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NMR Sample Preparation

03/19/2013 S.D.C

Location: 1232 Hach Hall

Contact: Shu Xu or Sarah Cady, 1234 Hach Hall

This handout covers a variety of considerations for good preparation of NMR samples. This covers the basic
preparation for most small-molecule organic compounds. Additional preparation procedures for large
molecules, polymers, biomolecules, inorganic molecules, etc. may be found in the literature.

1. Use the proper amount of material.

a. For small molecules (less than 1000 g/mol), typical 1H NMR spectra require 5-25 mg of material.
Typical 13C spectra require 50-100 mg of material. This amount of material will allow you to
obtain a 1H spectrum in a few minutes or a 13C spectrum in 20-60 minutes. When the amount of
material is doubled, the resultant signal will be doubled. Bear in mind that a very concentrated
sample will produce a quick 13C spectrum, but may result in a broadened 1H lineshape. Overly
concentrated samples can also be difficult to shim.
b. For larger molecules and polymers, the amount of material may need to be significantly greater.
Please consult literature affiliated with your molecule of interest for advice regarding sample
concentration in these situations.
2. Use the proper solvent and amount of solvent.
a. Deuterated solvents, in which 1H atoms are replaced with 2H atoms, are typically used in
solution NMR for a variety of reasons. These reasons include deuterium lock, shimming, and
providing an “invisible” background material that will not be observable in a 1H or 13C spectrum.
b. Common deuterated solvents are available in small quantities from ISU Chemistry Stores.
Common solvents include chloroform-D, acetone-D6, benzene-D6, deuterium oxide (D2O),
DMSO-D6, ethanol-D6, and methanol-D4. Additional less common deuterated solvents can be
ordered through Chemistry Stores from Cambridge Isotope Laboratories or Sigma-
Aldrich/Isotec.
c. Typical NMR samples contain 0.6-0.7 mL of deuterated solvent. Do not fill the NMR tube full of
solvent. This will dilute your sample, waste solvent, and make shimming more difficult.
3. Prepare your sample in a secondary vial.
a. Use a small vial to dissolve the solid sample and transfer it to the NMR tube with a glass Pasteur
pipette. Once the sample is in the NMR tube, effective mixing can be difficult. This also gives you
the opportunity to treat the sample with heat or vortexing in order to get complete dissolution.
If the sample contains significant solids, it is best to filter any particulate from the sample before
transferring to the tube. Solid particles will not show up in a solution NMR spectrum, and may
interfere with proper shimming.
4. Use clean, unscratched NMR tubes and clean caps.
a. Particulate stuck to the inside of the NMR tube or scratches/defects in the tube can interfere
with proper shimming. Use an NMR tube cleaner (either purchased or homemade) to clean
tubes after using.
b. Disposable NMR tubes are not appropriate for use on the high-field NMR instruments available
in the CIF – the low glass quality will not allow for adequately good shims. High quality NMR
tubes are available from Chemistry Stores or can be ordered from Wilmad Lab Glass or Norell
NMR Tubes.
5. Use an internal standard.
a. Residual 1H in deuterated solvents can often be used for spectral calibration. However, in
situations where an exact chemical shift is desired, or there is not solvent available for reference
(such as for 13C conducted in D2O or 31P), an additional internal standard must be used for
chemical shift calibration.
b. Internal standards such as TMS (for organic solvents) or DSS and TSP (for aqueous samples) will
give you an exact NMR reference. For nuclei other than 13C or 1H, additional standards can be
used such as phosphoric acid for 31P.
c. Internal standards can be added directly to the sample if desired. In this situation, just a drop of
TMS is often too much for one NMR tube. Add a drop of TMS to 5-10 mL of deuterated solvent
that can be used for several samples.
d. Alternatively, if you are concerned about an internal standard reacting with the compound of
interest, a capillary tube can be filled with an internal standard and placed in the NMR tube. This
situation is not appropriate if you are using the internal standard for quantitation purposes. For
quantitation, the internal standard must be added directly to the sample in order to achieve the
same filling factor in the coil.
6. Label your sample.
a. Permanent marker can be used to label the sample to ensure you do not mix up your NMR tube
with another NMR user, or it can be retrieved at a later time. If you use a sticker or tape to label
the tube, the sticker must be flush with the tube so it does not interfere with insert, eject or
spinning inside the magnet.
7. Air sensitive samples.
a. Some samples must be degassed to remove oxygen. Oxygen can be paramagnetic and broaden
lineshape or interfere with T1 relaxation measurements.
b. For degassing air-sensitive samples, J-Young Tubes can be directly attached to a vacuum line.