Issue 9: Let’s try doing quantitative NMR (2)

Practical pointers for sample preparation

Difference between the internal standard method and external standard method for sample preparation

It has been explained that both an internal standard method and an external standard method can be used to perform quantitative NMR.
For the sample preparation, the difference between the internal standard method and the external standard method is whether the reference standard for qNMR is contained within the same sample solution as the analyte; but, for both methods it is important to carefully weigh out the sample and select the reference standard for qNMR.

1) Internal Standard Method

  • Selection of a reference standard for qNMRl: The important points for the selection are a high purity, and a signal that does not overlap the signals from the analyte.
    If possible, it is best to choose a material with traceability, such as a National Standard Substance.
  • Weighing-out: A sample of about 0.6 ml is sufficient for one NMR measurement sample.
    Therefore, the amounts weighed out for both the reference standard for qNMR and the analyte are on the order of mg. Since errors in weighing have a large effect on the purity calculations, it is necessary to adequately consider the specifications of the balance that will be used, as well as the work environment.
  • Solution preparation: After precisely weighing out the reference standard for qNMR and the analyte, add a suitable solvent and dissolve the materials completely.
    Since the molar ratio is known, the amount of solvent does not need to be precise.

2) External Standard Method

  • Selection of a reference standard for qNMR: Since the measurements are performed with a sample solution that is separate from the analyte, it is not necessary to worry about overlapping the signals from the analyte.
    However, it is best to choose a material with a purity that is precisely known, because this becomes the reference for the quantification.
  • Weighing-out: In the same way as the internal standard method, the amounts weighed out are on the order of mg; so, it is necessary to adequately consider the specifications of the balance, as well as the work environment.
  • Solution preparation: The solution of the reference standard for qNMR which is standard solution will be the reference for the quantification, so, it is necessary to prepare precise molar concentrations. Consider the accuracy and calibration of the measuring flasks and pipettes, etc. that are used when performing this work.

For quantitative NMR, errors in the sample preparation are a major error factor for the entire measurement. It will depend on the precision that is required, but protocols should be established.
For more information about proper weighing out techniques, consider attending a lecture or seminar on balances.