qNMR: top tips for optimised sample prep

NMR spectra provide various types of information, usually focused on the identification of the compounds contained in a sample; however, it’s well-known that quantitative data can also be obtained

There are in fact two types of quantitative analysis in NMR:

  • relative quantitation: the measurement of the ratio of a target component contained within the sample being measured
  • absolute quantitation: the measurement of the actual amount of the target component contained within the sample being measured.

Absolute quantitation has been attracting attention recently because it has some unique characteristics and advantages compared with other analytical methods. Chromatography detects the characteristics of the molecule itself, such as the absorbance, refractive index and fluorescence.

This means that, for quantitative analysis, we need a standard substance that is identical to the component being quantified to have a reference or yardstick to measure the target molecule.

NMR detects the nuclei that form molecules. For instance, consider the hydrogen atoms in a molecule. Nearly all organic compounds contain hydrogen. As long as there is a proton in the molecule we are trying to quantify, we don’t need our reference yardstick to be a standard substance that’s identical to the target component; it may be any suitable compound. This is a significant advantage and is one of the reasons why qNMR has been gaining traction.

Key benefits of qNMR

NMR can be used for almost any organic compound that can be made into a solution. It can be applied to agrochemicals, pharmaceuticals, food additives, standard substances and other classes of chemical compounds.

Substances that are difficult to analyse quantitatively using chromatography when there is no standard substance available — such as new compounds or unknown materials — can be quantitatively analysed using qNMR. It’s even possible to use one reference substance for the quantitative analysis of many measurement targets.

There is no need to create calibration curves for qNMR and no conditioning is required to perform a measurement. For a low molecular weight compound, several milligrams are required to make a measurement … but each one can be done in about 10–15 minutes. If an appropriate protocol is followed, qNMR can be used to perform Si traceable purity assessments, ensuring the reliability of the results.

Analytical procedure

qNMR analysis begins with sample preparation, which is extremely important. There are two key considerations for sample preparation:

  • the selection of the reference material for quantification with NMR
  • the accuracy of the weighing.

For the selection of the reference material for quantification, there are several factors to consider. First, the signal from the reference material must not overlap the NMR signal of the analyte.

Secondly, the purity should be high and the known purity value must be accurate. Thirdly, it should be easy to weigh the sample accurately and precisely. This means that it should not sublime or absorb moisture as a solid.

Reference materials

There have been reports of many different materials used as reference materials for qNMR. The five reference materials listed below satisfy the requirements of qNMR analysis; they are all commercially available and relatively easy to handle:

  • 3,5-bis(trifluoromethyl)benzoic acid
  • maleic acid
  • dimethyl sulfone
  • 1,4-bis(trimethylsilyl)benzene-d4 (1,4-BTMSB-d4)
  • 3-(trimethylsilyl-1-propanesulfonic acid-d6 sodium salt (DSS-d6).

It is important, however, to choose a reference material and solvent that are suitable for the specific application.

Weighing quantities accurately

With qNMR, 1 mL is a sufficient volume for analysis — so the amount of sample used is a few milligrams. Therefore, the specification of the balance to be used should be considered and, if possible, there should be a fixed protocol for maintaining and using the balance.

At JEOL, we have a simple procedure as an example of sample preparation. We use ultramicro balances to enable the precise weighing of 1 mg quantities. Other items used are as follows:

  • standard substance
  • analysed material
  • sample bottle
  • deuterated solvent
  • aluminium boats for weighing
  • tweezers
  • Pasteur pipette
  • spatula.

First, tare the balance with the aluminium boat, then weigh the sample. Place the aluminium boat with the sample into the sample bottle. Repeat this weighing process for the standard substance and the analyte. Depending on the situation, it may not be appropriate to leave the tared aluminium boats in the bottle.

If so, only place the weighed contents into the bottle. After weighing out both materials, add a suitable amount of deuterated solvent and dissolve the solid materials completely. After everything is dissolved, transfer the solution to the NMR sample tube as soon as possible.

It’s also a good idea to initially prepare samples in triplicate. Not because the NMR measurement requires this step; rather, it tests the weighing skills of the technician as well as the entire chain of sample preparation procedures. As the operator gains skill — as verified by highly consistent results — this step can be relaxed, depending on the required level of accuracy and precision for a given analysis.

Summary

Nuclear magnetic resonance is an incredibly versatile analytical technique and its ability to generate quantitative measurements is increasing its popularity throughout many sectors. Owing to the nature of the analysis, when it comes to sample preparation for qNMR, there are two key considerations: the purity of the reference material used for the quantification and the weighed values.

Ensuring both are optimal will give you a great chance of realising fast, reliable and efficient analyses of an endless array of substances.

NB: This article will appear in the March 2019 issue of Manufacturing Chemist. A recent digital edition is available online.

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