*4.1. E*ff*ect of HKM Bu*ff*er on the DPPC Monolayer* Π*-A Isotherm*

In this work, we studied the optical and structural properties of a DPPC monolayer in the fluid condensed phase (<sup>Π</sup> = 30 mN·m−1). Looking at the <sup>Π</sup>-*A* isotherm and the corresponding *C*<sup>s</sup> <sup>−</sup><sup>1</sup> as a function of the surface pressure (Figure 2), we obtained information about the different phases of the lipid monolayer and its molecular packaging. The behavior of DPPC at the air/buffer interface is different from the one observed using pure water. Although this is an aspect that merits future work, there are different studies in the literature on the influence of mono and divalent cations on the behavior of DPPC at the air/water interface that agree with our findings: the formation of a liquid-condensed phase with higher lateral compressibility and shifted towards larger molecular areas than the same monolayer at the air/water interface. Complexation effects of mono-, but especially, divalent ions to amphiphilic molecules, such as phospholipids, are known to modify their orientation, morphology and packing. These aspects have been recently evaluated by Adams et al. by studying the influence of highly concentrated salt solutions on the behavior of DPPC monolayers at the air/water interface [15]. The Π-*A* isotherms reported, together with the Brewster angle microscopy images, showed that the presence of K+, Na<sup>+</sup>, or Mg2<sup>+</sup> causes an expansion of the isotherm towards higher values of area per molecule and a decrease in the compressibility modulus, especially in the latter case. This is in full agreement with what we observed here. These authors also demonstrated that the presence of high salt concentration disrupts lipid packing, resulting in an extension of the liquid-expanded phase. Since we observed a slight LE–LC coexistence phase, we thought that we were not in this situation. It is important to mention that the salt concentrations used in that study are higher than the ones that we used. However, the general trend of the expansion of the isotherm and the decrease of the compressibility modulus are indicators that the behavior of DPPC at the air/water interface is affected by the constituents of the HKM buffer. Besides, in this work, there were up to five different species that could affect the organization of DPPC at the interface. Indeed, Bringenzu et al. studied the interaction

of a protein with DPPC monolayers using a very similar buffer and they also observed an expansion of the isotherm [57].
