Aqueous Subphase Research Articles

Electrostatic and Structural Effects at the Adsorption of Polylysine on the Surface of the DMPS Monolayer

The effect of the adsorption of a polypeptide on the lateral interaction of dimyristoylphosphatidylserine molecules in different phase states on the surface of a 10 mM KCl aqueous solution has been studied. Changes in the surface pressure and Volta potential induced by the adsorption of large poly-D-lysine molecules (about 200 links in a chain) have been determined at different areas per lipid molecule in a monolayer. The adsorption of macromolecules noticeably increases the elasticity of the monolayer under lateral compression in the liquid expanded state of lipid and reduces the effective dipole moment from 0.48 to 0.38 D. These properties are in qualitative agreement with X-ray reflectometry data for the lipid monolayer obtained with synchrotron radiation with a photon energy of ≈70 keV. The electron density profiles perpendicular to the surface of the aqueous subphase have been reconstructed from reflectometry data within a model approach to the structure of an interface with two and three layers. These profiles indicate the existence of a wide diffuse polymer layer (150 ± 40) A in width at the interface of the monolayer in both the liquid expanded and liquid condensed states. A decrease in the area per molecule in the monolayer by a factor of 2 results in the doubling of the surface density of the macromolecule film. The adsorption of the polymer also affects the integral density of the layer of polar phospholipid groups, which decreases by a factor of ≈2 in the liquid expanded phase and by ∼30% in the liquid condensed phase.

Collapse Mechanisms of Nascent and Aged Sea Spray Aerosol Proxy Films

Sea spray aerosol (SSA) is highly enriched in marine-derived organic compounds during seasons of high biological productivity, and saturated fatty acids comprise one of the most abundant classes of molecules. Fatty acids and other organic compounds form a film on SSA surfaces, and SSA particle surface-area-to-volume ratios are altered during aging in the marine boundary layer (MBL). To understand SSA surface organization and its role during dynamic atmospheric conditions, an SSA proxy fatty acid film and its individual components stearic acid (SA), palmitic acid (PA), and myristic acid (MA) are studied separately using surface pressure–area ( Π − A ) isotherms and Brewster angle microscopy (BAM). The films were spread on an aqueous NaCl subphase at pH 8.2, 5.6, and 2.0 to mimic nascent to aged SSA aqueous core composition in the MBL, respectively. We show that the individual fatty acid behavior differs from that of the SSA proxy film, and at nascent SSA pH the mixture yields a monolayer with intermediate rigidity that folds upon film compression to the collapse state. Acidification causes the SSA proxy film to become more rigid and form 3D nuclei. Our results reveal film morphology alterations, which are related to SSA reflectivity, throughout various stages of SSA aging and provide a better understanding of SSA impacts on climate.

Fluorocarbon Exposure Mode Markedly Affects Phospholipid Monolayer Behavior at the Gas/Liquid Interface: Impact on Size and Stability of Microbubbles

Although most phospholipid-shelled microbubbles (MBs) investigated for medical applications are stabilized by a fluorocarbon (FC) gas, information on the interactions between the phospholipid and FC molecules at the gas/water interface remains scarce. We report that the procedure of introduction of perfluorohexane (F-hexane), that is, either in the gas phase above dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) Langmuir monolayers, or in the aqueous subphase, radically affects the compression isotherms. When introduced in the gas phase, F-hexane is rapidly incorporated in the interfacial film, but is also readily desorbed upon compression and eventually totally expelled from the phospholipid monolayers. By contrast, when introduced in the aqueous phase, F-hexane remains trapped at the interface. These dissimilar outcomes demonstrate that the phospholipid monolayer acts as a barrier that effectively hinders the transfer of the FC across the interfacial film. F-hexane was also found to significantly accelerate the adsorption kinetics of the phospholipids at the gas/water interface and to lower the interfacial tension, as assessed by bubble profile analysis tensiometry. The extent of these effects is more pronounced when F-hexane is provided from the gas phase. The size and stability characteristics of DMPC- and DPPC-shelled microbubbles were also found to depend on how the FC is introduced. As compared to reference MBs prepared under nitrogen only, introduction of F-hexane always causes a decrease in MB mean radius. However, while for DMPC this decrease depends on the F-hexane introduction procedure, it is independent from the procedure and most pronounced (from ∼2.0 μm to ∼1.0 μm) for DPPC. Introducing the FC in the gas phase has the strongest effect on MB half-life (t1/2 = ∼1.8 and 6.8 h for DMPC and DPPC, respectively), as compared to when it is delivered through the aqueous phase (∼0.8 and ∼1.7 h). Fluorocarbonless reference DMPC and DPPC bubbles had a half-life of ∼0.5 and 0.8 h, respectively. The effects of F-hexane on MB characteristics are discussed with regard to the interactions between phospholipids and F-hexane and monolayer fluidization effect, as revealed by the Langmuir and tensiometric studies.

