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NMR Basic Operation - 60 MHz Anasazi

Basic Experiment Setup and Basic NMR Spectrometer Operation Setup for Anasazi 60 MHz spectrometers:

For a description of the Anasazi 60 MHz spectrometers at the University of Wyoming, please click here.

Sample Preparation

Please prepare your samples in the designated area or under a hood, and not on top of the magnet.

The basic sample preparation procedure is similar to the one outlined for the Bruker instruments. Deuterated solvents are still recommended. They are not needed for "locking" but may increase the signal to noise ratio for diluted samples and the extra signals will complicate the spectrum. Proton-free solvents like carbon disulphide, CS2, or carbon tetrachloride, CCl4, or deuterated solvent such as deuterochloroform, CDCl3, or deuteroacetone, CD3COCD3, are popular. An internal standard such as 1-2 % tetramethylsilane (TMS) is recommended, as it facilitates to calibrate the spectrum that is more of an issues for these 60 MHz instruments than for the superconducting Bruker instruments. TMS has a proton chemical shift of 0 ppm. There are commercially available solvents that are spiked with TMS, but it is not difficult to prepare bulk solvent with 1-2% TMS, which can be stored for extended periods in a laboratory refrigerator. It is also possible to use solvent signals as an internal reference.

The chemical shifts (d) of solvent signals observed for 1H NMR and 13C NMR spectra are listed in the following table. The multiplicity is shown in parentheses as 1 for singlet, 2 for doublet, 3 for triplet, etc.

Solvent 1H NMR Chemical Shift 13C NMR Chemical Shift
Acetic Acid 11.65 (1) , 2.04 (5) 179.0 (1) , 20.0 (7)
Acetone 2.05 (5) 206.7 (13) , 29.9 (7)
Acetonitrile 1.94 (5) 118.7 (1) , 1.39 (7)
Benzene 7.16 (1) 128.4 (3)
Chloroform 7.26 (1) 77.2 (3)
Dimethyl Sulfoxide 2.50 (5) 39.5 (7)
Methanol 4.87 (1) , 3.31 (5) 49.1 (7)
Methylene Chloride 5.32 (3) 54.00 (5)
Pyridine 8.74 (1) , 7.58 (1) , 7.22 (1) 150.3 (1) , 135.9 (3) , 123.9 (5)
Water (D2O) 4.8

Signals for water occur at different frequencies in 1H NMR spectra depending on the solvent used. Listed below are the chemical shift positions of the water signal in several common solvents. Note that H2O is seen in aprotic solvents, while HOD is seen in protic solvents due to exchange with the solvent deuteriums.

Solvent Chemical Shift of H2O (or HOD)
Acetone 2.8
Acetonitrile 2.1
Benzene 0.4
Chloroform 1.6
Dimethyl Sulfoxide 3.3
Methanol 4.8
Methylene Chloride 1.5
Pyridine 4.9
Water (D2O) 4.8

Please keep in mind that the sensitivity of the 60 MHz spectrometers is much less than for the superconducting Bruker instruments, roughly about 100 times less as compared to the 600 MHz NMR instrument. Thus, about 100 times more sample is needed, or the data acquisition needs to take much longer, more than 100 times longer (the signal increases proportionally but the noise only by the square root, and the signal to noise ratio is important). As a rough guide, deposit about 25 to 50 mg of the sample into a clean glass vial, add about 2 ml of bulk solvent containing 1-2% TMS. Mix well, filter out any particulates or un dissolved materials using a Pasteur pipet packed with a small amount of glass wool, or centrifuge and take the supernatant. Add 700 ul of the filtered solution to a clean 5 mm NMR tube, and cap and label the tube. For the liquid samples, use about 700 ul spiked with 1-2 % TMS. As a general guide, a sample concentration of about 5% or 300 mM for proton 1H samples works fine. For 13C samples, a 100% concentration will give good spectra in 1 minute and 1000 mM samples in about 5 minutes. Please be aware that the spectra for the same compound acquired at 60 MHz may look more complicated than the spectra acquired at high field (300 MHz or up), especially for proton 1H spectra, as couplings may be more than first order. The difficulties encountered with interpreting complex 60 MHz spectra may tempt the user to study only simple molecules that can be studied at higher concentrations. However, it should not be forgotten that in the "good old days" of NMR a lot of biomolecules were studied at these frequencies that contained thousand of atoms.

