Stopped flow apparatus for time-resolved Fourier transform infrared difference spectroscopy of biological macromolecules in (H2O)-H-1

Stopped flow spectroscopy is an established technique for acquiring kinetic data on dynamic processes in chemical and biochemical reactions, and Fourier transform infrared (FT-IR) techniques can provide particularly rich structural information on biological macromolecules. However, it is a considerable challenge to design an FT-IR stopped flow system with an optical path length low enough for work with aqueous ((H2O)-H-1) solutions. The system presented here is designed for minimal sample volumes (similar to5 muL) and allows simultaneous FT-IR rapid-scan and VIS measurements. The system employs a micro-structured diffusional mixer to achieve effective mixing on the millisecond time scale under moderate flow and pressure conditions, allowing measurements in a cell path length of less than 10 mum. This makes it possible to record spectra in (H2O)-H-1 solutions over a wide spectral range. The system layout is also designed for a combination of kinetic and static measurements, in particular to obtain detailed information on the faster spectral changes occurring during the system dead time. A detailed characterization of the FTIR stopped flow system is presented, including a demonstration of the alkaline conformational transition of cytochrome c as an example.