Department of Chemistry

Teaching Laboratories

Shimadzu RF-5301 Fluorimeter operation guide for

students

General directions
Detailed instructions for use of the fluorimeter may be given in the lab script, consult
a demonstrator for assistance where necessary.

The fluorimeter cell holder has two positions; ‘Right angle’ and ‘Front surface’, see
photographs;

Right Angle Cell holder positions Front surface

Handle the fluorimeter cells with care;

 Always handle on the corners, or at the top and bottom

 Ensure that the cell is washed thoroughly after each solution and then rinsed
out with the new solution before taking its spectrum
 Ensure that the same cell face is used for each solution
 Ensure that the cell windows are clean, dry and not contaminated

Page 1 of 5 Fluorimeter Last updated: 21/06/2017

How to record fluorescence emission spectra
1. Ensure the sampling geometry is right-angle illumination by carefully rotating
the cell holder (if necessary). Please check with a demonstrator if you are
unsure.
2. In the software click on Configure and select Parameters; the Spectrum
parameters dialogue box will be displayed.
3. To run an emission spectrum, click on Spectrum type: Emission.
4. Use the UV absorption maximum, λmax, of your sample as the excitation
wavelength; click on the EX wavelength box and type in this value.
5. Click on the EM wavelength boxes and type in (for e.g.) Start 300 nm, end
600 nm. This selects the range of wavelength over which the emission
spectrum will be scanned. Details may be in your script.
6. Click on sensitivity: select Low.
7. Click Scan speed: Medium, and Slit width: EX 5 nm, EM 5 nm; dots will
appear against the selected parameters.
8. Click on the Recording range boxes, adjust the spectrum y-axis to a size
which fills the screen by typing values in, e.g. Low: 0 and High: 400. You can
change the range later if necessary if the spectrum goes off scale.
9. Click on Ok to return to the main application window.
10. Click on Start at the bottom of the window; the spectrum will be scanned and
displayed on the screen.
11. Save the spectrum obtained, type a filename into the box in the filename entry
window (e.g. abcd1) when it appears at the end of the scan, and choose the
Save option. Record this filename.
12. The main application window should now be displayed. Click on Manipulate
and select Peakpick. A window showing the channels containing the
emission spectrum will appear. Click on the channel you wish to analyze and
then select Ok. The spectrum will be redrawn with the peaks labelled
13. Click on the cross in the top right hand corner to close the peak window. The
main application window should now be displayed.

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How to record fluorescence excitation spectra
1. Click on Configure and select Parameters. The Spectrum parameters
dialogue box will be displayed.
2. To run an excitation spectrum, click on Spectrum type: Excitation.
3. Take the λem from the emission spectrum and use this value as the excitation
wavelength. Click on the EM wavelength box and type in this value. This
selects the wavelength of the light used to observe emission from the sample.
4. Click on the EX wavelength boxes and type in (for e.g.) Start 300 nm, End
600 nm. This selects the range of wavelength over which the excitation
spectrum will be scanned.
5. All the other spectrum parameters should be the same as those used for
obtaining the emission spectrum.
6. Click on OK to return to the main application window.
7. Click on Start at the bottom of the window; the spectrum will be scanned and
displayed on the screen.
8. Save the spectrum using a different filename, use Manipulate and Peakpick
as for the emission spectrum. Record the wavelength of excitation (λex) and
intensity
9. Click on the cross in the top right hand corner to close the peak window. The
main application window should now be displayed with both the emission and
excitation spectrum shown.

How to print out spectra

1. To print out the spectra it is useful to present both scans on the same plot. To
select the files you wish to display, go to the main application window, click on
Presentation and select Channel status.
2. The display option for each channel can be toggled between Y and N. Select
Y for both of your emission and excitation spectra, N for other files. Click on
OK
3. If you wish to change the x- and y-ranges for your graph on the screen, click
on Presentation and select Set limit. The Set limits dialogue box will
appear. Change any values by clicking on the appropriate box and entering
new values. Click on OK to return to the main applications window. The graph
will now be displayed with your new ranges.

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 4. To plot this data, click on Presentation and select Plot. The Plot layout
dialogue box will be displayed. In row A double click on graph; a cross will be
shown in the box and the Plot data dialogue box will appear. Type a title, if
you wish, in the Graph title box. The y-axis range may be altered, if you
wish. Click on OK.
5. Ensure that Screen is now selected in the Channel/f.name area of the Plot
layout dialogue box; this will allow a plot of the contents of the graph as they
appear on the screen. Click on the Quadrant boxes to ensure that only 1 and
2 are active (showing crosses); clicking on the boxes toggles between active
and inactive. Click on Print to obtain the plot.

How to record spectra of donor-acceptor mixtures

1. Set the sampling geometry to front-surface angle illumination by rotating the
cell holder clockwise until it clicks (see page 1)
2. In the energy transfer study you need only obtain emission spectra; therefore
Spectrum type = Emission.
3. Select the absorption maximum λmax of the donor as the excitation wavelength
and type this into the EX wavelength box.
4. Run the fluorescence emission spectra of all solutions between (for e.g.) 300
nm and 600 nm; type Start 300 nm, End 600 nm in the EM wavelength
boxes.
5. Click on sensitivity: select Low. Click Scan speed: Medium, Slit width: EX
5nm, EM 5 nm. A Recording range of 300 – 600nm is likely to be
appropriate.
6. Commence with the donor only solution and ensure that the peak is on scale,
then repeat the measurement for the remaining solutions and do not adjust
any instrument settings or the filter in the excitation beam for the D – A
mixtures.
7. Ensure that the cell is washed thoroughly after each solution and then rinsed
out with the new solution before taking its spectrum.
8. Obtain the emission intensity, i, at the peak of the donor emission for each of
the solutions
9. When you have obtained all the spectra, make a plot with all of these overlaid
on one chart.

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Instrument set-up guide for technicians / demonstrators
Turn the instrument on: see photo

Power switch

Turning on: XE Lamp switch to ON and Main switch

to I listen to lamp striking; ticking noise should stop
after a few seconds

Turning off: XE Lamp switch to OFF, wait 5-10 min to

cool, turn main switch to O

Turn on the pc and start the software

 Open the RFPC software
 Select ’Configure’ from the header menu and then ‘PC configuration’. In the
dialogue box choose the ‘Serial port’ setting box and connect to the number
which is suggested, then click ‘Ok’. Note that the PC sometimes connects to the
correct com port automatically
 Select ’Configure’ from the header menu and then ‘Instrument’. In the dialogue
box select the ‘On’ button against the spectrometer. A new dialogue box will open
with the progress of the instrument ready checks displayed
 Once all checks are complete (each box will go green against the tested
parameters) click ‘Ok’
The instrument is now ready for use and the PC communications established.

Page 5 of 5 Fluorimeter Last updated: 21/06/2017