During data acquisition, the sample tubes are usually spinning at around 45 Hz. The temperature of the sample is 29 Degree Celsius. Please keep this in mind.

Basic precautions

Due to their different designed as compared to the superconducting Bruker magnets, they are fewer issues. The 1 Gauss line (~ Earth's magnetic field) is within the magnetic case. A static magnetic field of 1 Gauss or less is considered unlikely to cause significant problems. Inside the case, there is still a strong magnetic field, though, so do not drop anything metallic inside the magnet and the probe. Please do not let the sun heat up the magnets (the temperature of the magnets should stay constant).

Basic NMR operation procedure

In contrast to the Bruker instruments (where data acquisition and processing can be done with the same program TOPSPIN), there are 2 programs for the Anasazi 60 MHz spectrometers: PNMR for spectrometer control, including data acquisition, and NUTS for processing, data reduction, display, and plotting. Both programs operate within the Microsoft Windows environment. NUTS stands for NMR Utility Transform Software. There are NUTS versions also for Mac and the Windows version runs fine on Linux under Wine. The NUTS manual in pdf format can be downloaded from here (7.5 MB). Sample data files can be found here.

In the following paragraphs the basic NMR operation procedure is described. For additional information, please consult the manuals or ask somebody who knows. It is not described in the manuals, but it is possible to modify the pulse programs, similar to the Bruker NMR spectrometers, to suit specialized tastes.

It is recommended to acquire a proton 1H spectrum first. In contrast to the superconducting Bruker magnets, the NMR spectrum tends to shift around much more and the use of a reference (internal or external). i.e., running a spectrum on a chemical with known chemical shift, so the software can compensate for the shift. A list of reference compounds with their expected chemical shifts is above.

Acquiring a proton 1H spectrum

In PNMR:

1. Open the PNMR program. The PNMR prompt, at the bottom left-hand corner of the PNMR program, indicates the nucleus that is set to observe. It should be H1> to observe proton. If not, type: nu H1 <enter>.

2. Insert the sample into the spectrometer. To do this, wipe the sample tube with a Kimwipe or other soft tissue, to remove fingerprints and other contaminants. Lift the lid on top of the magnet to reveal the sample compartment. Press the EJECT button at the nearby controls and watch any sample that may be inside the magnet. To eject, and remove the sample tube (if any) from the sample compartment, grasp the tube near the cap. Gently, using a Kimwipe, remove the spinner (the white holder around the tube) and set the old sample aside. While holding the spinner, gently insert the new sample into the spinner such that the bottom of the tube sticks out about a couple of centimeters, and insert the tube with the spinner into the black depth gauge on the front the magnet until the button of the tube hits the bottom of the gauge. The spinner will slide up the tube to the appropriate height. Insert the sample so it floats on top of the probe (where the previous sample came out) and press the INJECT button. Watch carefully how the sample is lowered inside the magnet and begins to spin. If it is not spinning, press the INJECT bottom again, and confirm that the tube is spinning. Repeat if necessary.

2. Type: shim <enter> to shim the magnet (make the magnetic field around the tube homogeneous) to optimize the resolution.

3. Modify the parameters such as NS (number of scans), sweep width, as desired, either by typing in NS 16, for example, or by using the pulldown menu. The gain is a number below 200. The current parameters are displayed on the bottom right.

4. Type: zg <enter> to acquire the data. A pop-up window appears and asks for a file name. Use the default name or, if you want to keep the data, insert a filename. The format year-month-day-time-samplename-initials of instrument operator is recommended for easy retrieval, for example 2016-01-02-1025-ethanol_AKG

5. To correctly position the spectrum (REFERENCE), type fo <enter> in the PNMR program, then type: value <enter>, where value is the expected chemical shift, e.g., 0 for TMS. Then it is possible to repeat step 4, i.e., acquire a spectrum and the spectrum will be shifted correctly.