Immobilization of urease in Langmuir-Blodgett films of di-ureasil hybrid compounds

Incorporation of enzymes in matrices as nanostructured films is a strategy to achieve molecular architectures able to enhance the efficiency of sensors and optoelectronic devices. In this present work, urease was inserted in the aqueous subphase of a di-ureasil hybrid compound (DUPTS) assembled as a Langmuir monolayer. Phosphotungstic acid (PWA) was introduced in the aqueous subphase in order to stabilize DUPTS by means of electrostatic interactions. Incorporation of urease in the Langmuir film could be attested with surface pressure and surface potential-area isotherms, rheological measurements, polarization modulation infrared reflection-absorption spectroscopy, and Brewster angle microscopy. The monolayers were transferred to solid supports as Langmuir-Blodgett films and characterized with infrared and fluorescence spectroscopies, proving not only the co-transfer of urease to the solid support, but also the conservation of part of its secondary structure. The enzyme activity could be detected towards urea hydrolysis enabling the urease/PWA/DUPTS supramolecular system to be employed for sensors as well as for other optoelectronic devices.

Surface Pressure of Charged Colloids at the Air/Water Interface.

Charged colloidal monolayers at the interface between water and air (or oil) are used in a large number of chemical, physical, and biological applications. Although considerable experimental and theoretical effort has been devoted in the past few decades to the investigation of such monolayers, some of their fundamental properties are not yet fully understood. In this article, we model charged colloidal monolayers as a continuum layer of finite thickness, with a separate charge distribution on the water and air sides. The electrostatic surface free energy and surface pressure are calculated via the charging method and within the Debye-Hückel approximation. We obtain the dependence of surface pressure on several system parameters: the monolayer thickness, its distinct dielectric permittivity, and the ionic strength of the aqueous subphase. The surface pressure scaling with the area per particle, a, is found to be between a-2 in the close-packing limit and a-5/2 in the loose-packing limit. In general, it is found that the surface pressure is strongly influenced by charges on the air side of the colloids. However, when the larger charge resides on the water side, a more subtle dependence on salt concentration emerges. This corrects a common assumption that the charges on the water side can always be neglected due to screening. Finally, using a single fit parameter, our theory is found to fit the experimental data well for strong- to intermediate-strength electrolytes. We postulate that an anomalous scaling of a-3/2, recently observed in low ionic concentrations, cannot be accounted for within a linear theory, and its explanation requires a fully nonlinear analysis.

Observation by optical second harmonic generation of the mean tilt angle of cyanine dyes during compression with a phase transition in a Langmuir-Blodgett trough

Optical second harmonic generation (SHG) signals from a cyanine dye during compression of a monolayer in a Langmuir-Blodgett trough were measured to investigate the mean tilt angle of the dye corresponding to the formation of an H-band. The pressure-surface area (π-A) isotherm for 3,3′-dioctadecyloxacarbocyanine molecules spread on an aqueous subphase showed a plateau region after a kink point in the isotherm characteristic, which suggests a phase transition from the liquid expanded (LE) phase to the liquid condensed (LC) phase. SHG signals from the monolayer were obtained as the surface area decreased for the input and output polarization combinations PP, MS, SP, PS, and SS, where the first and second letters denote the input and output polarizations, respectively, and M refers to the polarization midway between S and P. For polarization combination PP, the SHG signal increased steeply during compression, whereas for polarization combination MS, the signal increased gently. The SH field from the monolayer for polarization combination SP in the LC phase was almost inverted to the field in the LE phase. We estimated the mean polar angles from the SHG responses during compression. The angle decreased continuously by almost 5° from 28° with the growth of domains in the LC phase. Visible absorption spectra of monolayers transferred to glass substrates were also obtained. The area intensity of the peak in the spectra attributed to H-aggregates increased compared with that of the monomer as the surface area decreased in the LC phase. The results were consistent with the polar angle reduction in the LC phase observed by in situ SHG. The decrease of the angle in the LC phase probably allowed the cyanine dyes to pack together tightly and align side by side, promoting H-aggregation.

