Sample Preparation

Quantity

In general, the more the better. Typical concentrations range from 100 uM to 100 mM but it is possible to work outside this range too. Required volumes are 500 uL for 5 mm tubes and 35 uL for 1.7 mm tubes. Be aware that signal-to-noise increases as the square root of scans so halving your concentration will require an experiment four times as long to produce the same sensitivity.

Purity

In general the purer the better, but this depends on what you are trying to do. Confirming the result of a reaction may simply require detection of a characteristic peak and therefore not require purification. If you are trying to determine the structure of an unknown then your sample should be at least 85% pure and elute as a single peak from an LC. A biomolecular sample for 3D structure determination should show a single band on a gel.

Organic solvents

Solvents should be deuterated to reduce the size of their signals and the chance of obscuring signals from your sample. If you have exchangeable groups on your sample that you need to detect (e.g. -NH, -OH) then avoid using protic solvents such as D2O and methanol-d4. Acetonitrile-d3 and DMSO-d6 are a better choice. If possible avoid using mixtures of solvents. Mixtures are harder to lock onto and to shim.

Buffers

For biomolecular samples phosphate buffers are preferred as they introduce no extra signals, but Tris, HEPES, etc may be used with isoptopically labelled samples as the signals from the non-labelled compounds will be filtered out. The pH should be physiological or acidic to slow exchange of amides with the solvent. Salts are often added, but be aware that high concentrations (> 150 mM) will make uniform excitation of the entire frequency range more difficult and will increase sample heating.

Remove insoluble material

Your solution should be clear. Cloudy solutions indicate that the sample has not dissolved properly. Such solutions will be harder to shim and will give broader peaks. If your sample does not dissolve well consider filtration or centrifugation to remove the insoluble material.

NMR tubes

Use tubes of sufficient quality. Low grade, or disposable, tubes will be more difficult to shim and will give broader peaks than better quality tubes. Wilmad 535-PP-7, or equivalent, are acceptable. If using Shigemi tubes make sure they are the Bruker versions with a small bottom length of 8 mm. The longer Varian Shigemi tubes will damage Bruker probes.

The table below lists the model codes used by the different manufacturers.

Size Supplier Model
5 mm Wilmad 535-PP-7
5 mm Kontes 897241-0000
5 mm Norell S-5-600-7
5 mm New Era NE-UP5-7
5 mm Shigemi Sigma-Aldrich
Wilmad
Bruker 		BMS-005TB for water
CMS-005TB for chloroform
DMS-005TB for DMSO
MMS-005TB for methanol
1.7 mm, rack of 96 Bruker Z106462

Cleaning tubes

Do not use a brush. It is likely to create scratches on the inner surface of the tube which will lead to reduced magnetic homogenity, poorer shimming and broader lines. Generally, rinsing the tube with acetone, distilled water then methanol should be sufficient. A final rinse with deuterated water will exchange protons adsorbed on the glass. The NMR Facility has apparatus for rinsing 5 mm tubes that can be borrowed. Do not dry tubes in an oven. The heat will cause the tubes to warp so that shimming will become difficult and the bent tubes may end up damaging the probe. It is best to dry tubes under a vacuum.

Volume

Sufficient volume to fill the active detection space must be used. Using less will make the samples difficult to shim and lead to broader peaks. Using too much will dilute your sample and waste solvent.

The table below lists the preferred volume and fill heights of different sized NMR tubes. Please note that the SSPPS NMR Facility does not have any probes capable of handling 10mm NMR tubes, that 3 mm tubes require a special adaptor to fit a 5mm spinner, and that while 1 mm tubes can be used with the 1.7 mm probe this arrangement is not optimal.

Tube size Volume Fill height
1 mm 5 uL
1.7 mm 35 uL 30 mm
3 mm 160 uL 30 mm
5 mm Shigemi 280 uL
5 mm 500 uL 40 mm
10 mm 4000 uL 40 mm

Filling 1.7 mm NMR tubes

The easiest way is to use a syringe or pipette. Long, fine pipettes can be drawn from a standard glass pipette. Disposable syringes are used by several UCSD labs. These have the advantage of not requiring cleaning. Other UCSD labs use Hamilton gas syringes. These can be purchased with an extended narrow gauge needle (22S,112 mm). If reusing a syringe it must be cleaned carefully before reuse. Its also posible to use a pippettor fitted with gel loading tips, but these don't deliver the solution to the bottom of the tube. Also, if using organic solvents, the solvent may leach plasticizer and other small molecules from the tips into your solvent.

To reduce solvent evaporation from 1.7 mm NMR tubes polyoxymethylene balls can be used to plug the hole in the cap. They can be purchased from Bruker or other suppliers. Plugging the holes with parafilm is not a good idea, as the evaporating solvent extracts paraffin into your sample and onto the spinners.

Written by Brendan Duggan. Last modified 2022-Jul-22