NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy rightfully comes to our minds when we think about an ideal and the most powerful spectroscopic technique that provides comprehensive structural elucidation. It is a single sufficient technique that generates a complete characterization profile of a chemical substance with utmost accuracy.

 Considering the immense importance and the multifold applications of NMR spectroscopy, we have compiled for you everything you need to know about NMR spectroscopy in this single article. So, let’s start reading!

What is NMR spectroscopy     

Nuclear magnetic resonance (NMR) spectroscopy is an atomic absorption spectroscopic technique. It is based on the intrinsic property of a nucleus known as nuclear magnetic spin. In the absence of any external magnetic field (B₀), all the spin states of a given nucleus are of equal energy. In the presence of an applied B₀, some nuclei align with the field while some align against the field. As a result, their equivalent spin states split into two different energy levels. The nuclei then change their spin state by absorbing radiowaves of frequency equal to the energy gap between the two energy levels. The absorbed wavelength gives information about the chemical environment and thus the structure of the sample molecules.

Historical perspective of NMR spectroscopy

NMR was introduced back in 1946 by the combined effort of three researchers from Harvard University (Edward M. Purcell, Pound, and Torrey) and a trio (Felix Bloch, Hansen, and Packard) from Stanford University. Since then, there is no looking back in the popularity and advancements of NMR spectroscopy. Purcell and Bloch were jointly awarded a Nobel prize in Physics in the year 1952. However, the first NMR spectrometer became available for commercial use in the late 1950s.

What are radiowaves    

 Radiowaves are low-frequency, long-wavelength electromagnetic radiations that lie at the far-right end of the electromagnetic spectrum. Their frequency ranges from 200 MHz to 1 GHz while the wavelength of radiowaves lies between 30 cm to several thousand meters (m). The frequency of radiowaves is ideally compatible with the low energy requirements of NMR spectroscopy.

The chemistry behind NMR spectroscopy

Atomic nuclei consist of nucleons i.e., positively charged protons and neutral particles called neutrons. Thus, nuclei are charged species, the electric charges are continuously circulating. This means not only do the negatively charged electrons circulate a nucleus but the nucleus itself is also circulating on a fixed axis. Circulating electric charges also gives rise to a magnetic field. Thus, the nucleons possess an intrinsic magnetic spin, represented by a nuclear spin quantum number (I). The magnetic spin is also known as the intrinsic angular momentum of the nucleus. Two nucleons of opposite spins can pair up.

If a nucleus consists of an even number of protons and neutrons, all the nucleons will be spin-paired, having I=0. For example, in the ¹²C-nucleus, there are 6 protons and 6 neutrons, all spin paired so I=0. In contrast to that, if the nucleus consists of an odd number of protons, neutrons, or both then I ≠0. These nuclei behave as tiny magnets under an applied magnetic field. Each nucleus possesses 2I+1 spin states. Such nuclei and their chemical and electronic environments can be studied through NMR spectroscopy. ¹³C and ¹H are two of the most useful nuclei for  NMR spectroscopic analysis.

All the chemical molecules having atomic nuclei with a non-zero spin possess a specific magnetic dipole moment value (μ). μ is related to I by the equation given below.

μ=ץ I…………. Equation 1

The quantity ץ is known as the magnetogyric ratio. It is the ratio between the magnetic dipole moment of a molecule and the angular momentum or spin of the nuclei present in that molecule. It is measured in MHz/T, T= Tesla. Equation 1 can also be written as equation 2.

ץ1/2= π /h (μ/I)…… Equation 2

where 2π/h is reduced Planck’s constant. It can also be denoted by a symbol ћ. So,  equation 2 becomes

 ץ = ћ (μ/I)……. Equation 3

In addition to spinning about a particular axis, the nuclei also undergo rotational motion. When an external magnetic field is applied, the rotational axis is never aligned exactly parallel or anti-parallel to this field. This periodic wobbling is called precession which keeps the nuclei stable at all energy levels. The frequency associated with this precession is called Larmor frequency (ω) and it is related to the magnetogyric ratio as shown in equation 4. 

