Abstract
Laser-based infrared (IR) spectroscopy is an emerging key technology for the analysis of solutes and for real-time reaction monitoring in liquids. Larger applicable pathlengths compared to the traditional gold standard Fourier transform IR (FTIR) spectroscopy enable robust measurements of analytes in a strongly absorbing matrix such as water. Recent advancements in laser development also provide large accessible spectral coverage thus overcoming an inherent drawback of laser-based IR spectroscopy.In this work, we benchmark a commercial room temperature operated broadband external cavity-quantum cascade laser (EC-QCL)-IR spectrometer with a spectral coverage of 400 cm−1 against FTIR spectroscopy and showcase its application for measuring the secondary structure of proteins in water, and for monitoring the lipase-catalyzed saponification of triacetin. Regarding the obtained limit of detection (LOD), the laser-based spectrometer compared well to a research-grade FTIR spectrometer employing a liquid nitrogen cooled detector. With respect to a routine FTIR spectrometer equipped with a room temperature operated pyroelectric detector, a 15-fold increase in LOD was obtained in the spectral range of 1600–1700 cm−1. Characteristic spectral features in the amide I and amide II region of three representative proteins with different secondary structures could be measured at concentrations as low as 0.25 mg mL−1. Enzymatic hydrolysis of triacetin by lipase was monitored, demonstrating the advantage of a broad spectral coverage for following complex chemical reactions. The obtained results in combination with the portability and small footprint of the employed spectrometer opens a wide range of future applications in protein analysis and industrial process control, which cannot be readily met by FTIR spectroscopy without recurring to liquid nitrogen cooled detectors.
Highlights
Mid-infrared spectroscopy is a powerful and versatile technique for the analysis of structure and dynamics of polypeptides and proteins [1,2,3]
Fourier-transform IR (FTIR) spectrometers are commonly equipped with thermal light sources such as Globars that emit broadband and constant but rather low power radiation across the mid-IR range
For mid-IR spectroscopy of proteins, there is the pronounced challenge of the HOH-bending band of water near 1643 cmÀ1 that overlaps with the protein amide I band
Summary
Mid-infrared (mid-IR) spectroscopy is a powerful and versatile technique for the analysis of structure and dynamics of polypeptides and proteins [1,2,3]. Vibrations of the polypeptide repeat unit result in nine characteristic group frequencies in the mid-IR region that are referred to as amide bands. For mid-IR spectroscopy of proteins, there is the pronounced challenge of the HOH-bending band of water near 1643 cmÀ1 that overlaps with the protein amide I band. The optical path is restricted to < 10 mm for FTIR transmission measurements in order to avoid total IR absorption [2,3] These short path lengths lead to impaired sensitivity because of the lower band absorbances and limited robustness due to higher probability of cell clogging
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