Electron Paramagnetic

Resonance Spectroscopy User

Manual

Table of Contents
Xenon Software............................................................................................................................................. 2
Quick Intro To EPR ........................................................................................................................................ 7
EPR Consumables – X band ......................................................................................................................... 12
Running EPR Spectroscopy ......................................................................................................................... 14
EPR Data Collection..................................................................................................................................... 19
Simulating EPR Data .................................................................................................................................... 21
Safety .......................................................................................................................................................... 31

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Xenon Software

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3
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5
6
Quick Intro To EPR

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EPR Consumables – X band

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Running EPR Spectroscopy
1) Turning on the equipment
Turn on the chiller, then the electronics cabinet, wait 5-10 min

Turn on the magnet, wait 30 minutes for the field to be stabilized.

Once enough time has passed, and you are ready to start experiments, open the nitrogen tank, then
turn on the N2 gas and temp controller.

2) Sample preparation Techniques

Radical:

 Solids: Only need 3 grains of solid (like 3 grains of sand) in the EPR tube.
 Liquids: With low dielectric constant solvents you may fill the EPR tube to about 2cm in height.
 Liquids: With alcohols or acetonitrile, fill a capillary tube (generally used for melting point
apparatus) up to 3 cm in height, then drop this into the EPR tube.
 Liquids: With water, fill a miniscule capillary tube (the fine ones which is open on either end) by
dabbing a concentrated solution of your compound in water.

Paramagnetic Inorganics:

 Solids: Need approximately 0.5 cm of height in the EPR tube.

 Liquids: With low dielectric constant solvents you may fill the EPR tube to about 3cm in height.
 Liquids: Don’t use any high dielectric constant solvents

3) Inserting Sample – Always run DPPH first for calibrations

Safety: Ensure that the microwave is not on (ie. There are only 0’s displayed on the microwave
synthesizer).

From the top of the probe, there is a black plastic sample holder containing a white plastic insert (collet).
Swap out to the appropriate collet such that your tube will fit properly.

To align the sample height properly, estimate the distance to the middle of the EPR cavity with your
finger width, then translate that over to the EPR tube. Place the sample into the holder using your
height estimation.

4) Pre Tuning
Open Xenon Software on the Linux computer

Press the button “Switch to acquisition mode” (found in the top left of the window)

Click Tune the Probe:

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Icon looks like: in the bottom left side of the window (it is very small).

This opens a new window for the microwave bridge tuning

Note: We are optimizing the Q factor, which is difficult with high dielectric constant solvents (H 2O,
MeOH, etc)

The tune window should already be in standby mode.

Ensure the N2 Tank is open and the flow is working properly by looking at the manual flow indicator on
the electronic box (the metal bead should be floating).

5) Tuning (Must be done for each sample)

Click on Tune mode

Alter the attenuation from db max = 60, lower by steps of 10 db until the signal in the window to the left
displays a dip. Once you reach 30 db, start lowering by steps of 1. (60, 50, 40, 30, 29, 28, 27, etc). In
general, 20 db is a good place to stop.

Look over at the brass iris, at the top of it you should see a slit with a brass bead which indicates how
screwed in the iris is. Bring this bead to the top of the iris by pressing and holding the black “down
arrow” on the computer. Once this reaches the end of travel, you’ll notice the down arrow is greyed out.
Press and hold the up arrow for a second or two until the tune dip is displayed on the screen.

You should be able to see a small tune dip which we need to tune to. If you do not see one, use the
frequency slider until a dip comes into view.
Note: With the ER 4119HS the frequency slider should be far to the left, and with ER 4123D the
frequency slider should be far to the right.

To move the dip over use the black arrows of the frequency slider. Place the grey straight line to the
tune dip.

6) Optimization of Diode Current and Lock Offset

Click on Operate to switch to Operate mode. You’ll notice that the microwave counter now displays 9
344 000 000 Hz (for ER 4119HS grey) or 9 700 000 000 Hz (for ER 4123D blue) instead of 0. Do not

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remove your sample from the cavity during this time! If you want to remove a sample, change back to
Tune mode.

Look at the indicators on the bottom right of the “switch to acquisition mode” window (Diode current
and the Lock Offset). The goal here is to obtain a diode current of 200 mA and a Lock Offset of 0%.

