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Electrons can be regarded as small bar magnets with north and south poles, because they have own magnetic moments arising to 99% from their quantum- mechanical “spin” (called spin magnetic moment) (Lurie et al.; 2005; Swartz et al.; 1972). In general, stable chemical bonds consist of paired electrons with opposite spins, according to the Pauli exclusion principle (Swartz et al.; 1972). This is imperative in order to coexist in such close vicinity. The magnetic moment of paired electrons is erased (Lurie et al.; 2005). In contrast, unpaired electrons have a magnetic moment which can interact with the externally applied magnetic field B₀ during ESR measurements.

Figure 4-48 Electron spins without (left) and with externally applied magnetic field (right) (modified from (Mäder; 1998))

Two orientations of the electron spins in the presence of an externally applied magnetic field are possible (see Figure 4-48). Spins can either be aligned along the externally applied magnetic field or against it. This effect, known as the Zeeman splitting, results in two different energy states for the electron spins, a low energy state (m=-1/2) and a high energy state (m=+1/2) (see Figure 4-49). The energetic difference ∆E between these two states is dependent on the strength of the applied magnetic field B₀ and the molecular weight of the excited species. Due to the lower mass of electrons, the Bohr magneton is larger than that of protons and a higher energetic difference ∆E between the two states results which increase the sensitivity of ESR compared to NMR measurements (Mäder; 1998). In ESR investigations, electromagnetic radiation with frequencies between 0.3 - 10GHz are used to excite unpaired electrons (Lurie et al.; 2005). At the resonance conditions, the photon energy of the electromagnetic radiation matches the difference between the two energy states

E

∆ (see Figure 4-49). Electrons can now adsorb energy and jump from the low- to the high-energy state by flipping their electron spins by 180° (Lurie et al.; 2005).

B0

Figure 4-49 Electronic energy level diagram with resonance conditions (modified from (Mäder; 1998))

Since all physical systems prefer the low-energy state, electrons fall back at this level within 0.1 – 1µs and simultaneously release electromagnetic radiation at the same frequency as the excitation frequency (Lurie et al.; 2005). If many electrons pass through this scenario at the same time, the resulting electron magnetism is strong enough to alter the externally applied magnetic field by reflecting parts of the applied microwaves. This change is detected during ESR measurements (Swartz et al.; 1972) and is monitored either as adsorption spectrum (amplitudes of the reflected microwaves versus magnetic field strength) or as first derivative spectrum (slope of the adsorption spectrum against magnetic field strength) (see Figure 4-50) (Lurie et al.; 2005).

∆E: energy difference between the two spin states h: Planck constant

v: microwave frequency

g: g-factor or Zeeman splitting factor (e-= 2.0023; H+= 5.5856) µ: Bohr magneton (e-= 9.274*10-24JT-1; H+= 5.051*10-27JT-1) B0: applied magnetic field

mS: quantum number E mS= + 1/2 mS= - 1/2 hv B g E = = ∆

µ

Figure 4-50 Adsorption (top) and first derivative (bottom) ESR spectrum (modified from (Palmer; 1999))

In general, ESR spectra consist of more than one individual line, because unpaired electrons interact not only with the externally applied magnetic field, but also with the magnetic moments of nuclei (Katzhendler et al.; 2000b). The electron-nucleus interaction can take place between unpaired electrons and its own nucleus or a neighboring nucleus (Palmer; 1999). Since the nitrogen nucleus has a nuclear spin of 1, three orientations in the applied magnetic field are possible, resulting in three different magnetic moments (Katzhendler et al.; 2000b). Unpaired electrons in nitroxides are exposed to a magnetic field composed of the strong externally applied magnetic field and a weaker local magnetic field of the nitrogen nucleus. The consequence is a “hyperfine splitting” of the ESR signal e.g. a three line spectrum for nitroxides, because the resonance condition is satisfied at three different values of the externally applied magnetic field (see Figure 4-51) (Swartz et al.; 1972). Hydrogen nuclei in vicinity of the nitrogen nucleus also influence the ESR spectrum. Their effect

is far less pronounced than that of the nitrogen nucleus, due to a greater distance to the unpaired electron, and results in line broadening instead of additional hyperfine splitting (Lurie et al.; 2005).

