Improved IRRAS apparatus for studies of aqueous monolayer films: Determination of the orientation of each chain in a fatty-acid homogeneous ceramide 2

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 similar to 20 s- and p-polarized IRRAS spectra over a range of angles of incidence from 25 degrees to 70 degrees may be acquired from a single monolayer film in similar to3 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 degrees from the normal to the bilayer, while the base chain is tilted at 18 degrees. As a control, the average tilt angle for both ceramide 2 acyl chains has been determined from a fully proteated derivative to be 20 degrees. The precision of the derived tilt angle varies from similar to1-3 degrees. Second, the reduction of interference from water vapor in the 1500-1800 cm(-1) is demonstrated for sn-2-C-13=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.