Nuclear magnetic resonance spectroscopy/NMR: An introduction, principle with its instrumentation.

NMR (Nuclear Magnetic Resonance)

What is NMR spectroscopy?

What-is-NMR-spectroscopy
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NMR (Nuclear Magnetic Resonance) spectroscopy is the measurement and interpretation of the radiofrequency which induces the transitions in the nuclei by absorbing the radiofrequency waves and the spectra are known as the nuclear magnetic resonance spectra.

The atoms which contain the nuclei with the property are called the spin. The number of spins of the active spin nucleus is denoted by the spin quantum number and it is the property of the elements which are containing the odd mass or odd atomic number.

Introduction of NMR-

 The study of spin change at the nuclear level when radiofrequency energy is absorbed in the presence of a magnetic field is known as NMR spectroscopy.

When a proton (hydrogen) is studied, then it is called Proton Magnetic Resonance (PMR). When other nuclei like 13C, 19F, 35C1, etc., are studied, then It is called NMR. Generally, in practice, the study of Hydrogen (Proton) itself is called as NMR Spectra.

Nuclei with the odd mass number only give NMR spectra. the example I H, 13C, 19F 35Cl, etc., because they have asymmetrical charge distribution. Other nuclei like 12C 160, 32S 14N, 2H, etc., do not give NMR spectra because of symmetrical charge distribution, and their spin quantum number is an integral value.

The spin quantum numbers of the nuclei are in the following table-

ElementSpin quantum number (I)Number of spin states
1H1/22
2H13
13C1/22
14C13

History of NMR-

It is the phenomenon of nuclear magnetic resonance, which was first reported independently in 1964. It is discovered by two groups of physicists- Block, Hansen, and Packard at Stanford University who detected a signal from the protons from water. Other groups of physicists- Purcell, Torrey, and Pound at Harvard University observed a signal from the protons in paraffin wax.

Block and Purcell were jointly awarded the Noble Prize for physics in 1952 for this discovery. Since that time, the advances in NMR techniques leading to widespread applications in various branches of science resulted in the Noble Prize in chemistry in 1991. The applications of NMR in the field of clinical, solid-state, and biophysical sciences are really incredible.

Principle Of NMR-

Any proton or nucleus with an odd mass number of spins on its own axis. By the application of an external magnetic field (Ho), the nucleus spins on its own axis and a magnetic moment is created, resulting in a processional orbit, with a frequency called processional frequency, and the state is called as Ground State or Parallel orientation.

In this state, the magnetic field caused by the spin of nuclei is aligned with the externally applied magnetic field.

When the energy in the form of Radiofrequency is applied and when

 Applied frequency = Processional frequency,

absorption of energy occurs and an NMR signal is recorded.

The nucleus moves from the ground state to excited state because of the absorption of energy, which results in spin reversal or antiparallel orientation in which the magnetic field caused by the spin of nucleus opposes or aligned against the externally applied magnetic field.

Principle-of-NMR-spectrosopy

When the application of radiofrequency energy is stopped, then the nucleus returns to ground State or in parallel orientation. It should be noted that increasing the strength of the magnetic field does not cause the transition from the ground state to state, but it merely increases the processional frequency.

Without the application of the magnetic field, there are no two spin states and there is only one average spin. Hence radiofrequency radiation cannot be absorbed. Therefore, the application of a magnetic field and radio frequency is necessary to cause an NMR spectrum.

 

Basic Principle of NMR– When the energy in the form of radiofrequency is applied→ when applied frequency is equal to processional frequency→ Absorption of energy occurs→ Nucleus is in resonance→ NMR signal is recorded.

You may read- Spectroscopy.

Theory of NMR-

There are two energy states for a proton are in the magnetic field, in which lower energy state with the nucleus aligned in the same direction as B, and a higher energy state where the nucleus aligned against B.

The energy difference between these two states is applied when an external energy source matches, then energy is absorbed, which causes the nucleus to spin-flip from one orientation to another.

