**1. Introduction**

Synthetic in vitro lipidmono- and bilayers, as well as uni- andmulti-lamellar vesicles, can be considered as simple biomembrane models. For more than 20 years, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) has been widely exploited to mimic plasma membranes or lung surfactant, mostly in the form of monolayers at the air/water interface. Indeed, phospholipids with 16- and 18-carbon fatty acids chains are the most abundant in plasma membranes [1,2]. DPPC is, therefore, a well-studied lipid. Besides, DPPC has being used alone or in combination with other lipids, to study the interaction of biomembrane with proteins [3,4], anticancer [5–8] and antifungal [9] compounds, and small molecules of biological relevance, such as cholesterol [10], hormones [11], and antibiotics [12]. Its handling simplicity, relatively low price, and stability at room temperature as well as when exposed to air, make DPPC a versatile model system for biomedical research.

In general, phospholipid monolayers at the air/water interface can be characterized by different techniques such as microscopy (e.g., atomic force microscopy, AFM, and Brewster angle microscopy, BAM), scattering (e.g., neutron reflectometry, NR, and X-ray reflectometry, XRR), and ellipsometric and spectroscopic (e.g., polarization modulation infrared reflection–absorption spectroscopy) techniques, together with surface pressure-area (Π-*A*) isotherms. Currently, there are many outstanding examples of the usefulness of this combinatorial approach, exploited to study DPPC monolayers structure and properties [13,14] as well as their interaction with different cations [15], nanoparticles [16], graphite-based compounds [17], and molecules of biological relevance, such as proteins [4,18], small peptides [19], and antibiotics [12].

Different techniques have been used to study the structure and optical properties of lipid monolayers. Indeed, the refractive index of the film (*n*F) in combination with the reflectivity allows obtaining the thickness (*d*F) of the monolayers. Kienle et al. successfully used multiple beam interferometry to determine simultaneously *n*<sup>F</sup> and *d*<sup>F</sup> of DPPC and DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) supported monolayers at high surface pressures [20]. The results obtained by this technique were in good agreement with those obtained by XRR and AFM. Another approach to determine *n*<sup>F</sup> of monolayers at the air/water interface is looking at the minimum of the reflectivity as a function of the refractive index of the subphase. Pusterla et al. used this approach varying the concentration of glycerol or sucrose in the subphase, with known refractive indices [21]; the refractive index of the monolayer is equal to the one of the subphase when the reflectivity is minimum. Besides, knowing the refractive index and the reflectivity they calculated *d*F. The determination of the increment of the refractive index with concentration (d*n*/d*c*) constitutes another alternative to obtain *n*F. The subsequent application of the lipid density to the d*n*/d*c* provides *n*<sup>F</sup> [22]. However, *n*<sup>F</sup> can slightly vary with the surface pressure, especially from one phase to another, so it is necessary to take into account the physical state of the monolayer to know its *n*<sup>F</sup> [23]. NR has also been widely exploited to perform studies at the air/water interface to investigate the structure and properties of lipid monolayers [24–26]. This technique enables complete structural characterization of the monolayers, giving information about the chemical composition along the axis normal to the interface, the thickness of both polar headgroups layer and hydrophobic tails layer when a two-layer model is used to interpret the data. Through NR it is also possible to determine the hydration degree of the lipids, as well as the surface excess and the area per molecule at a certain value of surface pressure [27]. Hence, NR is a very powerful tool to study lipid monolayers at the air/water interface.

Traditionally, ellipsometry has been one of the most exploited techniques to study surfactant and lipid monolayers at the air/water interface [28,29]. It gives access to the determination of the thickness and the refractive index of films, and it is useful to investigate the interaction of monolayers with different molecules such as proteins [30,31] or nanoparticles [32,33] through time/spatial resolved experiments. Nevertheless, the simultaneous determination of the *n*<sup>F</sup> and the *d*<sup>F</sup> for Langmuir films at the air/water interface with *d*<sup>F</sup> << λ by the measurement of the phase shift at a fixed angle of incidence, can give inaccurate results due to the strong coupling of the parameters [29,34]. The combination of ellipsometry with other techniques that give access to the determination of one of the parameters allows the accurate determination of the other one by ellipsometry [35,36]. Benjamins et al. developed a method for the study of films at liquid interfaces by ellipsometry without assumptions of the thickness or the refractive index [37]. They demonstrated that the combination of measurements performed for the same system using D2O and H2O as the subphase, i.e., different refractive indices of the subphase, give enough additional information to determine the amount of material adsorbed.

In this work, we use the combination of two reflection techniques, NR and ellipsometry, and surface pressure measurements to determine the interfacial structure and the optical properties, including the refractive index anisotropy, of a condensed DPPC monolayer at the air/buffer interface (see Figure 1). Besides, we show how HKM buffer molecules (see composition below), widely exploited in biological assays, are responsible for the differences observed in the structure and density of DPPC monolayers.

**Figure 1.** (**a**) Structure formula of DPPC. The polar headgroup is depicted in green and the hydrophobic tails in violet following a two-layer model proposed in the main text. A green sphere and a violet cylinder are drawn to better show *V*m\_heads and *V*m\_tails, respectively. (**b**) The scheme representing the main electrostatic interactions between the phospholipids molecules with the ions composing the buffer: the magnesium cation is depicted as a red sphere, while the acetate anion is depicted as a green sphere. We deduce that these interactions have an influence on DPPC monolayer structure. Bottom panel compares neutron reflectometry (**c**) and ellipsometry (**d**) principles of measurement. In the case of NR, a two-layer model can be exploited in order to get both the thickness of the headgroups (*t*heads) and the one of the tails (*t*tails); indeed, the two-layer model is shown, depicting the heads in green and the tails in violet. Moreover, NR also gives information about the fraction of water per polar headgroup (*f* w\_heads) and the roughness of the interfaces (*r*), whose value depends on the water capillary waves. The angle of incidence of the neutron beam (θ) and the scattering vector, or momentum transfer, (*Q*Z) are shown. On the other hand, ellipsometry does not disentangle the contribution of the headgroup and the aliphatic chains to the thickness as NR. In addition, the roughness is considered to be equal to zero (as an ideal interface). The incoming and the reflected light beams, whose electric field is divided in parallel (*E*p) and perpendicular (*E*s) components, are shown. Besides, the angle of incidence (AOI) and the ellipsometric angle Ψ are shown.

This work reports complementary measurements from two different methods. On one hand, NR, which has been demonstrated to be a suitable technique for the investigation of the structure at the sub-nm scale of thin films, allowed us to determine the thickness of the aliphatic chains and the level of hydration of the polar headgroups of the DPPC monolayer. On the other hand, we propose a novel ellipsometric method to determine both the surface excess and the optical anisotropy shown by the DPPC monolayer in the condensed phase that depends on the orientation of the aliphatic chains.
