Title: External cavity-quantum cascade laser spectroscopy for mid-IR transmission measurements of proteins in aqueous solution
Language: English
Authors: Alcaráz, Mirta Raquel 
Schwaighofer, Andreas  
Kristament, Christian 
Ramer, Georg  
Brandstetter, Markus 
Goiccoechea, Héctor 
Lendl, Bernhard  
Category: Research Article
Issue Date: 2015
Journal: Analytical Chemistry
ISSN: 1520-6882
In this work, we report mid-IR transmission measurements of the protein amide I band in aqueous solution at large optical paths. A tunable external-cavity quantum cascade laser (EC-QCL) operated in pulsed mode at room temperature allowed one to apply a path length of up to 38 μm, which is four times larger than that applicable with conventional FT-IR spectrometers. To minimize temperature-induced variations caused by background absorption of the ν2-vibration of water (HOH-bending) overlapping with the amide I region, a highly stable temperature control unit with relative temperature stability within 0.005 °C was developed. An advanced data processing protocol was established to overcome fluctuations in the fine structure of the emission curve that are inherent to the employed EC-QCL due to its mechanical instabilities. To allow for wavenumber accuracy, a spectral calibration method has been elaborated to reference the acquired IR spectra to the absolute positions of the water vapor absorption bands. Employing this setup, characteristic spectral features of five well-studied proteins exhibiting different secondary structures could be measured at concentrations as low as 2.5 mg mL−1. This concentration range could previously only be accessed by IR measurements in D2O. Mathematical evaluation of the spectral overlap and comparison of second derivative spectra confirm excellent agreement of the QCL transmission measurements with protein spectra acquired by FT-IR spectroscopy. This proves the potential of the applied setup to monitor secondary structure changes of proteins in aqueous solution at extended optical path lengths, which allow experiments in flow through configuration.
DOI: 10.1021/acs.analchem.5b01738
Library ID: AC15520864
URN: urn:nbn:at:at-ubtuw:3-7633
Organisation: E164 - Institut für Chemische Technologien und Analytik 
Publication Type: Article
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