How to Prepare Samples for NMR
In NMR, unlike other types of spectroscopy, the quality of the sample
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
1) Use the Correct Quantity of Material. For 1H spectra of
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
concentrations, the peaks from common contaminants such as water and grease
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
Solid particles distort the magnetic field
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
2) Remove All Solid Particles.
3) Make Samples to the Correct Depth. In the magnet, the main
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.
For the Bruker spectrometers, the optimum height is 4 cm, or 0.55 mL.
For the Varian spectrometers, the height is 5 cm, or 0.7 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 minimise solvent loss through
4) Use Deuterated Solvents. Samples must be prepared using
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
Many deuterated solvents are available from the stockroom. The NMR lab does
supply you with solvents.
5) Use Clean Tubes and Caps. NMR tubes are available from the
and after use they should be rinsed with acetone or some other suitable
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.
6) 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
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
7) Use an internal reference. Usually, a small amount of
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
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
an internal reference. Remember that the chemical shift of water is highly
8) Degassing Samples. Some samples need to be degassed or have
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
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.
9-13-99, modified from Alan S.F. Boyd's document dated 4 October, 1995 (NMR
Services at Heriot-Watt University Chemistry Dept., Edinburgh, Scotland)
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