NMR Spectroscopy Working Principle

To understand the various complex physical structures around us, we need to understand their molecular structures first. Nuclear magnetic resonance spectroscopy, which is commonly known as NMR spectroscopy is an analytical technique that helps researchers or scientists to analyse the molecular structure, and study the biological, chemical or physical properties of the sample by observing its nuclear spin interaction. The NMR spectroscopy technique was first demonstrated by the two physicists Felix Bloch and Edward Mills Purcell in 1946, and for this achievement, they were jointly rewarded with the Noble Prize in Physics in 1952. The first commercial NMR spectrometer was manufactured in 1950, and until now, the NMR spectrometer is used as an essential tool by the researcher and chemists. The earlier spectrometers that were based on the permanent magnets used to be large, bulky, and expensive as compared to the modern spectrometers such as benchtop spectrometers and tabletop spectrometers. Some other methods such as X-ray diffraction and electron microscopy are also used to study the molecular structure but NMR spectroscopy is preferred over these methods because NMR spectroscopy is a non-destructive method and it also requires less sample preparation. Here in this article, we’ll understand the various aspects of NMR spectroscopy.

Basis of NMR

The nuclear magnetic resonance technique involves the absorption of electromagnetic radiations in the rf region (4-900MHz) by the nucleus of the atom. It is considered one of the most powerful methods to detect the number of Protons (hydrogens) in the compound. The theory of NMR spectroscopy is based on the spin quantum number. The spin quantum number (I) tells about the intrinsic angular momentum of the electron, and it is related to the mass number (number of protons and neutrons) and the atomic number (number of protons) of the atom. It is observed that the elements that have either an odd atomic number or an odd mass number show nuclear spin. For example, the H atom has an odd atomic mass and odd atomic number, and upon calculating, it has the spin equals 1/2, and it shows the NMR spectra, while the C atom has even atomic mass and even atomic number, and it has spin equals to zero, and it does not show the NMR spectra.

When the magnetic field is applied to the sample, the total number of possible orientations can be calculated by the formula, 2I+1, where “I” is the spin quantum number.

Now, as the spin quantum number of the Hydrogen is 1/2

The possible number of orientations will be,

2I+1, i.e., 2(1/2) + 1 =2

Hence, there are two possible orientations of the H atom, i.e., +1/2 and -1/2.

Principle of NMR Spectroscopy

Every element has an electrically charged nucleus, when the spins of the neutrons and the protons do not get paired in the nuclei, then the net spin of the nucleus will generate a magnetic dipole along the axis of the spin. The net magnitude of this dipole is referred to as the nuclear magnetic moment. The internal structure of the molecules is determined by the symmetry and distribution of the magnetic dipoles. The principle behind NMR spectroscopy is based on the spin of the nucleus that can generate the magnetic field. When there is no external magnetic field, the spins of the nucleus are arranged in a random manner; however, when an external magnetic field say Bo is applied to the sample, the spin present in the nucleus gets aligned in either with or against the direction of the applied magnetic field. The application of the external magnetic field results in the generation of energy differences (ΔE) between the excited state and the ground state. The transfer of the energy takes place at the wavelength which is equivalent to the radio frequency. When the spin comes back to the initial ground state it emits the absorbed radiofrequency, and this emitted radiofrequency gives the NMR signal of the corresponding nucleus. The emitted radiofrequency is proportional to the magnitude of the applied magnetic field.

Mathematically,

v = (γB0)/2π

Where v is the emitted radiofrequency, B0 is the applied magnetic field, and γ is the magnetogyric ratio.

The magnetogyric ratio is the ratio of the nuclear magnetic moment to the angular moment.

Components of NMR Spectrometer

1. Magnets

Magnets are a crucial part of NMR spectroscopy. The efficiency of the NMR spectrometer is largely dependent upon the size, strength and homogeneity of the applied magnetic field. The main types of magnets used in the spectrometer are given below,

1. Superconducting Magnets

Superconducting magnets are constructed from the coils made of superconducting wire; these magnets require to be cooled down during the operations. Superconducting materials can generate an intense magnetic field because, in their superconducting state, the wires surrounding them offer almost zero electrical resistance, hence the superconducting magnets are much more conductive than the average magnets. The major drawback of superconducting magnets is the loss of energy in the form of heat.

2. Permanent Magnets

These magnets always possess the magnetic field around them as they are made from the materials in which the atoms are permanently aligned to give a constant magnetic field. These magnets are less powerful than the superconducting magnets, but they are more preferred in the NMR spectroscopy, especially in the benchtop spectrometer for convenience purposes.

3. Resistive Magnets

Resistive magnets provide a much more dc magnetic field than the other two types of magnets. These magnets are usually copper sheets that consist of a number of cooling holes in them.

2. Sample Holder

The sample holder used in the NMR spectroscopy should be transparent to radio frequencies, chemically inert and durable. It is generally a glass or pyrex tube, which is about 6-7 inches long and has a diameter of about 1/8 inches.

