NMR Sample Preparation

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This page covers a variety of considerations for good preparation of NMR samples. All users must supply their own NMR tubes and solvents. Purchase of tubes and solvents is discussed below. This tutorial covers the basic preparation for most small-molecule organic compounds. Additional preparation procedures for large molecules, polymers, biomolecules, inorganic molecules, etc. may be found in the literature.

  1. Use the proper amount of material. 
    1. For small molecules (less than 1000 g/mol), typical 1H NMR spectra require 5-25 mg of material. Typical 13C spectra require 50-100 mg of material. This amount of material will allow you to obtain a 1H spectrum in a few minutes or a 13C spectrum in 20-60 minutes. When the amount of material is doubled, the resultant signal will be doubled. Bear in mind that a very concentrated sample will produce a quick 13C spectrum, but may result in a broadened 1H lineshape. Overly concentrated samples can also be difficult to shim. 
    2. For larger molecules and polymers, the amount of material needed may be significantly greater. Please consult literature associated with your molecule of interest for advice regarding sample concentration in these situations. 
    3. Broad lines regardless? Very viscous samples or samples that contain paramagetic ions (even in small quantities) can produce broad spectral lines. Gentle heating of the sample or removing residual paramagnetic ions may improve lineshape.
  2. Use the proper solvent and amount of solvent. 
    1. Deuterated solvents, in which 1H atoms are replaced with 2H atoms, are typically used in solution NMR for a variety of reasons. These reasons include deuterium lock, shimming, and providing an “invisible” background material that will not be observable in a 1H spectrum. 
    2. Common deuterated solvents are available in small quantities from ISU Chemistry Stores. Common solvents include chloroform-D, acetone-D6, benzene-D6, deuterium oxide (D2O), DMSO-D6, ethanol-D6, and methanol-D4. Other, less common deuterated solvents can be ordered through Chemistry Stores from Cambridge Isotope Laboratories or Sigma-Aldrich/Isotec
    3. Typical NMR samples contain 0.6-0.7 mL of deuterated solvent. Do not fill the NMR tube full of solvent. This will dilute your sample, waste solvent, and make shimming more difficult. 
  3. Consider preparing your sample in a secondary vial.
    1. You may want to use a small vial to dissolve the solid sample and transfering it to the NMR tube with a glass Pasteur pipette, especially if your sample is not easily solubilized. Once the sample is in the NMR tube, effective mixing can be difficult. This also gives you the opportunity to treat the sample with heat or vortexing in order to get complete dissolution.
    2. If the sample contains significant solids, it is best to filter any particulate from the sample before transferring to the tube. Solid particles will not show up in a solution NMR spectrum, and may interfere with proper shimming. 
  4. Use clean, unscratched NMR tubes and clean caps.
    1. High quality NMR tubes are available from Chemistry Stores or can be ordered in larger quantities directly from Wilmad Lab Glass or Norell NMR Tubes. Some suggested part numbers:
      1. Wilmad Part Number: 535-PP-7 (Called "Tubes, NMR, for 600 MHz 5mm" at Chem Stores, can be bought individually)
      2. FisherSci Part Number: K8972350000 (Called "Tubes, NMR, for 400 MHz 5mm" at Chem Stores, can be bought individually)
      3. Norell Part Number: 509-UP-7 (Not available through Chem Stores, pricing is for a 5 pack)
      4. Norell Part Number: 507-HP-7 (Not available through Chem Stores, pricing is for 5 pack)
      5. If you're looking at a specific tube you want to order in bulk, please don't hesitate to ask before ordering. 
    2. Particulate stuck to the inside of the NMR tube or scratches/defects in the tube can interfere with proper shimming. Use an NMR tube cleaner (either purchased or homemade) to clean tubes after using. 
    3. Disposable NMR tubes are not appropriate for use on the AVIII600, AVII600, or NEO400 NMR instruments available in the CIF – the low glass quality will not allow for adequate shimming.
    4. In general, we discourage the use of disposable NMR tubes, because the low tolerance of the outer diameter makes them slide around in the sample spinner and can result in more breakage inside the probe. However, the MR400 and DRX500 spectrometers do not seem to be as sensitive to lower quality NMR tubes. 
  5. Use an internal standard.
    1. Residual 1H in deuterated solvents can often be used for spectral calibration (calibration table). However, in situations where an exact chemical shift is desired, or there is not solvent available for reference (such as for 13C conducted in D2O or 31P), an additional internal standard must be used for chemical shift calibration.
    2. Internal standards such as TMS (for organic solvents) or DSS and TSP (for aqueous samples) will give you an exact NMR reference. For nuclei other than 13C or 1H, additional standards can be used such as phosphoric acid for 31P.
    3. Internal standards can be added directly to the sample if desired. In this situation, just a drop of TMS is often too much for one NMR tube. Add a drop of TMS to 5-10 mL of deuterated solvent that can be used for several samples. 
    4. Alternatively, if you are concerned about an internal standard reacting with the compound of interest, a capillary tube can be filled with an internal standard and placed in the NMR tube. This situation is not appropriate if you are using the internal standard for quantitation purposes. For quantitation, the internal standard must be added directly to the sample in order to achieve the same filling factor in the coil. 
    5. Indirect referencing can be used in lieu of an internal standard. 
  6. Label your sample.
    1. Permanent marker can be used to label the sample to ensure you do not mix up your NMR tube with another NMR user, or it can be retrieved at a later time. If you use a sticker or tape to label the tube, the sticker must be flush with the tube so it does not interfere with insert, eject or spinning inside the magnet. 
  7. Air sensitive samples.
    1. Some samples must be degassed to remove oxygen. Oxygen can be paramagnetic and broaden lineshape or interfere with T1 relaxation measurements.  
    2. For degassing air-sensitive samples, J-Young Tubes can be directly attached to a vacuum line.