NMR: Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance Spectroscopy is the most widely used method of structure determination present in modern chemistry. When used in conjunction with Mass Spectrometry and Infrared Spectroscopy, the three techniques make it possible to determine the complete structures of even the most complex molecules. Mass Spectrometry is used to determine the size of a molecule and its molecular formula and Infrared Spectroscopy helps identify the functional groups present in a molecule. NMR Spectroscopy is used to determine the carbon-hydrogen framework of a molecule and works with even the most complex molecules.

NMR is a spectroscopic technique which uses radio-frequency electromagnetic radiation and magnetic fields to determine the structure of organic compounds. Radio-frequency radiation is used to stimulate nuclei present within the molecule and from the information we obtain from doing this we can very accurately determine where the carbon atoms are located and where hydrogen atoms are located. The effect was first noticed in 1902 by P. Zeeman, a physicist, who won a Nobel Prize for noticing that nuclei of certain atoms behave strangely in a magnetic field. Fifty years later the physicists F. Bloch and E. Purcell put this idea to good use by constructing the first NMR spectrometer. They too received a Nobel Prize for this work.

The principle of NMR is based upon the spin of atomic nuclei in an external magnetic field. Many nuclei do not have the ability to spin in a magnetic field, but proton (1H) and an isotope of carbon (13C) can spin in a magnetic field, so they are the most widely used elements in NMR. When no magnetic field is present, the nuclear spins of magnetic nuclei are oriented randomly.

Once a strong magnetic field is introduced to the nuclei, they reorient their spins so their magnetic fields are either parallel or antiparallel to the alignment of the applied force. The orientation parallel to the alignment of the applied force is lower in energy and therefore favored. When the nuclei are irradiated with RF radiation the lower energy nuclei spin-flip to the higher state. When this occurs the nuclei are said to be in resonance, hence the name NMR. The nuclei reach the higher energy state in a negligible amount of time since they spin-flip at an infinitely high rate. Once the strength of the magnetic field which caused them to spin-flip returns to zero, the collection of nuclei become unstable and return to their original lower energy state. This process is called spin relaxation. The data from the spin relaxation is collected in the form of a modulated free induction decay (FID) signal. An FID is considered a time domain data set, because it shows the behavior of the spinning nuclei as time elapses.

A mathematical manipulation of the FID data, called a Fourier Transform, is carried out to convert the data from a time-domain model to a frequancy-domain model. The result is a spectrum which contains peaks that represent the activity of each nucleus or group of equivalent nuclei.

Instruments

A modern NMR instrument is composed of three basic components: the magnet, which houses the sample; the console which controls the magnet and recieves the signal; and the computer, which is used to analyze the data. Data acquisition using an NMR spectrometer involves careful sample preparation, knowledge of the individual NMR system available in a lab and training from an experienced user in order to learn specific acquisition procedures.

When a sample is placed between the poles of the NMR superconducting magnet, the nuclei adjust their spins either with the applied magnetic field (parallel) or against (antiparallel) the direction of the applied magnetic field. RF energy is introduced to the sample through the transmitter coil surrounding the sample tube. This causes the lower energy nuclei to spin flip to the higher energy state. Once the RF energy is no longer applied to the sample, the nuclei in the higher energy state reverse spin flip to the lower energy state. This process is called relaxation. When the nuclei relax back to the lower energy state, the excess energy is emitted. This energy release is detected by the receiver coils and recorded as a modular free induction decay (FID) signal.

Once data has been collected by the magnet, it is sent to the digital console as a FID. The digital console determines the frequency and intensity of each component wave of the FID, by performing a Fourier Transform of the data. These frequencies and intensities make up the peaks of an NMR spectrum. The digital console then performs other manipulations of the data, such as zero-filling and apodization. These calculations help to make the spectrum as detailed as possible and they eliminate imperfections caused by data loss when the FID is sent from the magnet to the digital console.

1H & 13C NMR

In 13C NMR, each carbon atom which is in a unique environment gives rise to a distinct peak in the spectrum. Many compounds which are partially symmetrical have equivalent atoms, which are effectively in the same chemical environment. Likewise, in 1H NMR, each non-equivalent proton is responsible for the presence of a distinct peak in the spectrum.