3. Radiofrequency Coil

The radiofrequency coils are used to transmit the magnetic field into the desired region; they induce the magnetic field when the electric current flows through them. They also detect the resulting NMR signal. Generally, the same coil is used at the transmission and the receiving end, but the use of a different coil at both ends is preferred.

4. Sweep Generator

A sweep generator is an electronic device that generates a waveform with a constant amplitude and a linearly varying frequency. The main role of the sweep generator in the NMR spectroscopy is that it allows the equal magnitude magnetic field to pass through the sample.

5. Radio Frequency Transmitter

The transmitter gives the radiofrequency pulses of the required power, frequency, and shape. It consists of the following components.

• Frequency generator: It is responsible for generating the frequency.

• Waveform generator:  It shapes the pulse as per the requirement.

• Gate: It switches the transmission of the pulses on and off at the desired time.

• Power Amplifies: It boosts the power of radiofrequency to the required value according to the Fourier transform NMR.

6. Radio Frequency Reciever

The important function of the radio frequency receiver is to convert the analogue NMR signal to the digital format. The suitable radio frequency receivers having the effective resolution and conversion bandwidth are preferred in the NMR spectroscopy.

7. Amplifier

The purpose of the amplifier is to amplify the obtained NMR signal, it improves the visibility of the NMR signals by adjusting the frequencies by using the upper pass and lower pass filters. Usually, the SRS 560 amplifier is used in NMR spectroscopy.

8. Recorder/Oscilloscope

It is a kind of computer that records and analyse the data received from the radio frequency receiver and display it on the screen.

NMR Spectroscopy Solvent

The preparation of the accurate sample in NMR spectroscopy is a crucial step. The three major types of samples used in NMR spectroscopy are liquid sample, solid sample, and gas sample. The liquid samples are analysed by putting around 0.5 ml of the liquid in the NMR sample tube. The solid samples are mixed with the suitable solvent for the analysis, usually, the 2-3 mg of sample is mixed with 0.5 ml of the solvent. The gas samples are analysed by concentrated them with the help of a suitable solvent. The prepared sample in the NMR spectroscopy is filtered to remove iron particles (if any) and is also degassed to remove the oxygen because both the iron and oxygen are paramagnetic, which may result in the undesired line broadening of the NMR spectra.

NMR spectroscopy provides better signals if the sample is in the liquid form; hence all the samples are usually converted into the liquid form to get the better NMR spectra. The type of solvent should be carefully selected, and it should follow the following requirements.

• The solvent should be chemically inert towards both the sample holder and the sample.

• It should have no or minimal absorption spectra so that the NMR signal of only the sample can be clearly obtained.

• The original sample should be recovered from the solvent upon distillation, in case the sample may require further testing.

• The types of solvents that are typically used in the NMR spectroscopy are Cs2 (carbon disulphide), CCl4 (carbon tetrachloride), CDCl3  (Deuteriochlorform), D2O (Deuterium oxide), and {C}_{6}{D}_{6} (Hexa deuteriobenzene)

The majority of solvents such as hydrocarbon consists of protons that are NMR active. Hence, to reduce the NMR signals of the hydrogen atoms of the solvent, the deuterated solvent is used. In deuterated solvents, 99% and above of the protons are replaced with the H2, i.e., deuterium.

Working of NMR Spectroscopy

The nuclei sample is placed in the magnetic field, and it is excited into the nuclear magnetic resonance by using radio waves. This results in the generation of the NMR signal. This NMR signal is detected with the help of the radio frequency receiver. The intermolecular magnetic field surrounding an atom varies the resonance frequency, which provides the structural details of the molecules. As every molecule has a different chemical environment, and have slightly different resonance frequencies, this results in the unique NMR spectra of each molecule.

NMR Spectrum

It is the plot of the intensity of the NMR signal with respect to the magnetic field with TMS (Tetramethylsilane) as the reference. NMR spectroscopy provides the unique and well-resolved spectra of the different functional groups.

Interpretation of the 1H-NMR Spectra

• The number of signals in the NMR spectra represents the presence of different kinds of protons in the sample.

• The position of the NMR signal represents the chemical shift and the magnetic environment of the proton.

• The splitting of the NMR signal (spin-spin coupling) represents the number of nearby nuclei (protons).

• The intensity of the NMR signal is directly proportional to the number of protons in the sample.

Chemical Shift

The chemical shift is the difference between the resonance frequency of the observed proton or hydrogen and the hydrogens of the tetramethylsilane (TMS). In NMR spectroscopy, TMS (at δ = 0 ppm) is considered as the reference compound.

Mathematically, the chemical shift is expressed as,

Chemical Shift= [(frequency of the signal – frequency of the reference)/ spectrometer frequency] × {10}^{6}

As we have discussed above that the external magnetic field is applied to the sample, and the frequency at which the nuclei achieve resonance is then measured. Nuclear magnetic resonance is a phenomenon in which the nuclei of the atoms absorbs a certain amount of energy, and re-emit it in the form of electromagnetic radiation. The absorbed energy is if a certain resonance frequency, which is based on the strength of the applied magnetic field, and the magnetic properties of the different isotopes of the atom. The electrons orbiting around the nucleus act as small magnetic dipoles, which may oppose the external magnetic field. Let’s understand the shielding and deshielding effect of protons.

