External reflection Fourier transform infrared spectroscopy has been applied to the <i>in situ</i> measurement of the infrared spectra of insoluble phospholipid monolayer films that were spread at the air/water interface. The spreading conditions for the phospholipids [1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)] were chosen to mimic those of liquid-condensed and liquid-expanded surface films, respectively. It is shown that the infrared reflection spectra can identify vibrations due to the hydrocarbon acyl chains, carbonyl ester, and phosphate groups for these monolayer films on water. Detailed examination of the frequencies and polarization properties of the C-H stretching region shows that the liquid-condensed DSPC acyl chains are in a rigid, mostly all-trans configuration, while the liquid-expanded DMPC acyl chains are highly fluid. The spectra demonstrate that the external reflection FT-IR experiment can differentiate between the physical conformation of monomolecular films when water is used as a reflective substrate.

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