Electron Paramagnetic Resonance (EPR) Spectroscopy

Inorganic Chemistry

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Overview

Source: David C. Powers, Tamara M. Powers, Texas A&M

In this video, we will learn the basic principles behind Electron Paramagnetic Resonance (EPR). We will use EPR spectroscopy to study how dibutylhydroxy toluene (BHT) behaves as an antioxidant in the autoxidation of aliphatic aldehydes.

Cite this Video

JoVE Science Education Database. Inorganic Chemistry. Electron Paramagnetic Resonance (EPR) Spectroscopy. JoVE, Cambridge, MA, (2017).

Principles

Fundamentals of EPR:

EPR is a spectroscopic technique that relies on similar physical phenomena as nuclear magnetic resonance (NMR) spectroscopy. While NMR measures nuclear spin transitions, EPR measures electron spin transitions. EPR is chiefly used to study paramagnetic molecules, which are molecules with unpaired electrons. Recall that an electron has a spin quantum number, s = ½, which has magnetic components ms = ½ and ms = -½. In the absence of a magnetic field, the energy of the two ms states are equivalent. However, in the presence of an applied magnetic field (B0), the magnetic moment of the electron aligns with the applied magnetic field and, as a result, the ms states become non-degenerate (Figure 1). The energy difference between the ms state is dependent on the strength of the magnetic field (Equation 1). This is called the Zeeman effect.

E = m2geμBB0    (Equation 1)

where ge is the g-factor, which is 2.0023 for a free-electron and µB is the Bohr magneton.

At a given magnetic field, B0, the energy difference between the two ms states is given by Equation 2.

ΔE = E½  — E = geμBB0 = hυ  (Equation 2)

An electron moves between the two ms states upon emission or absorption of a photon with energy ΔE =hυ. Equation 2 applies to a single, free-electron. However, similar to the way that the chemical shift in 1H NMR depends on the chemical environment of the H atom, electrons within molecules do not behave in the same way as an isolated electron. The electric field gradient of the molecule will influence the effective magnetic field, given by Equation 3.

Beff = B0(1 — σ) (Equation 3)

where σ is the effect of local fields, which can be a positive or negative value.

Plugging Equation 3 into Equation 2, we can define the g-factor for an unpaired electron in a given molecule as g = ge(1 - σ), which simplifies the overall equation to:

hυ = gμBB0 (Equation 4)

During the EPR experiment, the frequency is swept, most commonly in the microwave region ranging from 9,000-10,000 MHz, and the field is held constant at approximately 0.35 T, allowing for the calculation of g. Experimentally determining g using EPR provides information about the electronic structure of a paramagnetic molecule.

Figure 1
Figure 1. Splitting of magnetic moment states, ms, in the presence of a magnetic field.

Application of EPR:

In this experiment, we will use EPR spectroscopy to investigate the chemistry of antioxidants. O2, which comprises ~ 21% of the Earth's atmosphere, is a strong oxidant. Despite its potential to act as an oxidant, O2 is a ground state triplet and thus only reacts quite slowly with most organic molecules. One important, though often undesired, reaction mediated by O2 is autoxidation. In autoxidation chemistry, O2 initiates radical chain processes, which can quickly consume organic molecules. Figure 2 illustrates a common autoxidation, in which aldehydes are oxidized to carboxylic acids.

Preventing autoxidation chemistry is important to prevent decomposition of many common organic materials, such as plastics, and a large field has developed around identifying effective antioxidants to inhibit autoxidation. One mechanism by which antioxidants can function is by reacting with the radical intermediates to inhibit radical chain processes. Because radical species have unpaired spins, EPR is a valuable tool for understanding the chemistry of antioxidants. In this experiment, we will use EPR spectroscopy to explore the role of BHT as an antioxidant in the autoxidation of aliphatic aldehydes.

Figure 2
Figure 2. Aldehyde autoxidation proceeds via a radical chain mechanism.

Procedure

1. Autoxidation of Butyraldehyde

  1. Prepare a solution of butyraldehyde (100 mg) and CoCl2·6H2O (1 mg) in 1,2-dichloroethane (DCE) (4 mL) in a 20 mL scintillation vial. Add a magnetic stir bar and fit the vial with a rubber septum.
  2. Attach the barrel of a 1 mL plastic syringe to a short piece of rubber tubing. Insert the rubber tubing into a latex balloon and secure the balloon to the tube with a rubber band and electrical tape. Inflate a latex balloon with O2.
  3. Insert the needle of the O2 balloon into the reaction vial. Insert a second needle into the septum and purge the head-space of the reaction vessel with O2.
  4. Using a stir plate, stir the reaction at room temperature for 4 h under an O2 atmosphere.
  5. Concentrate the reaction mixture using a rotary evaporator and take an 1H NMR spectrum of the resulting oily residue in CDCl3.

