Parameters check box to include your experimental parameters in the report. Select the Graph check box to include a graph in the generated report.

Note: If Auto print is selected, the system will send the report information to the specified printer as well as displaying it in the Report area. However if Auto print is not selected, the report will be displayed in the Report area only. The Report area can be viewed by selecting the Report command from the View menu.

Set up the Peaks reporting options. Select Maximum peaks to report the peak with the largest peak threshold that exceeds the peak Threshold value. Select All peaks to report all peaks meeting the Peak type criterion and exceeding the Threshold value. Select the Peak type and specify the peak Threshold. If required, select the X-Y Pairs table check box. You can use the Actual Data Interval by which the data was collected or you can make the Cary Eclipse Interpolate the points to a new interval.

7. Set up storage of collected data before run -- Select the Auto Store tab to display the Auto store page. Use this page to set up whether the collected data is to be saved, and if so, when the Cary Eclipse should store the information. Select Storage option of On; Prompt at end.
Select the Auto convert option you require.If you select Select for ASCII (csv) or Select for ASCII (csv) with Log, then at the end of the data collection the system will automatically generate a report and store the data both in the Cary Eclipse format as well as ASCII XY pairs format in the current folder.

8. Set up email options -- Refer to How to email your results for instructions on setting up the email options.

9. Finish Setup -- Once you are satisfied with your method setup select OK to confirm any changes you have made and close the Setup dialog.

10. Zero the instrument -- Place the blank solution in the sample compartment of the Cary Eclipse instrument.

Click the Zero button to zero the system. Alternatively, select Zero from the Commands menu to perform a zero. When the result is zeroed, the word 'Zeroed' will appear in the Y display box in the top left corner of the Scan Application window.

11. Start the Scan run -- Select the Start button to commence a data collection. Alternatively, select Start from the Commands menu. The Sample Name dialog is displayed. Place the sample in the sample compartment of the Cary Eclipse instrument.

In the Sample Name dialog, enter the appropriate name for you sample and select OK. The scan will commence and the trace will appear in the Graphics area.

12. Save the collected data -- Once the run is finished, select the Save As command from the File menu. In the File name field, enter a file name for this scan run.