Try moving the iris. Click and hold the black up button and see if the diode current is moving at all. If
nothing happened, just move the iris bead back to the top by clicking and holding on the black down
arrow.

If the diode current is stuck at 400 mA, you’ll need to alter the bias or the signal phase and see if you can
observe any change in the mA. If there are no changes at all to the diode current, then you likely need to
increase the attenuation in steps of 1 until the diode current is close to 200 mA. Try the above steps
again. If the diode current is still stuck at 400 mA, then you have too much sample in your EPR tube.
Remove some and try again.

Using the black up and down arrows, adjust the Iris until the diode current is 200 mA.

Adjust the frequency by clicking left to decrease the %, or right to increase the %, until the lock offset is
~0%. +/- 2% is okay.

Switch the Reference Arm to OFF, then alter the signal phase to minimize the diode current. If this value
hits 0, it’s not yet minimized! Use the black up arrow on the Iris to be able to observe more diode
current, then continue altering the signal phase to reach an observable minimum.

Once this minimum is reached, use the black down arrow on the iris to bring the diode current down to
0.

Switch the Reference Arm to ON, then adjust the signal phase to maximize the diode current.

Once this maximum is reached, use the Bias to bring the diode current back to 200 mA.

Adjust the frequency until the offset is close to 0%, the tuning is now complete.

7) Running the Experiment (Collecting the data)

Check to ensure that we are in Operate mode

Maneuver back to the normal window (while keeping the tuning window open).

Fill out the necessary paperwork (Service form in blue, Name, Date, Sample). On the back of the form
you’ll write the sample DPPH and frequency (on the frequency count 9 340 000 000) the attenuation,
the B0 which is calculated as indicated in the front of the service sheet binder at the bottom of the page.

B0 = Frequency (in MHz, ie. 9340.8) * 0.7144775 / 2.0037, which should be approximately 3330 G.

In the window you can alter the Center field, the Sweep Width (max 5000), The Sweep Time (always
>30). If you scroll down, you can click the buttons to “set to radical measuring” or “set to
organometallics measuring” which alters the above conditions to a good starting approximation.

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In the signal channel you may set the Receiver gain, the number of scans, and the modulation
amplitude. Notice that these values are currently set at 20, 1, 1, respectively. You may need to turn the
receiver gain down if the sample is highly concentrated. If your sample is very dilute then you can
increase the receiver gain to 30 or 40 (max 60). For the first run, always select the number of scans as 1.

If your results are not phased properly (ie. The negative absorption occurs first) then under options (the
second tab from where you may alter the center field or sweep width), select the modulation phase and
change it to 180. Now you may select any number of scans as you'd like.

If using Simulation software WinSim, then complete the following:

Ensure that the number of points used to collect the data is 2 n (512, 1024, 2048, etc). This will allow
WinSim to open your data without errors. Play around with the sweep width until the number of points
is correct.

Now hit Play to acquire the spectrum.

Adjust the spectrum to increase the vertical scale using the Ix2 or I/2 buttons on the top left of the
window. If you like the results you are acquiring, then you can add more scans while the experiment is
running! Just increase the number of scans.

If using Simulation software WinSim, then complete the following:

Switch to the Processing Mode, then click Properties tab > Individual scaling. This will open a new
window. Select to alter the Y axis by checking that box, then in the Factor box enter “1E6” to multiply
the EPR signal. Hit apply. This will allow WinSim to open your data without errors.

Once you are happy with your spectrum, you MUST save it before running another, otherwise you will
overwrite the data! There is no autosaving in this program.

8) Saving
This will create the .DSC and .DTA files:

Click on Save To Disk and navigate to the folder of the user

Save as:
date_SampleAsWrittenOnForm_SampleABCD_SampleName_attenuation#_FirstFourDigitsOfFrequency

Note: Ensure the Filename box below contains the exact filename you are using in the New Title box
otherwise the program will fail.

This will create the .par and .spc files: Click on File>Export ESP, then save just like above to create the
.spc and .par file which is needed for the WinSim simulations.

Note: Ensure the Filename box below contains the exact filename you are using in the New Title box
otherwise the program will fail.

