Improved IRRAS Apparatus for Studies of Aqueous Monolayer Films: Determination of the Orientation of Each Chain in a Fatty-Acid Homogeneous Ceramide 2

Abstract

The performance of a new accessory for measuring IR reflection-absorption (IRRAS) spectra at various angles of incidence from monolayer films of lipids is demonstrated. The device is comprised of an external variable angle reflectance accessory and a self-contained computer-controlled unit for varying the angle of incidence, both units being coupled to a custom-designed Langmuir trough. This combination provides for excellent experimental precision and reduction of interference from H₂O vapor in the 1500–1800 cm⁻¹ spectral region. A set of ∼20 s- and p-polarized IRRAS spectra over a range of angles of incidence from 25° to 70° may be acquired from a single monolayer film in ∼3 h. The performance of the device is demonstrated for two lipids as follows. First, the instrument precision is demonstrated through determination of the orientation of each of the chains in a fatty acid homogeneous ceramide 2 derivative. Interestingly, the orientation (tilt angles from the bilayer normal) of the individual chains differ, as determined from an isotopically-labeled derivative where the fatty acid chain is perdeuterated. The fatty acid chain is tilted at 24° from the normal to the bilayer, while the base chain is tilted at 18°. As a control, the average tilt angle for both ceramide 2 acyl chains has been determined from a fully proteated derivative to be 20°. The precision of the derived tilt angle varies from ∼1–3°. Second, the reduction of interference from water vapor in the 1500–1800 cm⁻¹ is demonstrated for sn-2–¹³C=O-1,2 distearoylphosphatidylcholine, where spectra from the individual C=O bonds in the molecule are readily detected with good signal/noise and signal/water vapor ratios. The device provides a major advance in the determination of chain orientation by IRRAS and will facilitate the determination of functional group orientation.