Theory-of-NMR-spectroscopy
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The difference of energy between these two nuclear spin states coincides with the low-frequency radiofrequency region of the electromagnetic spectrum. When a charged particle like a proton spins on its axis, then it creates a magnetic field, so that the nucleus can be considered to be a tiny bar magnet.

 Generally, these tiny bar magnets are randomly oriented in space. However, in the presence of a magnetic field B, they are oriented with or against this applied field.  This arrangement is lower in energy because more nuclei are oriented with the applied field. The energy difference between the two states is very small in <0.1 Cal.

Types of Nuclear magnetic resonance spectroscopy-

There are two types of NMR spectroscopy are used to characterize the organic structure are-

1H NMR– It is used to determine the type and number of H (hydrogen) atoms in a molecule.

13C NMR- It is used to determine the type of C (carbon) atoms in the molecules.

Relaxation process- The process of transition from the excited state to the ground state where the absorbed energy or radiofrequency energy can be lost. It can be used for spectral assignment and the study of quadrupolar and paramagnetic interactions, and exchange dynamics. It is done by two ways are-

Relaxation-process-in-NMR-spectroscopy
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-Radiation emission – In this process, the emission of radiation is with the emission of radiofrequency radiation itself.

Radiationless transition -By its name, it is the process done without radiation. This process is done in two ways are-

1.Spin-Lattice process– In this process the energy is lost by means of translational/ vibrational/rotational energy. It is also called the longitudinal relaxation process.

2.Spin-spin process– In this process the energy is lost to the neighboring nuclei. It is also called a transverse relaxation process.

Parameters that are affecting NMR spectroscopy-

Shielding and deshielding-
Shielding-and-deshielding-in-NMR-spectroscopy
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The process of NMR is to apply an external magnetic field which is called B0 and measure the frequency at which the nucleus achieves resonance. The electrons orbiting around the nucleus generate a small magnetic field that opposes B0. In this case, we say that electrons are shielding the nucleus from B0.

Shielding- Higher the electron density around the nucleus, that becomes higher the opposing magnetic field to B0 from the electrons, or the greater the shielding.  Because the proton experiences lower external magnetic field, which needs a lower frequency to instate resonance, and hence, the chemical shift shifts upfield with lower ppms.

Or when a proton is present inside such a magnetic field or closer to an electropositive atom, a more applied magnetic field is required to cause excitation. Such protons are called as shielded protons. This effect is called a Shielding effect.

Deshielding– If the electron density around a nucleus decreases, the opposing magnetic field becomes small and therefore, the nucleus feels more the external magnetic field B0, and therefore it is said to be deshielded. Because the proton experiences a higher external magnetic field, that needs a higher frequency to instate resonance. Hence, the chemical shift shifts downfield with higher ppms.

Or when a proton is present outside such a circulating magnetic field or when it is attached to an electronegative atom. the less applied magnetic field is sufficient for excitation. Such protons are called deshielded protons and this effect is called a deshielding effect.

Chemical shift-

Theoretically for any organic compound, for all the protons present, only one NMR signal should be recorded. But this does not happen in practice since all the Hydrogen atoms are not in the same environment i.e. the magnetic field applied is not felt by all the Hydrogen atoms uniformly.

Chemical-shift-in-NMR-spectroscopy
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This is because of the presence or nearness to double bonds or triple bonds, presence or nearness to aromatic or heteroaromatic or alicyclic ring systems, electronegative atoms. etc. Because of the differences in the magnetic field felt by ‘H’ atoms, each proton has a different processional frequency and hence different range of applied frequency are required for excitation. Therefore, we get different peaks or signals for each kind of proton. Chemical shift is the difference between the absorption position of a sample proton and the absorption position of the reference compound. Chemical shift is measured in δ values. The value ranges from O to 10 δ for most compounds.

 Spin–Spin coupling-

The splitting of the lines in the NMR spectra because of the interaction between the spins of the neighboring nuclei in a molecule is known as spin-spin coupling. The spacing of the adjacent lines is the measure of spin-spin coupling and is known as the spin-spin coupling constants (J). It is expressed in cycles per second which depend on the structural relations between the nuclei.

