NUCLEAR MAGNETIC RESONANCE SPECTROMETER

ANASAZI NMR SPECTROMETER

OPERATION

DR. SUNDIN

Organic Chemistry Lab, UW-Platteville

INTRODUCTION: There are two "classes" of nuclear magnetic resonance (nmr) spectrometers, continuous wave (cw)spectrometers and pulse spectrometers. Using a cw spectrometer, the sample is placed in the magnetic field and subjected to a slowly swept frequency. The cw nmr spectrum consists of frequency peaks at which there is absorption of energy as well as the intensities of these absorptions. Using a pulsed spectrometer, the sample is placed in the magnetic field and is pulsed with high power radio frequency (rf) waves sufficient to simultaneously excite all nuclei in the sample. After the pulse, the nuclei, over time, return to the ground state and radiate the absorbed energy. The pulsed spectrometer collects this energy and produces the Free Induction Decay (FID) signal which is the sum of all nuclei radiating over time. The FID does not lend itself to direct interpretation. However, Fourier transformation converts the time data of the FID into frequency data which is interpreted just like the original cw spectra.

The Anasazi Eft-60 Spectrometer uses the magnet from a Varian EM360 cw spectrometer and all new electronics and software operating in a Windows environment to obtain both proton and carbon-13 nmr spectra. Upon placing the sample in the magnetic field, Anasazi's PNMR software controls the data acquisition parameters and obtains the FID. Acorn's NUTS software converts the FID into the frequency spectrum. The NUTS software can be used to process data from a wide variety of nmr spectrometers. A free, tutorial version of NUTS is available on the web at http://www.acornnmr.com/shareware.htm.

SAMPLE PREPARATION: Sampling takes place in a 5 mm diameter, thin walled, glass, cylindrical, nmr tube which is spun by a jet of air while the tube is in the probe. The spinning balances out inhomogenieties in the magnetic field. The solvent is usually carbon tetrachloride or deuterated chloroform with tetramethylsilane (TMS) added as an internal standard. Since only about 1% of the carbon nuclei are carbon-13, carbon-13 sample solutions must either be much more concentrated than hydrogen nmr samples or, lacking sample, many more scans must be made and the results averaged. For some samples, one can use the carbon-13 sample to obtain the proton nmr provided one drastically turns down the receiver gain.

For liquid carbon-13 samples, place about 1-2 cm of sample in a clean nmr tube. (If you doubt the cleanliness of the nmr tubes, clean nmr tubes by rinsing with acetone followed by rinsing with pure carbon tetrachloride (no TMS!).
Place about 2-3 cm of 10% TMS in carbon tetrachloride in the nmr tube. (Caution! Carbon tetrachloride is listed as a carcinogen. It should only be used in the hood. Do not get it on your hands nor breathe the vapors.)
Rotate to mix. If there are any particulates, the sample must be filtered through cotton in a Pasteur pipet.
You may use this sample to obtain the proton nmr spectrum in many cases provided you turn down the receiver gain (RG) PNMR. If that does not give a satisfactory spectrum, pour out (see the Tube Cleaning section) 50-75% of the sample (save or halogen wastes) and put enough carbon tetrachloride (no TMS!) in the tube such that you have 4 cm of solution in the tube.

PLACING THE SAMPLE IN THE PROBE: The nmr spectrometer is in Ottensman 307. (Safety Caution: when sitting at the spectrometer for long periods of time, the room lights might suddenly go out leaving you in the dark. Do not panic. Room 307 has so much equipment in it that you do not want to walk around in the dark. Instead, carefully stand up and wave your arms foolishly in the air to turn the lights back on again.)
The air pump should be running. If not, plug it into the outlet.
Open the smoked glass door on top of the magnet.
Rotate the left knob 180 degrees counterclockwise. You should hear a hissing sound and a tube should rise up out of the probe.
If the tube fails to stay up, rotate the left knob 90 degrees clockwise and then quickly rotate it 90 degree counterclockwise and catch the tube as it rises up out of the probe. (Caution, tubes are thin walled glass.)
Remove the tube from the white, Teflon spin collar and place that tube in the rack on the north wall.
Insert your nmr tube into the spin collar.
Place the tube in the Depth Gauge and carefully push down on the tube until it hits the bottom.
Remove the tube from the depth gauge and carefully wipe off the tube and spin collar with a clean KimWipe to remove fingerprints, dust, etc.
Place the clean tube in the probe. It generally will not sink all the way into the probe.
Rotate the left knob 90 degrees clockwise. The tube should drop to the bottom of the probe.
Rotate the left knob an additional 90 degrees clockwise. The tube should start spinning. It should be spinning fast enough so that you can no longer discern the spin rate. (Spinning too fast can cause vortexing and loss of signal quality!)

OBTAIN THE PROTON FID:
Your proton nmr sample should be in the probe.
Enter the PNMR program if needed. You may have to go to the Windows Start Menu to open PNMR or press <Alt+Tab> (do not type the <> characters!) to switch from NUTS (which the previous user used).
Check to see if the program listed at the top left of the screen is WinPNMR-H1 and the bottom left command line says H1>. (If not type NU H1 <Enter>.)
Check to see if the following parameters are correctly set: SI=8192; NS=1; RG=20; PW=16. If any parameter is not correct or you need to change to nonstandard parameters (eg., you are using a concentrated sample and you may want the receiver gain set at say 5, type RG 5 <Enter>).
To acquire FID, type zg <Enter>. An information box appears. When the box disappears, the scan is finished. After a few seconds the FID will be displayed. If the FID is in red, that scan is useless. Set RG to a lower value and type zg <Enter> again. Continually lower RG until the FID appears in yellow, or increase RG until the FID is in red and then back off of the RG until it is yellow again.