This will create the .txt file: Click File>Export ASCII, then click on the newly saved .DTA or .DSC file,
remove the .DTA extension and add the new extension .txt.

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Save it.

9) Switching to New Samples and Repeating

Before removing your sample, ensure the machine is in tune or stand by mode. Do not remove samples
when it is in operate as microwaves are currently being sent to your sample!

Insert your new sample.

Repeat Steps 3-8.

10) Billing
Staff: Fill out a yellow sheet and reference the blue sheet in the EPR folder.

User: Fill out the time sheet in the blue laboratory notebook, signing off on the amount of time the EPR
was in use.

11) Logging Out/Shut Down

Change from Operate to Tune.

Increase the attenuation slowly, from 20,21,22…..29,30,40,50,60.

Change from Tune to Stand-by.

Close the Tune window

Close Xenon through File>Exit

Save all data sets? Check NO, then hit okay

Do not log out. Leave the computer logged in for future experiments.

Turn off the magnet

Turn off the electronics cabinet (The large box with the large green and red button).

Turn off the chiller.

Close the nitrogen tank.

Wait until the manual nitrogen gauge drops to 0, then turn off the N 2 temp controller.

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EPR Data Collection

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Simulating EPR Data
1) Download Simulation Program WinSim
On your personal computer, go to:
https://www.niehs.nih.gov/research/resources/software/tox-pharm/tools/index.cfm

and download “EPR-WinSim.zip” and “WinSim.zip”. These are two different versions of the software,
wherein the newer version (2002) is used to simulate the EPR spectrum and the old version is used to
export the data properly to be able to plot a graph with Microsoft Excel.

2) Collect EPR Data

Use the SOP “Operating the EPR” as found on Confluence.

3) File Alteration Prior to Simulation

Take the EPR data from the computer and transfer the files via USB to your personal computer.

Open the parameter file for your spectrum (in WordPad) and alter the HCF, HSW, and RSWW values
displayed into the format: 1234.567
Note: Remove any numbers afterwards. We are essentially converting to the proper units.

Also alter the MF value to GHz (ex. 9.334) which should be a part of the filename.

Now the program will open your data without errors!

4) Simulation Part I
Open the program “Winsim2002”

Click File tab>Open, select filetype of .spc at the bottom right, then select your file with the .spc
extension.

Click Simulation tab > Parameters. A new window opens up:

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Ensure that the “Yes” option is selected on the left-hand side, otherwise the program won’t run
properly. On the right-hand side you’ll notice the columns, “Set” “Hyperfine” “Spin” “Number” “Opt”.

For each nuclei which splits the signal of your EPR, click the “Set” checkbox, then add in a Hyperfine
coupling (obtained from previous samples, or as an estimation from literature). Enter the spin of the
nuclei (31P = ½) and the number of these nuclei (ex. One phosphorus atom).
Note: The hyperfine coupling here is in Gauss!

Next click on “Area” on the left side then click “Set to Slider 1” at the bottom. A new window opens up:

You may click on “g-shift”, “Lineshape”, or “SimpleLW” and set these to sliders 2 and 3 as well.

Move these sliders and try to match the red simulation line to the experimental black lines as best as
you can.

Once satisfied, exit out of the slider window, then click Simulation tab > Optimize which opens a new
window:

Select the first option “Downhill Simplex” and click GO!

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A prompt will open up saying the EPR signal was properly optimized. Click and draw this window to
display the Correlation window just beneath it!

Click “Yes” to continue. The goal here is to continually optimize using the Simulation tab > Optimize
function as well as the sliders until the “Correlation for Simulation 1.” is as close to 1.00 as possible.
Usually, a value of 0.95 is excellent enough.

As a first attempt, click Simulation tab > Optimize and select “LMB1”. Then Click GO ! If the Correlation
value decreases, when the prompt asks “Keep these parameters”, click No. Then try another
optimization with “Downhill Simplex”. If the Correlation ever increases, ensure that you click Yes from
the prompt window to save the new parameters.

Use the sliders from before to optimize the Correlation. Once satisfied, get a Print Screen of the
Correlation number, the parameters and the spectrum. Open Paint and paste the screen, then save it so
that you have a record of these values. You’ll need them for the next steps.

