History of NMR

NMR Nobel Prize Laureates

A brief historical account of the Nobel Prize Laureates clearly shows the track of the discovery, development, and applications of NMR spectroscopy.

Otto Stern, USA: Nobel Prize in Physics 1943, “for his contribution to the development of molecular ray method and his discovery of the magnetic moment of the proton”

Isidor I. Rabi, USA: Nobel Prize in Physics 1944, “for his resonance method for recording the magnetic properties of atomic nuclei”

Felix Bloch, USA and Edward M. Purcell, USA: Nobel Prize in Physics 1952, “for their discovery of new methods for nuclear magnetic precision measurements and discoveries in connection therewith”

Richard R. Ernst, Switzerland: Nobel Prize in Chemistry 1991, “for his contributions to the development of the methodology of high resolution nuclear magnetic resonance (NMR) spectroscopy

Kurt Wüthrich, Switzerland: Nobel Prize in Chemistry 2002, “for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution”

Paul C. Lauterbur, USA and Peter Mansfield, United Kingdom: Nobel Prize in Physiology or Medicine 2003, “for their discoveries concerning magnetic resonance imaging”

NMR Timeline…

1945/6: NMR first observed by two independent groups. Purcell (Harvard) (Paraffin, 30 MHz); Bloch (Stanford) (water, 8 MHz)

1950: First pulsed experiment

1951: Observation of chemical shifts

1952: Purcell and Bloch share Nobel Prize for physics

1950’s: Commercial spectrometers available

1970: FT technique introduced, pioneered largely by Ernst. 13C NMR routine

1971: 2D NMR proposed by Jeener

1970’s – 1990’s:

  • Explosion in experimental techniques in one, two or more dimensions.

 

  • Availability and development of superconducting magnets allows use of much higher fields.

 

  • Concurrent advances in computing technology allows for data manipulation in reasonable amounts of time.

1999: State of the art applications include solving the 3D structures of proteins up to 40 kD using 2, 3 and 4D NMR.

Safety

5 Gauss line

5 Gauss is the strength of a magnet that has a strong enough magnetic force to act upon objects. The 5 gauss line describes the distance from the centre of the magnet where a magnetic field strength of 5 gauss is experienced. In our NMR Facility, this is approximately 40 cm from the edge of the magnets.

Cryogens

The cryogens used are liquid N2 and liquid He:

Temperature: N2: -196 and He: -269 deg. C
Colour: none
Toxicity: very low
Fire hazard: non combustible
Volume Expansion (from normal boiling point to room temp.): 700x

The main risks are of burns when handling cryogens and of asphyxiation if a magnet quenches. These are minimized by only allowing very experienced staff to fill the magnets with liquid nitrogen and liquid helium.

The magnet cryostats are only filled using stainless steel transfer lines to reduce risk of rupture. At least two staff must be present during refilling and appropriate safety clothing must be worn (gloves and eye protection). Refills must be continously attended. It is particularly important that the person filling the magnet, once trained, should do so on a very regular basis so as to be very familar with the required routine. Magnet quenches (the rapid release of gaseous cryogens from the cryostat into the room) should trigger the fire alarm and thus open the smoke vents thereby preventing any risk of asphyxiation due to the large volume expansion.

In the event of a quench personnel should evacuate the area (a quench warranting evacuation would be obvious by the noise of the escaping gas and clouds of vapor).
Access to the NMR rooms is strictly limited to the NMR staff during refills and any major maintenance. There is a potential danger of asphyxiation during N2 fills because the density of N2 is higher than of O2.

The magnet cryostats continuously expel a small quantity of gaseous He and N2 into the air. This does not present a hazard since during everyday use the air is constantly changed in the NMR rooms by the air-conditioning system.

Medical implants, electronic

Medical implants that have electronics (such as pacemakers) are set and/or reset through the use of magnetic fields. Thus exposure to magnetic fields can cause such devices to operate in an unintended manner or stop working altogether. People with such implants should never enter the NMR Facility. Signs are posted at all entrances to that effect.

Medical implants, non-electronic

Medical implants such as pins, surgical clips, etc. may be magnetic and maybe subject to the same forces described above. People with such implants should stay outside the 5 gauss line described above.

Safety rules for working in large magnetic fields.

1) People with medical implants should check with facility personnel before entering the NMR Facility.

