High resolution and sensitivity are critical in nuclear magnetic resonance (NMR) spectroscopy. Both can be overdoubled when using heteronuclear decoupling. This enables the severance of overlapping signals for analyzing diluted components in reaction mixtures.
Advancements in the chemistry field directly relate to our profound comprehension of chemical reactions. This is best studied using NMR, which provides insights into molecules at the atomic level.
This article discusses two pivotal features of high-quality NMR spectra: The sensitivity or signal-to-noise ratio (S/N) and the spectral dispersion directly associated with NMR spectrum resolution.
It is vital to exploit both in NMR spectroscopy. While 2D NMR is frequently employed to enhance the resolution over 1D experiments, this can negatively affect the S/N and signal-to-artifact ratios.
As demonstrated in heteronuclear decoupling, resolution and sensitivity can sometimes be improved without drawbacks.
1H NMR Sensitivity
NMR spectroscopy may theoretically appear less sensitive than other frequently employed analytic approaches, but in practice, particularly when studying small organic molecule samples, the contrary is true.
The 1H nucleus is highly sensitive and commonly present in organic molecules. This enables even traces of compounds to be easily identified, irrespective of their substance class. NMR can reveal all substances containing protons, ensuring no such signals or substances are missed.
This can be difficult with other methods, which may only be sensitive to one substance class and are more invasive, potentially altering the sample during analysis.
An NMR signal must be substantially bigger than the noise level to investigate. A minimum requisite for an S/N level might be 3:1. In standard 1H NMR experiments of small organic molecules. However, the S/N ratio can vary from several hundred to thousands.
Heteronuclear and Homonuclear Decoupling
In NMR spectroscopy, adjacent nuclei communicate via chemical bonds. The spectrum observes this contact as “couplings” that divide spectral lines into doublets, triplets, and more sophisticated multiplets.
Homonuclear couplings can be seen between nuclei of the same kind (e.g., 1H-1H couplings) and heteronuclear couplings between different nuclei (e.g., 1H-13C).
While couplings contain invaluable data, they also reduce the signal intensity and the spectral dispersion. “Decoupling” is a series of radio frequency pulses applied during the free induction decay (FID) that essentially turns off the coupling between spins, causing clean and intense signals and spectra with enhanced resolution.
Heteronuclear decoupling works by irradiating one nucleus (e.g., 13C) while recording another (e.g., 1H). It can, therefore, be applied for the entire duration of FID.
Heteronuclear decoupling is simple to employ and can be advised for most uses. Alongside 1D spectroscopy, decoupling technology can produce spectra with extremely low artifact levels and is normally used on modern NMR spectrometers like the AvanceCore.
Homonuclear decoupling varies drastically from heteronuclear decoupling and is not discussed in detail in this article. In homonuclear decoupling, the same nucleus is irradiated and decoupled concurrently, causing a conflict of objectives:
- Optimal decoupling is acquired if a substantial proportion of the FID time is linked to the decoupling sequence.
- High S/N is only acquired if a substantial proportion of the FID time is linked to data acquisition.
Homonuclear decoupling, therefore, reduces the S/N ratio and can establish spectral artifacts.
S/N and the Carbon Spectrum
A frequently documented NMR spectrum is the 1D 13C carbon spectrum, which is less sensitive than the 1H spectrum. Proton decoupling during the FID acquisition can boost its sensitivity (Figure 1).
Figure 1. Aliphatic cutout of three signals from a 13C spectrum of a sample of 21 mg Ibuprofen in 0.6 ml DMSO-d. The first spectrum (A) was recorded without proton decoupling, using 1024 scans, while the second spectrum (B) was recorded with proton decoupling, using only 16 scans. Image Credit: Bruker BioSpin - NMR, EPR and Imaging
Proton decoupling decreases the number of spectral lines while retaining the integral, causing enhanced dispersion and S/N ratio. The spectrum (Figure 1B) reveals three lines—the number of 13C atoms in this spectral area.
Utilize the AvanceCore spectrometer to record a 13C spectrum with proton decoupling by following these steps:
- Read the parameter set C13CPD by entering “rpar C13CPD” in the TopSpin command line.
- Read the pulse power parameters by entering “getprosol”.
- Begin the experiment by entering “zg”.
- Upon acquisition completion, process the spectrum by entering “efp” then “apk”.
