NMR Sample Preparation
In nuclear magnetic resonance (NMR), unlike other types of spectroscopy, the quality of the sample has a profound effect on the quality of the resulting spectrum. Follow these few simple rules to ensure that the sample you prepare gives a spectrum in which useful information is not lost or obscured.
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 6,000 times less sensitive than 1H. A good rule of thumb is to provide as much material as needed to give a saturated solution. If about 0.2 to 0.3 millimoles can be dissolved in 0.55 mL, the spectrum will take no more than about a half 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 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: 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 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 is done as seldom as possible, your samples must be prepared so that they physically resemble the test sample. After filtration, they must be made up to a similar depth. For the Bruker spectrometers, the optimum height is 4 cm, or 0.55 mL. 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 minimize 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 stockroom. The NMR lab does not supply you with solvents.
Use proper tubes and caps
NMR tubes are available from the chemistry research stockroom. Minimum length is 7". Minimum quality is 507PP (Wilmad product number) for 400 MHz. 528-quality is preferred for 500 MHz instruments. You must not use NMR tubes with a chipped or broken top because they are dangerous, and very likely to splinter lengthwise. 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 vapor effectively, and solvent peaks will appear in your spectrum. Tubes must be capped, and caps should be treated the same way as tubes. Sample Tube Grade Affects the Resolution has helpful and important information.
Label your samples
Labels on your samples are 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.
Use an internal reference
Usually, a small amount of reference is added to the solvent by the supplier. However, the amount of tetramethylsilane (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. Think about adding 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.
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, 4 October, 1995, NMR Services at Heriot-Watt University Chemistry Dept., Edinburgh, Scotland