Infra-red spectroscopy

Bruker Equinox 66 FT-IR spectrometer with thermostated, purged sample compartment.

Infra-red spectroscopy, together with Raman spectroscopy, is a powerful vibration technique that allows to observe directly vibrations of individual atoms and groups of atoms. This technique is based on the ability of matter to absorb a very low energy photons (heat) whose frequency matches internal vibration of molecules.

IR spectroscopy is a very inclusive technique because it relies on direct absorption independent of electronic (Raman) or spin (EPR) resonance. As the result a vast range range of vibrations can be detected and analyzed, including those that are not associated with particular chromophore.

We use IR spectroscopy as a probe to study molecular changes that occur in biological samples during metalloradical catalysis. As with other techniques, the range of samples vary from redox active enzymes to model proteins to simple, isolated redox elements and components. Currently we are particularly interested in vibration al changes associated with oxidation of amino acid side chains as opposed to protonation/deprotonation events, and redox transitions of common cofactors, such as hemes and their ligands.

Because majority of bi logical catalysis takes place in aqueous environment, most of our IR measurements are done in water-based solvents. IR water absorption requires that most aqueous samples are measured using a reaction-induced difference method, where IR spectra before and after the reaction are compared revealing only the vibrations that undergo changes during the reaction. This method allows us to increase sensitivity dramatically compared to absolute absorption, but imposes additional requirements, such as very short sample pathlengths (0.01 mm) to reduce absorption by water. To keep background stable we maintain stringent temperature control (>0.01^oC) and avoid mechanical perturbations.

Janis cryostat in the IR beam with auxiliary optical fiver UV/Vis absorption probe.

The need of thermal and mechanical stability requires us to use non-invasive methods of reaction initiation, i.e. without mixing in reagents. Photolysis using various sources of visible and UV light is a common method in reaction-induced difference spectroscopy, such as in ligand dissociation measurements. For vibrational measurements on transient forms, such as reactive radical species, IR spectroscopy can be combined with mid- and far-UV photolysis and cryogenic immobilization in frozen solutions. Furthermore, such IR measurements can be simultaneously with electronic absorption measurements using optical fiber sampling system. Such combinational approach facilitates optimization of IR measurements and allows to carry out direct correlation between optical and vibrational assignments.

Thin layer transmission electrochemical cell in the infrared beam.

Electrochemistry is an attractive alternative to photolysis in controlling redox reactions. Recently we undertook to advance the practical usability of this approach by developing a novel thin layer transmission electrochemical cell. Besides a significant increase in stability and reliability, the X1 cell provides great flexibility by using most available solid electrode materials from modified gold mesh to optically transparent electrodes. Boron-doped diamond film electrodes on Si substrate, continuously developed by Swain's group, are particularly beneficial for biological IR applications due to small background absorption in the infra-red region and robust electrochemical properties.