Recently developed dynamic step-scan FT-IR spectroscopy combined with software-based digital signal processing (DSP) was applied for infrared rheo-optical measurements of isotactic polypropylene and regenerated <i>Bombyx mori</i> silk fibroin film undergoing sinusoidal mechanical strain. Comparative measurements of dynamic infrared spectra of polypropylene film using the DSP-based method and two lock-in amplifiers (LIAs) indicated that a high signal-to-noise ratio (SNR) advantage is attained by the DSP method compared with the LIA method. With the DSP technique, dynamic spectra of <i>Bombyx mori</i> silk fibroin film with high SNR were successfully recorded in the whole mid-infrared region in only 30 min. The dynamic spectra revealed that stress-induced dynamic reorientation in fibroin film is mainly localized in the segment with β-sheet conformation and is almost synchronous with the applied mechanical strain. Two-dimensional (2D) correlation analyses of the dynamic spectra showed that (1) the broad amide I band is resolved into three components whose positions are dependent on secondary structures, (2) the dynamic behavior of tyrosine residues was extracted by separation of the feature due to its aromatic side chains from the broad envelope of the amide I band in the 2D asynchronous map, and (3) the dynamic behavior of the amide A and B modes is the same as that of the amide II mode, when the mechanical strain is applied to fibroin film.

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