OBTAIN THE PROTON SPECTRUM:
Press <Alt+Tab> to enter the NUTS program (You may have to go to the Windows Start Menu if you are the first user of the day.)
Press <Ctrl+F1> which references TMS, peak picks, integrates, and plots automatically after you input your name, comments, and click OK. (After obtaining proton spectra in this manner several times, you may want to consult the manual to learn how to do these, and other things, manually. See Integration below.)
Save your spectrum in the appropriate subdirectory (if you are in Chemistry 361 and you are saving a proton spectrum, use c:\Eft\data\361\H1\YourFileName).
Either obtain the Carbon-13 spectrum or remove your nmr tube and either run another sample, let the next user put their sample in, or place a standards tube in the probe.
Dispose of your sample and clean the nmr tube (see below).
If you are the last one to use the nmr for the day, unplug the air pump and close the WinPNMR and NUTS software on the computer.

OBTAIN THE CARBON-13 FID:
Your carbon-13 nmr sample should be in the probe.
Enter the PNMR program if needed. You may have to go to the Windows Start Menu to open PNMR or press <Alt+Tab> to switch from NUTS (which the previous user used).
Check to see if the program listed at the top left of the screen is WinPNMR-C13 and the bottom left command line says C13>. (If not type NU C13 <Enter>.)
To set the field offset one needs to acquire a new proton spectrum). Type zgh <Enter> to get the proton nmr spectrum.
Press <Alt+Tab> to enter NUTS.
Type A2. Note that <Enter> is not needed for most NUTS commands.
Place the cursor over the TMS peak (close to zero ppm) and press the left mouse button. The apparent position of the TMS peak will appear below.
Remember that number including sign.
Press <Alt+Tab> to enter PNMR.
Type fo <Enter>.
A data box appears. Type the current position (include sign) of the TMS peak and then the desired position of the TMS peak (0).
Check to see if the following parameters are correctly set: SI=16384; NS=76; RG=100; PW=16, RD=2.36, DP= ~22.
To acquire data, type zg <Enter>. This may take over 5 minutes if you are averaging 76 scans. After a few seconds the first FID will be displayed. The FID will grow as each scan is completed. If the FID turns red, those scans are useless. Press <Ctrl+K> to stop scanning. Set RG to a lower value and type zg <Enter> again. Repeat the process until the FID stays yellow when the last scan is completed.

OBTAIN THE CARBON-13 SPECTRUM:
Press <Alt+Tab> to enter NUTS.
Press <Ctrl+F3> . An input box will request a value for line broadening. Enter typically 1.
Plot data by typing pl.
Save your spectrum in the appropriate subdirectory (if you are in Chemistry 361 and you are saving a carbon-13 spectrum, use c:\Eft\data\361\C13\YourFileName).
Remove your nmr tube and either run another sample, let the next user put their sample in, or place a standards tube in the probe.
Dispose of your sample and clean the nmr tube. (See below.)
If you are the last one to use the nmr for the day, unplug the air pump and close the WinPNMR and NUTS software on the computer.

OTHER EXPERIMENTS: After you feel comfortable with manual integration, phasing, peak picking, etc., you can consult the manual for 2-D and DEPT experiments.

MANUAL INTEGRATION AND VALUES: Automatic integration of the proton spectrum will also integrate the TMS reference and impurities. It also will sometimes group together as one unit two different sets of peaks. It also leaves you with the task of calculating the number of protons for each group of signals.
Enter the NUTS program and retrieve your saved proton FID.
Type ID. Note that <Enter> is not needed for most NUTS commands.
Type C. (This clears most of the previous integration.)
Place the cursor just to the left of the group of peaks, or peak, to integrate and double click.
While holding the mouse button down, move the cursor just to the right of the group of peaks, or peak.
Release the mouse button and click once. This should give you an integration of that groupof peaks, or peak.
If you are not satisfied with the integration, type L, which erases the last integration. Redo the integration.
Repeat the process for each group of peaks, or peak.
Press <Enter> to get out of the integration mode.

To change the integration values, in NUTS, type ID.
Choose a peak or group which you believe you know the number of protons it represents. (The smallest peak might be 1.00H or a sharp singlet to the left of TMS might be a methyl with 3.00H, etc.)
Place the cursor over this peak or group.
Click and hold the mouse button.
Type V.
Release the mouse button. A data box appears. Type in the relative value for that peak or group.
Click OK.
The relative values of all integrations should change.
If the values are satisfactory, press <Enter>. If not, repeat the process.
Press <Enter> to get out of the integration mode.
Plot the spectrum by typing pl.

CLEAN NMR SAMPLE TUBE:
If you want to save your sample, pour the contents of the sample tube into a 5 mL conical vial (hood!).
Rinse the tube with either ether or methylene chloride and add the rinse to the conical vial.
Evaporate off the solvent in the hood.
Rinse the tube twice with a small amount of acetone and dispose of the rinses in the halogen waste container.
Place the tube inverted in the clean tube container.
Rinse cap with acetone and place in the clean cap container.

If you do not need to save the sample, pour the sample into the halogen water container.
Rinse the tube three times with a small amount of acetone and dispose of the rinses in the halogen waste container.
Place the tube inverted in the clean tube container.
Rinse cap with acetone and place in the clean cap container.

NOTE: These instructions assume that the nmr spectrometer has been properly set up and tuned prior to your lab section.

Version 5, March 8, 2001

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