The use of monomolecular Langmuir films and multilamellar systems as models of biological membranes is widespread in the literature. However, several examples highlighted some different properties of monolayer and multilayer systems like miscibility, phase equilibrium, hydration or dynamics. Our work contributes to this question by comparing the lipid ordering in monolayers and stacks of flat bilayers. We have considered the monolayer spread at the air–water interface and the liquid-crystalline gel phase formed by 1,2-distearoylphosphatidylcholine (DSPC) at 20 °C. Grazing Incidence X-ray Diffraction (GIXD) and Small-Angle X-ray Scattering (SAXS) coupled with Wide-Angle X-ray Scattering (WAXS) were employed to analyse the ordering of DSPC chains in these model systems. Results show that the ordering of DSPC in monolayers and stacked bilayers is indeed different. The lattices in monolayers and stacked bilayers are distorted in different directions and at different rates. Additionally, we incorporated an unsaturated neutral lipid (1-stearoyl-2-arachidonoyl-glycerol, SAG) in DSPC monolayers and multilayers. Despite the same miscibility range, we found the same difference between monolayers and stacked bilayers in the mixed system DSPC–SAG as for pure DSPC. The in-plane structure of self-assembled lipids must be then considered with care when comparing monolayers and stacks of bilayers.
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