2. Using BHT as an Antioxidant for the Autoxidation of Butyraldehyde

Set up two vials as described below. One will be used to analyze the product distribution and one will be used in step 3 for EPR spectroscopy.

  1. Prepare a solution of butyraldehyde (100 mg) and CoCl2·6H2O (1 mg) in DCE (4 mL) in a 20 mL scintillation vial. Add BHT (10 mg) to the solution. Add a magnetic stir bar and fit the vial with a rubber septum.
  2. Attach the barrel of a 1 mL plastic syringe to a short piece of rubber tubing. Insert the rubber tubing into a latex balloon and secure the balloon to the tube with a rubber band and electrical tape. Inflate a latex balloon with O2.
  3. Insert the needle of the O2 balloon into the reaction vial. Insert a second needle into the septum and purge the head-space of the reaction vessel with O2.
  4. Using a stir plate, stir the reaction at room temperature for 4 h under an O2 atmosphere.
  5. Concentrate the reaction mixture using a rotary evaporator and take an 1H NMR spectrum of the resulting oily residue in CDCl3.

3. Measuring EPR Spectra

  1. Turn the EPR spectrometer on and let the instrument warm up for 30 min. Set up an EPR acquisition with the following parameters: center field 3,345 G, sweep width 100 G, sweep time 55 s, time constant 10 ms, MW power 5 mW, modulation 100 kHz, and modulation amplitude 1 G.
  2. Measure an EPR spectrum of an empty EPR tube to ensure that there are no background signals from either the EPR tube or the instrument resonator.
  3. Prepare a solution of BHT in DCE in an N2-filled glove box. Transfer 0.5 mL of the solution to an EPR tube and measure the EPR spectrum of BHT using the acquisition parameters set up in step 3.1.
  4. Transfer 0.5 mL of the BHT-added reaction solution from Step 2 to an EPR tube and acquire and EPR spectrum using the acquisition parameters set up in Step 3.1.

Electron paramagnetic resonance, or EPR, spectroscopy is an important technique for the characterization of paramagnetic compounds, such as compounds with unpaired electrons.

EPR has many important applications in the study of organic radicals, paramagnetic inorganic complexes, and bioinorganic chemistry.

This video will illustrate the basic principles behind Electron Paramagnetic Resonance, the use of EPR to study dibutylhydroxy toluene and its antioxidant behavior in the autoxidation of aliphatic aldehydes, and discuss a few applications.

EPR is a spectroscopic technique that is used to study molecules with unpaired electrons by measuring electron spin transitions.

An electron has a spin quantum number of 1/2, which has magnetic components of either +1/2 or -1/2.

In the absence of a magnetic field, the energy of the two spin states is equivalent. However, in the presence of an applied magnetic field, the magnetic moment of the electron aligns with the applied magnetic field and, the spin states become non-degenerate.

The energy difference between the spin state is dependent on the strength of the magnetic field. This is called the Zeeman effect.

At a given magnetic field, the energy difference between the two spin states is given by ΔE.

An electron moves between the two spin states upon emission or absorption of a photon with energy ΔE. However, this equation applies to a single, free-electron, and does not account for the fact, that electrons within molecules do not behave in the same way as an isolated electron does.

The electric field gradient of the molecule will influence the effective magnetic field, which, if plugged into this equation, defines the g-factor for an unpaired electron in a given molecule in this simplified overall equation.

During an EPR experiment, the frequency is swept, while the field is held constant, allowing for the calculation of the g-factor providing information about the electronic structure of a paramagnetic molecule.

In this experiment, EPR spectroscopy is used to study anti-oxidants. Oxygen, which is a strong oxidant, is a ground state triplet and thus reacts quite slowly with most organic molecules. One important, though often undesired, reaction mediated by oxygen is autoxidation, where O2 initiates radical chain processes.

This can lead to quick consumption of organic molecules and decomposition of many organic materials, such as plastics. Therefore, identifying effective antioxidants to inhibit autoxidation has become an important research field.

