Beyond structural elucidation, introduction to qNMR – Part I

Over the last few years, more and more analytical and industrial laboratories have started employing quantitative nuclear magnetic resonance (qNMR) spectroscopy as a tool for content assignment (due to its superb structural elucidation abilities) and quantification of purity in a sample. This is due to the increase in regulations being imposed by governments onto the pharmaceutical and environmental sectors. It has been previously demonstrated that qNMR spectroscopy can give results with less than 1% uncertainty and possibly down to 0.1% if the right conditions are met.^[1,2] In this blog post I would like to introduce several topics that are relevant for the qNMR experiment.

NMR spectroscopy is inherently quantitative and non-destructive, which make it an ideal technique for purity studies. There are only two major requirements that need to be met for analysis by NMR spectroscopy:^[3]

1. The sample is fully soluble in a solvent (usually deuterated).
2. The compound is NMR active (i.e. the compound contains NMR active nuclei).

For the most part, NMR spectroscopy is usually conducted using ¹H as the active nucleus, however, other NMR active nuclei such as ¹³C, ¹⁹F, and ³¹P can be used as well utilizing the same methodology.^[3]

To be able to quantify a compound, a calibrant is needed and it can either be internal or external. With an internal calibrant, the sample and the calibrant are weighed out and co-dissolved to form a single solution. With an external calibrant, the compound and the calibrant are separated. This set-up requires an NMR tube with the sample and a co-axial NMR tube insert with the calibrant. While both internal and external referencing have their merits, internal calibrants are more precise and result in lower levels of uncertainty.^[4]

After deciding which referencing method to use, there are a few things that need to be considered when conducting qNMR experiments:

1. Compatibility

The chosen calibrant and sample should be inert to one another as well as the solvent. Another thing one should keep in mind is the overlap in signals. To be able to correctly quantify the sample in question, having clear and distinct signals between the calibrant and the sample is a must. It is recommended to use a reference sample that has a few signals (the fewer the better) and is soluble in various solvents.^[4]

2. Precision

To ensure the numbers obtained are as accurate as possible, weighing should be conducted in a metrological manner. By eliminating as much uncertainty as you can, you will ensure that more accurate values are obtained. Try to avoid materials that are volatile and hygroscopic as error will be added due to weighing. Reference compounds should also be of high purity to decrease uncertainty. Many reference samples suitable for qNMR experiments are available on the market.^[5]

3. Instrument Settings

When conducting qNMR experiments, it is crucial to have a sufficiently long interscan delay ensuring all the nuclei have relaxed before the next pulse is applied. This is directly related to the T₁ relaxation time of the sample and the calibrant. Since T₁ relaxation times can vary depending on the mixture and concentration, it is recommended to determine the T₁ relaxation times for all signals. Once the longest T₁ relaxation time is determined, the delay is set to the longest T₁ relaxation time multiplied by 5-7 times.^[4]

After all these steps have been completed and an acceptable spectrum has been obtained, the purity of the sample can be determined using the following equation:

Where:
• P = Purity
• I = Integral Area
• N = Number of Protons
• M = Molar mass
• m = mass of compound used
• S = sample
• R = reference

To further reduce the uncertainty of the measurement, the qNMR experiment should be performed in multiplicates.

I hope that this blog post has given you a good introduction into qNMR experiments. Stay tuned for more in-depth blogs pertaining to topics of qNMR.

References

^[1]Weber, M.; Hellriegel, C.; Rueck, A.; Wuethrich, J.; Jenks, P.; Obkircher, M. Analytical and Bioanalytical Chemistry 2014, 407, 3115-3123.
^[2]Giraudeau, P. Magnetic Resonance in Chemistry 2016, 55, 61-69.
^[3]Chauthe, S.; Sharma, R.; Aqil, F.; Gupta, R.; Singh, I. Phytochemical Analysis 2012, 23, 689-696.
^[4]Quantitative NMR https://www.sigmaaldrich.com/analytical-chromatography/analytical-standards/application-area-technique/organiccrm.html (accessed Feb 26, 2019).
^[5[Bernstein, M.; Fraga, S.; Sánchez, E. What is qNMR and why is it important? - Mestrelab Resources http://resources.mestrelab.com/what-is-qnmr/ (accessed Feb 26, 2019).