Beyond Structure Elucidation - Introduction to qNMR Part II - Calibrants

In my previous blog post, I introduced several concepts that are relevant to the qNMR experiment. In this blog post, I will talk about how to select a suitable calibrant as well as the difference between using an internal and external calibrant.

Internal vs. External Calibrants

When conducting qNMR experiments, one of the first things that needs to be considered is how the calibrant is employed to quantitate your sample. There are advantages and disadvantages to each technique, and depending on your particular system, one method might be preferable over the others. The two different methods are:

1. Internal Calibrants

Internal calibrants are the most widely used referencing method for qNMR experiments. This method allows for the most precise and accurate results when compared to the other methods available.^[1] It involves co-dissolving both the internal calibrant and the sample of interest into a deuterated solvent to form a single homogenous solution. It is crucial that the mass of both the calibrant and the sample are measured metrologically to minimize the error in the final results. Based on the masses of the materials and the absolute areas of their respective signals, the amount of analyte present can be determined. Although this method allows for high accuracy and precision, there are a few drawbacks to using an internal calibrant. The first drawback is that the masses of both the calibrant and analyte must be weighed out accurately for each solution. This step consumes time and effort as well as the use of relatively expensive referencing materials. Another factor that needs to be considered is the method development required. Lots of time and energy needs to be invested to find a suitable combination of calibrant, analyte, and solvent for which no signals overlap, the compounds are inert to each other, and their stability is not compromised. I will address this problem in more detail later in this blog post.

2. External Calibrants

When looking at external calibrants, it was found that the values obtained were not as precise and accurate as using an internal calibrant however the technique allows you to overcome some of the drawbacks associated with using internal calibrants.^[1] The major difference between using an internal calibrant or an external calibrant would be the separation of the calibrant and the analyte into different solutions. Since the sample and the calibrant are separated and not mixed together, it is possible to use the same standard multiple times and obtain accurate results. This reduces the amount of calibrants used as well as time since compatibility issues are easier to solve and the calibrant only needs to be weighed out once. There are 2 different variations of using an external calibrant.

i. Co-axial Insert

The first variation of an external calibrant is with the use of a co-axial insert.^[1] The reference sample is present in the co-axial insert while the sample solution is present in the outer NMR tube. Unfortunately, the signal from the calibrant still appears in the spectrum with the analyte and creates a potential for peak overlap.

FIGURE 1. AN ILLUSTRATION OF A CO-AXIAL WITH ITS CAP (STRIPED) AND NMR TUBE ADAPTOR CAP (BLACK) IS SHOWN IN PANEL A. PANEL B REPRESENTS THE CO-AXIAL INSERT AND NMR TUBE FULLY ASSEMBLED.[2]

ii. NMR Spectrometer Calibration

This variation of external calibration erases the drawback seen in the first variation. In this method the absolute integral of the calibrant is used to calibrate the instrument’s response under specific conditions.^[1] The absolute integral is obtained from the NMR spectrum of the calibrant only. Based on this set calibration, the absolute integrals of the analyte are determined and thus the mass and/or purity can be calculated. One of the benefits of using this method is that compatibility between the calibrant and the sample is no longer an issue. Since the materials are separated, there will be no unwanted reactions or peak overlap between the calibrant and the analyte. Another advantage is that this single calibration can be used for multiple samples for long periods of time as long as the calibration conditions are met.^[1] This method also grants you the ability to save time. Since most calibration materials are small and symmetric, it usually means they have longer spin-lattice delay times than those of the analyte and require a longer acquisition time (delay times will be talked about in more detail at a later date). Since the analyte and the calibrant are now separate, the use of an external calibrant will allow for more rapid data collection since samples of the analyte can be run at shorter acquisition times than that of the calibrant and analyte combined. However, for this method to be used correctly, the stringent conditions in which the NMR spectrometer is calibrated must be met and deviation from these conditions could result in more errors in the measurement. This is because this variation relies on the assumption that peak areas are consistent between different spectra as long as they are recorded under exactly the same conditions.

It should be noted that using external calibrants can result in a higher degree of error. While internal calibrants rely on only mass, external calibrants rely on concentration so extra error is added due to volumetric measurements during sample preparation.

Electronic Signal Calibrant

Although internal and external calibrants are the main methods used to quantify your NMR sample, a newer method exists. This method is referred to as Electronic Reference To access In-vivo Concentration (ERETIC).^[1] Rather than spiking the sample with a specific amount of calibrant, ERETIC introduces a new resonance using a signal generated from another channel in the NMR spectrometer. There are many advantages to using ERETIC. If employed properly, ERETIC can present both accurate and precise values when compared to the expected value of the sample. Since ERETIC generates an electronic signal, it is possible to adjust the intensity of this signal as well as its chemical shift in the spectrum, allowing for the quantification of virtually any sample. ERETIC shows great promise, however, only a few literature studies have utilized this new technique.^[3]

Selecting a Calibrant for the Perfect Combination

When selecting a qNMR calibrant, there are a few things that should be kept in mind in order to achieve accurate and precise results.

1. The calibrant should be of high purity. By using high purity calibrants, less overlap will occur due to impurities.

2. The calibrant should be non-hygroscopic and non-volatile. Compounds that are hygroscopic or volatile are hard to weigh out and as a result, increases error when trying to determine mass.

3. The calibrant should be free of water. Having any residual water in the calibrant will affect the baseline of the spectrum.

4. The calibrant should be small and symmetric. This allows the calibrant to have minimal signals and thus reduces the chances of overlapping signals.

5. The calibrant should be soluble in multiple deuterated solvents. This allows for a wider range of potential combinations.

There are traceable certified calibrants available on the market for ¹H qNMR experiments.^{[4] 19}F and ³¹P qNMR reference materials are also available but there aren’t as many options available.

Once a suitable calibrant is found, it is important to ensure that the solvent, the sample, and the calibrant are compatible with one another. When I talk about compatibility, I am referring to their stability and how inert the calibrant, the sample, and the solvent are towards one another. To check their compatibility, it is recommended that you run NMR experiments at time 0 and repeat these experiments throughout an extended period to ensure no unexpected reactivity occurs.^[4] General knowledge of reactivity between functional groups will be quite helpful for this step. When checking for compatibility, one should also make sure that the signals of interest are distinct and have no overlaps with other signals. It is quite common to have to try multiple systems before finding the desired setup.

Hopefully this blog post has given you some insight on how to choose an appropriate calibrant as well as the differences between different kind of calibrants.

References
^[1]Cullen, C.; Ray, G.; Szabo, C. Magnetic Resonance in Chemistry 2013, 51, 705-713.
^[2]Henderson, T. Analytical Chemistry 2002, 74, 191-198.
^[3]Lumsden, M. Quantitative NMR without Internal Standards https://cdn.dal.ca/content/dam/dalhousie/pdf/Diff/nmr3/NMR%20Experiments/eretic.pdf (accessed Apr 10, 2019).
^[4]Quantitative NMR https://www.sigmaaldrich.com/analytical-chromatography/analytical-standards/application-area-technique/organiccrm.html (accessed Feb 26, 2019).