Time-resolved step-scan FT-IR spectroscopy: Focus on multivariate curve resolution

The present paper describes the application of step-scan FT-IR spectroscopy in combination with chemometric analysis of the spectral data for the study of the photocycle of bacteriorhodopsin. The focus is on the performance of this instrumentation for time-resolved experiments. Three-dimensional data-spectra recorded over time-are studied using various factor analysis techniques, e.g., singular values decomposition, evolving factor analysis, and multivariate curve resolution based on alternating least squares. Transient intermediates formed in the time domain ranging from 1 mus to 6.6 ms are clearly detected through reliable pure time evolving profiles. At the same time, pure difference absorbance spectra are provided. As a result, valuable information about transitions and dynamics of the protein can be extracted. We conclude first that step-scan FT-IR spectroscopy is a useful technique for the direct study of difficult photochemical systems. Second, and this is the essential motivation of this paper, chemometrics provide a step forward in the description of the photointermediates.