A quantum cascade laser setup for studying irreversible photoreactions in H2O with nanosecond resolution and microlitre consumption

Abstract

Time-resolved infrared spectroscopy on irreversible reactions requires in general an exchange of sample for thousands of acquisitions leading to high sample consumption. Here, we present a setup employing a modern quantum cascade laser (QCL) as a probe light source to record time-resolved difference spectra of irreversible photoreactions in H₂O. The combination of the focused QCL with a pressure-tolerant flow cell and a micrometre stage orthogonal to the flow allowed us to drastically reduce the sample consumption. We investigated the irreversible photoreduction of the cofactor flavin mononucleotide (FMN) in H₂O, which is a common reaction taking place in biological photoreceptors. A broad time range from 20 nanoseconds to 1 second was accessible, because the approach minimized any signal drift by the flow. Kinetics were recorded at 46 selected wavenumbers consuming 12 microlitres for a complete dataset. The tuning range of 1490–1740 cm⁻¹ included relevant carbonyl vibrations and the region of strong water absorption at around 1650 cm⁻¹. A continuous dataset in the spectral dimension was generated by applying a fit with a sum of Lorentzians. Subsequent global analysis allowed us to resolve reference spectra and kinetics of the photoreaction proceeding from the triplet excited state via the intermediate flavin anion radical to the product, the fully reduced state of FMN. Accordingly, the neutral radical state is not populated in the disproportionation. The approach strongly facilitates the spectroscopic access to irreversible reactions of flavin-containing photoreceptors and photoenzymes with high time resolution and small sample consumption.