Implementation of simultaneous ultraviolet/visible and x-ray absorption spectroscopy with microfluidics.

X-ray spectroscopies are uniquely poised to describe the geometric and electronic structure of metalloenzyme active sites under a wide variety of sample conditions. UV/Vis (ultraviolet/visible) spectroscopy is a similarly well-established technique that can identify and quantify catalytic intermediates. The work described here reports the first simultaneous collection of full in situ UV/Vis and high-energy resolution fluorescence detected x-ray absorption spectra. Implementation of a fiber optic UV/Vis spectrometer and parabolic mirror setup inside the dual array valence emission spectrometer allowing for simultaneous measurement of microfluidic flow and mixing samples at the Photon-In Photon-Out X-ray Spectroscopy beamline is described, and initial results on ferricyanide and a dilute iron protein are presented. In conjunction with advanced microfluidic mixing techniques, this will allow for the measurement and quantification of highly reactive catalytic intermediates at reaction-relevant temperatures on the millisecond timescale while avoiding potential complications induced by freeze quenching samples.