Evanescent electric field amplitudes in thin lipid films for internal reflection infrared spectroscopy

Internal reflection infrared spectroscopy is a commonly used technique for determining the conformation and orientation of polypeptides in thin films such as supported lipid membranes. Accurate determinations of molecular orientation by this technique depend on accurate calculations of evanescent electric field amplitudes. These calculations, in turn, depend on film thickness and assumptions about the dielectric behavior within such films. Different assumptions lead to either two-phase or three-phase approximations for calculating electric field amplitudes, and both approximations are widely used. To test the accuracy of these approximations, we have examined a series of trisubstituted benzenes for their suitability as an internal orientation reference standard. We find that 1,3,5-tri-tert-butyl-benzene is absorbed into supported lipid membranes and exhibits an infrared absorption dichroism that is well described by the two-phase approximation but is inconsistent with the three-phase approximation. These results, and an analysis of published results from other laboratories, suggest that the effective dielectric of lipid films does not remain constant as film thickness decreases from the thick film regime to the thin film regime. This renders the three-phase or "thin film" approximation inaccurate for monolayer films and has broad implications for the determination of molecular orientation in supported lipid membranes.