Save the project by clicking File tab > Save Project, and name it appropriately. The reason we use paint
to save a backup is that these project files sometimes save improperly and can not be opened at a later
date.

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4) Simulation Part II
Open the older version of WinSim

Click File tab > Open > Spectrum to open a new window. Select Bruker SPC and Experiment then click on
Files >>. Change the file type on the bottom right to Bruker (*.spc) and locate your .spc file that you
opened for the WinSim 2002 software.

Click on Display tab > Rescale, to get a zoom on your EPR signal.

Click on Simulation tab > Parameters… and copy over the “Area”, “g-shift”, “Lineshape”, “Simple LW”,
and all values under the Hyperfine section that you acquired with the previous optimization. This is why
we saved the data in Paint earlier.

Ensure that the “Yes” option is selected under “Calculate this species”, otherwise it’ll fail. Now click
Simulate. You’ll notice a red simulation line joins your experimental data now. It should line up nicely,
but if it doesn’t we can optimize it here as well.

Click Simulation tab > Correlation to display the Correlation value for this simulation in the bottom left-
hand corner.

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If needed, click Simulate tab > Optimize and select either of the “LMB1” or “Simplex” options and see if
the red simulation line fits your black experiment line well.

Once satisfied, Print Screen with the spectrum, correlation number, and parameters in view and paste to
Paint. Save the file for later reference.

Save the project file as well under File tab > Save > Project, but know that it may not reopen later.

Before exiting the software, we’ll need to copy the data to be able to paste into Excel for graphing
purposes. Click Edit tab > copy data.

5) Graphing in Excel
Open Excel

Start a Blank Workbook

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Select the A2 box, then paste the data. This data is the Simulated data (the red line that we obtained
from before).

The first column is the Gauss value, the second column is the intensity of the peaks, and the third
column is useless data. Delete the third column.

Open the “raw” data in Excel, which is the .txt file obtained from the Xenon software (not the .rdc. or
.spc file). A new window will open:

Select the option “Fixed Width” which is underneath the currently selected “Delimited”. Then hit
“Next>”

Hit Next again to move onto the next screen, then click “Do not import column (skip)”. Then click Finish.

A new window opens up with the Gauss and Intensity data of the raw data. Copy and paste this data
into the first Excel file (containing the simulated data) and plot both data sets on a single graph.

A Nice Trick:
If you simply plot both data sets into a single graph, they’ll overlap and you won’t be able to see the
differences in the two sets. To have both data sets on one graph, just create an intensity shift of +10 for
the Raw data and + 20 for the Simulated data. To do this, continue reading.

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Create a “intensity Shift Raw” and “Intensity Shift Simulated” cell and enter the values 10 and 20,
respectively. Also create a “Shifted Intensity” column beside the intensity data for the simulated and
raw data as shown below.

Click on the cell C3 (the first of the Shifted Intensity cells) and type ‘=’ then click on B3 (the first of the
Simulated’s Intensity data) type ‘+’, then click on the Cell for the Intensity Shift Simulated.

Click on the C3 cell (the first of the Shifted Intensity cells) and alter the code at the top from “=B3+I2” to
“=B3+$I$2”. This will ensure that the next manipulation doesn’t shift the value we’re using from the
“Intensity Shift Simulated”.

Click on the bottom right-hand corner of the cell and drag down to the bottom of the Simulated
Intensity data to copy this cell’s parameters into the ones below.

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When you unclick, the data will fill out.

Repeat this step for the Raw intensity data.

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Now we’re all set up to graph this!

Unser the Insert tab, select Insert Scatter (X,Y)… Then select Scatter with Smooth Lines.

Right click on the empty graph and click Select Data.

Now select all the data to graph your EPR signal raw and simulated data, ensuring that as the “y-axis”
you select the Shifted Intensity Data for each.

Alter the values in the “Intensity Shift Raw” and “Intensity Shift Simulated” cells now to obtain both
spectra above the y=0 line, and ensuring that the spectra do not overlap. Delete the Y axis and the grid
lines as they have no real significance here. Here is my end result:

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Some journals require the x-axis to be Gauss, others milliTesla, and others g-value. Just pay attention to
what the journal wants and change the units of the x-axis accordingly.

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Safety

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