Besides the permanent magnetic electron-nucleus interactions, orbital-orbital coupling effects occur as well, which depend on the symmetry of the electron orbital (Assenheim; 1966). This effect, known as the orbital magnetic moment, contributes the remaining 1% to the total magnetic moment of electrons (Swartz et al.; 1972) and is displayed in the ESR spectrum shape. If electrons of a spherical orbital (s-orbital) take part, isotropic interactions occur and the ESR spectrum is composed of identical lines. If a conical orbital (p-orbital) participates, the ESR signal is influenced by the three different planes (x, y and z). An anisotropic environment results, reflected by different, broadened line shapes which superpose to the finally observed spectra (see Figure 4-51 bottom). These anisotropic effects can only be averaged out, if the detected electron can tumble very fast (see Figure 4-51 top) (Palmer; 1999).

Summarizing the presented influences on electrons shows that the spectrum shape and width provide information about the molecule structure (Swartz et al.; 1972).

4.4.1.2 Samples

A main advantage of ESR measurements is that a high variety of samples can be examined without any pre-treatment. All states of aggregation (liquid, solid or gaseous) can be studied. Furthermore, samples can be investigated without destruction and they must not be transparent or homogenous, as in other commonly used spectroscopic techniques. Due to the high sensitivity of this method, concentrations down to the nanomolar region can be detected (Mäder; 1998). Samples for ESR measurements must have almost only one unique property: they must be paramagnetic, that means they must contain free electron spins arising from unpaired electrons. Otherwise they are ESR “silent” and no signal can be detected. In general, two kinds of paramagnetic samples are possible. On the one hand, samples which already contain unpaired electrons, e.g. oxygen, metals and transition metals or where radicals are

introduced by e.g. γ-irradiation (Mäder; 1998). Especially free radicals often exist only at a small number and have very short life times. Consequently, they can only be detected at low temperatures or with spin trapping techniques (Lurie et al.; 2005). On the other hand, stable free radicals can be incorporated into ESR silent diamagnetic samples as spin probes (physical incorporation) or spin labels (chemical incorporation) (Mäder; 1998). Due to their higher stability, measurement at room temperature is possible. Spin probes, like nitroxides can either be used directly, e.g. to simulate small model drugs, or can be used to label larger molecules, like polysaccharides or proteins (Lurie et al.; 2005). Pyrrolidine derivates, e.g. 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA), are more stable than piperidine radicals, e.g. 4-hydroxy-2,2,6,6- tetramethyl-1-piperidinyloxy (TEMPOL) (Mäder; 1998; Yoshioka et al.; 1995). Limitations in sample selection are the size, restricted by the cavity dimensions, and the water content due to non-resonant dielectric loss (Lurie et al.; 2005). Samples of approximately 3mm in diameter and 10mm in length are normally used in X-band spectrometers. The water content is often the limiting factor if biological samples are examined. Water is electrically conductive, strongly interacts with the applied microwaves and limits the penetration depth to approximately 1mm for X-band spectrometers (Mäder et al.; 1996). Consequently, often only the outer surface of the sample can be investigated (Katzhendler et al.; 2000b). To overcome these limitations, low frequency spectrometers have been developed. The penetration depth is increased to e.g. 16mm for L-band spectrometers (1GHz) (Mäder et al.; 1994) and direct in vivo studies of mice become possible (Lurie et al.; 2005; Saito et al.; 1997). A decrease in frequency goes along with a lower splitting of the energy levels which results in a remarkable decrease in sensitivity (Mäder et al.; 1994).