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Electron Paramagnetic Resonance (EPR) Electron Spin Resonance (ESR)

M s = +½. M s ±½. D B pp. D E=h n =g b B. Energy. M s = -½. B = 0. B > 0. Magnetic Field (B). h Planck’s constant 6.626196 x 10 -27 erg.sec n frequency (GHz or MHz) g g-factor (approximately 2.0) b Bohr magneton (9.2741 x 10 -21 erg.Gauss -1 ) B magnetic field (Gauss or mT).

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Electron Paramagnetic Resonance (EPR) Electron Spin Resonance (ESR)

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  1. Ms = +½ Ms ±½ DBpp DE=hn=gbB Energy Ms = -½ B = 0 B > 0 Magnetic Field (B) h Planck’s constant 6.626196 x 10-27 erg.sec n frequency (GHz or MHz) g g-factor (approximately 2.0) b Bohr magneton (9.2741 x 10-21 erg.Gauss-1) B magnetic field (Gauss or mT) Electron Paramagnetic Resonance (EPR) Electron Spin Resonance (ESR) Electron Magnetic Resonance (EMR) EPR ~ ESR ~ EMR What is EPR? • hn = gbB • = (gb/h)B = 2.8024 x B MHz • for B = 3480 G n = 9.75 GHz (X-band) • for B = 420 G n = 1.2 GHz (L-band) • for B = 110 G n = 300 MHz (Radiofrequency) EPR is the resonant absorption of microwave radiation by paramagnetic systems in the presence of an applied magnetic field

  2. Electron S (½) Nucleus I (½) Ms MI +½ +½ -½ MS=±½ DE1 DE2 B -½ -½ +½ DE1 = gbB + a/2 DE2 = gbB - a/2 DE1 – DE2 = a Hyperfine Coupling a “doublet” E = gbBSz + (hA0)SzIz E = gbBSz + (a)SzIz (hA0 (Hz) -> a (G) via g-factor) Selection Rule DMS = ±1 (electron) DMI = 0 (nuclear)

  3. Electron S (½) Nucleus I (1) Ms +½ MS=±½ B -½ DE1 = gbB + a DE2 = gbB DE3 = gbB - a Hyperfine Coupling MI +1 a +0 -1 DE1 DE2 DE3 -1 “triplet” +0 +1 E = gbBSz + (hA0)SzIz E = gbBSz + (a)SzIz (hA0 (Hz) -> a (G) via g-factor) Selection Rule DMS = ±1 (electron) DMI = 0 (nuclear)

  4. Resonance position (g-factor) • Multiplet structure (hyperfine network) • Line-shape (line-width, symmetry, etc) • Intensity (amplitude/area) serve as “finger prints” in the identification & quantitation of the radical under investigation!

  5. What do we do with EPR? We can detect & measure free radicals and paramagnetic species • High sensitivity (nanomolar concentrations) • No background • Definitive & quantitative Direct detection primary species, detected as is e.g.: semiquinones, nitroxides, trityls primary species are detected intact as spin-adduct, ‘spin-trapping’ Species: superoxide, hydroxyl, alkyl, NO Spin-traps: DMPO, PBN, DEPMPO, Fe-DTCs Indirect detection(secondary radicals) Spin-formation : hydroxylamines (Dikhalov et al) Spin-change : nitronylnitroxides (Kalyanaraman et al) Spin-loss : trityl radicals

  6. Can we use EPR to measure free radicals from biological systems (in vivo or ex vivo)? Yes! Radicals from intact tissues, organs or whole-body can be measured. But there is a catch! Biological samples are aqueous and undergo ‘non-resonant’ absorption of microwave energy (microwave cooking!) and hence poor penetration depth. The frequency of the instrumentation is reduced to overcome this problem! What is the optimum frequency? - depends on sample size

  7. What else can we do with EPR? Instead of “spying on free radicals”, we can use free radicals as “spying probes” to obtain functional information from biological systems • A known free radical probe is infused or injected into the animal • The change in the EPR line-shape profile, which is correlated to some physiological function, is then monitored. • The measurements can be performed in real-time and in vivo to obtain ‘functional information’.

  8. Oxygen, pO2 Acidosis, pH Viscosity Molecular motion width Splitting Redox status Cell viability Tissue perfusion amplitude Functional parameters from an EPR spectrum In vivo EPR spectroscopy is capable of providing useful physiologic and metabolic (functional) information from tissues Oxygen, pO2 Redox status Acidosis, pH Thiols (GSH) Cell viability Viscosity Tissue perfusion Molecular motion

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