ω=ץB₀………. Equation 4

4 basic components of an NMR spectrometer

The NMR spectrometer consists of the following main components:

• Permanent magnet
• Sample tube
• Radio frequency oscillator
• Radio frequency detector

The permanent magnet is usually a superconducting material that requires a very low temperature (around 4 K) to function properly. Therefore, it is situated in a cylindrical chamber called a probe. The probe consists of a cooling system made up of an inner jacket filled with liquid helium which is further encapsulated in an outer jacket filled with liquid nitrogen and multiple thermal isolation layers. 

Now, let’s see how these components work together to carry out a successful NMR spectroscopic analysis.

What is the working principle of NMR spectroscopy

Step I: Sample preparation              

• The sample is prepared in a solvent that does not contain any NMR active nuclei.
• Popular solvent choices for NMR spectroscopy are deuterium oxide/ heavy water (D₂O),  tetrachloromethane (CCl₄), and deuterochloroform (CDCl₃).
• About 20 mg sample is dissolved in 0.5 mL of this solvent. 
• A drop of tetramethyl silane (TMS) is added as the reference standard.  TMS i.e., (CH₃)₄Si is a volatile, inert chemical compound that produces a strong singlet peak on the NMR spectrum. This peak is used as a reference peak. It does not interfere with any other peak obtained on the NMR spectrum.
• The sample solution so prepared is then filled into a glass tube (8.5 cm long and 0.3 cm in diameter). The glass tube is held fixed between the poles of the permanent magnet using saddle coils.  
• The coils are attached on one side to the radio frequency oscillator while on the other side to the radio frequency detector.
•  The magnet creates an external magnetic field (60-100 MHz) around the sample tube while the oscillator supplies radiowaves for the nuclear spins to flip.

Step II: Interaction of matter with applied B₀

• Under the influence of an external magnetic field, the magnetically active nuclei present in the sample change their spin orientation.
• Some nuclei adopt a preferred orientation and align with B₀ acquiring a low energy level. While the remaining nuclei adopt a less favored orientation and achieve a high energy state by aligning against B₀.
• This splitting of degenerate energy levels into two different spin orientations and energy states is called Zeeman splitting.

• A specific energy difference (∆ E) exists between the two spin orientations. Due to the small energy difference (usually 0.2 J/mol), the low-energy spin state is generally more populated. For instance, only 1 out of 30,000 nuclei will be spinning at a higher energy than the ground state while the other 29,999 spins at the lower energy level. But that 1 nucleus makes all the difference.
• The nuclei spinning at a lower energy level need to absorb energy equal to ∆ E in order to flip to the higher energy spin state. The process is called magnetic resonance and the energy absorbed by spinning nuclei is known as resonance energy.
• This energy is thus supplied by the radiowave frequency according to equation 5.

Resonance energy = ∆E =hf …….. Equation 5

where h = Planck’s constant (6.626 x 10^-34 m² kg/s)

• ∆ E can also be related to the external magnetic field (B₀) and to the magnetogyric ratio (ץ) as shown in equation 6.

∆ E= ћ ץ B₀ …… Equation 6      

• Thus, the strength of the applied magnetic field also influences the ∆E value.

Step III: Energy absorption followed by relaxation

• Spinning nuclei absorb radio frequency equal to ∆E from the radio frequency oscillator and flip their spin state from a low energy level to a higher energy level.
• Energy absorption is followed by a relaxation step. Spin relaxation occurs as the spinning nuclei return to their base position by emitting energy.
• The emission occurs at the same frequency that was absorbed for flipping the spin state in the first instance.

• The emitted energy is received by the radio frequency detector as an electrical signal called free induction decay (FID).
• This signal is then amplified and recorded by the recorder. The spectrometer functioning in this manner is known as a continuous wave NMR spectrometer.
• If a Fourier transformation mathematical operation is performed on the raw signal to generate a characteristic peak on the NMR spectrum, such a spectrometer is then known as the FT-NMR spectrometer just like an FTIR spectrophotometer.

Step IV: Collecting the NMR spectrum

• The NMR spectrum is obtained as a plot of energy absorbed against chemical shift measured in ppm.
• The TMS peak is denoted as the reference peak and assigned an arbitrary value of 0.
• All the peaks are measured relative to this reference peak. The chemical shift value increases from right to left on the NMR spectrum.
• The peaks on the NMR spectrum shift depending upon the strength of the applied magnetic field as well as the chemical environment of the sample. It is unlike UV-Vis spectroscopy or IR spectroscopy where absorption peaks are located at some specific wavelengths.

