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 lipid s is demonstrated. The device is comprised of an external variable angle re flectance accessory and a self-contained computer-controlled unit for varyi ng the angle of incidence, both units being coupled to a custom-designed La ngmuir trough. This combination provides for excellent experimental precisi on and reduction of interference from H2O vapor in the 1500-1800 cm(-1) spe ctral 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 acquire d from a single monolayer film in similar to3 h. The performance of the dev ice is demonstrated for two lipids as follows. First, the instrument precis ion is demonstrated through determination of the orientation of each of the chains in a fatty acid homogeneous ceramide 2 derivative. Interestingly, t he orientation (tilt angles from the bilayer normal) of the individual chai ns differ, as determined from an isotopically-labeled derivative where the fatty acid chain is perdeuterated. The fatty acid chain is tilted at 24 deg rees from the normal to the bilayer, while the base chain is tilted at 18 d egrees. As a control, the average tilt angle for both ceramide 2 acyl chain s has been determined from a fully proteated derivative to be 20 degrees. T he precision of the derived tilt angle varies from similar to1-3 degrees. S econd, 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.