A detailed study is performed using infrared reflection absorption spectroscopy (IRRAS) to characterize the molecular behaviour of proteins at and near the air/water interface of protein solutions. IRRAS spectra of #-casein solutions in H2O and D2O show spectral shifts and derivative-like features not commonly observed in monomolecular layer systems. They can be fully understood using optical theory. Fair agreement between experimental and simulated IRRAS spectra over a broad spectral range (4000-1000 cm-1) is obtained using a stratified layer model. An attenuated total reflection and transmission spectrum is used to represent the protein extinction coefficient in H2O and D2O, respectively. It is shown that the derivative-like features observed result from the reflective properties of the proteins themselves. Furthermore, both concentration and film thickness could be fitted. At high protein concentrations (100 mg/mL) the spectrum is that of a single homogeneous protein solution. At 0.1 mg/mL, #-casein is accumulated at the surface in a thin layer of approximately 10 nm thickness, with a concentration about 2500 times higher than in the sub-phase. At an initial concentration of 10 mg/mL, the concentration in the surface layer is about 15 times higher than in the sub-phase, while the thickness is about 30 nm.
Meinders, M. B. J., van den Bosch, G. G. M., & de Jongh, H. H. J. (2001). Molecular properties of proteins at and near the air/water interface from IRRAS spectra of protein solutions. European Biophysics Journal, 30(4), 256-267. https://doi.org/10.1007/s002490000124