Concentration and Volume

The optimal concentration depends on the nucleus: For routine 1H NMR, 5-10 mg is typical for medium-sized (MW 50-400) organic molecules; for 13C NMR, which is 5700 times less sensitive, about 30-40 mg of sample is best if your molecule is soluble enough to get this amount in less than 1 mL. Too high a concentration can cause problems: It may cause overloading of the receiver in 1H spectra (this can be fixed, though); it will weaken the lock signal because there is less solvent present; and it can increase the viscosity of the solution, which will lead to broader peaks. The lower limits of concentration depend on field strength and the type of probe: For 1H you can get a good spectrum of an organic molecule with as little as 0.1 mg (500 MHz or higher); for 13C with a probe optimized for 13C detection you can get away with as little as 2-3 mg. The sample volume should be about 0.65 mL, which gives a 4.0 cm depth in a standard 5-mm NMR tube. Smaller volumes will require that you position the tube on the spinner turbine very carefully to center the sample volume in the probe coils of the spectrometer. Small-volume samples will also require a lot more time to shim properly. Too large a volume wastes solvent, reduces concentration, and can sometimes cause problems with spinning the sample because the weight of the sample and turbine is larger. The sample tube should be of high quality to avoid wobbling and breakage in the probe. Cheap or damaged tubes can lead to a broken tube in the probe, which can cost thousands of dollars in repairs and possibly lead to weeks of downtime for the spectrometer. NMR tubes with cracked or broken tops should be discarded or "sawed off" in a glass shop—they are dangerous and can lead to very serious injuries to the hands when capping or removing caps. If there are any solid "specks," chunks, or crystals in the sample solution, it should be filtered through a plug of glass wool placed in a disposable pipette. These chunks can degrade the linewidth and quality of your spectrum, even though solid material is "invisible" in the liquid NMR experiment. Fine crystals or powders may have no effect if they are evenly distributed throughout the sample volume or if they collect in an area above or below the probe coil. Cloudiness indicates that the sample molecule is only marginally soluble ("unhappy") in the solvent, which leads to aggregation of solvent molecules into large (but still microscopic) globs that tumble slowly in the solvent, leading to broad NMR peaks. Adding a cosolvent may solve the problem; for example, CD3OD in CDCl3 for molecules too polar for CDCl3, or CD3CN in D2O for molecules too nonpolar for D2O. If you add a cosolvent, be sure to measure it accurately and keep track of the volume ratio of the solvents used. You will need to report this ratio in the literature because chemical shifts depend on the ratio of solvents used. Also, you will need to use tetramethylsilane (TMS) as a reference because residual solvent peaks no longer will have the standard chemical shifts in a mixed solvent. If TMS is not used, at least report the solvent peak and chemical shift used as a reference.

3.1.3 Chemical Shift Reference

A standard is usually added to provide a sharp peak of known chemical shift in the NMR spectrum, in a region of chemical shifts that does not interfere with the sample peaks. For organic solvents, TMS is ideal because its XH chemical shift is upfield of nearly all organic signals, it gives a strong, sharp singlet, and its volatility makes it easy to remove. TMS is not soluble in D2O, so a related sodium salt (sodium 2,2',3,3'-d4-3-trimethylsilylpropionate or TSP) is used as the standard. The solvent 13C peak is usually used as the 13C chemical shift reference because the TMS peak is usually too weak (see below). Since D2O does not contain carbon, you will need to add a standard (such as methanol, acetonitrile, or dioxane) to the sample for a 13 C reference. A very common error is to add too much of a standard— this makes the standard peak dominate the spectrum and limits the sensitivity and dynamic range of the sample peaks as the receiver gain has to be reduced to accommodate the huge standard signal. The best way to add a standard is to "spike" a bottle (100 g) of deuterated solvent with a single drop of standard and mark the bottle accordingly. Fourier-transform NMR is very sensitive, and the standard peak can be very small and still be easily detected.

3.1.4 Sample Recovery

Unlike many other analytical techniques, NMR is a nondestructive test. You can recover your sample by removing the solution from the tube and evaporating the solvent. Samples in organic solvents such as CDCI3 can be removed from the tube by simply inverting the NMR tube into a vial and touching the top of the tube on the bottom of the vial to start the flow of solvent; rinse the tube with organic solvent. D2O has too much surface tension to be poured out of an NMR tube; Wilmad sells extra-long disposable glass pipettes that will reach all the way to the bottom of the NMR tube. With care it is possible to remove relatively volatile solvents in the NMR tube. The tube is clamped in a fume hood at a 45° angle and a long glass pipette is introduced so that the end is near the top of the solvent. A very gentle stream of dry air or nitrogen is introduced into the pipette and as the solvent evaporates the pipette is moved down to keep it near the solvent level. This is usually done when NMR data are needed in a different solvent for comparison with literature data—chemical shifts are solvent dependent and must be compared in the same solvent. Care of NMR tubes is important: They should be washed with solvents and water only, and dried with a stream of dry air or nitrogen. NMR tubes can be cleaned by repeatedly filling them with solvent (CHCl3, acetone, methanol, or water) and emptying. Aldrich sells an NMR tube cleaner that uses pump or aspirator vacuum to pull solvent into the tube. Never use paramagnetic solutions (e.g., chromium-containing glass cleaners) to clean NMR tubes. Drying the tubes is very important—residues of nondeuterated solvent can ruin your spectrum or cause you to make erroneous assignments. Drying NMR tubes in a drying oven not only is ineffective at removing solvents but also warps the tubes. The best method is to invert an extra-long glass pipette and run a slow stream of clean, dry air or nitrogen through it, and place the inverted NMR tube on the pipette so that the pipette reaches all the way to the bottom of the tube. A few seconds or minutes of gas flow should flush out all of the solvent residues without any heating. One final caveat: storing NMR samples in D2O in the freezer will crack or weaken the glass as the "water" expands.

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