Nuclear Magnetic Resonance Spectroscopy

Reference to this article: ConductScience, Nuclear Magnetic Resonance Spectroscopy (2019). doi.org/10.55157/CS20191121

Two molecules may have the same number and type of atoms, but their properties would change depending on how they are arranged (i.e., the bonds linking them and their orientation). As an example, Ethanol and Dimethyl ether, both have one oxygen, two carbon, and six hydrogen atoms, but the structures and properties of both these compounds are different. Ethanol exists as a liquid and Dimethyl ether, on the other hand, is a poisonous gas ^[1]. Thus, it is crucial for scientists to identify the exact structure of the compounds in order to understand their properties. Nuclear Magnetic Resonance (NMR) spectroscopy is a technique used to precisely identify the molecular structure of the compound ^[2]. NMR results for unknown compounds can be scanned with respect to a library of known compounds to reveal the identity ^[3].

What do the individual letters “NMR” mean?

1. Nuclear: NMR technique is concentrated around the properties of the nucleus of an atom. A closer examination of an atom reveals that it has a dense core that is made up of protons and neutrons. This core is called the nucleus of an atom. The number of protons in the nucleus is used to identify which element the atom is out of the 118 identified elements of the periodic table ^[4]
2. Magnetic: A nucleus having an odd number of protons creates a magnetic field. ¹H and ¹³C are commonly used in NMR since these have an odd number of protons. If we consider ¹H, it has only one proton and no neutron thereby possessing this property of spin [5]. When a sample of these protons is placed in a magnetic field (say of strength B₀), they will either align with (α(alpha) state) the magnetic field or against it (β(beta) state). The energy of the α spin state is lower than that of the β spin state^{[6, 7]}.
3. Resonance: The proton in the α spin state can be converted to the higher energy β spin state by applying external radiofrequency energy. If these nuclei fall from the β spin state to the α spin state, it also emits radiofrequency energy^[6]. The electrons (which surround a certain nucleus in the compound) for instance, would create a phenomenon known as diamagnetic shielding ^[7]. This simply means that the electrons would shield the nucleus from the effect of the magnetic field applied by the NMR machine. Due to this shielding, different amounts of radiofrequency energies would be required for different nuclei to change their spin state from α spin state to the β state. When all the nuclei are flipping between either state, they are said to be in resonance.

The energy difference ΔE between the two spin states is determined by the equation

ΔE= hV

h= Planks constant

V= frequency of the energy that we use on protons in α spin state.

We can further breakdown the equation as

ΔE= hV = h [γ/2π * B₀ ]

where γ = Gyromagnetic ratio of the nucleus in the study.

Let’s look at the terms associated with NMR

Resonant Frequency:  This is the frequency at which the phenomenon of resonance occurs in the protons of the sample. Using the procedure described earlier, we would generate an NMR spectrum which will consist of various peaks, representative of energy necessary to bring each of the nuclei in the compound, in resonance. Since all the nuclei will need different energies from the radio frequency light to shift to the β spin state, this will result in different peaks on the graph.

Chemical shift: The interpretation of the NMR data is crucial for understanding the structure of the molecule in question. The NMR spectrum data, which is in the form of peaks on a graph, depicts the position of the signal from the spinning protons. Protons behave differently under the applied magnetic field depending on whether they are in an aliphatic, aromatic, or aldehydic electronic environment (Figure 1). Chemical shift in NMR represents the resonant frequency which is plotted on the NMR spectrum graph with respect to a reference compound ^[7]. Chemical shift is denoted by δ value and is represented by a scale from 0 to 10. The unit of chemical shift is PPM (parts per million). A commonly used reference compound in NMR is TMS (tetramethylsilane), which has a chemical shift 0.

J coupling: This is also called spin-spin coupling. It is the interaction that takes place between hydrogen atoms in a given molecule. This coupling causes the splitting of lines in NMR spectrum. The coupling constant is denoted by letter J. The distance between two adjacent H atoms would influence the value of the J constant. The coupling constant increases as this distance decrease. Furthermore, the orientation of the H atoms also has an effect on the spectral split. The J constant will be more if the H atoms are in Cis orientation than in trans orientation ^[8].

The chemical shift, J coupling, and resonant frequency parameters are different for each proton in the molecule being investigated. Therefore, in order to nail down the exact structure of the molecule, we need to generate NMR spectrum with high resolution and sharp peaks.

What can we do to get NMR peaks at high resolution?

We can do that in two ways:

1. Shimming the magnet: The NMR instrument is capable of reaching a resolution capability within a few Hertz, and this is really an exceptional achievement. Let’s say NMR picks up a peak at 600,000,000 Hz and then picks up another peak at 600,000,009 Hz. This is a difference of just 9 Hz. This difference is in the 9^th decimal place and is very subtle. This is where the process of shimming comes into play. The sample is spun at around 20 revolutions per second so that it experiences the same magnetic field inhomogeneity throughout. The challenge is then to also maintain a homogenous magnetic field at the long axis of the sample tube (also called the Z-axis).  This is done by shim coils. These coils compensate for the differences in the applied magnetic field to create a uniform magnetic field which is experienced by all the protons in the sample ^[9]
2. Locking the magnetic field: Another problem with the NMR machine is that the magnetic field may slightly change over time and this phenomenon is called magnetic field drift. The instrument corrects this by locking on to the resonant frequency of deuterium in the solvent (e.g., deuterated chloroform). The instrument measures the deuterium frequency over and over again and sets it to a certain value (in Hertz). This phenomenon is called locking and it prevents loss of resolution in the NMR spectrum ^[10].