Toluene, for example, has four different kinds of protons arranged symmetrically in the molecule, so its H-spectrum contains a peak for each non-equivalent group of protons. The methyl protons, H5, H6 and H7 produce a single peak at 2 ppm. The single peak at 3.7 ppm corresponsds to the methyl protons, H9, H10 and H11.

Methylacetate contains 3 non-equivalent carbon atoms, thus its C-spectrum shows 3 distinct peaks. The peak at 20 ppm corresponds to the methyl carbon C1. The methyl carbon, C5, has a peak at 52 ppm. It is further downfield than C1 because it is bonded to a highly electronegative oxygen atom. The carbonyl carbon, C2, produces a peak at 173 ppm, since it is bonded to two oxygen atoms.

Instructions for NMR Spectroscopy Using Bruker IconNMR and XWINNMR/TopSpin

The following instructions provide a step-by-step guide for sample preparation and data acquisition using two software packages provided by Bruker, a maker of NMR instruments. IconNMR is a UNIX software package that provides a simple, step-by-step layout for acquiring data. ICONNMR also has an automation feature which allows the operator to place a sample in the instrument and then set up a series of different acquisition experiments for that sample which the software automatically runs consecutively. XWINNMR is a UNIX software package which allows an operator the most control of individual acquisition and instrument control parameters, but is a more complex software package to learn and master.

IconNMR

  1. The first window on the computer screen is the avance200 lock-on window. Click on the computer icon above the user name that is desired.
  2. After double clicking twice on the username the window asks for a password to be entered at the bottom of the window. Enter in the password given to by the instructor. The desktop should now be displayed on the screen.
  3. The Winterm Window should already be open with the cursor waiting at the avance200 1% prompt. (If the window is not opened click on OPEN UNIX SHELL under desktop on the small menu window located at the top right hand corner of the screen.)
  4. At the avance200 1% prompt type xwinnmr.
    The XWIN-NMR Version 2.1 on avance200 Window should be open, this is the main window for running xwinnmr.
  5. Type iconnmr at the command line in the XWIN-NMR window. The main screen for Icon NMR appears with five icons lined up at the bottom of the screen.
  6. Click on AUTOMATION. This brings up the Username Window, where the operator must identify a username. Double click on user name desired.
  7. The Password-Check Window appears for the user to enter a password. Enter the password given by the instructor.
  8. The main window for Icon NMR should now appear. This window is where each sample run is entered and recorded into the computer for analysis.
  9. Down the left hand side of the main window there is a column labeled holder number. Each holder number corresponds to a new sample to be run. Clicking on the holder number opens that holder for an experiment to be entered.
  10. Once clicking on the holder number a series of columns are now available for the operator to change: Disk, Name, Exp. No., Solvent, Experiment, and finally Note.
  11. The disk dive should have a default to the z:/ drive and does not need to be changed.
  12. The Name (file name) is defaulted to the date of the experiment, and is recommend that the date remains the file name.
  13. The Exp. No. (Experiment Number) has a default of 10 and will automatically increment for each additional run by a factor of 10.
  14. For the solvent column, there is a little arrow to the right hand side of the column, which displays (when clicked on) the entire list of deuterated solvent to choose from. (Note: for most non-polar compounds deuterated chloroform is the most common solvent, CDCl3).
  15. To choose an experiment, there is a small arrow on the right hand side of the Experiment column, which displays the entire list of experiments, when clicked. Select the appropriate experiment desired.
  16. To identify each run, the name of the compound can be written in the Note column by clicking on the small icon to the left hand side of the column. (Examples: “2-bromopentane” or “Unknown A”).
  17. Once all of the information is entered into the computer, click the SUBMIT button located at the bottom left corner of the main window. This makes the computer read the information entered in by the operator.
  18. At the top right hand corner of the main window the green GO button becomes available. Click on the green GO button to start the experiment.
  19. The Initialize-Run Window appears to ask for the injection/ejection type. The default is manual injection/ejection. Click the GO/START button to say yes to injection/ejection type.
  20. The inject/eject Window appears. Click OK to eject the sample already present in the instrument. Replace this sample with your sample, and click on OK again in the inject/eject Window. Watch the sample descend down into the instrument on the cushion of air.
  21. Icon NMR automatically runs through the locking, shimming, auto gain, and acquisition process. Icon NMR also automatically prints out a hard copy of the spectrum for analysis.

