Use of polarized infrared external reflection spectroscopy to study phospholipid monolayers at the air-water interface

The pressure dependence of phospholipid monolayers at the air-water interface has been investigated using polarized infrared external reflection spectroscopy under carefully controlled environmental conditions. Results show that a splitting of the methylene symmetric and antisymmetric stretching vibrations is observed in the polarized spectra that has not been previously reported for unpolarized monolayer spectra. The splitting of the C-H bands results in sub-bands identifiable with ordered and disordered conformations. The intensities of these sub-bands correlate the particular thermodynamic state of the monolayer. The splitting of these bands qualitatively tracks the formation of these domains upon compression as previously seen in epiflourescence microscopy. As the surface pressure of the phospholipid monolayer film increases, the band resulting from the liquid domain structures in the film disappears. Curve fitting based on these results indicates that we can now determine the mole fraction of either solid or liquid phase domains as a function of surface pressure. These results have been observered at both 30 and 60 degree angles of incidence. The splitting of the methylene hydrocarbon vibration, and the presence of individual bands corresponding to particular conformational states has not previously been observed in polarized infrared external reflection spectra of monolayers at the air-water interface.