2) All magnetic objects should be kept outside the 5 gauss line of each magnet. This includes keys, wallets, pocketknives, tools, etc. ASSUME ANY PIECE OF METAL IS MAGNETIC UNTIL PROVEN OTHERWISE.

3) Keep electronics outside of the 5 gauss line.

4) Use only non-magnetic tools inside the 5 gauss line.

5) When in doubt, ask NMR Facility personnel. Each NMR system costs at least $500,000. Do not risk damage to the instruments or harm to your self. If you have any questions, get assistance from Facility Staff.

6) No one is to be present in the NMR Facility unless they have received training and are authorized to use the system.

Magnetic fields can generate large attractive forces on ferromagnetic objects. Such objects include, but are not limited to, most tools, high-pressure gas cylinders, pocketknives, key rings, and most electronics. Any such object that gets too close to the magnet will be drawn towards the magnet with great force. A best case scenario is simply lost time and expense of removing the object from the magnet. Worst case is the death of the user or innocent bystanders that could occur in two ways. First, in object pulled with great acceleration towards the magnet could strike someone, causing injury or death. Second, the object striking the magnet could cause the magnet to quench (i.e., become resistive). This vaporizes the magnet cryogens (helium, nitrogen), which displace air and can result in asphyxiation.

The most common danger from an object impacting a magnet is that the magnet could quench. A quench can result in severe and expensive damage to the magnet, in addition to the dangers of asphyxiation. If a quench occurs, evacuate the room immediately. A developing quench can de detected by visible (and/or audible) emission of cryogenic gas from the magnet.

Should any metallic object strike the magnet, get NMR Facility staff immediately. Do NOT attempt to pull the object off yourself.

Wallets, credit cards, watches, magnetic media…

While not strictly a safety issue, large magnetic fields can wipe out the magnetic information on ATM and credit cards and magnetic media like computer discs. Keep your ATM and credit cards and magnetic media outside the 5 gauss line. Mechanical watches may also be permanently affected by large fields and thus should also be kept outside the 5 gauss line. Digital watches are usually okay within high magnetic fields, although some may have magnetic material. Each user is responsible for knowing if their watch is magnetic.

Sample Preparation

How to Prepare Samples for NMR

In NMR, unlike other types of spectroscopy, the quality of the sample has a profound effect on the quality of the resulting spectrum. So that the sample you prepare gives a spectrum in which useful information is not lost or obscured, you must follow a few simple rules.

Use the Correct Quantity of Material

For 1H spectra of organic compounds (except polymers) the quantity of material required is about 5 to 25mg. It is possible to obtain spectra from smaller quantities, but at very low concentrations, the peaks from common contaminants such as water and grease tend to dominate the spectrum. 13C is six thousand times less sensitive than 1H, and a good rule of thumb is to provide as much material as will give a saturated solution. If about 0.2 to 0.3 millimoles can be dissolved in 0.7ml, the spectrum will take no more than about half an hour to record. If the quantity of material is halved, the data accumulation time will be quadrupled. You should be aware that if you make up a sample at high concentration for 13C, and then record a 1H spectrum from it, the increased solution viscosity may result in a spectrum that has broader lines than you would get from a more dilute solution.

Remove All Solid Particles

Solid particles distort the magnetic field homogeneity because the magnetic susceptibility of a particle is different from that of the solution. A sample containing suspended particles thus has a field homogeneity distortion around every single particle. This causes broad lines and indistinct spectra that cannot be corrected. So that there are no solid particles in your samples, you must filter ALL samples into the nmr tube. You should filter samples through a small plug of glass wool tightly packed into a Pasteur pipette. If the plug is not tight enough, the filtration will be ineffective; if it is too big, some of your sample will remain trapped in it. Do not use cotton wool, since most NMR solvents dissolve material from it which can easily be seen in 1H spectra. After filtration the sample should be as clear as water though, of course, not necessarily colourless.

Make Samples to the Correct Depth

In the magnet, the main field direction is vertical, along the length of the sample. Each end of the sample causes a major distortion of the field homogeneity which is corrected using the spectrometer’s shim controls. A partial correction is done for every sample, and takes a few minutes. A complete correction takes many hours using a high quality test sample. So that this lengthy task need be done as seldom as possible, your samples must be prepared so that they physically resemble the test sample so, after filtration, they must be made up to a similar depth. This must be between 4.5cm and 5.5cm. Shorter samples are very difficult to shim, and cause considerable delay in recording the spectrum. Samples that are too long are also difficult to shim and are a waste of costly solvent. You should check your sample depth using a ruler. After preparation, you should ensure that the cap is pushed fully onto the tube to minimise solvent loss through evaporation.