Processing the spectrum

In NUTS:

1. Open the spectrum using the OPEN menu button and select the file.

2. A. Automatic processing: Press <CTFL+F1>

OR

2.B Manual processing (recommended)

Useful commands for manual processing:

Expand selected region: zo <enter>, select region by dragging left mouse

Phasing:  pe <enter> for phasing (move the mouse horizontally for one order and vertically for the other one; automatic phasing by following windows menu)

fit baseline (normally only necessary for 13C): fb <enter>

Integrate data: id <enter>, and to select: two clicks of left mouse button for left boundary, then one left click for right boundary, to assign a relative integral value place cursor on integral, click left mouse button, press <v> and enter number), The integral display position can be adjusted by invoking a slider: z <enter>

Peak picking: automatically (type: pp <enter>), or manually (type: dp <enter>

To finish any of these operations type <enter>. Plot data by typing: pl <enter>. If the spectrum shows strong spinning sidebands, i.e. extra peaks around the main peak at the spinning speed spacing (such as 45 Hz), then a shimming calibration needs to be performed (ask the instructor or manager to do this).

Explanations of Selected NUTS commands:


ZZ -- Reads default data file pnmr.fid into NUTS and displays the FID.

BC -- Baseline Correct. Applies baseline flattening algorithm and improves integration accuracy.

LB -- Line Broadening. Used with EM.

EM -- Exponential Multiply. Applies exponential smoothing function to FID using LB factor. Can improve S/N (signal to noise) ratio.

FT -- Fourier Transform the FID.

PH -- PHase manual adjustment. Hold left mouse button and move mouse left or right. Press 'Enter'
to exit Phase mode.

PS -- Phase Same. Phases spectrum using current values.

AP -- Automatically Phase the spectrum.

ID -- Integrate Display. Allows manual peak integration and numerical value adjustments.

AI -- Automatic Integration of the peaks in the spectrum.

SZ -- Set Zero. Sets the offset of the peak in a Zoom region to zero.

O -- Adjust peak Offset. Hold the left mouse button and position vertical line on peak, then press 'o'.
Enter offset value.

PP -- Peak Picking. Displays peak tick marks and offsets on spectrum. Used with MH and RM.

MH -- Minimum Height. The smallest peak height allowed for Peak Picking.

RM -- Multiples of RMS. Adjusts discrimination of peaks with small splittings. Use PP after changing
RM.

ZO -- Zoom mode. Hold left mouse button and sweep across peak from baseline to baseline. Right-click highlighted region to Zoom. Right-click to return to previous view. Press 'Enter' to exit Zoom mode.

Acquiring and processing a carbon 13C spectrum

The procedure is essentially the same as for acquiring a proton 1H spectrum, but the PNMR prompt needs to say: C13> first (type, if needed: nu C13). Shim and acquire the spectrum as above. The processing procedure is similar to the processing procedure of a proton 1H spectrum. A useful macro to run is Aii_c13_1D.mac. This macro provides a calculated chemical shift scale, does an automatic peak pick, and sets the display range from 220 to -10 ppm. To invoke this macro, press <ctrl+F3>, then select the macro. There are many other macros that may also be useful.

Acquiring and processing of DEPT spectrum

Make sure that the C13> prompt is shown in the PNMR program (type, if necessary: nu c13 <enter>). Type: dept <enter>. After data acquisition, the program asks whether the data should be processed automatically. Press "Yes" or "No" as desired. A useful macro to invoke is aii_dept.mac. A typical line broadening is LB = 0.5 Hz (type 0.5 <enter> or some other value. The macro applies an appropriate chemical shift scale, sets display for 220 to -10 ppm window, and displays a stacked plot of the DEPT45, DEPT90, and DEPT135 spectra. The spectral amplitude can be adjusted with the mouse wheel or Page Up / Page Down, or arrow keys, or vertical slider. To print the stacked plot, <p>. To exit the stacked plot, type <enter>. To redisplay stacked plot from the base level NUTS prompt, use the command sp.