Shielding of Proton

If the density of the electron around the nucleus is high, the opposition of the external magnetic field by the electrons will also be high, hence the greater will be the shielding of the protons as protons will experience the less external magnetic field. As a result, a lower frequency will be needed to achieve the resonance and the NMR signals are upfield (towards the right) in the spectrum.

Deshielding of Proton

If the density of the electrons around the nucleus is low, the opposition to the external magnetic field becomes less, hence the protons will experience more magnetic field, and the protons are said to be de-shield. As the protons are experiencing a high magnetic field, hence high frequency will be needed to achieve the resonance, and the downfield shift (towards the left) in the chemical shift is observed.

Factors affecting the Chemical Shift

1. Electronegative Groups

When an electronegative group such as bromine, iodine, and chlorine is attached to the carbon-hydrogen bonding system, they pulled the electron density towards themselves, which reduces the density of electrons around the protons, and de-shielding is observed, which results in an increase in the chemical shift.

2. Magnetic anisotropy of π-systems

The term anisotropy means non-uniform, the magnetic anisotropy implies the non-uniform magnetic field. When the magnetic field is applied to the π-electron systems such as alkenes, alkynes, carbonyl, aromatics, the π system induces its own magnetic field, which results in the magnetic anisotropy, i.e., the shielding and deshielding of the protons. Benzene is the simplest organic chemical compound that shows this effect.

3. Hydrogen bonding

The values of the chemical shifts get also changes due to the protons, which are involved in the hydrogen bonding. The more hydrogen bonding implies the more will be the deshielding of the proton, hence the chemical shift value shifts to higher.

Spin-Spin Coupling

The spin-spin coupling is referred to the interaction between the spins of the nearby nuclei, which may result in the splitting of the NMR spectrum. This phenomenon is also known as spin-spin splitting. The splitting of the NMR signal is related to the number of the equivalent hydrogen atom in the neighbouring nuclei. Here are some rules that are being applied to spin-spin coupling.

The spin-spin coupling is not observed in the chemically equivalent protons, and it is only found in the non-equivalent protons. Consider the example as seen in the image below,

Here, the He couples with the proton Hb as they are non-equivalent; however, Ha and Hb do not couple as they are equivalent.

Generally, the protons present on the adjacent carbon atoms will couple, and the protons that are far away by four or more bonds do not couple.

Advantages of NMR Spectroscopy

• It is a non-destructive method, and the examined sample can be obtained back into its original form after distillation or other processes, which can be used for further examination.

• Commonly available techniques for molecular structure analysis are usually applicable for the solid or the liquid phase sample; however, the NMR technique can not only be used for the solid or the gases phase samples but can also be used for the gaseous phase.

• The NMR spectra reveal a large number of details about the structure of the molecule, such as chemical shift, spin-spin splitting, relative peak size, type of functional group attached, and other detailed information about the chemical environment of the molecule.

• NMR spectroscopy can be used for the analysis of both the simple and the complex molecules as the size of the molecule is not a problem in the case of NMR spectroscopy.

• The NMR spectra also provide information about the physical properties of the sample such as phase changes, diffusion, conformational exchange, and solubility.

• The traditional methods of molecular analysis such as chromatography, or colour reagents tests are replaced by NMR spectroscopy due to the convenience of the NMR spectroscopy process.

Applications of NMR Spectroscopy

• The most basic application of NRM spectroscopy is molecular structure analysis.

• NMR spectroscopy is used to check the presence of impurities in the given sample.

• As NMR spectroscopy can provide real-time data analysis of the chemical processes, so it is used by the researchers to examine the photosynthesis process in the algae and the plants to understand the survival rate of the particular crop in the different climatic conditions.

• It is used in the pharmaceutical sector for drug analysis and quality control.

• It is used in the analysis of nucleic acids, cells and biofluids.

• The applications of NMR spectroscopy is also seen in the development of the batteries. NMR benchtop spectrometer is used to get the molecular details of the potential of the battery for storing the energy.
• The NMR spectroscopy is used in the MRI techniques.

• It’s not only used to determine the structure of the molecule but also to compare the obtained NMR spectra of the unknown compound with the spectral libraries.

• NMR spectroscopy is considered one of the best methods for analysing the structure of organic compounds. It is used in the food sectors to determine the amino acid profile, lipid fractions, structure of the protein, and organic acids.

Disadvantages of NMR spectroscopy

• The NMR spectroscopy requires comparatively a larger amount of sample to give the NMR signal than the other conventional methods.

• The NMR spectroscopy of solids needs some special devices (magic-angle spinning machine) to give well-resolved NMR spectra.

• NMR spectroscopy also gives the NMR spectra of a large number of unwanted impurities present in the nuclei sample.