Instrumentation of Nuclear magnetic resonance spectroscopy-

The NMR instrument is consisting mainly of different components that are described as-

Schematic-diagram-of-NMR-spectrometer
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Magnet pole– The magnetic field in NMR is generated by the superconducting magnet. At first, a low temperature is needed for stainless steel or aluminum wall which contains liquid nitrogen. An inner wall contains a superconducting coil immersed in liquid helium. Then a bore is fitted with the shim coils and spinner assembly to spin the NMR sample tube.

Radiofrequency transmitter- It is used to apply a radiofrequency radiation example-60MHz, 99MHz, 100MHz, 220MHz, 300MHz, 400MHz depending on the capacity or resolution of the instrument.

Sweep Generator- To vary the strength of the applied magnetic field, i.e. to sweep the magnetic field. A field strength of 14,092 gausses, 21, 140 gausses, or 23,490 gausses, etc., is used, depending on the Radiofrequency region employed.

It is mainly used to resonate with the nucleus and thus producing the equal frequency of the applied radiofrequency radiation. This can be achieved by passing the current through the coils around the magnet pole pieces or through a Helmholtz coil holding the sample. So, in a slow sweep leads to saturation effects and a fast sweep results in ringing.

Sample cell- sample test tube which is about 25cm long and 5mm outer diameter is kept inside the sample cavity and is spun at 30rps (revolutions per second) so as to provide a uniform magnetic field, to the sample solution. Generally, glass tubes are used as sample holders. They should possess the following Characteristics are-

-Chemically inert

-Durable

-Transparent.

Radiofrequency receiver (detector or amplifier)- This is mainly used for the detection of the radiofrequency signal by two methods. They are absorption and dispersion. In the absorption method of detection, the Wheatstone bridge is used. The main principle is the absorption of the applied radiofrequency is detected by using the Wheatstone bridge. In other methods, a receiver coil is used. These coils are set at right angles to each other to the sample.

Recorder and integrator- The signals obtained from the receiver are recorded and integrated by the recorders. Generally, an electronic integrator is used for this purpose. A combination of the radiofrequency and magnetic field strength of 14,092 gausses is used. For high-resolution instruments, other combinations are used. In practice, radiofrequency is kept constant and the strength of the magnetic field is varied since vice versa is difficult to achieve.

Solvent Requirements-

When analyzing organic compounds for the nature, type, number, and environment of Protons (Hydrogen), the solvent used in the NMR spectroscopy should not contain Hydrogen atoms.

Hence, we use solvents like carbon tetrachloride (CC14), Deuterated Chloroform (CDC13), Deuterated water (D20), Deuterated Methanol (CD30D), Deuterated dimethyl sulphoxide (CD3) SO, Deuterated acetic acid (CD3COOD), Deuterated trifluoroacetic acid (CD3COOD), etc. Also, the solvents should have the following properties-

-Chemical inertness

-Magnetic isotropy (magnetically neutral)

-Volatility (to facilitate sample recovery)

-Absence of Hydrogen atoms    

-Easily available and inexpensive

Reference standard-

A reference standard should possess the following characteristics are-

-Chemical inertness

-Magnetic isotropy (magnetically neutral)

-Give a single sharp peak

-Easily recognizable peak

 -Miscible with a wide range of solvents

-Volatility – to facilitate recovery from valuable samples.

The reference standard, Tetra Methyl Silane (TMS) at 0.5% concentration is used normally and it is added to the sample. As it is added to the sample itself, it is called as the internal reference.

Tetra-Methyl-Silane-(TMS)-in-NMR-spectroscopy

 TMS has 12 protons which are uniformly shielded because of the highly electropositive nature of silicon at the Centre. Hence these 12 protons give a single sharp peak at 0 δ, which requires maximum magnetic field than Protons of most of the organic compounds.

