Electron Spin Resonance Spectroscopy (ESR)

Introduction:-

• Electron paramagnetic resonance (EPR) or Electron Magnetic Resonance (EMR) or otherwise known as electron spin resonance (ESR) spectroscopy is the resonant absorption of microwave radiation by an unpaired electron of an atom or molecule (paramagnetic species) when placed in a strong magnetic field.
• ESR is built on the idea that molecules, ions, atoms or molecular fragments with an odd amount of electrons have distinctive magnetic properties. 
• The electron is spin-dependent and as a result of spin, there is a magnetic moment. 
• In 1944 when it was discovered by E.K. Zavoisky, EPR spectroscopy has been utilized as a highly precise and useful technique to study different types of paramagnetic species found in liquid and solid states. 
• It is a powerful technique used to study.
• Free Radicals: Atoms, molecules or ions containing one unpaired electron either in the Solid, Liquid or Gaseous Phases.
• Transition Ions Including Actinide Ions: These routinely may have up to five or seven unpaired electrons, 
• Various ‘Point’ Defects in solids: Localized Imperfections 
• Systems with More than One Unpaired Electron: Triplets state systems, biradicals and multiradical, 
• Systems with Conducting Electrons: Semiconductors and metals 

Active Or Inactive ESR Molecules

Why ESR use in Biosciences

• Malignancy: Free radicals can interact with DNA and other macromolecules, leading to molecular damage, mutation and carcinogenic effect. 
• Aging process: Free radicals may be a cause of the aging process in the form of the weakened immune system. Some inflammatory diseases like rheumatoid arthritis, cataract, etc.
• Oxidation of –SH groups in protein: This leads to loss of biological activity of proteins and some enzymes. 
• Myocardial infarction: Ischemic myocardial cells, i.e cells suffering from oxygen lack, may produce free radicals such as superoxide and hydroxyl radicals. They damage myocardial cells by causing lipid peroxidation. Breakage of DNA strands and oxidation of groups in proteins. This may lead to death. 

Principle of Electron Spin Resonance (ESR):-

• The phenomenon of electron spin resonance (ESR) is based on the fact that an electron is a charged particle. 
• It spins around its axis and this causes it to act like a tiny bar magnet. 
• Every electron has a magnetic moment and spin quantum number s = ½ with magnetic components ms = + ½ or ms = -1/2 . 
• When a molecule or compound with an unpaired electron is placed in a strong magnetic field, the spin of the unpaired electron can align in two different ways creating two spin states ms = ± ½. 
• The alignment can either be along the direction (parellel) to the magnetic field which corresponds to the lower energy state ms = – ½ , and Opposite (antiparallel) to the direction of the applied magnetic field ms = +½

• The two alignments have different energies and this difference in energy lifts the degeneracy of the electron spin states. The energy difference is given by:                      ∆ E = E+1/2– E-1/2 = hv = gßB Where,
• h = Planck’s constant (6.626 x 10-34 J s-1) 
• v = the frequency of radiation 
• g = the g-factor which 
• ß = Bohr magneton (9.274 x 10-24 J T-1) 
• B = strength of the magnetic field in Tesla

           Fig: Splitting of the energy level.

Fig:-Instrumentation of Electron Spin Resonance (ESR) Spectrometer 

Instrumentation of Electron Spin Resonance (ESR) Spectrometer

• KLYSTRONS 
• Klystron tube acts as the source of radiation. 
• It is stabilized against temperature fluctuation by immersion in an oil bath. 
• The frequency of the monochromatic radiation is determined by the voltage applied to klystron. 
• It is kept a fixed frequency by an automatic control circuit and provides a power output of about 300 miliwatts.
• WAVE GUIDE OR WAVEMETER 
• The wave meter is put in between the oscillator and attenuator. 
• To know the frequency of microwaves produced by klystron oscillator. 
• The wave meter is usually calibrated in frequency unit (megahertz) instead of wavelength. 
• Wave guide is a hollow, rectangular brass tube. It is used to convey the wave radiation to the sample and crystal. 
• ATTENUATORS 
• The power propagated down the wave guide may be continuously decreased by inserting a piece of resistive material into the wave guide. This piece is called variable attenuator.
• It is used in varying the power of the sample from the full power of klystron to one attenuated by a force 100 or more. 
• ISOLATORS 
• It’s device which minimizes vibrations in the frequency of microwaves produced by klystron oscillator. 
• Isolators are used to prevent the reflection of microwave power back into the radiation source. 
• It is a strip of ferrite material which allows micro waves in one direction only. 
• It also stabilizes the frequency of the klystron. 
• SAMPLE CAVITIES 
• The resonant cavity containing the sample. 
• Rectangular TE120 cavity and cylindrical TE011 cavity have widely been used. 
• In most of the ESR spectrometers, dual sample cavities are generally used. This is done for simultaneous observation of a sample and a reference material. 
• Since magnetic field interacts with the sample to cause spin resonance, the sample is placed where the intensity of magnetic field is greatest. 
• CRYSTAL DETECTORS 
• Silicon crystal detectors, which converts the radiation in D.C has widely been used as a detector of microwave radiation.
• MAGNET SYSTEM 
• The resonant cavity is placed between the poles pieces of an electromagnet. 
• The field should be stable and uniform over the sample volume. 
• The stability of field is achieved by energizing the magnet with a highly regulated power supply. 
• The ESR spectrum is recorded by slowly varying the magnetic field through the resonance condense by sweeping the current supplied to the magnet by the power supply. 
  
