Photolysis

Light is what makes the world colorful and allows us to see its splendor. What we perceive as difference in color originate from difference in the energy of photons that travel through space and matter. As visually pleasant as sunshine can be, each photon in the visible spectrum carries significant amount of energy, which varies from about 3 eV for violet light to 2 eV for red light. For comparison this energy is higher than energy of all but most stable chemical bonds. When absorbed by a chemical or an organism, the energy of a photon can be quite significant and lead to a variety of photochemical reaction - chemical reactions due to excitation by a photon.

A narrow-band UV photolysis light produces blue fluorescence in frozen glassy sample.

Luckily, light visible to the human eye does not cause significant photochemistry in living organisms, mostly because few molecules can absorb it. Heme proteins in animals and chlorophylls in plants are the most common exceptions. Ultra-violet light, however, carries higher energy - between 3 eV and 7 eV for near to far UV light - and can be readily absorbed by biological materials leading to host of photochemical reactions from sunburn to mutations and cancer. Energy of UV light is sufficient to eject an electron from the outer shell of amino acids and nucleotides generating highly reactive radical species. At room temperature radicals readily engage in uncontrolled reactions leading to irreversible chemical alterations.

Light is a potent chemical agent which can initiate chemical reactions in predictable and controlled manner. Key advantages of light over conventional chemicals are that light are that light can be delivered to a solid sample quickly, uniformly, and non-invasively. These properties of light make it an ideal tool for studying and manipulating various chromophores, including biological systems.

We use light extensively throughout our studies on metalloradical enzymes and models. In addition to spectroscopic sampling, we use light to initiate and control chemical reactions. We use visible light to photolyze relatively low energy bonds of strong chromophores, such as ligand dissociation from iron ion in hemes. In some cases ligand dissociation can be reversible (O2 or CO dissociation in globins), in other cases photodissociation can be followed by a sequence of transient steps (O2 reaction with oxidase). In these examples light plays a role of a stimuli followed by other chemical events.

In cryogenic photolysis time is defined only by exposure. Between exposures sample can be studied as thoroughly as necessary

Higher energy, ultra-violet light can be used to initiate more difficult chemistry involving higher energy bonds. A common UV photolytic reaction involves photon ejection from a chromophore with concomitant formation of a radical. This reaction can be carried out at room as well as at cryogenic conditions. At low temperatures where all processes associated with diffusion stop, light becomes the sole factor controlling progress of the reaction. Cryogenic reaction no longer occurs in real time, but is controlled by exposure to photolytic light and stops in the dark. This allows great flexibility in controlling reaction rates and thorough examination of sample between exposures.

Photomax light source with Hg/Xe arc lamp provides highest light density for photolysis.

We use several light sources depending on experimental needs. Most photolytic experiments are carried our using broad, continuous light sources. For photolysis across visible region and near- to mid-UV regions we use output of Hg/Xe arc lamp which produced high density source with broad spectrum. For mid- to far UV regions (below 260 nm) we use high-power deuterium light source, whose output increases at shorter wavelengths until it reaches transmission limit of quartz at ~180nm. Both of our light sources are equipped with dispersion optics that allows us to select a 5-50 nm narrow band spectral line to match absorption of a particular chromophore. Light sources are equipped with several shutters which are controlled by computer and are automatically timed and synchronized to data acquisition.

Dispersion optics selects desired wavelength from the spectrum of arc lamp while shutters control timed exposure.

Other light sources include pulsed and continuous wave lasers with output form far red to UV region.