The Electron Spin Resonance (ESR) : Principle Theory And Applications

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Introduction

Also known as….       

Electron Paramagnetic Resonance (EPR)

Electron Magnetic Resonance (EMR)

What Is ESR???

  • It is a branch of absorption spectroscopy in which   radiation having frequency in microwave region is absorbed by paramagnetic substance to induce transition between magnetic energy level of electron with unpaired spin.
  • Magnetic energy splitting is done by applying a static  magnetic field.

ESR Phenomenon Is Shown By …………..

  • Atoms having an odd number of electrons.
  • Ions having partly filled inner electron shells.
  • Molecule that carry angular momentum of electronic   origin.
  • Free radicals having unpaired electrons.
  • Molecule with paired electrons and zero magnetic field  

-Diamagnetic

  • Molecules with unpaired electrons and magnetic moment-

- Paramagnetic

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Fig 1

Basic Principle Of ESR……………

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Fig 2

The unpaired electrons are excited to a high energy state under the magnetic field by the absorption of microwave "music." The excited electron changes its direction of spin and relaxes into the ground state by emitting phonons (song).

Microwave absorption is measured as a function of the magnetic field by ESR spectroscopy.

Theory & Working Of ESR

A molecule or atom has discrete (or separate) states, each with a corresponding energy.

Spectroscopy is the measurement and interpretation of the differences in these energies

The energy differences, DE ,can be measured, because of an important relationship between  DE and the absorption of electromagnetic radiation.

According to Planck's law, electromagnetic radiation may be absorbed if

DE= hv 

Where,

h is Planck's constant and

v is the frequency of the radiation.

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Fig 3

Transition associated with the absorption of electromagnetic energy

For a single electron

·The two spin states have the same energy in the absence of a magnetic field.

·The energies of spin states diverge linearly as the magnetic field increases.

Therefore,

·Without a magnetic field, there is no energy difference to measure.

·The measured energy difference depends linearly on the magnetic field.

The energy differences studied in EPR spectroscopy are due to the interaction of unpaired electrons in the sample with an external magnetic field produced by the EPR spectrometer. This effect is called Zeeman Effect.

A strong magnetic field B0 is applied to a material containing paramagnetic species.

Electron is a charged particle with angular momentum and hence possess magnetic moment.

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Fig 4

By virtue of it’s charge and spin electron act as a bar magnet and can interact with an external magnetic field B0.

Now this magnetic moment can be detected by its interaction with B0.

If field is zero magnetic moment of odd electron have randomized direction, but in presence of B0 individual magnetic moment arising due to electron spin of the unpaired electron can be oriented in two ways.  

It will have a state of lowest energy when the moment of electron, µ is aligned along the magnetic field (parallel) and a state with highest energy when µ is aligned against the magnetic field (antiparallel).

The two states are labelled by the projection of electron spin, Ms, on the direction of the magnetic field. Because the electron is a spin 1/2 particle, the parallel state is designed as Ms= -1/2 and the antiparallel state is Ms = + 1/2. From quantum mechanics, we obtained the most basic equations of EPR

E = gµBB0Ms = ±1/2 gµBB0

DE = EH – EL = 1/2 gµBB0 – (-1/2 gµBB0)= gµBB0

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Fig 5                                 

The equation describing the absorption (or emission) of microwave energy between two spin states is

∆ E = hv = gµBB0

where:

∆E is the energy difference between the two spin states

h is Planck constant

v is the microwave frequency

g  is the proportionality factor which is a function of electron’s environment.

It is called as              Zeeman splitting factor,

Spectroscopic splitting factor,

Lande’s splitting factor,

Gyromagnetic ratio.

Value of g is not constant.

For a free electron its value is 2.0023 ( @2 )

B0 is the applied magnetic field.

µB is the electron Bohr magneton, a natural unit of electronic magnetic moment.

µB = eh/ 4pmc    = 0.9723 ´ 10-25 erg/gauss

Application of magnetic field provides a magnetic potential energy which splits the spin state by an amount proportional to magnetic field(Zeeman effect).

Now if radiation is supplied to sample of frequency such that energy of each quantum is equal to the difference in energy between the electron state resonance occurs.

For a given magnetic field the spin can be made to flip to opposite direction when they absorb radiation at a corresponding resonant frequency.

These spin flips can be considered as transition between states that become separated in energy when the magnetic field is applied.

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Fig 6

Relaxation And Saturation

CIn order to maintain steady state conditions there must be some mechanism known as relaxation.

by this electrons that have been exited to higher energy  level can lose energy and return to lower level.

CIn absence of relaxation saturation occurs.

in which continuous absorption of energy by electron present  

in lower state leads to equal population in both state –  Saturation Condition.

§ No further absorption.

§ No further resonance.

§ No further signal.

§ Broadening in signal.

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Fig 7

·Relaxation time should be sufficiently rapid to prevent saturation of upper energetic level at the same time sufficiently slow to yield narrow spectral peaks.