One mechanism by which antioxidants can function is by reacting with the radical intermediates to inhibit radical chain processes. Because radical species have unpaired spins, EPR is a valuable tool for understanding the chemistry of antioxidants.

Now let's look at how EPR spectroscopy is used to explore the role of dibutylhydroxy toluene, as an antioxidant in the autoxidation of aliphatic aldehydes.

Let's start with the autoxidation of butyraldehyde in absence of an antioxidant. Using a 20 mL scintillation vial, dissolve 125 mL of butyraldehyde and 1 mg of CoCl2·6H2O in 4 mL of 1,2-dichloroethane. Add a magnetic stir bar and seal the vial with a rubber septum.

Attach the barrel of a 1 mL plastic syringe to a short piece of rubber tubing. Insert the rubber tubing into a latex balloon and secure with a rubber band and electrical tape. Then inflate the balloon with oxygen gas.

Insert the needle of the oxygen filled balloon into the vial. Insert a second needle through the septum, and purge the solution with oxygen gas for five minutes. Once purged, withdraw the second needle, and place the vial on a stir plate, stirring the reaction for 4 hours at room temperature.

When the reaction is finished, concentrate the mixture using a rotary evaporator. Then, dry the residue on a high-vacuum line for 1 hours, and acquire a 1H-NMR in deuterated chloroform.

Now let's compare the reaction if carried out in presence of the antioxidant dibutylhydroxy toluene, or BHT. Prepare two identical samples, by dissolving CoCl2·6H2O and butyraldehyde in 1,2-dichloroethane using a 20-mL scintillation vial. Add the antioxidant to each solution, followed by a stir bar, and fit each vial with a rubber septum.

Similar to the previous reaction, use a balloon to purge the solution in the vials with oxygen, then stir the reactions under oxygen atmosphere for 4 hours at room temperature. After 4 hours, concentrate one of the mixtures using a rotary evaporator for a 1H-NMR. Dry the sample on high vacuum, and use this sample to obtain a 1H-NMR. The other reaction will be used for EPR.

Turn on the EPR spectrometer and let the instrument warm up for 30 min. On the computer, tune the empty cavity of the EPR instrument to make sure there are no contaminants in the instrument.

Set up an EPR acquisition with the parameters stated in the text. Measure an EPR spectrum of an empty EPR tube to ensure that there are no background signals from either the EPR tube or the instrument resonator.

Then, use BHT and prepare a solution in 1,2-dichloroethane in a N2-filled glovebox. Transfer 0.5 mL of the solution to a 2 mm EPR tube, capping it with a plastic EPR-tube cap. Measure the EPR spectrum of BHT using the acquisition parameters set up previously.

Now, use the BHT containing reaction and prepare an EPR solution following the same procedure as for the BHT sample. Acquire an EPR spectrum using the acquisition parameters set up previously.

Now, let's compare the reactions with and without the BHT antioxidant using the NMR and EPR data.

The autoxidation of butyraldehyde affords butyric acid. The 1H-NMR spectrum obtained from the reaction shows the lack of an aldehydic C-H resonance and the presence of the resonances expected of butyric acid.

In contrast, the NMR obtained from the reaction mixture with added BHT displays signals consistent with butyraldehyde, with no butyric acid present. From these data, it is shown that BHT has served as an antioxidant in the aldehyde autoxidation.

The role of BHT in inhibiting aldehyde autoxidation is illuminated by the EPR spectra obtained of BHT and of BHT added to the aldehyde autoxidation reaction.

BHT is a diamagnetic organic molecule, meaning that there are no unpaired electrons. Accordingly, the EPR spectrum of BHT displays no signals. In contrast, the EPR spectrum of the autoxidation reaction in which BHT was added displays a strong four-lined pattern, consistent with an organic radical.

This spectrum arises because the O-H bond of BHT is weak. In the presence of radicals generated during autoxidation, the hydrogen transfer from BHT quenches the radical chain mechanism and generates a stable oxygen-centered radical.

Electron paramagnetic resonance spectroscopy is an analytical method, which is often used in organic and inorganic chemistry to gain additional information, aside of the common methods such as NMR or IR spectroscopy.

For example, EPR can be used to study biological systems such as the metabolism of cyanobacteria. The cyanobacteria are suspended in a solution containing trityl radical, and placed in an imaging probe. The sample is irradiated with light and the radical concentration measured with respect to time.

This study showed that the trytil concentration decreased under light, but remained constant in darkness, demonstrating that metabolic activity is light dependent.

