The knowledge of the assay of a substance is crucial information and of interest in many applications. For example in pharmaceutical or chemical laboratories, in the production of reference materials, or to follow the conversion of reagents in reactors. In the past decades quantitative nuclear magnetic resonance spectroscopy (qNMR) has been established as a standard method for assay determination, as it takes advantage of the fact that NMR provides a linear signal response. This results in the NMR signal intensity being proportional to the number of nuclei, which allows for an easy one-point calibration against a known standard. The type of qNMR method, which we present in this blog post, utilizes an internal standard as a reference. The other method, using an external standard, will be presented in an upcoming blog post.
For optimum performance of the internal standard qNMR method it is required that the reference material has a known assay, the integrated signals are not overlapping, and that analyte and standard are sufficiently soluble in the desired solvent. One example of this is the determination of the assay of diclofop-methyl with dimethyl sulfone as internal standard. Diclofop-methyl is a post-emergence herbicide used, for example, for wild oats and wild millets. As it can be absorbed by the soil it is important to have a reference material with known assay with which one can determine the contamination of soil samples.
Figure 1: Structures and molecular weights of diclofop-methyl (left) and dimethyl sulfone (right).
The sample was provided to us by HPC Standards GmbH, a manufacturer and distributor of high-purity analytical standards for residue analysis. For the analysis, a sample of 34.9 mg diclofop-methyl, 4.9 mg dimethyl sulfone and 500 µL methanol-d4 was prepared, homogenized, and transferred into a standard 5 mm NMR tube.
Figure 2 shows the acquired proton spectrum of this sample including the integrals of dimethyl sulfone (calibrated to 600 for 6 protons) and diclofop-methyl, which were used for the assay determination. The spectrum was recorded with carbon decoupling to avoid any errors in the calculations due to overlapping carbon satellites from neighbouring signals. As can be seen in the spectrum, the signal for dimethyl sulfone does not overlap with other signals and can be integrated separately.
Figure 2: The 1H{13C}-NMR spectrum of dimethyl sulfone and diclofop-methyl in methanol-d4 measured on a Spinsolve 80 MHz Carbon spectrometer (32 scans, 30 s repetition time, 16 min total measurement time).
To demonstrate the good quality of the spectra and the reproducibility achievable with the Spinsolve NMR spectrometer the experiment was repeated 63 times. Figure 3 shows all measurements superimposed, where all processing parameters were kept the same for all measurements. It can be seen in figure 3 that no changes in phase, line shape or line width were observed over a course of about 20 hours.
Figure 3: Superimposed plot of the 63 measurements and zoom of the superimposed integrated signals for diclofop-methyl.
For all 63 measurements the assay of diclofop-methyl was calculated according to the following equation with W as the weight of the substances, M as the molar masses, Int as the integral values and N as the number of protons integrated for each signal respectively.
The excellent robustness and reproducibility of the Spinsolve NMR spectrometers are confirmed by the results of these repetitive measurements as shown in figure 4. The mean value of the assay is 99.77 % with a standard deviation of 0.06 % and in full accordance with the expected value.
Figure 4: The results of the assay determination of diclofop-methyl for the 63 repetitive measurements.