The Department of Chemistry and Biochemistry
The Michael Faraday Laboratories
Northern Illinois University
DeKalb, IL 60115-2862
NMR Sample Preparation Tips
In NMR, unlike other types of spectroscopy, the quality of the sample has a profound effect on the quality of the resulting spectrum. To obtain useful results, you must follow a few simple rules.
1. Sample Tube
Only good-quality NMR sample tubes should be used. The Bruker Avance 500 MHz machine requires the use of e.g., a Wilmad 528-PP tube or better.
The tube is quite fragile. It is precision-made from thin glass to specific dimensional tolerances. Because of the thin glass, it may break very easily. Take special care in removing and replacing the tube cap. The bottom of the NMR tube should be touched as little as possible to avoid making the glass dirty. A dirty NMR tube will lead to poor resolution spectra and a dirty instrument. Always clean the lower portion of the NMR tube when preparing your sample. Afterwards, handle the tube by the top only.
2. Sample Volume and Quantity
For conventional samples in organic solvents, the best results will be obtained using a sample volume of 0.6 - 0.8 ml for a 5mm NMR tube. Smaller volumes can be used but will require much more shimming. Samples that are too long are also more difficult to shim and are waste costly solvent. The preferred solvent for NMR studies is CDC13, so use it if solubility permits. If not, use C6D6, acetone-d6, methanol-d4, DMSO-d6, D20, CD2Cl2, … If unsure of solubility, test it in a small vial with the corresponding non-deuterated solvent.
For 1H spectra of organic compounds (except polymers) the quantity of material required is about 5 to 25 mg. 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. For a list of common impurities check H. E. Gottlieb et al., "NMR Shifts of Common Laboratory Solvents as Trace Impurities", J. Org. Chem. 1997, 62, 7512-7515, which can be found next to the solvent list.
13C is less sensitive than 1H (the natural abundance of 13C is only 1.1%), 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 ~ 0.7 ml, the spectrum should 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.
3. Sample Quality
Solid particles distort the magnetic field homogeneity because the magnetic susceptibility of a particle is different from that of a 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.
If undissolved material or ferromagnetic contaminants are present, the solution must be filtered.
You should filter samples through a small plug of glass wool tightly packed into a Pasteur pipette. If the plug is not tight enough, 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 clear.
4. Sample Solvents
Samples must be prepared using solvents that contain deuterium instead of hydrogen. The NMR signal from the deuterium nuclei is called the NMR lock and is used by the spectrometer for stabilization.
Solvents should be kept dry and free from impurities. Molecular sieves can be used to dry solvents; these create particulates which must be removed using the filtering method described above.
5. Use Clean Tubes and Caps
After use, NMR tubes 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 will reshape and ruin NMR tubes. Tubes placed in an oven in a beaker, flask, or tube rack can bend, increasing Camber (lack of straightness). Bent tubes may still fit the spinner turbine, but can damage or break inside the NMR probe insert resulting in costly repairs. They are also difficult to shim.
Tubes must be capped to slow down solvent evaporation, 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 can easily splinter lengthwise.
6. Sample Labeling
This is best done cutting a small hole in a piece of paper and inserting the NMR tube. Then, print all information onto this piece of paper. If you need to label the tube directly, use a permanent marker on the top of the tube, or on the cap. Do NOT use a sticker or a piece of tape which will leave a flap. Remember that the tube will spin at 20 Hz while it is in the magnet.
7. Sample Referencing
For conventional 1H and 13C NMR, our NMR equipment does not need TMS or any other reference materials in the NMR tube. Locking on the solvent will also calibrate the spectrum.
However, if you would like to add an internal standard, 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. Add a drop of TMS to a new bottle of CDCl3 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.
For samples in D2O, DSS or TSP is used as an internal reference. All of these standards are available from the NMR lab.