Quantitative orientation measurements in thin lipid films by attenuated total reflection infrared spectroscopy

Quantitative orientation measurements by attenuated total reflectance (ATR) infrared spectroscopy require the accurate knowledge of the dichroic ratio and of the mean-square electric fields along the three axes of the ATR cry stal. In this paper, polarized ATR spectra of single supported bilayers of the phospholipid dimyristoylphosphatidic acid covered by either air or wate r have been recorded and the dichroic ratio of the bands due to the methyle ne stretching vibrations has been calculated. The mean-square electric fiel d amplitudes were calculated using three formalisms, namely the Harrick thi n film approximation, the two-phase approximation, and the thickness- and a bsorption-dependent one. The results show that for dry bilayers, the acyl c hain tilt angle varies with the formalism used, while no significant variat ions are observed for the hydrated bilayers. To test the validity of the di fferent formalisms, s- and p-polarized ATR spectra of a 40-Angstrom lipid l ayer were simulated for different acyl chain tilt angles. The results show that the thickness- and absorption-dependent formalism using the mean value s of the electric fields over the film thickness gives the most accurate va lues of acyl chain tilt angle in dry lipid films. However, for lipid monola yers or bilayers, the tilt angle can be determined with an acceptable accur acy using the Harrick thin film approximation. Finally, this study shows cl early that the uncertainty n the determination of the tilt angle comes most ly from the experimental error on the dichroic ratio and from the knowledge of the refractive index.