An instrument has been developed to acquire polarized attenuated total internal reflection (ATIR) mid-IR spectra of supported monomolecular films <i>in situ</i> at the lipid/water (L/W) interface. The monolayer film is applied to the treated surface of a trapezoidal germanium crystal as it is immersed into the subphase of a specially designed Langmuir-Blodgett trough. This approach offers several advantages over the study of monolayer films at the air/water (A/W) interface by external reflection techniques, including (1) an improved signal throughput and multiple reflections, both contributing to an improved signal-to-noise (S/N) ratio; (2) versatility with respect to the chemical nature of the surface; and (3) the ability to use standard spectral subtraction methods to compensate for absorption of the aqueous subphase. The enhanced sensitivity of this approach facilitates the study of <i>in situ</i> interactions of peptides with fully hydrated lipid membrane surfaces.

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