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M. Alcaraz, A. Schwaighofer, C. Kristament, G. Ramer, M. Brandstetter, H. Goicoechea, B. Lendl:
"External-Cavity Quantum Cascade Laser Spectroscopy for Mid-IR Transmission Measurements of Proteins in Aqueous Solution";
Analytical Chemistry, 87 (2015), 13; 6980 - 6987.

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.

http://dx.doi.org/10.1021/acs.analchem.5b01738