NMR Service

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. If you follow a few simple rules, the sample you prepare will
give a spectrum in which useful information is not lost or obscured. Your sample must be made up in
a 5mm NMR tube, unless you have arranged to provide it free of solvent.
1) 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 a saturated solution. If you can dissolve about 0.2 to 0.3 millimoles
in 0.7ml, the spectrum will take no more than about half an hour to record. If the quantity of material
is halved, the necessary data accumulation time will be quadrupled. You should also 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. If you wish to observe polymers or other nuclei, please contact me to discuss
quantities.
2) 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. Thus, a sample
containing suspended particles has a field homogeneity distortion around every single particle, causing
broad lines and indistinct spectra. To remove solid particles from your samples, you must filter ALL
solutions into the NMR tube. You should filter 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 will
dissolve material from it which can easily be seen in 1H spectra. After filtration the sample should be
clear though, of course, not necessarily colourless.
3) 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 5cm and 5.5cm, and requires about 0.6 to 0.7ml solvent.
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. 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.
4) Use Deuterated Solvents. Samples must be prepared using solvents that contain deuterium in
place of hydrogen. The NMR signal from the deuterium nuclei is used by the spectrometer for
stabilisation and is called the NMR lock. Deuterochloroform is available from the stores, and a small
selection of other deuterated solvents is kept for trial purposes in Room G.20. If you have a regular
need for deuterated solvents other than CDCl3, you should order your own supply. Please see me for
up-to-date information about prices and suppliers. Because they are extremely costly materials, they
must be used with great care to minimise waste. You must not use deuterated solvents to do solubility
trials. If you keep a 1ml calibrated all-glass syringe solely for use with deuterated solvents, you will
simplify handling and reduce the risk of contamination. Do NOT use mixtures of deuterated solvents,
or mixtures of deuterated with non-deuterated solvents, without taking advice.
5) Use Clean Tubes and Caps. 5mm NMR tubes are available from the Stores. After use, they
should be rinsed with acetone or some other suitable solvent, preferably using a tube washing device.
If your tube is badly contaminated you should use solvent with a doubled-over pipecleaner. Tubes
should then be dried with a blast of dry air or nitrogen. You must NOT dry tubes in a hot oven, firstly
because it does not effectively remove solvent vapour, so solvent peaks will appear in your spectrum,
and secondly because tubes distort significantly at the temperatures used in many ovens, and become
dangerous to use. Tubes must always be capped, and caps should be cleaned 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. It is very easy to trim off the damaged part yourself, see me if you don't
know how to do this.
6) Label Your Samples. Samples must be labelled, preferably with just your User Code and a
Sample Code. Do not clutter up a necessarily small space with structures or anything else. You
should write directly on the NMR tube with an solvent-based felt pen. If it does not write legibly then
the tube is dirty. You should be aware that new tubes are usually covered with a film of grease from
the manufacturing process, and should be cleaned.
7) Do Not Add TMS. 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. Even one drop of TMS
in a sample causes serious problems due to distorted baseline and exceeded dynamic range. Suppliers
can provide, on request, solvents with a small amount of TMS (about 0.03%) already added and, if you
need it, you should specify this when you order solvents. Usually, the residual protons in the
deuterated solvent are used as a secondary reference.
8) 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.
 DE
11 January 2008
 NMR Data Naming
Conventions
So that you may find your NMR data in the archive, all files from the AC200 are named in
accordance with this pattern:

aaabcccc.ddd
aaa User Code, generated by the registration program.
b Observed nucleus, e.g. H or C, inserted automatically.
cccc User’s Sample Code, provided by you.
ddd Experiment serial number, generated automatically.

Because of limitations imposed by the computer on the AC200, you must use only upper-case
letters and numbers. You must not use more than four characters. You must not use lower case
letters, or punctuation characters such as / : ( ] '. You should not include your initials in a Sample
Code. There are two reasons for this. Firstly, because the computer puts your User Code at the
beginning of the data file name, so your personal identity is automatically attached to the file.
Secondly, because you are limited to only four characters to identify each sample, and later on you may
need all of these to provide enough distinct Sample Codes. If you use Sample Codes more than once,
you should remember that any earlier data file is destroyed by any later one with the same name. You
may use a Sample Code again if you are observing a different nucleus, since the nucleus is made part
of the file name. However, the solvent you use is not made part of the filename, so if you record
spectra of a sample in different solvents, each one should have a different Sample Code. Here are
some Sample Code examples:

47/2 incorrect, because it contains '/'.

35b incorrect, because it uses lower case 'b'.
*** incorrect, because it uses '*'.
34A57 incorrect, because it uses more than four characters.
5 correct
B6UD correct
SKG correct

You are strongly recommended to use a simple numbering system, e.g. 0 to 999, with
resubmission of the 'same' sample designated by a letter suffix, A to Z. Sample Codes don't need any
leading zeroes, but you may find them useful. It's generally unnecessary to use more than one or two.
You should be careful to avoid confusion between letter 'I' and figure '1', letter 'O' and figure '0', letter
'S' and figure '5', and letter 'Z' and figure '2'. The AC200 automation system will detect some mistakes
in a Sample Code entry, but not all.
For DPX400 data sets there are a few differences. Sample codes may be up to eight characters
long and are case-insensitive. All Sample Codes will be changed to lower case for the data set name,
and will be printed in upper case in the spectrum title. The experiment number is no longer an integral
part of the name, but is the name of an individual directory inside the data set. All the other
restrictions and advice given above still apply.
DE
11 January 2008