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Guide to Operation of

Bruker EleXsys E500 EPR spectrometer

By Lingchao Zhu and Robert Shanks Last updated: July 31, 2019 (HH)

I. Intro

This is a simple and brief guide to operation of our Bruker EleXsys E500 EPR spectrometer. It includes information on powering up the system, inserting and tuning the sample in the cavity, logging into your account and connecting to the spectrometer, setting up experiments, changing and optimizing parameters, finishing up on the spectrometer, as well as powering down the system. For more extensive information on operation and optimization of parameters, please see the Elexsys E 500 User’s Manual: Basic Operations and Elexsys E 500 User’s Manual: Advanced Operations. Copies are available in the EPR room, 2230 Chemistry.

EPR CIC staff: Heike Hofstetter Room2201B Robert Shanks Room2130 Check out our website for more information: epr.chem.wisc.edu

II. Powering up the system

1. Push the “Electr On” button on the magnet power supply. The green LED in the switch will light and the five Error indicator LEDs (on right side) will all light red. Wait approximately 5 seconds until all red Error LEDs have extinguished. (Should any red Error LED remain lit, stop and find CIC personnel) See Figure 1.

Figure 1. Magnet Power Supply

2. Press and hold the “Power On” switch until you hear the electrical contactor engage. You should also note that the heat exchanger pump should be running. (This powers on the electromagnet and pumping of cooling water for the magnet and the microwave bridge.) See Figure 1.

3. Press the green power button on the Elexsys console. The green LED on the microwave bridge will light after approximately 10 seconds and begin to blink. After approximately 20 seconds the green LED on the bridge lights continually. Do not attempt to connect to the spectrometer while the LED on the microwave bridge is blinking. See Figure 2.

Electr On Button Power On Button

Five Error Indicator LEDS

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Figure 2. Elexsys console ON(green)/OFF(red) button and microwave bridge

4. Log on to the acquisition PC (mambo) using your standard login & password. 5. Open the Xepr program by single clicking on the Xepr icon (make sure you don’t open two

versions of the program – check on the toolbar at the bottom). 6. Go to top Menu Bar, Acquisition -> Connect to Spectrometer, then enter “localhost” and click OK

to connect to the spectrometer console. Once connected, the “Monitoring panel” at the bottom of the Xepr software will display, allowing you to view different functions of the spectrometer. See Figure 3.

The magnet power supply should always be turned on before the Elexsys console is powered on and inversely the Elexsys console should be turned off before the magnet power supply is powered down.

Figure 3. Xepr software display

Microwave Bridge Light

Acquisition

Monitoring Panel

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III. Inserting and tuning the sample Here it is assumed that the appropriate cavity is installed in the magnet and the software is pointing to the calibration file that corresponds to this cavity. For details on proper installation of a cavity, see 7-13 through 7-25 of the Basic Operation Manual, or consult CIC EPR staff. It is ESSENTIAL that the cavity does not become contaminated with paramagnetic junk! Do NOT leave the cavity open to the outside world and make sure that anything you put into it is clean. Clean the outer surface of your tube with a Kimwipe and the appropriate solvent prior to inserting into the cavity. There are solid collet plugs that should be installed whenever there is no sample in the cavity. Remember: accidents do happen; if you break a sample tube in the cavity, PLEASE report it directly to CIC staff so that the necessary cleaning and recalibration can be done before others' experiments are corrupted.

1. Unscrew the top blue collet cap of the cavity, and place an appropriately sized collet for your sample in place of the solid collet that’s already there. See Figure 4.

2. Replace the screw and don’t tighten all the way until after you’ve inserted the sample. See Figure 5. Be careful when you insert the sample tube. The space of magnet is limited and sample tube is easy to break!

Figure 4. No sample status for EPR cavity

3. Sample depth can be roughly judged by holding the sample in the approximate position prior to inserting it into the cavity. You can also adjust the sample depth by pushing or pulling the bottom white collet support - you will need to loosen the blue collet cap at the bottom. Make sure your sample is in the middle area of cavity (black part). See Figure 5. Adjust accordingly.