·Ratio of numer of electrons in upper energy level to those in lower energy level is given by BOLTZMANN LAW.

n1/n2 = exp – DE / kT   = exp – gµBB0 / kT

Spin Spin Splitting (Hyperfine Splitting)

Magnetic resonance can also occur without an external magnetic field from interaction of the electron and nuclear spin. Such resonance produces fine and hyperfine structure of atomic spectra.

The nuclei of atoms in a molecule or complex often have magnetic moment which produces a local magnetic field at the electron. The interaction between an unpaired electron an nuclei with non zero nuclear spin is called the hyperfine  interaction. This leads to splitting of the ESR line and is known as hyperfine spectrum

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Fig 8

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Fig 9: Diagrammatic presentation of hyperfine splitting due to one proton.

Applications:

All application of ESR is based on three aspects, which are,

  1. Study of free radicals,
  2. Investigation of molecules in the triple state, and
  3. Study of inorganic compounds.

Study of free radicals

Even in very low concentration of sample ESR can study via free radicals. It is also applied in determination of structure of organics and inorganics free radicals. The intensity of ESR signal is directly proportional to the no. of free radicals present. Hence using ESR we can measure relative concentration of free radicals.

Investigation of molecules in the triple state

A triple state molecule has a total spin S=1 so that, its multiplicity can be given as 2S+1=3. While free radicals with S=½ has an odd no. of unpaired electrons. A triple state molecule has an even no. of electrons two of them unpaired. In triple state molecule the unpaired electrons must interact whereas in diradical, the unpaired electrons do not interact for they are a great distance apart.

Study of inorganic compounds

ESR is very successful in the study of inorganic compounds. The ESR studies may be used in knowing the exact structures of solvated metal ions. ESR is used in the study of catalysts. ESR is used in the determination of oxidation state of metal. eg. Copper is found to be divalent in copper protein complexes whereas it is found to be monovalent in some biologically active copper complexes.  The information of unpaired electrons is very useful in various aspects in applications of ESR. Like, Spin labels, Structural determination, and Reaction velocities and reaction mechanisms.

Spin labels:

Groups with unpaired electron can be attracted to macromolecules such as protein and membranes to obtain a great deal of information obtain their structure. The nitroxide molecules bound to macromolecules are called spin labels. This spin labels are stable molecules that possessing an unpaired 2p electrons. A commonly used TEMPOL (2,2,6,6-tetramethyl piperidinol-n-oxyl. The hyperfine structure of an ESR spectrum is a kind of fingerprint that helps to identify the free radicals presents in the sample. Spin labels give very useful information about the molecules to which they are bound.

Also, we get information like, The rate of motion of macromolecules to which they are bound, or the amount of thermal motion in a membrane in which they have been inserted. The spin label can give information about the polarity of its environments.

Structural determination

The ESR technique cannot be applied to determine molecular structure because the information obtained from the superfine structure is mostly about the extent of delocalization and Fermi contact interaction. It does not tell us about the arrangement of the atoms in the molecule although the symmetry of the molecule can be sometimes deduced from the sets of equivalent nuclei. In certain cases ESR is able to provide useful information about the shape of the radicals.

Reaction velocities and reaction mechanisms:

A large no of organic reactions are known which proceed by a radical mechanism. Most of the radicals formed during organic reaction are not stable but are very reactive. The ESR spectroscopy can be used to study very rapid electron exchange reactions.

The various applications of ESR spectroscopy are grouped in to two categories.

1.Analytical applications

2.Biological applications

Analytical applications:

Mn+2 ions can be measured and detected even when present in trace quantities. The method is very rapid and can be measured in aqueous solution over the range from 10-6 M to 0.1M. ESR method has proved to be a rapid and convenient method for determination of Vanadium in petroleum products. ESR can also be used to estimate Cu(II), Cr(II), Gadolinium(III), Fe(III) and Ti(III). The ESR spectroscopy has been used to estimate polynuclear hydrocarbons, which are first, converted in to radical cations and then absorbed in the surface of an activated silica-alumina catalyst.

Biological systems:

From the ESR studies of variety of biological system such as, leaves, seeds, and tissue preparation, it is found that a definite, correlation exists between the concentration of free radicals and the metabolic activity of the plant material. ESR has studied the presence of free radicals in healthy and diseased tissues. Most of the oxidative enzymes function via one electron redox reaction involving the production of either enzyme bound free radicals or by a change in the valence state of transition metal ion. This has been conformed by ESR studies. Much of the ESR work on photosynthesis has been carried out with photosynthetic bacteria. The oxidation of bacteriochlorophyll in vitro produces an ESR signal.

Modern biotechnology:

ESR being effectively used to revealed both structure and functional information. It  is very useful in modern biotechnology. There are tree branches of modern biotechnology in which ESR is applied,

a) Molecular biotechnology

b) Medical biotechnology

c) Classical biotechnology

Specific features of ESR in modern biotechnology are:

ü Selectivity

ü Specificity

ü Non-invasiveness

ü Sensitivity

ESR in molecular biotechnology :

DNA: ESR is used to investigate the nucleotide-centered free radicals in DNA, either produced by irradiation, or indirectly by other free radicals. ESR is applied to analysis of DNA hydration and the process of the hole or electron transfer from the hydration layer to DNA due to water ionization, and to the analysis of DNA repair by DNA photolyase, by detection of flavin radical formation. ESR is useful in analysis of Reverse Transcriptase (RT) inhibition by polynucleotide.

