Infrared absorbance spectroscopy of aqueous proteins: comparison of transmission and ATR data collection and analysis for secondary structure fitting

Marco Pinto Corujo, Meropi Sklepari, Dale L. Ang, Mark Millichip, Andrew Reason, Sophia C. Goodchild, Paul Wormell, Don Praveen Amarasinghe, Viv Lindo, Nikola P. Chmel, Alison Rodger*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    17 Citations (Scopus)
    62 Downloads (Pure)


    Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path-length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers-Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm−1 and 1700 cm−1 and a self-organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.

    Original languageEnglish
    Pages (from-to)957-965
    Number of pages9
    Issue number8
    Publication statusPublished - Aug 2018
    EventInternational Conference on Chiroptical Spectroscopy (16th : 2017) - Rennes, France
    Duration: 11 Jun 201715 Jun 2017
    Conference number: 16th

    Bibliographical note

    Copyright the Author(s) 2018. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.


    • attenuated total reflectance
    • ATR correction
    • refractive index
    • depth of penetration


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