Mass Spectrometry & Spectroscopy
Optimising measurement conditions for accurate qNMR analysis Ronald Crouch, NMR Applications Consultant, JEOL USA, Inc and Takako Suematsu NMR Application Chemist, JEOL Resonance Inc
NMR spectra provide various types of information, usually focused on the identifi cation of the compounds contained in a sample. However, it is well-known that quantitative information can also be obtained. There are in fact 2 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: 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 over other analytical methods. Chromatography detects the characteristics of the molecule itself, like the absorbance, refractive index, and fl uorescence. This means that for quantitative analysis, we need a standard substance that is identical to the component that is being quantifi ed in order to have a reference or yardstick for measuring the target molecule.
NMR detects the nuclei that form molecules. Consider 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 is identical to the target component; it may be any suitable compound. This is a signifi cant advantage and is one of the reasons that qNMR has been attracting attention.
Key benefi ts 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 diffi cult to analyse quantitatively using chromatography where there is no standard substance available, such as new compounds, or unknown materials, can be quantitatively analysed using qNMR. It’s 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 for performing a measurement. For a low molecular weight compound, several milligrams are required to make a measurement, but each measurement can be completed in about 10- 15 mins. If an appropriate protocol is followed, qNMR can be used to perform SI traceable purity assessments, therefore the reliability of the results can be assured.
Figure 2: NMR spectrum of ethyl crotonate acquired using measurement conditions optimised for quantifi cation
NMR measurement
Measurement conditions for routine proton NMR are not suitable for quantitative analysis.
Figure 1 shows an NMR spectrum of ethyl crotonate acquired with ordinary, routine conditions, meaning the default measurement conditions on JEOL NMR instruments. They allow us to collect proton spectra quickly as required at many NMR laboratories. Figure 2 was acquired using measurement conditions optimised for quantifi cation.
If we compare the peak areas, in Figure 1, from the left, the values are 0.92, 0.93, 1.96, 3.0 and 2.97. For a structural analysis, this can be interpreted as 1 to 1 to 2 to 3 to 3, but you can see that there is an error in the integral values of as much as 8%. This is unacceptable in high-precision qNMR analysis.
In comparison, in Figure 2, you can see that the area values and the proton counts match. The error is around 1%. This demonstrates the importance of using quantitative conditions when performing measurements for quantitative analysis.
But what are quantitative conditions? Figure 1: NMR spectrum of ethyl crotonate acquired with ordinary, routine conditions
Figure 3 shows a comparison of the typical parameters for routine conditions and for quantitative conditions. The routine conditions are the default settings of the JEOL instrument for proton measurement. The quantitative conditions are based on the conditions specifi ed in the Japanese Pharmacopoeia. The parameter differences can be very broad, but there are 6 specifi c parameters that should be considered.
INTERNATIONAL LABMATE - APRIL 2019
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