Multi-Pathlength Mid-IR Spectroscopy of Proteins in Aqueous Solutions Using an External Cavity Quantum Cascade Laser

Mid-infrared spectroscopy is a powerful technique for probing molecular structure and composition, yet its application to aqueous systems remains limited by strong solvent absorption and the dynamic range of mid-IR detectors, which restrict the usable optical pathlength. Here, we present a quantum cascade laser-based spectroscopic setup that enables simultaneous acquisition of absorption spectra measured in transmission across a continuum of optical pathlengths using a custom-made wedge-shaped flow cell and a pyroelectric detector array. This design eliminates the need for mechanical adjustments or sample dilution, allowing direct analysis of complex liquid samples. We introduce the pathlength-to-noise ratio as a novel metric for determination of the optimal pathlength region for every wavenumber. By weighing detector signals based on this ratio, we achieve robust integration of multi-pathlength data into a single coherent mid-IR absorption spectrum. The system was validated using aqueous protein solutions of bovine serum albumin and β-lactoglobulin in the range of 1 to 25 mg mL^-1. Characteristic amide I and II bands were successfully resolved, and secondary structure differences were detected. This work establishes a flexible approach to mid-IR absorption spectroscopy, aiming to eliminate the limitations of detectors with a small dynamic range and to get a better understanding of the optimal pathlength for complex samples.