Let’s try doing quantitative NMR In the previous seven issues we have introduced an overview, including the background of quantitative NMR. From this issue we will present practical content. <Preparation> Before actually performing quantitative NMR measurements, there are some preparations required beforehand. These are (1) selecting a quantitative measurement method (internal standard method or external standard method), (2) determining the sample preparation conditions (selection of solvent and reference standard for qNMR, the stability of the sample solution), and (3) confirming that signals suitable for quantification appear on the 1 H-NMR spectrum. Here, we will briefly introduce the selection of the quantitative measurement method. For the internal standard method the reference standard for qNMR is prepared in the same solution with the analyte. For the external standard method the reference standard for qNMR and analytes are prepared separately. A point to consider when deciding which method to choose is whether accuracy or recovery of the sample has a higher priority. If you want to emphasize accuracy, the internal standard method is recommended. On the other hand, if a precious material that is a single isolated substance is being analyzed, the external standard method is probably better. ( Fig2. Internal standard and external standard method) <Analytical Scheme of Quantitative NMR> Quantitative NMR with either the internal standard method or the external standard method has three steps: Sample preparation → NMR measurement → Analysis. (Fig1. qNMR analysis flowchart) 1) Internal standard method (Fig.3 Procedure for the internal standard method) Sample Preparation: Confirm the precision of the balance to be used, precisely weigh the reference standard for qNMR and the analyte. It is recommended to create 3 samples from the same lot. NMR measurement: Perform 1H measurement under optimized conditions for quantification. One of the parameters to be noted is the pulse repetition time setting. Set a sufficiently long repetition time based on T1 (longitudinal relaxation time). We recommend that you perform three measurements on each qNMR sample. Analysis: When processing the FID, some processing can be done automatically, but it is recommended that you check by visual inspection.

2) External standard method (Fig.4 Procedure for the external standard method) Sample Preparation: Confirm the accuracy of the balance to be used, precisely weigh the reference standard for qNMR and the analyte separately. In this case, it is important to know the precise molar concentration of the reference standard for quantification. NMR measurement: Measurement is performed under quantitative conditions, but calibration such as temperature and pulse width measurement is necessary to compare the spectrum of the reference solution and the spectrum of the analytical sample solution. Analysis: As with the internal standard method, visual inspection after processing is necessary. When calculating, depending on the measurement parameters of each qNMR sample, the calculations must include the calibration factors.

Flowchart for quantitative measurement using NMR

Consider experiment conditions

Sample preparation

NMR measurement

Processing /Analysis/ Confirmation

1. Assignment of spectrum for analyte 2. Stability of the analyzed sample 3. Selection of reference material, solvents, concentration

Quantitative NMR

Fig.1 qNMR analysis flowchart

Internal Standard Method vs. External Standard Method

Internal standard method:

0 1 2 3 4 5 6 7 8 9 10 11 ppm

One sample measured Comparison within the spectrum

Analyzed sample

Reference material

NMR spectrum from an internal standard method （image)

External standard method: example, PULCON

Solution of the analyzed sample

Reference material solution

0 1 2 3 4 5 6 7 8 9 10 11 ppm

0 1 2 3 4 5 6 7 8 9 10 11 ppm

2 samples measured (Also 360°pulse measurement)

Comparison between spectra

NMR spectrum of the analyte (image)

NMR spectrum of the quantitative reference material (image)

Fig.2 Internal standard and external standard method

Preparation of the balance Measure the minimum weight of the balance.

Preparation of sample for quantitative NMR Weigh out the reference material and the analyte using a micro- or ultramicro-balance. Completely dissolve both the reference material and the sample in the selected solvent.

As a basic procedure, make 3 samples for each lot.

Processing The acquired data is processed and the integrated values of the target signals are extracted.

Analysis The integrated values and weighted values are loaded into the calculation program and the

purity and concentration are calculated.

Confirmation Check the data and results using various software programs.

Quantitative NMR measurement Set the sample solution and perform quantitative measurements.

As a basic procedure, make measurements 3 times for each of the 3 samples, non-consecutively. (total of 9 data acquisitions)

qNMR

Sample preparation

NMR measurement

Processing/Analysis/ Confirmation

Fig.3 Procedure for the internal standard method

Preparation of the balance Measure the minimum weight of the balance.

Preparation of sample for quantitative NMR Separately weigh out the reference material and the analyte using a micro- or ultramicro-balance. Completely dissolve the reference material in a solvent to make the reference

solution. Completely dissolve the sample in a solvent to make the sample solution.

Calibration Set the reference solution and specify the temperature. Then, pulse width setting and

measurement using the qNMR conditions is performed. If necessary, create a calibration curve.

Processing The acquired data is processed and the integrated values of the target signals are extracted.

Analysis The integrated values and weighted values are loaded into the calculation program and the

purity and concentration are calculated.

Confirmation Check the data and results using various software programs.

Quantitative NMR measurement Set the sample solution and specify the temperature. Then, pulse width setting and

measurement using the qNMR conditions is performed.

qNMR

Sample preparation

NMR measurement

Processing/Analysis/ Confirmation

Fig.4 Procedure for the external standard method