Time-Resolved FT-IR Absorption Spectroscopy Using a Step-Scan Interferometer

TLDR

The study implements time‑resolved step‑scan FT‑IR spectroscopy on a commercial interferometer, enabling full‑reaction coverage that facilitates difference‑spectra analysis and extraction of absorbance time courses at selected wavenumbers. The method was validated on bacteriorhodopsin, showing sub‑0.01 absorbance‑unit spectral changes, outperforming conventional flash‑photolysis by providing multiplexed monitoring, with mirror stability better than ±1.5 nm ensuring reliable detection of small changes.

Abstract

The implementation of time-resolved step-scan FT-IR spectroscopy with a commercial interferometer is described. With the use of the photo-reaction of the biological system bacteriorhodopsin as an example which exhibits infrared spectral changes smaller than 10 −2 absorbance units, the quality of the method is demonstrated. A comparison with conventional flash-photolysis experiments with a monochromatic infrared monitoring beam clearly demonstrates the multiplex advantage. The advantage of covering the total time course of the reaction allows for a variety of data analysis, such as forming difference spectra between intermediates of the reaction and the deduction of time courses of absorbance changes at selected wavenumbers. The mirror stability is better than ±1.5 nm, which is sufficient for the reliable measurement of small absorbance changes.