White solid collet

Blue collet cap

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Figure 5. Sample inserted in EPR cavity

4. Open the tuning panel by clicking on the tuning icon on Monitor panel(see Figure 3) and you will immediately notice that the microwaves are in “stand by” mode(indicator shows green)

5. Click on the “tune” button and in a few seconds, the tuning display will show on the left 6. Choose “Auto Tuning” by clicking on “up” or “down” and the spectrometer will automatically tune

your sample. When autotuning is done, the “stop” button will become green and the “operate” button will also become green. See Figure 6.

7. To tell that your sample is fully tuned, the diode current should be at the center (at 200 mA) and the lock should also be at the center (at 0%), both will be displayed in the Monitoring Panel. “Operate”, “Levelled” and “Calibrated” bars should be all green; (See Figure 6) if not, repeat the autotuning and if that also doesn’t work, you will need to manually tune (see pages 7-1 to 7-11 of the Basic Operations Manual).

8. You may want to “touch up” the tuning, by doing “fine tuning” after autotuning. You do not need to be in “tune” mode to fine tune. It is best to stay in “operate” mode. You should also re-tune the sample when you change the parameters, such as microwave power.

Figure 6. Tuning panel and fully-tuned monitor panel

Bottom white collet support

Three green bars

Most effective area for sample location (in black)

Auto Tuning

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IV. Acquiring a spectrum

1. Create a “new experiment” by clicking on in furthest left part of monitoring panel then enter name. You can also enter the sample info at “Acquisition -> Set Sample Info”. See Figure 3.

2. Click on “Parameters to Hardware” icon to get into the interactive parameters setting mode (in this mode parameters will be immediately activated on the spectrometer).

3. Open Acquisition Parameters by clicking on the parameters icon and parameter panel will show up. See Figure 7.

4. Check “signal channel” parameters: make sure that the calibrated button is green. Check “field” parameters: here you define the center of the magnetic field (in Gauss), the sweep width and the number of points acquired (typically 1024, unless want higher resolution).

5. Check “microwave” parameters: start with an attenuation of 25dB, which corresponds to 0.62 mW. 6. Check “scan” parameters: use 1 scan to start with, can do more scans with signal averaging once

you’ve optimized signal acquisition.

7. Acquire the spectrum by clicking on the run icon 8. Center the spectrum: Acquisition -> Tools -> Center tools (or use the icon); then drag the tool to

where you want the center of the magnetic field to be; then click to run new spectrum with

new center field. To remove the tool, click on the “Remove Tool” icon 9. To zoom in on a section of your spectrum, L click and drag over this section (click on “FS” icon

at top to get back to the full screen view. See Figure 8. 10. To measure peak-to-peak Gauss, put cursor on spectrum line (cursor will become a +) and

Left&Right mouse click simultaneously & drag to a different part of the spectrum line. 11. To change to another sample after your previous experiment, click the “standby” button in the

tuning panel first to make sure the attenuation>60dB, then safely change the sample and re-tune the instrument. See Figure 6.

Figure 7. Parameter Panel

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V. Saving & Exporting the data

1. Go to top Menu Bar: File -> Save and enter the new filename for the data set. The program will also associate “Title” with the same dataset and typically the “title” will be identical to the experiment name

2. Your data will be stored in /home/user(your login)/XeprFiles/Data and two files get created: filename.DSC (parameter file – can view as text) and filename.DTA (actual data – open in Xepr only).

3. For ASCII: Go to File -> Export ASCII, then enter a filename that is NOT identical to the original file name (as used for saving in part V). This file may then be directly read into Excel or any other program that handles ASCII files.

4. For ESP: Go to File -> Export ESP, then enter a new filename. This will create two files: *.par (the parameter file) and *.spc (the spectrum). You may open this data in WinEPR, however you need to first multiply the data by a large number (1e6) before exporting as ESP.