Acquiring and processing of COSY or HETCOR spectrum

Make sure that the H1> prompt is shown in the PNMR program (type, if necessary: nu h1 <enter>). Type: cosy <enter> or hetcor <enter> and follow the prompts. Recommended is 2 seconds for relaxation delay for cosy and hetcor, 4 scans or multiple or 4 for number of scans. Make sure that the C13> prompt is shown in the PNMR program (type, if necessary: nu c13 <enter>). Press: dept <enter>. After data acquisition, the program asks whether the data should be processed automatically. Press "Yes" or "No" as desired. To process the data manually with macros, press <ctrl+F5> and open the appropriate macro Aii_cosy.mac for COSY and <ctrl+F6> and Aii_hetcor.mac for HETCOR. Choose borders by pushing "Yes" or "No", adjust data display by typing: mh <enter>. For COSY, the MH values range 2-4, for HETCOR MH value range 15-25. Look for off-diagonal peaks. Zoom by typing: zo <enter> and select expansion region with mouse, or type <f> and enter start and end of zoom for both dimensions. To print contour plot, type: p1 <enter>, to exist 2D contour plot, type: 1d <enter>.

Relaxation time (T1) guide

This can be done either for 1H or 13C. Adjust the prompt in PNMR as desired. Type: invrec <enter> and follow the prompts.The relaxation delay RD must be at least 6 times the longest expected T1 to get an accurate measurement. There is default list t1list for the file containing delay times, or new ones can be used. Type: <enter> to show the list. Acquire data by typing <enter>. For processing in the NUTS program, type: a1 <enter>. This executes the processing commands in link a1. Use link a3 and set s1 to the last one (7) is processing stored spectra from an earlier experiment. Use LB ~ 1 for C13. Zoom and trim phase by zo <enter> and pe <enter>, or ap <enter>. Process T1 data with macro Aii_t1.mac (press <ctrl+F7> and open). Enter data acquisition parameters. Display stacked plot with arrow keys or vertical slider, print stacked plot: z <enter>, and to exit press <enter>. Calculate T1 value by entering zoom mode: zo <enter>, select peak to be measured by clicking and holding left mouse bottom to left of peak and drag to right. Selected peak is shown in red. Expand selected peak: <ctrl+E>. Get relaxation data by integrating selected peak for each spectrum in series and save results: gr <enter>. Show X-Y plot of peak integration vs. time: dr <enter>, optimize fit: o <enter> (select "T1 31R" method). Print data: p <enter>.


Kinetics Experiment guide

First run spectrum on reference sample and use fo command to properly position the spectrum. Prepare and equilibrate reactions to same temperature as magnet.

In the PNMR program, type: kinetic <enter> and follow the prompts. For the initial delay use approximately 20% of the expected half-life of the reaction such that about half of the spectra will be taken before the mid-point of the reaction. All times are in seconds. For number experiments, try 20. For the time between mixing and starting NMR acquisition, the PNMR program counts down the time, starting at the moment the mixing starts (at a later prompt), 30-60 seconds to mix may be appropriate. Keep pressing <enter> until the end of the list of delays. For the pulse width, the program either requests the 90 degree pulse width or reads it from the pulse file. Follow prompts and press <enter> when ready to mix. In the NUTS program, process first spectrum by typing a1 <enter>, and run kinetics macro via <ctrl+F9>, follow prompts, and adjust stacked plot amplitude with Page Up / Page Down, arrow keys, or vertical slider. Follow gr <enter>, dr <enter> commands as outlined for the relaxation time (T1) guide. Choose fit function: f <enter>, optimize fit, o <enter>. Exit data reduction by pressing <enter>. Repeat for other regions. Use NUTS d1 command to list delay times.


Changing Samples and Finishing up

Remove the sample by pressing the EJECT button, take out the sample, remove the spinner, insert the next sample or the standard H2O spectrum into the spinner and insert the tube with spinner into the magnet as elucidated above.


Page created by Alexander Goroncy.


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