NMR Sample Preparation and Spectra Acquisition

Check an NMR tube and cap out of the stockroom.
Clean the tube with reagent grade acetone using the NMR tube cleaner,

Place the open end of an
nmr tube over the 4 mm tube
and pour a few milliters
of reagent acetone into
the 10 mm tube while the
aspirator is turned on.
Acetone will be aspirated up
through the nmr tube to clean it.

in the laboratory hood as demonstrated by your instructor. Be sure to draw air through the tube for a few minutes to evaporate all of the acetoneAcetone will give a signal at ~2.1 ppm and its presence will typically result in a reduced sample grade..
Place 0.100 g of the compound to be analyzed in a scrupulously clean, dry semi-micro test tube and add 0.80 mL of deuterochloroform (CDCl₃ 1% v/v TMS internal standard)You must recap the bottle tightly after use - Volatile!. with a 1.0 mL syringe.
Using a clean disposable pipet transfer all of the sample solution to an NMR tube and cap securely.

If any undissolved solids are present in your sample, they must be removed before a satisfactory spectrum can be obtained.
Pack a small piece of cotton firmly in a disposable pipette to act as filter and transfer your solution with another pipet through this filter directly into the NMR tube.
If your sample does not dissolve in chloroform, consult your instructor for alternate solvents.

When you arrive in B124 check the monitor (you may need to turn the monitor on) on the NMR system to ascertain if the instrument is in use by examining the tool bar:
The stop button is active and the instrument is in use - do not disturb.

OK to use the instrument. The stop button is grayed.
If the floating message, Press Control-ALT-Delete to log on is displayed, Press Control-ALT-Delete and log on using your normal campus username and password. The NMR control software will automatically startup when you logon.
There should be a sample spinning in the magnet at all times and it must be removed by:

Turn spinner air knob labeled SPIN CONTROL clockwise until closed(about 1/4 turn)(sideways-the spinner tachometer will register 0 or some very large(<100) number), and
turning up the EJECT CONTROL (counterclockwise-probably all the way) until the sample floats at the top of the spinner tower and remove the NMR tube from the spinner turbine by carefully 'inching' the tube out of the turbine. Be careful, NMR tubes are very thin glass and break easily, usually resulting in severe lacerations.

Place your sample in the turbine with the same careful 'inching' procedure and adjust the tube's position in the turbine with the spinner gauge attached to the magnet. The bottom of the tube should extend all the way to the bottom of the spinner gauge, but no further. Be very careful to position the tube properly - if it is too far into the turbine, it may break when placed in the magnet.
Thoroughly wipe the outside of the tube and spinner with a KimWipe and allow the sample to float at the top of the spinner tower. Try to hold the sample so that it just floats with no rotation or oscillation. As soon as it is steady, carefully turn the EJECT CONTROL down (clockwise) until it is off.
Turn the spinner air control about 1/2 turn counterclockwise ((horizontal) - parallel to the floor). The sample should begin to spin as indicated by a reading on the spinner tachometer. Turn the spin control clockwise until the arrow on the knob is at the 2 o'clock position. The tachometer reading should stabilize somewhere between 15 and 60 RPS. If it does not spin or the spin rate is very erratic, consult your instructor or Dr. Badger.
Using the mouse select Scripts | Acquisition | Routine Spectra from the scripts menu of the NTNMR application.
You should insert the sample notebook number and compound description in the text box that has the rbadger (or your logon) in it. The default collection and processing selections are usually satisfactory for routine samples. It is recommended that autoshim always be selected whenever a new sample is inserted in the magnet. If you wish to collect carbon-13 data, select the C13 option above.
After the data has been collected, remove your sample from the magnet and place one of the sealed samples in the magnet as described above.
If you logged on to the instrument, it is recommended that you log off when done.
Please turn off the monitor when through.
You can access your data from any web browser via the nmr log. You will need software on your system that will open and process JCAMP FID data files. It is also possible to save data in other file formats. Consult Dr. Badger for details.
Processing your data with MacNUTS (for Macintosh) or PCNUTS (for Windows) in B124 or A113 Science is virtually identical once you download the raw data from the nmr log (http://chemdept-nmr.uwsp.edu/nmr/log.cgi). Just click on your data file and let the browser save it in your documents folder or other logical location. We have the 'Pro' version which can do almost anything with nmr data you want to do. See the really complete documentation at the Acorn nmr site (yep, they are probably NUTS)
I have a couple of sample data files for 3-methyl-1-butanol (aka isoamyl alcohol or isopentyl alcohol) here (3-methyl-1-butanol H1 and 3-methyl-1-butanol C13 right click on these links and select save link as) that you can use for practice.
Start up the nuts application by double clicking the application icon or selecting it from the start menu.
Click the OK button to close the startup window and type im (text in bold should be typed as shown - the command will be immediately executed) on the keyboard (menu selections and buttons are available for some of these commands, but the keyboard is really faster).
From the dialog (that appears after im) select the file you downloaded from the nmr log.

