Quantitative Orientation Measurements in Thin Lipid Films by Attenuated Total Reflection Infrared Spectroscopy

Abstract

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 crystal. In this paper, polarized ATR spectra of single supported bilayers of the phospholipid dimyristoylphosphatidic acid covered by either air or water have been recorded and the dichroic ratio of the bands due to the methylene stretching vibrations has been calculated. The mean-square electric field amplitudes were calculated using three formalisms, namely the Harrick thin film approximation, the two-phase approximation, and the thickness- and absorption-dependent one. The results show that for dry bilayers, the acyl chain tilt angle varies with the formalism used, while no significant variations are observed for the hydrated bilayers. To test the validity of the different formalisms, s- and p-polarized ATR spectra of a 40-A lipid layer were simulated for different acyl chain tilt angles. The results show that the thickness- and absorption-dependent formalism using the mean values of the electric fields over the film thickness gives the most accurate values of acyl chain tilt angle in dry lipid films. However, for lipid monolayers or bilayers, the tilt angle can be determined with an acceptable accuracy using the Harrick thin film approximation. Finally, this study shows clearly that the uncertainty on the determination of the tilt angle comes mostly from the experimental error on the dichroic ratio and from the knowledge of the refractive index.

The First Studies of a Tetrathiafulvalene‐σ‐Acceptor Molecular Rectifier

“…The associated order parameters with respect to the film normal (P 2 ) were then calculated by using mean-square electric field amplitudes obtained from the Harrick thin-film equations. [27] The values of R ATR , P 2 , and the average angle g between the transition moment and the surface normal for the films transferred at the highest pressure (25 mN m À1 ) are given in Table 1 for the major bands. It is important to remember here that P 2 should be equal to zero for an isotropic sample (g = 54.98, the magic angle), and to one and À0.5 for perfect orientation of the transition moments parallel (g = 08) or perpendicular (g = 908) to the surface normal, respectively.…”

Application of Polarization Modulation Infrared Reflection Absorption Spectrosocpy Under Electrochemical Control for Structural Studies of Biomimetic Assemblies

“…These experimental challenges of in situ IRRAS were solved by using either a cell in Kretchmann configuration and the phenomenon of total reflection of the IR light [22] or a thin electrolyte layer cell for experiments performed under condition of the external reflection of the IR beam [16]. In the last years attenuated total reflection infrared spectroscopy (ATR IRS) [23][24][25][26][27][28] and IRRAS tech-niques [9,11,29,30] have developed into powerful methods to study in situ the structure and its changes in biomolecules present in various supramolecular assemblies on the electrode surfaces. Obviously studies of the structure and activity of biomolecules require a strict fulfilment of experimental conditions such as the composition, ionic strength and pH of the aqueous electrolyte solution, temperature or chemical nature of the used solid substrate.…”

Determination of Anisotropic Optical Constants and Surface Coverage of Molecular Films Using Polarized Visible ATR Spectroscopy. Application to Adsorbed Cytochrome c Films

“…An anisotropic extinction coefficient can affect the values one calculates for the electric field, which will ultimately affect the molecular anisotropy one determines for the dichroic layer. In addition to this limitation, transfer-matrix calculations presented in the literature for ATR applications 4,5 have focused mainly on the determination of the Cartesian components of the electric field, which are essentially used as auxiliary variables for subsequent calculations of either (a) the polarized absorbance using anisotropic optical constants determined (or known) by other means or (b) the anisotropic optical constants from polarized ATR data. In configurations where the thickness of the absorbing layer cannot be neglected, the electric field is no longer constant across this layer, and an average needs to be evaluated to obtain accurate absorbance results, which makes the overall calculation cumbersome as already pointed out by Axelsen.…”