Abstract
Absorption spectroscopy of a solution containing an analyte solute typically uses the transmission through a solvent as a reference based on the assumption that the solvent does not absorb light. However, when a solvent absorbs light, the resulting absorbance becomes lower and often negative because of the reduced number of solvent molecules due to the space taken by solute molecules. The solvent exclusion (SE) effect is problematic for accurate quantitation and analysis across various absorption spectroscopies, especially near the broad, strong water absorption peaks. The commonly practiced method of subtracting a scaled reference water spectrum seems ideal; however, this requires extensively broad spectra that are not available with the combination of flow cell chamber thickness and currently available high-intensity laser sources. In this work, we present a simple volumetric SE correction method using partial specific volume (PSV) to successfully retrieve the solute-only absorption spectrum of globular proteins in aqueous solutions. We demonstrate this method with spectra acquired with an in-house-developed, state-of-the-art quantum cascade laser (QCL) spectroscopy system with solvent absorption compensation (SAC) for unprecedented molecular sensitivity. Additionally, we discuss the effectiveness of this simplified PSV-based SE correction for globular solutes and small-molecule solutes using a model consisting of a solvation shell and a solute core.