Deuterated solvents in NMR spectroscopy

The solvent has to be carefully chosen so that it does not contribute to the NMR spectrum. Deuterium (²H) is an isotope of hydrogen which has a neutron in the nucleus and does not have a spin, unlike ¹H.  The most common solvent used in NMR spectroscopy is deuterated chloroform ^[11].

Sample handling in NMR spectroscopy

The quality of the sample determines the quality of the NMR spectrum generated.

1. Usually, 5-25 mg of starting material is needed in the case of proton NMR.
2. The sample should be homogeneously suspended to get a sharp NMR spectrum.
3. NMR tube should be pre-washed with acetone to ensure cleanliness.
4. The sample is degassed to get rid of oxygen which has paramagnetic properties.
5. Test the solubility of the sample in the proper deuterated solvent.

Variations of NMR Spectroscopy

1. 1-Dimensional NMR spectroscopy: This method is a first step in the characterization of the structure of a molecule plots intensity vs frequency (which is the chemical shifts in ppm). The signal is acquired after excitation with radiofrequency waves.
2. 2-Dimensional NMR spectroscopy: This method is used to further map the coupling between H atoms. The intensity is plotted as a function of two frequencies rather than just one. The data is represented as contour plots (much like the topographical maps). In simple terms, a 2D NMR experiment involves a series of 1D NMR acquisitions ^[12].
3. Biomolecular NMR spectroscopy: This method refers to using the NMR technique in studying biological material in conditions which are close to those in vivo.
4. Solid-state NMR spectroscopy: It is now possible to observe drugs in action in membrane proteins with an advancement in the Biomolecular NMR spectroscopy technique. This is known as Solid-state NMR ^[13].

Instrumentation for NMR Spectroscopy

NMR spectrometer has the following components:

1. a) A powerful magnet with a homogeneous magnetic field: Its job is to align nuclear spins in the sample.
2. b) A sample holder.
3. c) Shims: These are coils that maintain a uniform magnetic field.
4. d) Locks: These are coils that transmit current and maintain a constant resonance frequency of deuterium in the solvent of the sample.
5. e) A transmitter to emit radiofrequency waves: The radiofrequency causes a flip in the nuclear spins.
6. f) A receiver and amplifier: The flip induced by radiofrequency is detected by the receiver and the signal is amplified for ease of visualization.
7. g) A computer: To analyze signals from the detector and convert it into an NMR spectrum.

How to operate a 1H NMR machine

1. The first step is sample preparation.
2. The sample (in NMR tube) is placed in a sample holder. The height of the sample should be adjusted by the depth gauge according to the manufacturer’s guidelines.
3. The sample tube is introduced in the magnet chamber.
4. The rest of the process is monitored by a software.
5. The solvent (which was used to prepare the sample) is selected in the software.
6. NMR machine is then primed to other parameters such as temperature, gain, force tuning, angle of pulse, etc.
7. If everything runs without any technical hiccups, we end up getting a NMR spectrum.
8. TMS peak at 0 ppm acts as a marker for calibrating the NMR spectra.
9. The number of unique proton environments could be identified by unique peaks in the spectrum.
10. The area of the peak corresponds to the number of protons.
11. The spin-spin coupling causes the peaks to split into sub-peaks.
12. The information from chemical shifts and the area under the peaks are then used to determine the proton environment.

Commonly used NMR machines and models

Decades of technological advances in NMR machines has led to the advent of modern machines which are fast and give robust data. There are several models of NMR machines to choose from depending on what the purpose of your analysis is. In academia, for example, the most common ones used for regular purposes are 300 MHz Bruker Avance, 400 MHz Bruker Neo and 500 MHz Agilent ProPulse.

Application of NMR Spectroscopy

NMR is used:

1. To determine the purity of a sample and its molecular structure ^[7].
2. To quantify the percentages of different isoforms of the molecules in the sample ^[14].
3. To measure reaction kinetics ^[15].
4. To analyze biofuels ^[16].
5. To determine edible oil composition, fat, and water content analysis in the food industry ^[17].
6. To do metabolomics and identify products in body fluids ^{[18, 19]}.

Advantages of NMR spectroscopy

1. It is a high precision technique.
2. It is reproducible.
3. The sample can be recovered fully and can be used for other applications.
4. Changes in metabolic reactions can be monitored over time ^[20].
5. The data acquisition and the downstream data analysis is fast.
6. No ionizing radiation is involved in the process, which limits the users and the samples to exposure to radioactivity.

Disadvantages and Limitations of NMR spectroscopy

1. NMR spectroscopy requires expensive equipment.
2. Determining the structures for higher molecular weight molecules is a problem.
3. NMR is less sensitive than Mass spectroscopy (MS) since it requires a sample amount in milligrams, whereas it is a picogram from MS ^[21].
4. Great care must be taken when using ferromagnetic materials in close proximity to NMR because of the strong magnet, as they could potentially become dangerous for the machine and user ^[22].
5. Ionic states of elements cannot be deciphered using NMR.
6. Hydrogen atoms in a molecule having similar resonant frequency could be hard to resolve.
7. Only nuclei having magnetic moments could be analyzed.

Conclusion

NMR spectroscopy is miles ahead from its 8-decade old counterpart for which it bagged the Nobel prize in 1943. The modern NMR machines are spearheading research in various disciplines such as structural biology, food industry, polymer science, etc.

References

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