Nuclear Magnetic Resonance (NMR) Spectroscopy has had a massive impact on the structural determination of organic molecules and biomolecules, becoming a standard tool for organic chemists, biochemists, and structural biologists.
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NMR spectroscopy is a powerful technique for determining the structure of organic molecules – and biomolecules – in solution, with samples typically dissolved in deuterated solvents. Solid-state NMR is also possible, although it does not provide a level of information as high as in a solution.
Spectrometers can be very expensive since they require big superconducting magnets that generate strong, homogeneous magnetic fields. Skilled personnel is also required to operate and maintain the instruments. Nevertheless, in the last few years smaller and more affordable benchtop spectrometers have also become available.
The real strength of NMR spectroscopy lies in the fact that signals in NMR spectra can be identified and assigned to the respective atoms in the molecule, with a level of structural information so high that is rivaled only by X-ray crystallography.
The principles behind the “resonance” phenomenon
NMR spectroscopy relies on the magnetic property of the atomic nucleus. Some atoms, with an odd number of protons or neutrons (i.e. 1H, 13C, 11B, 15N, 31P), behave as if they are positively charged spheres spinning on an axis. Such spinning generates a very small magnetic field.
If placed in a strong external magnetic field, these nuclei will align their magnetic fields with the external one (just like the needle of a compass) at a particular frequency, in a phenomenon called “resonance”. The resonant frequency is characteristic for every nucleus and it also depends on the chemical environment.
Modern NMR spectrometers generate pulsed radio frequencies that excite at once all the nuclei in a sample. A coil then detects the radiation emitted when the nuclei return to their original state, and the instrument converts via Fourier-transform the signal — called Free Induction Decay (FID) — from the time domain to the frequency domain, resulting in the final spectrum.
Depending on the type of nucleus several types of experiments and spectra are possible. However, the most commonly used technique for the structural elucidation of organic molecules is 13C NMR and, more importantly, 1H NMR, which can produce a spectrum in a few seconds.
Who are the neighbors? Chemical shift and spin-spin splitting
The resonant frequency can vary slightly depending on the position of the atom in the molecule and the surrounding atoms. Bonding electrons generate their own magnetic field, influencing the overall external field. This subtle variation affects the position of the signals in the spectrum, known as “chemical shift”, which is measured in parts per million (ppm) and indicated by the Greek letter delta (δ).
The chemical shift can thus provide information on the position of an atom within the molecule and/or the presence of neighboring electronegative atoms or unsaturated groups (double and triple bonds). In 1H NMR, a hydrogen next to an oxygen has a characteristic chemical shift (4.0-4.5 ppm), and so does a hydrogen next to a carbonyl group (2.0-3.0 ppm) or on an aromatic ring (6.0-8.0 ppm).
Another important source of information can be extracted from the so-called “spin-spin splitting”, which is observed when two protons Ha and Hb with different chemical shifts are on two adjacent carbons (HaC-CHb). The own magnetic field of Hb can align with or against the external magnetic field.
These two situations change the magnetic field experienced by Ha and therefore affect its resonant frequency. The result is that the signal of Ha (and Hb respectively) appears as two peaks of the same intensity (a doublet). The difference in Hertz between the two peaks is called the coupling constant (J).
The analysis of the coupling constants can give information on the conformation of the molecule. Hydrogens in equatorial positions on a carbohydrate backbone will have a different J than those in axial position. Likewise, depending on the J value it is possible to determine whether two hydrogens are in a cis or trans relationship.
When there are multiple neighboring hydrogens, more complex splitting occurs. In general, with n neighboring nuclei, the Ha resonance peak will split into n+1 peaks, with an intensity ratio determined by Pascal’s triangle.
Getting the “full picture” – 13C NMR and bidimensional NMR
1H NMR is an extremely informative technique and it works well for simple molecules. However, for more complex cases, 1H NMR spectra may not be sufficient to determine the molecule’s structure. Therefore, 13C NMR is often performed alongside. The principles of this technique are the same as 1H NMR, although there are some particular differences.
Firstly, the abundance of 13C is very low (1.1% of total carbon) therefore experiments require a little longer. Since coupling with other 13C is negligible due to the very low abundance, there is no splitting, and signals in the spectra appear as single lines. Finally, while typical 1H spectra range between 0-12 ppm, the 13C spectral window is larger (0-220 ppm).
Aliphatic carbons produce signals around 0-30 ppm, while carbons next to heteroatoms (N, O, or halogens) have chemical shifts between 60 and 80 ppm. Very characteristics are the signals of carbonyl groups, such as esters, amides, and carboxylic acids (160-180 ppm) and aldehydes and ketones (190-200 ppm).
More advanced bidimensional (2D) techniques complete the set of experiments for structural identification. 2D NMR spectra present two chemical shifts scales (horizontal and vertical). When two nuclei are close to each other, signals appear as spots at the intersection of the two chemical shifts.
Among the various techniques available, 2D COSY (correlation spectroscopy) reports correlations between 1H and 1H and is used to identify adjacent protons. 2D HSQC (heteronuclear single quantum correlation spectroscopy) instead provides information on 1H and 13C that are directly bonded. These two types of experiments allow the identification of all protons and carbons within a molecule.
How to overcome the limitations of NMR
- Blümich, B. (2016). Introduction to compact NMR: A review of methods. TrAC Trends in Analytical Chemistry, 83, 2-11.10.1016/j.trac.2015.12.012
- Mlynarik, V. (2017). Introduction to nuclear magnetic resonance. Anal Biochem, 529, 4-9.10.1016/j.ab.2016.05.006
- Wishart, D. S. N., Alex M. (1998). Protein chemical shift analysis: A practical guide. Biochemistry and Cell Biology, 76, 153-163.https://doi.org/10.1139/o98-038
- Lindon, J. C. 2005. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY TECHNIQUES Multidimensional Proton, Elsevier.https://doi.org/10.1016/B0-12-369397-7/00413-1