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NMR spectroscopy
NMR sample preparation for liquid-state NMR

NMR sample preparation

When making an NMR sample, ensure that you use the correct NMR-tube and sample volume. A range of deuterated and and some non-deuterated solvents are possible.

NMR tubes

NMR-tubes comes in various lengths, diameters, and shapes. The most common lengths are 10.4 cm (4") and 17.8 cm (7"), while the most common diameters are 3 mm and 5 mm. In addition to the standard cylindrical tube you can encounter the Young tubes for atmospheric control and the Shigemi tubes for low volumes. Never use broken or partly broken NMR tubes.

500 MHz routine instrument

For this instrument the 17.8 cm NMR-tubes with 5 mm outer diameter should be used, e.g. Wilmad 528-PP-7. When purchasing new tubes, we recommend the “Unracked 7" NMR-tubes with coded closed caps” (Z172600) which can be obtained from Bruker Labscape or Cortecnet. These tubes are inexpensive (~30 NOK/tube) and comes fitted with the special SampleJet cap, which is required when using the 600 MHz and 850 MHz. These caps are compatible with the 500 MHz.

Specialized Young tubes may also be used with the 500 MHz. However, it is important to ensure that the slots next to a Young tube is vacant.

600 MHz and 850 MHz instruments

The 600 MHz and 850 MHz instruments are equipped with SampleJet sample changers. The SampleJet can accommodate both 3 mm and 5 mm tubes, and both long (7"/18 cm) and short (4"/10 cm) tubes. While stored in the SampleJet the long tubes and Rack 1 on the 600 MHz are kept at room temperature. All other racks are refrigerated (5 °C). Bruker SampleJet caps with code must be used. We recommend using "Unracked 4" NMR-tubes with coded closed caps" (Z172599 or Z172597), which can be obtained from Bruker Labscape or Cortecnet.

Limitations

Specialized NMR-tubes, such as Young- and Shigemi tubes are not compatible with the SampleJet. To use these tubes the sample needs to be inserted manually, which require additional training or assistance from the staff.

All the rack on the 850 MHz are cooled to 5 °C, which is incompatible with the solvent DMSO. If your solvent is DMSO, either use long tubes and spinners or use the 600 MHz. At the 600 MHz, long 3 mm tubes have to be used with spinners (position 97, 98, and 99).

Labeling of NMR tubes

Long NMR tubes can be labeled on the top with a permanent marker. Never put markings on the short 10 cm NMR tubes. Use the code on the cap for tracking the sample.

Sample volume

We recommend 600 µL for a 5 mm NMR tube and 200 µL for a 3 mm NMR tube. Newer go below 4 cm (550 µL for a 5 mm tube and 160 µL for a 3 mm tube. Over or underfilling the tube can lead to poor shimming. If you have less than 160 µL and cannot dilute your sample, you need to use the special Shigemi tubes.

Solvent

To avoid signals from the solvent, a deuterated solvent should be used. There is a range of possible deuterated solvents, the most common being CDCl3, DMSO-d6, C6D6, MeOD, CD2Cl2, and D2O. Mixtures are also possible. Contact the staff if you want to use a solvent which is not available.

It is also possible to used H2O as a solvent. In this case you need to add 10% D2O for lock. When using a protonated solvent, the solvent signal needs to be suppressed. This puts some limitation on the choice of NMR experiments. If a buffer is required, preferable use a phosphate buffer as this does not contain any covalently attached protons. Keep the ionic strength to a minimum, as high concentrations of salt will reduce the sensitivity.

Additives

Consider adding a chemical shift reference (TMS for organic solvents, DSS/TSP for water), or use a solvent which already contains it. When using water as a solvent, it is prudent to include 0.02% NaN3 to prevent bacterial growth.

Other factors

  • The sample should be particle free
  • Sample concentration: typically in the millimolar range. For 1H the detection limit is round 1 µM.