Proton decoupling is pivotal for obtaining clean carbon spectra with high S/N ratios, while dispersion is key in acquiring 1H spectra, as detailed below.
Dispersion in Experiments
The 13C spectrum usually has a bigger dispersion than the 1H spectrum, meaning signals in the 1H spectrum will more likely overlap. Due to the 1H experiment’s excellent sensitivity, dispersion often becomes the restrictive factor in practical applications (Figure 2).
Figure 2. Aliphatic region of the 1H spectrum of Ibuprofen. The first spectrum (A) provides an overview of the region, while the second spectrum (B) has the intensity scaled to the level of the 13C satellites. Image Credit: Bruker BioSpin - NMR, EPR and Imaging
Ibuprofen is a compelling example as it resembles many other small organic molecules. In daily laboratory procedures, NMR samples are frequently prepared utilizing 0.1–100 mg of the test substance, causing NMR spectra like the one shown in Figure 2.
Observing the aliphatic region overview (Figure 2A), the signals are clear and well-resolved. Smaller signals become noticeable when scaling the peak intensities. Figure 2B illustrates the spectrum at the intensity level of the 13C satellites (around 0.55 % of the central signal).
More small signals become visible at this intensity level. Symmetric signals involving intense signals are 13C satellites from the coupling between proton and carbon atoms. Those satellites are smaller because of the low natural profusion of 13C which is around 1.1 %.
Two 13C satellites for each central signal fill approximately two times the spectral range of the central signal. In the first estimate, they can reduce the spectral dispersion by a factor of 0.66.
Figure 2B shows that the 13C satellites could potentially overlay and conceal many other small signals, hindering the analysis of signals from residual organic solvents like ethyl acetate, acetonitrile, etc., and compounds similar to Ibuprofen (e.g., byproducts).
The example of Ibuprofen is how dispersion can limit the sensitivity of the 1H experiment. Heteronuclear decoupling can improve dispersion by removing the 13C satellites.
Heteronuclear Decoupling for Improved Dispersion in the 1H Experiment
As mentioned, many signals show 13C satellites that can present invaluable data concerning the molecules under investigation in carbon-containing molecules. Yet 13C satellites also drastically lower the spectral dispersion, which can be restrictive.
Heteronuclear decoupling can overcome these restrictions, removing the 13C satellites, lowering spectral overlay, and easily detecting signals from diluted impurities.
Detailing decoupled and non-decoupled spectra is a valuable investigational approach. Evaluating the resulting spectra assists in differentiating 13C satellites from other small signals, facilitating continued analysis from the decoupled spectrum with enhanced resolution, as shown below.
Figure 3. Aliphatic region of the 1H spectrum of Ibuprofen zoomed to the baseline. The first spectrum (A) is a conventional 1H spectrum recorded without heteronuclear decoupling. The second spectrum (B) was recorded with heteronuclear carbon decoupling, which causes the 13C satellites to vanish and increases the spectral dispersion. Image Credit: Bruker BioSpin - NMR, EPR and Imaging
Follow these steps to document a 1H spectrum with 13C decoupling on an AvanceCore spectrometer:
- Read the parameter set P_PROTON_IG by entering “rpar P_PROTON_IG” in the TopSpin command line.
- Create the shaped pulses for 13C decoupling by entering “wvm -a”.
- Read the pulse power parameters by typing “getprosol”.
- Initiate the experiment by entering “zg”.
- Upon acquisition completion, process the spectrum by entering “efp” then “apk”.
The explained process utilizes low-power adiabatic bilevel decoupling for carbon. Further reading: “Perspectives of adiabatic decoupling in liquids” (Kupce et al. JMR 318 (2020) 106799).
This decoupling technology employs a low power level to allow reasonably long acquisition times while generally preventing warming of the sample and NMR coil.
This makes collecting an FID lasting two seconds possible, producing a spectrum with NMR signals as precise as those in the standard 1H spectrum without decoupling. This is evident when comparing the small signal at 1.6 ppm (Figure 3).
In Figure 3B, the spectral line of that signal is as intense as the one in Figure 3A. Heteronuclear decoupling, therefore, improves the dispersion without reducing the S/N, making it an essential technique for routine 1H NMR spectra acquisition.
This information has been sourced, reviewed and adapted from materials provided by Bruker BioSpin - NMR, EPR and Imaging.
For more information on this source, please visit Bruker BioSpin - NMR, EPR and Imaging.