VI. Creating Experiments from Data Sets and Saving & Loading Experiments

You can’t just load a data set and automatically start running another spectrum because the parameters don’t automatically transfer to the console. You need to create or load an “experiment”. You can create a new experiment by following Part IV 1-6. You can also create experiments from previous data sets.

1. Load the desired data set: File -> Load (and select from appropriate folder and list). You can load multiple data sets and Xepr will give you different handle number.

2. Click on the “create experiment from data set” icon , then enter the handle number (this is the handle number that the data set was loaded into). The handle number is the number listed before the data name. See Figure 8.

3. Click on the “Experiment Table” icon , then click on “Save” and enter a new filename to store the current experiment. A new file with a *.exd extension will be created and put in /home/user(you)/XeprFiles/Acquisition folder. You can change title by type new name in “Save as” column.

4. To load a previously saved experiment, go to the “Experiment table” and click on “Load”. Then select your stored experiment from the list of titles or filenames.

Figure 8. Full screen button and handle number

This example shows the handle number is 1.

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VII. Get Parameters from Data Set or Experiment You will need to have an experiment created or loaded before carrying out the following steps. The new parameters will show immediately in the Parameter Panel. See Figure 7.

1. Click to get parameters from data set, which has been loaded and given handle number. Choose the handle number related to the data set you want to get in the new window. Remember, you can have multiple data sets loaded and they will all be given different handle numbers. See Figure 8.

2. Click to get parameter from experiment you have loaded before. Choose the experiment you

want in the drop-down list of new window. You can always check “Experiment Tool” , which will show all the experiments you loaded.

VIII. Powering down the system

1. When you are done with the acquisition, make sure the microwave bridge is in “stand by” mode in the Tune Panel to remove your sample! Please insert a blank collet on the top of the cavity, in place of the sample. See Figure 4 and 6.

2. Go to Acquire -> Disconnect from spectrometer and click “OK”; if you’ve properly disconnected, the panel at the bottom of the software interface will disappear. See Figure 3.

3. Exit the Xepr software program (File -> Exit) 4. Log out of your account on “mambo” by either right-clicking on the background -> logout OR

clicking on the (bottom left corner) -> Leave -> Logoff (top choice) 5. Turn off the console: press the red button with the “o” (now green and red buttons are same depth) 6. Turn off the magnet on the magnet power supply: press the red “off” button (you will hear the

cooling water stop circulating) See Figure 1. 7. Then press the “Electr On” with the green LED on the magnet power supply to turn it off. All the

LEDs will go dark.

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Reference: Suggested parameter sets for unknown samples (Taken out of Elexsys E 500 User’s Manual: Basic Operations, Chapter 8, Table 8-1) Parameter Organic Radicals Metal Ions

Microwave Power (mW) 10 10 Center Field (G) 3480 3100 Sweep Width (G) 300 6000 Receiver Gain (dB) 70 70 Modulation Frequency (kHz) 100 100 Modulation Amplitude (G) 4 4 Time Constant (ms) 327.68 327.68 Conversion Time (ms) 327.68 81.92 Resolution in X (points) 1024 4096 Caution! It is important to know how each parameter affects the resulting spectrum. Following is a brief description of the most important parameters. See the Elexsys E 500 User‟s Manual for more details on these. Quick Guide of EPR Parameters

Parameter Value Note Modulation Frequency

Generally 100Khz If it is sharp line(<35mG), use lower value to avoid distortion

Modulation Amplitude

~ 1/4 of the narrowest EPR signal line width

Lower MA→higher resolution but lower sensitivity

Modulation Phase

0

Harmonic 1 1 → 1st derivative Receiver

Gain Start with 60dB

Conversion Time

Start with 5.12 ms Amount of time spends integrating one data point

Time Constant

Start with 1/4 of Conversion Time If it is low S/N, use the value of Conversion Time

