Fourier transform infrared (FTIR) spectroscopy


Fourier transform infrared (FTIR) spectroscopy is a highly valuable technique to monitor protein structure in the liquid and dried, e.g., lyophilized state.

FTIR spectra can be used to identify a wide range of compounds by comparing the measured spectra to spectral databases. For proteins, FTIR spectra from wavenumbers 1,700-1,500 cm-1 can be used to determine structural properties. Measuring protein absorbance over these wavenumbers gives two absorption bands, conventionally called Amide I and Amide II and lying between wavenumbers 1,700 - 1,600 cm-1 and 1,600 - 1,500 cm-1, respectively. 

The Amide I band is due to C=O stretching vibrations of the peptide bonds, which are modulated by the secondary structure (α-helix, β-sheet, etc.). Secondary structural content can be obtained by comparing the measured spectra to the spectra obtained for proteins with known secondary structures.

The Amide II band is due to C-N stretching vibrations in combination with N-H bending. Amide II absorbance can be used, for instance, to report on protein unfolding based on the extent of hydrogen (H) exchanged for deuterium (D) in H-D exchange experiments.

Water can interfere with FTIR measurements of protein samples, because it strongly absorbs in the Amide I region. Consequently, FTIR is best suited for lyophilized (freeze-dried) protein samples. Nevertheless, measurements can be obtained for protein samples in solution, but a relatively high (typically >5 mg/mL) protein concentration is required. Both liquid (transmission, attenuated total reflection (ATR)) and solid (ATR) samples can be analyzed at Coriolis Pharma.

Contact us

Need more information? Contact our experts with your questions today.