What are the nuclear screening effect and chemical shift

The electrons present around a spinning nucleus lead to a screening effect. They can influence and change the effect of the applied magnetic field experienced by the targeted nuclei. The electrons form a current loop centered around the positively charged nucleus, the secondary magnetic field produced by this current loop opposes the effect of B₀. Greater the number of electrons surrounding a nucleus, the stronger their screening or shielding effect. This leads to a lower ∆ E value and thus lower resonance frequency that needs to be absorbed to change the nuclear spin state which creates a new peak on the NMR spectrum.

The screening constant (α) is a small quantity measured in ppm. It is inversely proportional to ∆ E. It is related to the resonance frequency as shown in equation 7.

……Equation 7

The screening effect is maximum for an individual atom surrounded by freely circulating electrons. The free circulation gets hindered by chemical bonding in the presence of multiple positive centers in a molecule. So, the screening effect gets reduced and resonance frequency increases. This shows that not only the electron density of an individual atom, but the electronic environment of the surrounding atoms also affect the resonance frequency of a spinning nucleus. The presence of highly electronegative elements such as oxygen (O) and chlorine (Cl) in the vicinity of target nuclei leads to an opposite of the shielding effect i.e., electronic deshielding.

Strongly shielded nuclei give a peak closer to the reference signal on the NMR spectrum while heavily deshielded nuclei give a peak farthest away from the reference signal. Greater the deshielding, the greater the leftwards shift of the NMR peak from the reference signal. This is called chemical shift (δ) as it depends on the chemical environment of the spinning nuclei in a complex sample mixture. On the other hand, the intensity of an NMR peak tells us the concentration of the targeted nuclei in the sample.

What is NMR spectroscopy used for

• NMR spectroscopy is used in research and development for the characterization of newly synthesized polymers, pharmaceutical drugs, and other important chemical compounds.
• It provides information about different atoms present in a molecule and the chemical bonding present between these atoms. In this way, it reveals the structure as well as the chemical composition of a sample.
• In addition to the structure of a polymer, NMR spectroscopy also provides information about its molecular weight, monomer ratio, phase changes, tacticity, etc.
• NMR spectroscopy forms the basis of magnetic resonance imaging (MRI). Radiowaves penetrate the soft tissues of the human body and produce images that are then used to study the structure and function of these tissues.

• Biochemical pathways can be studied by performing NMR spectroscopy on biological molecules (amino acids, proteins, and carbohydrates) containing NMR active ¹H, ¹³C,  ¹⁵N, ¹⁹F,  ³¹P etc., nuclei.

Find other applications of analytical spectroscopic techniques here.

Advantages of NMR spectroscopy

• NMR spectroscopy is a highly sensitive and the most accurate structure determination technique.
• It is a non-destructive, non-invasive spectroscopic technique.
• It can be used to study a wide variety of samples in all solid, liquid, and gaseous states.
• NMR spectroscopy gives both qualitative as well as quantitative information.

There are two main types of NMR spectroscopy namely ¹³C-NMR spectroscopy and proton NMR spectroscopy, more about these two types in our subsequent articles in the spectroscopic series.

References

1. Greenbaum, N. and R. Ghose (2010). Nuclear Magnetic Resonance (NMR) Spectroscopy: Structure Determination of Proteins and Nucleic Acids. eLS.

2.Mishra, R. K., J. Cherusseri, A. Bishnoi and S. Thomas (2017). Chapter 13 – Nuclear Magnetic Resonance Spectroscopy. Spectroscopic Methods for Nanomaterials Characterization. S. Thomas, R. Thomas, A. K. Zachariah, and R. K. Mishra, Elsevier: 369-415.

3. Singh, M. K. and A. Singh (2022). Chapter 14 – Nuclear magnetic resonance spectroscopy. Characterization of Polymers and Fibres. M. K. Singh and A. Singh, Woodhead Publishing: 321-339.

4. Webb, A. (2012). “Increasing the Sensitivity of Magnetic Resonance Spectroscopy and Imaging.” Analytical Chemistry 84(1): 9-16.