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The EBS department is equipped for spectroscopic characterization of biological free radicals and metalloprotein complexes using a combination of approaches, including electron paramagnetic resonance, vibrational, and magnetic circular dichroism spectroscopies.
Electron Paramagnetic Resonance (EPR) Spectroscopy
Electron Paramagnetic Resonance (EPR) spectroscopy provides detailed information on species containing unpaired electrons (including free radicals and transition metal ions). Distinct spectra associated with different paramagnetic species can be resolved in complex biological or environmental samples, contributing to their characterization. The EPR spectra of well-defined samples can yield information on identity (element type), electronic structure (including oxidation state assignment) and bonding (including identification of neighboring atoms through detection of superhyperfine interactions).
Instrumentation available for EPR spectroscopy within EBS includes a Bruker E-500 X-Band EPR spectrometer equipped with a Super X microwave bridge and a variety of resonators (both high sensitivity (SHQ) and dual mode (DM) resonators) and both an Oxford Instruments E-900 helium cryostat and a nitrogen flow system for temperature control (3.8-300 K). Optical access to the DM resonator allows photochemical generation of paramagnetic species for both kinetic and spectroscopic investigation. (Whittaker, Moënne-Loccoz)
Vibrational Spectroscopy (Raman and FTIR)
Vibrational spectroscopy provides information on molecular structures based on detection of vibrational modes through absorption (FTIR) or Raman scattering of light. The observed vibrational frequencies may be compared with spectra of model complexes or the results of theoretical calculations to define the geometric structure and bonding in molecules. These methods can be made specifically sensitive to individual components in a sample. For example, FTIR spectroscopy combined with photochemical excitation gives information on the structures of photolabile species. Resonance Raman spectroscopy gains selectivity from resonance enhancement of the Raman scattering for excitation within an absorption band of a chromophore.
Instrumentation available for vibrational spectroscopy within the EBS department includes a Perkin-Elmer System 2000 FTIR spectrometer equipped with helium cryostat allowing operation to 4 K, and a resonance Raman spectrometer based on a custom McPherson 2061/207 spectrograph (0.67-m focal length, 600 groove grating, 7 cm-1 spectral resolution) using Coherent Inova 302 krypton ion, argon ion and He/Cd ion lasers, Kaiser optical super-notch filters, and a Princeton Instruments (LN-1100PB) liquid nitrogen-cooled CCD detector. (Moënne-Loccoz)
Magnetic Circular Dichroism Spectroscopy
Magnetic circular dichroism (MCD) spectroscopy serves as a complementary approach to EPR spectroscopy. Whereas EPR spectroscopy detects microwave transitions within the electronic groundstate of paramagnetic molecules, MCD spectroscopy detects electronic transitions between ground and excited states for these species. In a strong magnetic field (generally in the range of several Tesla (1 Tesla = 10000 Gauss)), circular polarization is induced in these electronic transitions, allowing spectra associated with paramagnetic species (including metalloprotein complexes and metalloenzyme active sites) to be sensitively detected. The characteristic magnetic field and temperature dependence of the MCD spectra can yield electronic structural details, including information on ground state splittings.
Instrumentation available for MCD spectroscopy within the EBS department includes a custom spectrometer based on an Aviv Associates Model 41 DS scanning CD monochromator (190 � 2000 nm) covering the entire spectral range from the UV to the NIR with photomultiplier and InSb photovoltaic detection. Magnetic field perturbations are provided by an Oxford Instruments SM4-6T superconducting magnetocryostat (magnetic field range 0-6 T, temperature range 1.5-200 K). (Whittaker)
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