Set-up for time-resolved step-scan FTIR spectroscopy of noncyclic reactions

Abstract

We have established a novel technique, which allows the application of time-resolved step-scan FTIR difference spectroscopy on noncyclic reactions. Cyclic reactions are ideally suited for the step-scan technique. However, it is difficult to apply the step-scan technique to noncyclic reactions, because the investigated process has to be repeated at about 1000 sampling positions of the interferogram. Consequently, to investigate noncyclic systems the sample has to be renewed at every sampling position. In the presented novel approach the IR-beam and the excitation laser-beam are focused to a very small diameter of 200 μm. Thereby, only a small segment of the sample, which has an overall diameter of 15 mm, is excited and probed. By moving the sample, which is mounted on an x-y-stage, to different nonexcited segments the reaction can be repeated until a complete interferogram data set is recorded. In so far as the typically used flow cells are concerned their optical pathlength is too large to perform difference spectroscopy. We use 4 μm thin films to depress the water background absorption of biological samples. As test, the well known photo-cyclic reactions of bacteriorhodopsin are measured. No systematic errors appear in the difference spectra. Because of intensity loss by the IR-microscope the signal-to-noise ratio is about 5 times less as compared to conventional step-scan measurements. For the first time, the technique is then applied to a noncyclic reaction, the photolysis of caged ATP. The successful performance with 10 μs time-resolution now opens the door for many new applications of step-scan FTIR measurements to noncyclic reactions.