Sweep Time Conversion Time × Number of points

OffSet 0

Center Field Center of EPR spectrum

Sweep Width whole width of EPR spectrum

Number of points

Start with 1024 At least 10 data points with the narrowest EPR signal line

Microwave Power

Start with the lower value(e.g. 0.6mW) Signal intensity is proportional to √P

when saturation doesn't occur

Lower attenuation→higher power→may heat the sample

Number of Scans

S/N is proportional to square root of Number of Scans

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Microwave Power: Be careful! High power may generate heat in your sample. An excessive power setting, P, can cause a decrease in, or elimination of, the EPR signal due to sample saturation. This can especially be a problem for samples with relatively long relaxation times (those with sharp resonance lines). Signal intensity is proportional to √P in the region where saturation does not occur. It can be a good idea to perform an experiment in which the integrated intensity is measured as a function of power microwave power; the linear region of a plot of I vs. √P indicates suitable power levels. Modulation Frequency: Sharp lines can be broadened by excessive values for modulation frequency (MF). This is usually not a problem except for very sharp lines (<35 mG), and 100 kHz MF is generally used. For a sample with very sharp lines, use a lower MF to avoid line shape distortion. If your sample has a very long relaxation time (T1), use MF-1>>T1. Modulation Amplitude: Increasing the modulation amplitude (MA) will, in general, increase the EPR signal to a point. Excessive values, those greater than the line width will broaden and distort the resonance line. A good “rule of thumb” is to use a modulation amplitude that is approximately ¼ of the width of the narrowest EPR line you are trying to resolve. Keep in mind that there is always a compromise that must be made between resolving narrow lines and increasing your signal to noise ratio. If you have a very weak signal, you may need to sacrifice resolution (e.g. by using a higher MA) in order to even detect the signal. However, if you have a high signal to noise ratio, you may choose to use a much lower MA in order to maximize the resolution. Receiver Gain: The best signal-to-noise ratio (S/N) is obtained with an optimal receiver gain setting. Too low a setting will result in poor S/N, and an excessive setting will result in a spectrum that is clipped off at the top and bottom. Starting with 60dB is a good option and you can increase it if the signal is too low. Conversion Time: The noise, N, in an EPR spectrum is inversely proportional to the square root of the conversion time; therefore, noise can be filtered out to some extent via longer conversion time values. The conversion time is actually the amount of time the analog-to-digital converter spends integrating at one field position (data point) before moving to the next one. If you need to detect lines that are very intense as well as well lines that are very weak within the same spectrum, you will need to use a sufficiently long conversion time. If the conversion time is too short, the smaller signals will be lost in the steps of the digitizer. Time Constant: The time constant (sometimes referred to as the response time) also filters out noise, N, but here the relationship is N~1/τ, where τ is the time constant. However, if the time constant is too large, the signal itself can be filtered out; a rule-of-thumb for undistorted line shapes is to make sure that the time needed to scan through an EPR signal (or narrowest peak line) is >10τ. A time constant that is ¼ that of the conversion time will guarantee that your spectrum is not distorted. For samples limited by a low signal to noise ratio, try time constant τ≈ conversion time for better S/N. Center Field: The center of your EPR spectrum. Sweep Width: The whole width of your EPR spectrum. Number of points: The number of data points will be taken in the range of sweep width. A general rule is to make sure that you have at least 10 data points within the narrowest EPR signal line that you are trying to resolve. This means that for EPR signals with very narrow lines you will need to increase the number of data points that are collected for a given sweep width. However, if the lines of your EPR signal are sufficiently wide, increasing the number of data point will not yield any additional information, but will only result in longer sweep times. Sweep Time: Experiment time of 1 scan = Conversion time × Number of points

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Harmonic: EPR spectra are commonly acquired as the first derivative of the absorption curve. So usually choose 1 here. Sometimes, the hyperfine structure of the EPR signal is not resolved in the first harmonic spectrum (1st derivative). Then you will need the second harmonic spectrum to help resolve some of the structure of the EPR signal by changing the value to 2.