A Study of Binary Phospholipid Mixtures at the Air-Water Interface Using Infrared Reflection-Absorption Spectroscopy and 2D IR âν Correlation Analysis†

Infrared reflection-absorption spectroscopy (IRRAS) in combination with isotopic substitution and a modified 2D IR correlation analysis procedure were used to study the dynamics of phospholipid acyl chain structure in both an ideal and a nonideal binary monolayer mixture spread at the air-water (A/W) interface. The ideal mixture consisted of a 1:2 molar ratio of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC-d62), whereas the nonideal mixture consisted of a 1:2 molar ratio of 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (DLiPC) and DPPC-d62. The IRRAS spectra were interpreted using standard 2D IR methods as well as a modified two-dimensional correlation analysis called âν correlation analysis. In a âν correlation analysis, the effective phase angle (âe) is determined for the signal variations in a discrete set of dynamic spectra at each spectral frequency. The âe values are then used to quantitatively establish the relative rates of intensity change (i.e., the temporal relationships) as well as the degree of coherence between signal variations. In this study, similar âe values are observed for the methyl and the methylene stretching bands in the DPPC component of the ideal mixture. This same behavior is observed for the methyl and the methylene stretching bands of the DPPC-d62 component in the ideal mixture and the DPPC-d62 component in the nonideal mixture. In contrast, dissimilar âe values are observed for the methyl and the methylene stretching bands of the DLiPC component of the nonideal mixture. This observation can be explained by considering the location of gauche defects in the acyl chains of the individual components. As the mixed monolayer is compressed, conformational ordering takes place throughout the acyl chains of DPPC and DPPC-d62, whereas ordering in the condensed phase DLiPC component is localized near the terminal methyl groups of the acyl chains. Methyl (2940 cm-1) and methylene (∼2890 cm-1) Fermi resonance bands are clearly resolved from the fundamental bands in the C-H stretching region of the âν plot for the nonideal mixture. Average âe values for the symmetric and antisymmetric methylene stretching vibrations were used to determine the relative rates of conformational ordering in the individual components of the two monolayer systems. The relative rates of ordering are summarized as DPPC in the ideal mixture (âe ) 220°) ≈ DPPCd62 in the ideal mixture (âe ) 218°) > DPPC-d62 in the nonideal mixture (âe ) 194°) > DLiPC in the nonideal mixture (âe ) 170°). This study demonstrates for the first time that methyl stretching modes in IRRAS spectra can be used as in situ indicators of structural dynamics in the acyl chains of a Langmuir monolayer.