Molecules with unpaired electrons can be challenging to characterize with NMR only, thus EPR spectroscopy is frequently used to analyze organic radicals in more detail. Experimental EPR spectra delineate the g-factor of the unpaired electron, providing information about the electronic structure of the paramagnetic center.

Furthermore, the nuclear spins of the nuclei with the unpaired electron, as well as neighboring nuclei, influence the magnetic moment of an electron, giving rise to additional splitting of the spin states and multiple lines in the EPR spectrum. The resulting hyperfine and super-hyperfine coupling provides further information about the electronic structure of the molecule

You've just watched JoVE's introduction to electron paramagnetic resonance spectroscopy. You should now be familiar with the principles of EPR, autoxidation, an autoxidation reaction, and various applications of EPR spectroscopy. As always, thanks for watching!

Results

The autoxidation of butyraldehyde affords butyric acid. The 1H NMR spectrum obtained from the reaction carried out in Step 1 shows the lack of an aldehydic C-H resonance and the presence of the resonances expected of butyric acid. In contrast, the NMR obtained from the reaction mixture from step 2 (with added BHT) displays signals consistent with butyraldehyde, with no butyric acid present. From these data, we observe the butyraldehyde has served as an antioxidant in aldehyde autoxidation.

The role of BHT in inhibiting aldehyde autoxidation is illuminated by the EPR spectra obtained of BHT and of BHT added to the aldehyde autoxidation reaction. BHT is a diamagnetic organic molecule, meaning that there are no unpaired electrons. Accordingly, the EPR spectrum of BHT displays no signals. In contrast, the EPR spectrum of the autoxidation reaction in which BHT was added displays a strong four-lined pattern, consistent with an organic radical. This spectrum arises because the O-H bond of BHT is weak and in the presence of radicals generated during autoxidation, H-atom transfer from BHT quenches the radical chain mechanism and generates a stable O-centered radical.

Applications and Summary

In this experiment, we explored the role of antioxidants in inhibiting autoxidation chemistry. We probed the mechanism of inhibition using EPR spectroscopy, which revealed that BHT serves as an antioxidant by quenching reactive radical intermediates via H-atom transfer.

Molecules with unpaired electrons can be challenging to characterize by NMR and thus EPR spectroscopy frequently provides useful and complementary information regarding these species. EPR spectroscopy is an experimental technique that is frequently used to detect and characterize organic radicals. In addition, paramagnetic inorganic complexes also frequently display EPR spectra that can be instructive for characterization. Experimental EPR spectra delineate the g-factor of the unpaired electron, which provides information about the electronic structure of the paramagnetic center. In addition, the nuclear spins of the nuclei with an unpaired electron as well as neighboring nuclei also influence the magnetic moment of an electron, giving rise to additional splitting of the ms states and multiple lines in the EPR spectrum. The resulting hyperfine and super-hyperfine coupling provides further information about the electronic structure of the molecule.

In addition to characterizing open-shell organic and inorganic species, the exquisite sensitivity of EPR spectroscopy is critical to application to bioinorganic systems, where the concentration of metal cofactors is low. EPR spectra are routinely used in bioinorganic chemistry to provide direct information about the structures and oxidation states of metal ions at the heart of enzymes.

1. Autoxidation of Butyraldehyde

  1. Prepare a solution of butyraldehyde (100 mg) and CoCl2·6H2O (1 mg) in 1,2-dichloroethane (DCE) (4 mL) in a 20 mL scintillation vial. Add a magnetic stir bar and fit the vial with a rubber septum.
  2. Attach the barrel of a 1 mL plastic syringe to a short piece of rubber tubing. Insert the rubber tubing into a latex balloon and secure the balloon to the tube with a rubber band and electrical tape. Inflate a latex balloon with O2.
  3. Insert the needle of the O2 balloon into the reaction vial. Insert a second needle into the septum and purge the head-space of the reaction vessel with O2.
  4. Using a stir plate, stir the reaction at room temperature for 4 h under an O2 atmosphere.
  5. Concentrate the reaction mixture using a rotary evaporator and take an 1H NMR spectrum of the resulting oily residue in CDCl3.

2. Using BHT as an Antioxidant for the Autoxidation of Butyraldehyde

Set up two vials as described below. One will be used to analyze the product distribution and one will be used in step 3 for EPR spectroscopy.