Use Deuterated Solvents

Samples must be prepared using solvents that contain deuterium in place of hydrogen. The NMR signal from the deuterium nuclei is called the NMR lock and is used by the spectrometer for stabilization. Many deuterated solvents are available from the suppliers. The NMR lab does not supply you with solvents.

Use Clean Tubes and Caps

NMR tubes are available from the stores, and after use they should be rinsed with acetone or some other suitable solvent, then dried with a blast of dry air or nitrogen. Do NOT dry tubes in a hot oven because it does not remove solvent vapour effectively, and solvent peaks will appear in your spectrum. Tubes must be capped, and caps should be treated the same way as tubes. You must not use NMR tubes with a chipped or broken top because they are dangerous, and very likely to splinter lengthwise.

Label Your Samples

This is best done with a permanent marker directly on the top of the tube, or on the cap. If you use a sticker or a piece of tape, your label must stick smoothly on the tube. Do not leave a flap. Remember that the tube has to spin at 20Hz (1200rpm) while it is in the magnet

Use an internal reference

Usually, a small amount of reference is added to the solvent by the supplier. However, the amount of TMS or any other reference material that is required for a 1H spectrum is far less than can be added after the sample has been prepared. One drop of TMS in a sample causes serious problems due to distorted baseline and exceeded dynamic range. Even the standard amount of TMS added to a bottle of CDCl3 is too much. I typically will add about 2-3 mLs of CDCl3 containing TMS to a bottle that does not contain TMS and then use that bottle for sample preparation. This provides a small TMS signal; you never want your reference signal to be taller than your solvent signal. Alternatively, the residual protons in the deuterated solvent may be used as a secondary reference. For samples in D2O, DSS or TSP is used as an internal reference. Remember that the chemical shift of water is highly temperature dependent.

Degassing Samples

Some samples need to be degassed or have oxygen removed. The only effective way of doing this is by using the Freeze-Pump-Thaw technique, at least three cycles. It is sometimes sufficient to flush the space above the sample surface with nitrogen. This should be done with great care to avoid blowing the solution out of the tube. Do not bubble nitrogen through the solution in an NMR tube. This wastes costly solvent through evaporation, and is not an effective method of removing oxygen.

Modified from Alan S.F. Boyd’s document dated 4 October, 1995 (NMR Services at Heriot-Watt University Chemistry Dept., Edinburgh, Scotland)

Solvent Selection

Once the sample has been sufficiently purified and dried, the next step is to choose a suitable solvent. Since deuterium is by far the most popular lock nucleus the sample is usually dissolved in a deuterated solvent (a deuterated solvent is one in which a large proportion, typically more than 99%, of the hydrogen atoms have been replaced by deuterium). Commonly used deuterated solvents are acetone-d6, benzene-d6 and chloroform-d though many other solvents are available.

Factors to be considered when choosing a solvent are:

1. Solubility:

Clearly the more soluble the sample is in the solvent the better. This maximizes the amount of sample within the sensitive volume which increases the sensitivity of the experiment. High solubility is particularly important if only small quantities of the sample are available.

2. Interference of solvent signals with the sample spectrum:

The solvent itself will inevitably produce NMR signals which will obscure regions of the spectrum. These ‘residual solvent peaks’ should not overlap with signals from the sample.

3. Temperature dependence:

For experiments above or below room temperature the solvent‘s melting and boiling points are also important factors. Furthermore the solubility of the sample is likely to vary with temperature.

4. Viscosity:

The lower the solvent viscosity, the better the resolution of the experiment.

5. Cost:

Clearly for routine NMR where many samples need to be measured, the solvent‘s cost is an important consideration. As a rule of thumb, the price increases with the number of deuterated atoms.

6. Water content:

Almost all NMR solvents contain water traces. Also many are hygroscopic (they absorb water from the atmosphere) and hence the longer they are stored the more water they contain. The presence of a water (HDO) peak will only serve to degrade the quality of NMR spectra. The water level in the solvent can be greatly reduced by filtration through a drying agent or by storing the solvent over molecular sieves.

The choice of solvent for a particular sample will be the best compromise between the various advantages and disadvantages of each.