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.

1Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy are analytical techniques employed for the analysis of protein secondary structure. The use of CD spectroscopy is limited to low protein concentrations (<2 mg ml −1 ), while FTIR spectroscopy is commonly used in a higher concentration range (>5 mg ml −1 ). Here we introduce a quantum cascade laser (QCL)-based IR transmission setup for analysis of protein and polypeptide secondary structure at concentrations as low as 0.25 mg ml −1 in deuterated buffer solution. We present dynamic QCL-IR spectra of the temperature-induced α-helix to β-sheet transition of poly-L-lysine. The concentration dependence of the α-β transition temperature between 0.25 and 10 mg ml −1 was investigated by QCL-IR, FTIR and CD spectroscopy. By using QCL-IR spectroscopy it is possible to perform IR spectroscopic analysis in the same concentration range as CD spectroscopy, thus enabling a combined analysis of biomolecules secondary structure by CD and IR spectroscopy.Structural analysis of biomolecules is of great importance in biology and biochemistry for characterising folding properties of proteins and polypeptides, as well as monitoring dynamic changes upon perturbation. Analytical methods used for investigation of biomolecule secondary structure include X-ray crystallography, nuclear magnetic resonance (NMR), circular dichroism (CD), as well as Raman and Fourier transform infrared (FTIR) spectroscopy 1 . Both NMR spectroscopy and X-ray crystallography are capable of providing structural information at atomic levels of resolution. However, NMR spectroscopy is restricted to relatively small biomolecules (≤ 40 kDa) at high concentrations and X-ray crystallography requires the availability of high-quality crystals of proteins, which is particularly demanding for membrane proteins [2][3][4] . In contrast, CD, Raman and FTIR spectroscopy are considered as low resolution techniques that provide overall structural information. Due to straightforward sample preparation and fast acquisition time, these methods are routinely used for rapid determination of secondary structure of proteins and for monitoring dynamic changes of protein structure. Due to their respective characteristics, infrared (IR) spectroscopy provides more dependable estimates of antiparallel β -sheets, whereas CD spectroscopy gives more confinable predictions of α -helix structures 5 . Regarding the complementary information provided, joint application of both methods would deliver most reliable results 6,7 . CD spectroscopy is a technique based on the difference in the absorption of the left-and right-handed circularly polarized light when it is in contact with the optically active compounds, or chromophores, present in the sample. In proteins, the most relevant chromophore is the amide group which absorbs in the far-UV region (180-240 nm). Their electronic transitions (n→ π*, π → π*) give signals at 220 and 190 nm. The periodic alignments of the amide groups in the polypeptide backbone lead to exciton co...