  1. Prepare a solution of butyraldehyde (100 mg) and CoCl2·6H2O (1 mg) in DCE (4 mL) in a 20 mL scintillation vial. Add BHT (10 mg) to the solution. Add a magnetic stir bar and fit the vial with a rubber septum.
  2. Attach the barrel of a 1 mL plastic syringe to a short piece of rubber tubing. Insert the rubber tubing into a latex balloon and secure the balloon to the tube with a rubber band and electrical tape. Inflate a latex balloon with O2.
  3. Insert the needle of the O2 balloon into the reaction vial. Insert a second needle into the septum and purge the head-space of the reaction vessel with O2.
  4. Using a stir plate, stir the reaction at room temperature for 4 h under an O2 atmosphere.
  5. Concentrate the reaction mixture using a rotary evaporator and take an 1H NMR spectrum of the resulting oily residue in CDCl3.

3. Measuring EPR Spectra

  1. Turn the EPR spectrometer on and let the instrument warm up for 30 min. Set up an EPR acquisition with the following parameters: center field 3,345 G, sweep width 100 G, sweep time 55 s, time constant 10 ms, MW power 5 mW, modulation 100 kHz, and modulation amplitude 1 G.
  2. Measure an EPR spectrum of an empty EPR tube to ensure that there are no background signals from either the EPR tube or the instrument resonator.
  3. Prepare a solution of BHT in DCE in an N2-filled glove box. Transfer 0.5 mL of the solution to an EPR tube and measure the EPR spectrum of BHT using the acquisition parameters set up in step 3.1.
  4. Transfer 0.5 mL of the BHT-added reaction solution from Step 2 to an EPR tube and acquire and EPR spectrum using the acquisition parameters set up in Step 3.1.

Electron paramagnetic resonance, or EPR, spectroscopy is an important technique for the characterization of paramagnetic compounds, such as compounds with unpaired electrons.

EPR has many important applications in the study of organic radicals, paramagnetic inorganic complexes, and bioinorganic chemistry.

This video will illustrate the basic principles behind Electron Paramagnetic Resonance, the use of EPR to study dibutylhydroxy toluene and its antioxidant behavior in the autoxidation of aliphatic aldehydes, and discuss a few applications.

EPR is a spectroscopic technique that is used to study molecules with unpaired electrons by measuring electron spin transitions.

An electron has a spin quantum number of 1/2, which has magnetic components of either +1/2 or -1/2.

In the absence of a magnetic field, the energy of the two spin states is equivalent. However, in the presence of an applied magnetic field, the magnetic moment of the electron aligns with the applied magnetic field and, the spin states become non-degenerate.

The energy difference between the spin state is dependent on the strength of the magnetic field. This is called the Zeeman effect.

At a given magnetic field, the energy difference between the two spin states is given by ΔE.

An electron moves between the two spin states upon emission or absorption of a photon with energy ΔE. However, this equation applies to a single, free-electron, and does not account for the fact, that electrons within molecules do not behave in the same way as an isolated electron does.

The electric field gradient of the molecule will influence the effective magnetic field, which, if plugged into this equation, defines the g-factor for an unpaired electron in a given molecule in this simplified overall equation.

During an EPR experiment, the frequency is swept, while the field is held constant, allowing for the calculation of the g-factor providing information about the electronic structure of a paramagnetic molecule.

In this experiment, EPR spectroscopy is used to study anti-oxidants. Oxygen, which is a strong oxidant, is a ground state triplet and thus reacts quite slowly with most organic molecules. One important, though often undesired, reaction mediated by oxygen is autoxidation, where O2 initiates radical chain processes.

This can lead to quick consumption of organic molecules and decomposition of many organic materials, such as plastics. Therefore, identifying effective antioxidants to inhibit autoxidation has become an important research field.

One mechanism by which antioxidants can function is by reacting with the radical intermediates to inhibit radical chain processes. Because radical species have unpaired spins, EPR is a valuable tool for understanding the chemistry of antioxidants.

Now let's look at how EPR spectroscopy is used to explore the role of dibutylhydroxy toluene, as an antioxidant in the autoxidation of aliphatic aldehydes.

Let's start with the autoxidation of butyraldehyde in absence of an antioxidant. Using a 20 mL scintillation vial, dissolve 125 mL of butyraldehyde and 1 mg of CoCl2·6H2O in 4 mL of 1,2-dichloroethane. Add a magnetic stir bar and seal the vial with a rubber septum.

Attach the barrel of a 1 mL plastic syringe to a short piece of rubber tubing. Insert the rubber tubing into a latex balloon and secure with a rubber band and electrical tape. Then inflate the balloon with oxygen gas.