XWINNMR

  1. The first window on the computer screen is the avance200 lock-on window. Click on the computer icon above the user name that is desired.
  2. After double clicking twice on the username the window asks for a password to be entered at the bottom of the window. Enter in the password given to by the instructor. The desktop should now be displayed on the screen.
  3. The Winterm Window should already be open with the cursor waiting at the avance200 1% prompt. (If the window is not opened click on OPEN UNIX SHELL under desktop on the small menu window located at the top right hand corner of the screen.)
  4. At the avance200 1% prompt type XWINNMR.
  5. The XWIN-NMR Version 2.1 on avance200 window should be open, this is the main window for running xwinnmr.
  6. Using the command line, located at the bottom of the window, enter ej for eject sample. (Note: The instrument must always have a sample within the sample chamber at all times.) A few seconds later a sample will be pushed up to the top of the instrument by a cushion of air.
  7. Before retrieving the sample remove watches, wallets, keys, or any other metal object from your pockets. (Note: The magnet can demagnetize anything with a magnet strip.)
  8. Remove the sample from the instrument by lifting straight up. Remove the plastic adaptor that is around the sample. Place the plastic adaptor around your sample and make sure the depth of the NMR tube is correct by the use of the depth gage. (Note: The bottom of the NMR tube should be touching the white platform.)
  9. Place the sample on the cushion of air in the instrument.
  10. At the command line in the XWIN-NMR window type ij for inject sample. Watch the sample go into the instrument and wait for the ‘click’ sound.
  11. Using the mouse, select WINDOWS on the menu bar. Under Windows select LOCK. The Lock window appears with the signal at the bottom of the window.
  12. Again, under Windows from the menu bar and select the BSMS PANEL, which brings up the BSMS display.
  13. Click on LOCK from the BSMS Panel, which opens the Lock Panel. Click on AUTO LOCK in the Lock Panel. The sample signal, in the Lock Window, is no located near the top of the window. (Note: This is an indication that the NMR has successfully locked onto the sample signal.)
  14. Close the Lock Panel and click SHIM on the BSMS Panel, which opens the Shim Panel. Within the Shim Panel a series of Z icons are bold faced (Z, Z2, Z3). Click on the Z icon and notice the -/+ button at the bottom of the window is now bold faced. Adjust the shims by clicking on the -/+ button several time with either the right mouse button or the middle mouse botton.
  15. Notice what happens to the sample signal in the Lock Window. Adjusting the shims moves the sample signal up or down within the Lock Window. Adjust the shims so the sample signal is at the highest point in the window.
  16. Adjust the shims even greater by clicking on the Z2, and Z3 icons and follow the same procedure. (Note: Shims must be adjusted for every sample.) Once the shims are adjusted, close the Shim Panel and reopen the Lock Panel by clicking on LOCK again in the BSMS Panel.
  17. Click on AUTO GAIN to adjust the sample signal back to the original position the signal started at before adjusting the shims. Then close the Lock Panel, and the BSMS Panel.
  18. Type rpar, read parameters, at the command line in the XWIN-NMR window. the rpar window comes up with a list of all the different spectra the NMR can collect. Double click on the spectrum desired. Select COPY ALL FILES at the bottom of the window.
  19. Type eda at the command line in the XWIN-NMR window, which brings up the edpar window. This window allows the operator to edit the acquistion parameters, such as pulse width, number of scans per run, etc. Scroll down the window until you reach the solvent parameter. Underneath the solvent parameter is an icon that reads false, click on the icon to change to true. Adjust all other parameters as desired, and then click save at the bottom of the window.
  20. Type edc at the command line in the XWIN-NMR window to bring up the edc window, which allows the operator to change the informational parameters and save the data collected. Click SAVE when finished.
  21. Type zg at the command line in the XWIN-NMR window to start collecting data.
  22. When the acquisition is finished, type em, ft, apk to convert the time-domain spectrum to a frequency-domain spectrum.
  23. Click on INTEGRATE, located on the left-hand side of the XWIN-NMR window. Click the right mouse button and the mouse icon becomes a small arrow pointing straight down and only runs along the spectrum. Click the middle mouse button on either side of each peak to determine the integration.
  24. Click RETURN at the bottom left hand corner of the XWIN-NMR window, and click SAVE INTEGRATION.
  25. Type plot at the command line in the XWIN-NMR window to print a hard copy of the spectrum.