NMR Spectroscopy in Structural Analysis of Organic Compounds

Nuclear magnetic resonance (NMR) spectroscopy is utilized extensively in molecular identification, in particular that of organic compounds within the field of organic chemistry, the pharmaceutical industry, and the life sciences. This article will discuss the basics of NMR spectroscopy, along with some of its variations and their specialized application.

Image Credit: Anwarul Kabir Photo/Shutterstock.com

Image Credit: Anwarul Kabir Photo/Shutterstock.com

Understanding NMR Spectroscopy

Atomic nuclei containing an odd number of protons and neutrons, such as 1H, 13C, 19F, and 31P, exhibit magnetic properties. When a magnetic field is applied to them, they may align with it in the low-energy state or against it in the high-energy state.

Within NMR spectroscopy, the sample is exposed to an increasingly strong magnetic field. At particular frequencies, energy is absorbed as the constituent magnetic atoms within the sample switch to the high-energy state.

The specific frequency at which this occurs is dependent on the chemical and structural environment surrounding the magnetic atom; a 13C atom bonded with three other carbon atoms will express a differing chemical shift to one bonded with one, for example, and this parameter is usually reported in Hz as frequency shift relative to tetramethylsilane, a molecule containing four equivalent carbons, the chemical shift of which is set as zero.

The Power of NMR in Organic Chemistry

NMR was discovered by Isidor Isaac Rabi, who was credited with a Nobel prize for his work in 1944. Since then, NMR spectroscopy has become the gold standard in the identification of small organic compounds as well as larger and more complex structures such as proteins, often in combination with other structural characterization techniques such as X-ray crystallography. Numerous forms of specialized NMR spectroscopy with specially engineered radiofrequency emitters and antenna assembly (probes) are employed in modern analysis, often suited particularly to the detection of specific elements. 

As briefly discussed, the specific resonance frequency of the atom is influenced by its chemical environment, in particular, the presence of adjacent electron-withdrawing or donating groups. Where two atoms, or protons in the case of 1H NMR spectroscopy, are magnetically inequivalent, i.e., one possessing a +1/2 spin state and the other -1/2, then a phenomenon known as spin-spin coupling occurs, causing a splitting of the relevant peak on the NMR spectrum.

This phenomenon can be used to more deeply characterize the molecule under investigation than only resonance peaks associated with the respective atom would, as it can be used to infer the spatial arrangement of the molecule, indicating the number of hydrogen atoms bonded to each carbon in a chain, for example.

NMR Spectroscopy Techniques

One-dimensional NMR spectroscopy is the standard method and is sufficient to distinguish various functional groups of small molecules. Larger molecules or complex mixtures may possess multiple instances of the same functional group in slightly differing chemical environments adjacent to differing functional groups, for example. Thus, it can be difficult to separate the peaks and interpret the generated spectra.

2D NMR spectra are plotted on two axes instead of one and thus offer differentiation between otherwise overlapping peaks. There are multiple sub-types of both 1D and 2D NMR, which differ in their application of the external magnetic field. For example, COSY (homonuclear COrrelation SpectrocopY) is a 2D NMR method that acts to decouple atoms from one another and prevent peak splitting, aiding molecular characterization when combined with information obtained from said splitting.

Another broadly employed sub-type of NMR is NOESY (Nuclear Overhauser Effect SpectroscopY), wherein resonant atoms are able to couple through close spatial positioning rather than only through bonds. This is useful in the analysis of large molecules with variable conformation, such as proteins, where the folding and bending of the molecule can be appreciated through intra-molecular interactions.

Since NMR spectroscopy is sensitive to 13C and, in particular, 1H, solvents without these atoms present must be used to dissolve the sample under investigation, as they would be present in such number as to occlude any resonance associated with the sample completely. To get around this problem, deuterated solvents are used in NMR spectroscopy, which, instead of containing 1H possesses only deuterated hydrogen (2H), which is not magnetically sensitive. 

Applications in Real-world Scenarios

NMR spectroscopy is considered the gold standard technique in the determination of small organic molecule structures. Combined with techniques such as X-ray crystallography, it is used extensively in the fine characterization of proteins and other large biomolecules. All industrial and research fields involving molecular synthesis, characterization, or analysis tend to utilize NMR spectroscopy extensively unless another more specific method is called for, and owing to the wide variety of NMR sub-types, it is useful in the characterization of a wide variety of compounds.

Advancements and Innovations

Numerous advancements in NMR equipment, data handling and interpretation, and fundamental methodology have been made to NMR spectroscopy in recent decades, which have allowed much broader analysis of a wider range of classes of substances with increasing levels of structural complexity.

For example, solid-state NMR is used to characterize the atomic arrangement of solids, thus avoiding many of the aforementioned issues with compatible solvents from the beginning. One potential advantage of solid-state NMR over solution-state NMR is that anisotropic spin interactions are better appreciated, which are otherwise averaged out by free rotation. Anisotropic interactions can be avoided if desired, however, by utilizing magic angle spinning, wherein the sample is spun at the “magic angle,” around 54.7°, relative to the X-ray emitter and detector.

This type of analytical setup is typically utilized in the analysis of tissue samples by NMR spectroscopy and is generally utilized in high-resolution NMR spectroscopy, which allows detailed spatial resolution during the analysis of complex substances.

For example, the spatial distribution of biomolecules and pharmaceuticals can be determined in vivo animal and human tissues, allowing drug interactions and the formation of metabolites to be observed in real-time. NMR spectroscopy is also increasingly deployed by lab-on-a-chip devices, which operate with sample volumes of only a few microliters and may be conveniently deployed in front-line diagnostics and testing applications.

Further Reading

Last Updated: Nov 7, 2023

Michael Greenwood

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Michael Greenwood

Michael graduated from the University of Salford with a Ph.D. in Biochemistry in 2023, and has keen research interests towards nanotechnology and its application to biological systems. Michael has written on a wide range of science communication and news topics within the life sciences and related fields since 2019, and engages extensively with current developments in journal publications.  


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