Once your file is imported(just say yes to overwrite if it complains that some file already exits in its temporary storage area) you are ready to process it. Now the fun begins.
General processing steps:

Process	Proton nmr	Carbon nmr	Proton Example	Carbon Example
baseline correct (bc)	It doesn't look like it does much, but it is critical to proper phasing and integration.	Again not much visual change, but important in removing the 'center glitch'.
Fourier transform (ft)	Converts from the time domain to the frequency domain. It's beginning to look a lot like an nmr spectrum.	Same as proton. The spectrum is usually much noisier. Tools are available to improve it. Talk with your instructor.
spectrum phasing	It is best to hand phase (phase expanded (pe) the proton since getting a flat baseline is critical to proper integration.	Since we don't integrate survey carbon spectra, an autophase (ap) is sufficient.
Set the reference	We usually set the reference by locating the right-most peak (TMS) and setting it to zero ppm.	We can set TMS (right-most peak) or we can set the center line of the deuterochloroform triplet to 77.0 ppm.
peak picking (pp)	Pretty much what it sounds like. We can adjust the minimum height (mh) variable (the default is 10 % of the strongest peak) to control the number of peaks.	Since the noise level of carbon spectra is usually larger than proton, a minimum height of 10 % usually works fine.
integration (id)	Now is when that hand phasing pays off, but we frequently need to perform a baseline fix (bf) to obtain good integration.	We won't integrate carbon spectra unless we are really desperate and then special techniques that require a lot of instrument time are required.
plotting (pl)	Pretty much what it sounds like. Exactly what you see on the screen is what you will get. So you need to be sure that you have expanded the region of interest and turned the peak and integration displays on appropriately.	Same as proton.

Now that you have seen an overview, let's get down to the nitty-gritty of doing the processing. Below is a step-by-step set of instructions with some tips thrown in for good measure along the way.

bc - executes a base line correction because we use two analog to digital converters.
ft - perform the fast Fourier transform to convert our data from the time domain to the frequency (ppm) domain.
zo - zoom mode so we can select regions for phasing purposes.

Drag across a large peak(or peaks) in the spectrum to select and press 1 to set the phase 1 region.
Drag across another peak(or peaks) (far away if possible) and press 2 to set the phase 2 region.
Press return to exit the zoom routine.
Useful info - pressing f while in the zoom mode will bring up a dialog box that will let you type in numerical limits to the zoom region.
It is perhaps worth noting that ctrl-e will expand the last zoom selection you made and ctrl-f will give the full scale spectrum.

pe - starts the phase expanded routine - region 1 should be displayed.

Drag back and forth with the left mouse button to obtain a symmetrical peak with the same base line on the left and right edge. Note: drag in this context means hold the mouse button down while moving the mouse. Use the up arrow or page up keys to increase the intensity so you can see small differences.
Drag back and forth with the right mouse button (phase region 2 should be displayed) to obtain symmetrical peaks with the same base line on the left and right.
OK, now that we have done it the hard way, let me mention the easy way to phase a spectrum:
- ap - autophase the spectrum immediately after doing the Fourier Transform.
- Why didn't I tell you that from the beginning? Because you almost always should hand phase a proton nmr to get good integration and autophasing just doesn't work perfectly.
- It does work just fine for carbon spectra since we don't usually integrate. Try the ap and see how it works.
Press return to exit the phasing routine.

You may have to press ctrl-f to see the full spectrum and you may have to type mf to rescale the spectrum intensity.
Setting the reference: just in case press return a couple of times to be sure you are at the base level. Identify an appropriate reference peak (TMS[0.0 ppm] for proton, the center line of the deuterochloroform triplet[77.0 ppm] for carbon) and drag the cross hair on top of the peak.
While holding the left mouse button down press the o key. An offset dialog will appear in which you should key the chemical shift of the reference peak you have selected.
pp - peak picking using the default minimum height of 10 % usually works well, but you can enter mh which will bring up an edit dialog so you can change mh - the minimum height threshold for peak picking which is a percentage of the largest peak in the spectrum. You will need to type pp again to see the effect. Experiment a bit - it's lots of fun.
If you are processing a routine carbon spectrum the only thing left is printing.
pl - brings up a printing dialog. I usually select landscape printing and let it print.
If you are processing a proton spectrum then you will probably want to obtain the relative integration of peaks in your sample.
zo - enter the zoom routine and select the region that includes all of the peaks you want to integrate. So you should omit TMS and the downfield region that has no signals.
Press return to exit the zoom routine.
ctrl-e - this means hold the ctrl key and press the e key which will cause an expansion of the spectrum to the region you selected.
bf - base line fix does some simple base line flattening and untilting that gives better integration.
id - enter the integration routine which will usually show a full window integration trace. You could use a ruler to measure trace height changes to get relative integration, but there is a better way.
Click the left button once - a vertical cursor appears.
Place the cursor to the left of a peak(or peaks if they are too close to separate) and click again. I usually like to include a very small amount of flat base line. A vertical marker will appear.
Place the cursor to the right of the peak(or peaks) and click again. An integration trace for the peak you have selected will appear.
Repeat these three clicks until you have integrated all of the peaks of interest.
Left click once and place the cursor inside one of your traces.
Press the v key and an integral relative values dialog box will appear. If you have no idea how many hydrogens the peak you have selected represents, enter 1 as a guess. If you are right, the rest of peaks should turn out to be nearly integer ratios when you press ok. If you are wrong, just perform this process again entering a different integer. Integration is never perfect, just get as close as you can to integer ratios. Relative integration can be very bad for impure samples or compounds with spin systems that have very different spin-lattice relaxation times.
Press return to exit the integration routine. You may find that your peak picks or integrals don't show up at the base level. Ctrl-i will toggle the integrals on and off; ctrl-p will toggle peak picks on and off.
Once you get the screen looking the way you want it, press pl to send it to the printer.
I recommend quitting the program (you can save your data when it complains if you are so inclined) as it tends to remember the previous processing session which can really confuse the next person.

Last update: Friday, 25-Aug-2006 10:16:09 CDT