There are two general types of NMR instruments are-

1. Continuous-wave NMR instrument

2. Fourier transform NMR instrument

Continuous-wave or CW NMR instruments- CW-NMR instruments are of less costly and lesser maintenance when compared to other NMR spectrometers. It is consisting of a console, magnet, and two orthogonal coils of wire which are used to receives the radiofrequency waves.

It detects the resonance frequencies of nuclei in a sample placed in a magnetic field by sweeping the frequency of radiofrequency radiation through a given range and directly recording the intensity of absorption as a function of frequency. The spectrum is generally recorded and plotted simultaneously with a recorder synchronized to the frequency of the radiofrequency source.

Fourier transform NMR instruments– The sensitivity of the NMR is less, so to increase that it is combined with Fourier transform principle.  FT-NMR spectrometer consists of a console, a magnet, and a coil of wire.  This coil of wire acts as a transmitter and receiver for the radiofrequency. But the main disadvantage of this is it is time-consuming which takes 2–8 min and the advantages are high sensitivity, higher resolution, and minimized noise ratio also.

In FT-NMR spectroscopy, the sample is subjected to a high-power short duration pulse of radiofrequency radiation contains broadband of frequencies and causes all the spin-active nuclei to resonate all at once at their Larmor frequencies.

Immediately following the pulse, the sample passes a signal called free induction decay (FID). This signal is modulated by all the frequencies of the nuclei return to equilibrium (intensity as a function of time) is recorded, digitized, and stored as an array of numbers in a computer.

The data of Fourier transformation affords a conventional (intensity as a function of frequency) representation of the spectrum.

You may read- Electron Spin Resonance spectroscopy.

Applications of NMR-

1.Structure elucidation of organic compounds-

Organic compounds invariably have Hydrogen atoms in their structure and the environment of each proton is not the same. Hence from NMR spectra, the following can be known about the structure.

-Types of protons– From the no. of peaks recorded, no. of types of protons can be known. (eg.) Two peaks mean two types of protons and 3 peaks mean 3 types of protons. etc.

The environment of protons– Whether the proton is shielded or deshielded, from the position of the peaks. Shielded protons require a high magnetic field and deshielded protons require a low magnetic field.

(eg.) Aliphatic proton – 0-2δ (High magnetic field).

 Aromatic proton – 6-9δ (low magnetic field).

-No. of protons of each type- From the intensity of peaks, if the peak is of more height/area, then more protons of that type is present. If the peak is of less height/area, then less no. of protons of that type is present.

-No. of adjacent protons- From the multiplicity of peaks. As a general rule, if one adjacent proton is present, then doublet (two peaks of the same height) is recorded. If two adjacent protons are present triplet (3 peaks in the ratio of 1:2:1) is recorded. If three adjacent protons are present, then quartet (4 peaks in the ratio of 1:3:3:1) is recorded. Hence the no. of adjacent protons can be known.

Absorption position of some protons

Aliphatic/alicyclic – O – 2δ                                   Aromatic & heteroaromatic-6-9δ

Acetylenic – 2 – 3δ                                               Aldehydic- 9-10δ

Olefinic – 4.5 – 7.5 5

2. Investigation of dynamic properties of the molecules like conformational isomerism, molecular asymmetry, hydrogen bonding, keto-enol tautomerism, etc., can be done.

3.Determination of optical purity.

4.Study of molecular interactions like micelle formation and drug macromolecule or drug-receptor interactions.

 5.Quantitative analysis

-Assay of components- Single component or multicomponent without separation of components, can be quantitatively estimated. The specific peak for each component is identified and the peak area/height ratio given by integral value is found using standard and sample and the quantity can be estimated.

-Surfactant chain length determination- This can be determined from the proportion of Hydrogen atom in the poly-oxyethylene chain.

-Hydrogen analysis- Percentage of Hydrogen in the compound can be determined.

-Iodine value- which is a measure of double/triple bond can be known from the proportion of olefinic protons.

-Moisture analysis- Since water (H20), can give a characteristic peak, the % can be known from the peak ratio of water peak and component peak.


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