Electron Paramagnetic Resonance (EPR) Spectroscopy

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 m_s = ½ and m_s = -½. In the absence of a magnetic field, the energy of the two m_s 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 m_s states become non-degenerate (Figure 1). The energy difference between the m_s state is dependent on the strength of the magnetic field (Equation 1). This is called the Zeeman effect.

E = m₂g_eμ_BB₀    (Equation 1)

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

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

ΔE = E_½  — E_-½ = g_eμ_BB₀ = hυ  (Equation 2)

An electron moves between the two m_s 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.

B_eff = 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 = g_e(1 - σ), which simplifies the overall equation to:

hυ = gμ_BB₀ (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. Splitting of magnetic moment states, m_s, 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. Aldehyde autoxidation proceeds via a radical chain mechanism.

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.

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 m_s 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.