RNA: ESR was employed to structure dependent molecular dynamics of Trans Activator Responsive (TAR) RNA of HIV-1. ESR is also used to determine the map of protein-RNA interactions between RNA and ribonuclease P from E.coli.

Protein structure and dynamics: The free radical damage of proteins in the field of research is still waiting for the complete exploration. Example of the ESR investigations of the interactions between ligands and target protein is the study on the ion siderophore complex and it’s binding to site directed spin labeled ferric enterobactin receptor responsible for iron uptake by enterobacteria.

Activity of enzymes: ESR can effectively screen potential inhibitors interaction with the enzymes with high speed. Now a days, ESR is used in the analysis of enzymatic activity of nitric oxide synthetase (NOS), the main enzymes delivering NO in biological systems.

Membranes: The existence of phospholipid bilayers in biological systems is confusing from the point of view of evolutionary biology. The model of Fluid Mosaic appears too simple to satisfactorily represent the details of membrane structure and the respective functions. The common view of the architecture of membrane has changed by the recent ESR evidence of the existence of structural domains stabilized by membrane proteins in the form of “rafts”.

Glycobiology: Spin labeled sugars, sugar residues, and spin labeled components interacting with sugar applied in two basic fields of carbohydrate research: Sugar metabolism (degradation and transport), Structural biochemistry of glycoproteins and membranes.

o  ESR is employed to analyze the process of sugar transport in bacteria.

o  ESR was applied to the analysis of the influence of diabetes on the properties of erythrocytes showing the decrease in erythrocyte deformability due to the non-enzymatic glycation of hemoglobin.

oThus, structural investigation often reveals medical aspects.

ESR in medical biotechnology:

Activation and transport of drugs: ESR is useful in several pharmacological investigations like interactions between DNA binding drugs and DNA. ESR may be used to characterize some herb derived products which act by increasing the level of free radicals and other reactive species produced during light induced oxidative stress of the cell. In vivo ESR experiments revealed that multimellar liposomes enhance the topical delivery of hydrophilic compound, drugs used to be more effective when applied in liposomes then in solutions.

Imaging: ESR imaging is a valuable tool for spatially resolved redox mapping of living tissues. Redox status of tumor tissues is significant for understanding tumor physiology, and for determining the effects of chemotherapy and radiation.

ESR in classical biotechnology:

Plant biotechnology: ESR is helpful even at developing artificial photosynthesis, which is biggest biotechnological challenge for the mankind.

Food production and storage: Commercially, ESR is used to analyze shelf life of beer and wine. It is based on free radicals generated in beer or wine due to the action of light, or spontaneously during the process of storage, contribute to the degradation and flavor changes of product. The level of free radical would depend on antioxidants presents in the solutions. Therefore antioxidant capacity of beer or wine helps to predict stability. Similar approach is applied to other food products, such as oils or milk. ESR measurement revealed also photosensitizing action of the important milk ingredient, vitamin-B2, which may affect quality of the product. ESR also used in food science, and the field is hydration, water diffusion, and small molecule mobility in food systems, or sugar-water systems used to model much more complicated systems.

References:

  1. Chatwal G. R. and Anand S. K., Electron spin resonance spectroscopy, in Instrumental methods of chemical analysis, by Anand A. and Arora M., 5th edition, Himalaya publishing house, 2002, pp 2.245 – 2.245.
  2. http://www.infoplease.com/ce6/sci/A0831159.html
  3. http://www.chm.bris.ac.uk/emr/Phil/Phil_1/p_1.html
  4. http://hyperphysics.phy-astr.gsu.edu/hbase/molecule/esr.html
  5. www.ptbf.am.wroc.pl/v261_175.pdf

About Authors:

Rashmin B. Patel

Mr. Rashmin B. Patel is currently working as a lecturer, department of Pharmaceutical Chemistry, A R College of Pharmacy, Vallabh Vidyanagar, Gujarat; India. He completed his B.Pharm and M.Pharm from A R College of Pharmacy, Vallabh Vidyanagar, Gujarat; India. His areas of interest in research includes New Drug Delivery systems & Pharmaceutical Analysis.

Mrs. Mrunali R. Patel

Mrs. Mrunali R. Patel is currently working as a lecturer, Department of Pharmaceutics & Pharmaceutical Technology, Indukaka Ipcowala College of Pharmacy, New Vallabh Vidyanagar, Gujarat; India. She completed her B.Pharm and M.Pharm from A R College of Pharmacy, Vallabh Vidyanagar, Gujarat; India. Her area of interest in research includes New Drug Delivery systems and Pharmaceutical Analysis.

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