COMBINED INFRARED AND P-31 NMR SPECTROSCOPIC METHOD FOR DETERMINING THE FRACTIONAL COMPOSITION IN LANGMUIR-BLODGETT-FILMS OF BINARY PHOSPHOLIPID MIXTURES

A method has been developed to determine the exact fractional composition of binary mixtures of phospholipids at the air/water (A/W) interface by infrared spectroscopy in combination with P-31 NMR spectroscopy and Langmuir-Blodgett surface chemistry. This procedure utilizes the wavenumber shift observed upon the synthetic replacement of hydrogen with deuterium in the lipid acyl chains to separate the vibrational bands due to each component in mixtures of deuterated and normally proteated lipids. Classical Langmuir-Blodgett monolayer transfer techniques are used to transfer the binary lipid mixture from the A/W interface onto Ge crystals, where their IR spectra may be obtained with the use of attenuated total reflectance (ATR) infrared spectroscopy. The ratio of the integrated areas for the symmetric C-H and C-D stretching vibrations in the IR spectrum of the transferred monolayer film is used to determine fractional composition empirically; these vibrational intensities can be related to fractional composition in the following way: First, the integrated area ratios of the symmetric C-H and C-D stretching vibrations are obtained from the IR spectra of a standard series of binary mixtures of proteated and deuterated lipids at varying mole ratios. Second, these integrated area ratios obtained from the IR spectra of standards are correlated with the exact mole fraction of each phospholipid component in the mixture. The exact mole fractions are determined by the intensities of the phospholipid peaks obtained from high-resolution, solution-phase P-31 NMR signals of the same set of standard mixtures that was used to prepare the IR samples. High-resolution P-31 NMR spectroscopy in the presence of a line-narrowing reagent is a method that can provide an independent spectroscopic means of determining mole ratios in a binary solution of phospholipids. This IR-NMR comparison method essentially establishes a calibration curve for the IR C-D:C-H integrated intensity ratio in the binary mixture versus mole fraction as determined by NMR. Third, the IR C-D:C-H ratio in monolayer films of the binary phospholipid mixture transferred from the A/W interface to Ge crystals may be used to calculate the mole fraction of each component on the basis of the IR-NMR calibrations established in the IR spectra of the binary mixture standards. This technique was used to measure the fractional composition of binary mixtures of phospholipids at the A/W interface containing acyl chain perdeuterated DPPC (i.e., DPPC-[d62]) in combination with acyl chain proteated DPPG. The Langmuir-Blodgett technique was used to transfer monomolecular films of DPPC-[d62]: DPPG mixtures to a Ge ATR crystal at 40 mN m-1. The mole fraction of each component was estimated from the relative intensities of the symmetric C-D and C-H stretching bands by the use of ATR-infrared spectroscopy in conjunction with the line-narrowed P-31 NMR spectra of the phospholipid solutions. This model system was chosen for study due to its relevance in evaluating the proposed ''squeezing out'' mechanism of pulmonary surfactant physiology, in which the surfactant is proposed to reduce the surface tension of the air/alveolar lining by excluding all components except DPPC from the surface layer upon compression. Using this combined infrared/P-31 NMR calibration method, we find no evidence for the exclusion of any surface component upon compression of this mixed film.