Insert the needle of the oxygen filled balloon into the vial. Insert a second needle through the septum, and purge the solution with oxygen gas for five minutes. Once purged, withdraw the second needle, and place the vial on a stir plate, stirring the reaction for 4 hours at room temperature.

When the reaction is finished, concentrate the mixture using a rotary evaporator. Then, dry the residue on a high-vacuum line for 1 hours, and acquire a 1H-NMR in deuterated chloroform.

Now let's compare the reaction if carried out in presence of the antioxidant dibutylhydroxy toluene, or BHT. Prepare two identical samples, by dissolving CoCl2·6H2O and butyraldehyde in 1,2-dichloroethane using a 20-mL scintillation vial. Add the antioxidant to each solution, followed by a stir bar, and fit each vial with a rubber septum.

Similar to the previous reaction, use a balloon to purge the solution in the vials with oxygen, then stir the reactions under oxygen atmosphere for 4 hours at room temperature. After 4 hours, concentrate one of the mixtures using a rotary evaporator for a 1H-NMR. Dry the sample on high vacuum, and use this sample to obtain a 1H-NMR. The other reaction will be used for EPR.

Turn on the EPR spectrometer and let the instrument warm up for 30 min. On the computer, tune the empty cavity of the EPR instrument to make sure there are no contaminants in the instrument.

Set up an EPR acquisition with the parameters stated in the text. Measure an EPR spectrum of an empty EPR tube to ensure that there are no background signals from either the EPR tube or the instrument resonator.

Then, use BHT and prepare a solution in 1,2-dichloroethane in a N2-filled glovebox. Transfer 0.5 mL of the solution to a 2 mm EPR tube, capping it with a plastic EPR-tube cap. Measure the EPR spectrum of BHT using the acquisition parameters set up previously.

Now, use the BHT containing reaction and prepare an EPR solution following the same procedure as for the BHT sample. Acquire an EPR spectrum using the acquisition parameters set up previously.

Now, let's compare the reactions with and without the BHT antioxidant using the NMR and EPR data.

The autoxidation of butyraldehyde affords butyric acid. The 1H-NMR spectrum obtained from the reaction shows the lack of an aldehydic C-H resonance and the presence of the resonances expected of butyric acid.

In contrast, the NMR obtained from the reaction mixture with added BHT displays signals consistent with butyraldehyde, with no butyric acid present. From these data, it is shown that BHT has served as an antioxidant in the aldehyde autoxidation.

The role of BHT in inhibiting aldehyde autoxidation is illuminated by the EPR spectra obtained of BHT and of BHT added to the aldehyde autoxidation reaction.

BHT is a diamagnetic organic molecule, meaning that there are no unpaired electrons. Accordingly, the EPR spectrum of BHT displays no signals. In contrast, the EPR spectrum of the autoxidation reaction in which BHT was added displays a strong four-lined pattern, consistent with an organic radical.

This spectrum arises because the O-H bond of BHT is weak. In the presence of radicals generated during autoxidation, the hydrogen transfer from BHT quenches the radical chain mechanism and generates a stable oxygen-centered radical.

Electron paramagnetic resonance spectroscopy is an analytical method, which is often used in organic and inorganic chemistry to gain additional information, aside of the common methods such as NMR or IR spectroscopy.

For example, EPR can be used to study biological systems such as the metabolism of cyanobacteria. The cyanobacteria are suspended in a solution containing trityl radical, and placed in an imaging probe. The sample is irradiated with light and the radical concentration measured with respect to time.

This study showed that the trytil concentration decreased under light, but remained constant in darkness, demonstrating that metabolic activity is light dependent.

Molecules with unpaired electrons can be challenging to characterize with NMR only, thus EPR spectroscopy is frequently used to analyze organic radicals in more detail. Experimental EPR spectra delineate the g-factor of the unpaired electron, providing information about the electronic structure of the paramagnetic center.

Furthermore, the nuclear spins of the nuclei with the unpaired electron, as well as neighboring nuclei, influence the magnetic moment of an electron, giving rise to additional splitting of the spin states and multiple lines in the EPR spectrum. The resulting hyperfine and super-hyperfine coupling provides further information about the electronic structure of the molecule

You've just watched JoVE's introduction to electron paramagnetic resonance spectroscopy. You should now be familiar with the principles of EPR, autoxidation, an autoxidation reaction, and various applications of EPR spectroscopy. As always, thanks for watching!

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