Several advantages of the near-infrared (NIR) technique in the characterization of the secondary structure of regenerated <i>Bombyx mori</i> silk fibroin are demonstrated. Silk fibroin films with thicknesses ranging from 50 to 300 μm are suitable for the NIR measurement. The bands due to hydrogen-bonded water are independent in the NIR spectra and facilitate our investigation of the amide region. Analysis of the combination modes of amide groups in the NIR spectra could lead to a profile of conformation ratio of silk fibroin, which was supported by nuclear magnetic resonance (NMR) observations.

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