*4.2. Influence of HKM Bu*ff*er on the Structural Parameters of DPPC Monolayers at the LC Phase*

By means of NR, we reported a total thickness of the DPPC monolayer of 23.5 Å, in agreement with literature values of DPPC in the condensed phase [20,58]. The thickness of the aliphatic chains was found to be 15 Å by NR. This was close enough to the thickness obtained from ellipsometry (*ca.* 14 Å) corresponding to the best fit. Assuming a length of 19 Å of an aliphatic chain of 16-CH2 groups in trans configuration [59], we obtained a tilting angle of 37◦, a value that is higher than the value expected for DPPC at the LC phase (27◦ at <sup>Π</sup> <sup>≈</sup> 30 mN·m<sup>−</sup>1). This agrees with a decrease in the packing density of DPPC monolayers in the presence of divalent salts from the HKM buffer (see Figure 1b). The average refractive index *n*<sup>F</sup> = 1.547, found here agrees with previous values reported by Ducharme et al. [23] and Thoma et al. [35] on the DPPC monolayer at the air/water interface. Ducharme et al. rationalized this increase of the refractive index of lipid monolayers to the existence of condensed phases. In addition, the value reported here for the anisotropy was slightly smaller to the one reported by Thoma et al. (Δ*n* = 0.05). It is well known that an increase in anisotropy is observed for DPPC monolayers going from LE to LC. It is rationalized by the increase of all-trans configurations for the alkyl chains. In our case, the reduced anisotropy found can also be explained by the effect of the buffer on the packing density of the alkyl chains.

The values obtained from the fitting of both NR (Table 2) and ellipsometry yields a surface excess, Γ, or molecular area (*A* = 1/(NA × Γ)) [60], which is consistent with the one extrapolated from the isotherm (see Figure 2a) at <sup>Π</sup> <sup>=</sup> 30 mN·m<sup>−</sup>1. The value found of 55.2 Å2 is slightly larger in comparison with the value for DPPC in water at a similar surface pressure [27]. This has been previously attributed to the effect of the buffer molecules on the monolayer. The calculation of both volume fraction and cross-sectional area profiles with the distance to the interface, (Figure 3c,d, respectively) allows us to rationalize the structure of the DPPC monolayer in real-space, even unraveling the contribution from the aliphatic chains facing the air and the solvated polar headgroups in contact with the buffer. We observed that the solvation of the polar heads by water molecules was about 30% of the volume fraction of the layer (see, cyan curve in Figure 3c) in agreement with previous studies on DPPC/water interfaces [27].