  
  
• MODULATION COIL 
• The modulation of the signal at a frequency consistent with good signal noise ratio in the crystal detector is accomplished by a small alternating variation of the magnetic field. 
• The variation is produced by supplying an A.C. signal to modulation coil oriented with respect the sample in the same direction as the magnetic field. 
• DISPLAY DEVICES 
• In order to observe the signal a system is connected different devices can be used. 

Application of Electron Spin Resonance (ESR)

• ESR Spectrometry is one of the main methods to study transition metal containing metalloproteins. 
• To determine the rate of catalysis. 
• To know about the active site geometry. 
• To study denaturation and protein folding. 
• In studies related to enzyme – ligand interaction. 
• In biological systems. 
• Study of free radicles.
• Spin label. 
• Study of inorganic compounds. 
• Reaction velocity and mechanism. 
• Study of naturally occurring substances such as minerals with transition elements , minerals with defect ( e.g; Quartz), Hemoglobin (fe), Petroleum , Rubber etc. 
• Conducting electrons

Advantages

1. Sensitive Detection: ESR spectroscopy can detect unpaired electrons, making it highly sensitive to free radicals and paramagnetic species even at low concentrations. 
2. Quantitative Analysis: It allows for quantitative analysis of radicals and paramagnetic species, providing information about their concentration and behavior in a sample. 
3. Non-destructive Technique: ESR spectroscopy is non-destructive, meaning it does not alter the sample during analysis, allowing for repeated measurements. 
4. Wide Applicability: It is applicable to a wide range of samples, including solids, liquids, and gases, making it versatile for various research fields like chemistry, biology, and material science. 
5. Structural Information: ESR spectra provide information about the electronic structure of molecules, helping in the determination of molecular structure and electronic environments.

Disadvantages

1. Limited Sensitivity for Some Species: While ESR is highly sensitive to certain paramagnetic species, it may have limited sensitivity for certain types of radicals or when dealing with diamagnetic materials. 
2. Complexity of Interpretation: Interpreting ESR spectra can be complex, especially for samples with overlapping signals or complex molecular structures. 
3. Equipment Cost and Maintenance: High-quality ESR spectrometers can be expensive to purchase and maintain, requiring specialized equipment and expertise. 
4. Sample Preparation: Sample preparation for ESR spectroscopy can sometimes be time-consuming and require specific handling techniques to ensure accurate results. 
5. Limited Depth of Analysis: ESR spectroscopy typically provides information about the surface or near-surface of a sample, limiting its depth of analysis compared to other techniques like NMR spectroscopy. 

Conclusion

Electron Spin Resonance (ESR) spectroscopy is a powerful technique used to study paramagnetic species, such as free radicals and transition metal ions, by detecting the absorption of electromagnetic radiation due to electron spin transitions. Its conclusion often involves identifying the presence of paramagnetic species, determining their electronic structure, and elucidating their chemical environment and interactions. ESR spectroscopy is particularly valuable in fields like chemistry, biochemistry, and materials science for understanding reaction mechanisms, studying radical intermediates, and characterizing materials with unpaired electrons. 

References

• https://www.biosciencenotes.com/free-radical/
• https://science.howstuffworks.com/life/cellularmicroscopic/free-radicals.htm 
• https://www.jeol.com/products/science/esr.php#:~:text=ESR%20is%20a%20method%20for,%2C%20nature%2C %20environment%20and%20behavior. 
• http://www.nou.ac.in/econtent/Msc%20Chemistry%20P aper%20IX/MSc%20Chemistry%20Paper-IX%20Unit6.pdf • https://www.youtube.com/watch?v=vjJlVOVqsPo