Driving Coordination Polymer Monolayer Formation by Competitive Reactions at the Air/Water Interface.

We have developed a novel approach enabling us to follow and facilitate the formation of two-dimensional coordination polymer monolayers directly at the air/water interface without the need of complex instrumentation. The method is based on the use of a surface active ligand that, when spread at the air/water interface, progressively undergoes hydrolysis with consequent gradual decrease in surface pressure. Notably, if the aqueous subphase contains metal ions capable of coordinating the ligand, coordination competes with hydrolysis, resulting in a lower surface pressure decrease. As a consequence, the formation of the coordination polymer monolayer can be verified simply by surface pressure measurements. Competition between hydrolysis and coordination was investigated as a function of the main experimental parameters affecting the two reactions, enabling the formation of stable coordination polymer monolayers with controlled density. Finally, the formation of continuous rigid 2D layers was confirmed by compression isotherms and ex situ morphological characterization. This work will simplify the verification of coordination polymer monolayer formation; thus, it will boost the synthesis of novel and innovative 2D materials.

Capacity of cholesteryl hemisuccinate in ion pair/phospholipid complex to improve drug-loading, stability and antibacterial activity of clarithromycin intravenous lipid microsphere.

Current research was to evaluate the capacity and molecular interaction of ion pair/phospholipid complex during preparation of clarithromycin intravenous lipid microsphere (CLA-LM) for improving drug-loading, stability and antibacterial activity. The optimum pH range for the presence of ion pair formed by CLA and cholesteryl hemisuccinate (CHEMS) was found to be between 6.4 and 8.2. CLA-LM prepared by ion pair/phospholipid complex possessed improved drug-loading (5-10 mg/ml), plasma stability, storage stability (24 months at 4 ± 2℃) and better activity for multi-drug-resistant Mycobacterium tuberculosis (MIC = 0.058 μg/ml). The release profile in vitro was pH-sensitive and greater in more acidic condition, which was effective for pathological targeting. CLA-LM presented controlled release and low drug leakage in plasma. Langmuir monolayer showed that the incorporation of CHEMS obviously improved interfacial molecular interactions of the mixed monolayer. The ordering and condensing effect of CHEMS resulted in higher collapse surface pressure and smaller limiting area, as well as reduced compression modulus. Close aggregate network distribution with particular micro domains in atomic force microscopy images reflected the enhanced miscibility and thermodynamic stability. The change of the spatial location and protonation degree of CHEMS resulted from the aqueous subphase pH value from 6 to 8 was observed. More acidic environment allowed for better molecular interaction and interface behavior. These results indicated that CHEMS enhances the phospholipid packing to achieve stable CLA-encapsulation in phospholipid interface and identify the preference of ion pair/phospholipid complex as a valuable delivery strategy to develop CLA formulations for improving drug-loading, stability and antibacterial activity.

Ultrathin film of carboxylated graphene at air-water and air-solid interfaces

Ultrathin films of graphene and functionalized graphene is drawing scientific attentions due to its potential for industrial application. Due to the ease of processing and device fabrication, and enhancement in the molecular-specific interaction during sensing, graphene can be chemically modified to yield functionalized graphene. The ultrathin Langmuir–Blodgett (LB) films of nanomaterials show enhanced performance as compared to that of thick film. Such enhancement is attributed to the remarkable increase in surface-to-volume ratio and some ordering of the nanomaterials in the LB film. In this article, we report our studies on Langmuir film (LF) and LB film of carboxylated graphene (G-COOH) at air-water (A/W) and air-solid interfaces, respectively. The LF of G-COOH at the A/W interface exhibited gas and liquid-like phases. The liquid-like phase was found to be stable and reversible. The LF of G-COOH at the A/W interface is found to interact strongly with urea in the aqueous subphase via electrostatic interaction. This indicated that the LB film of G-COOH can be employed for sensing urea in aqueous medium. A functional layer of LB film of G-COOH is fabricated onto quartz wafers of a quartz crystal microbalance and the sensing performance towards urea in aqueous medium was studied. The sensing performance was compared with spin coated film of G-COOH. The lowest detectable concentration of urea using LB film of G-COOH (8.3 µM) is about 5 times lower than that of spin coated film (41.6 µM) and at the same time the sensitivity due to LB film (42.5 ng/cm2/µM) is about 3 times better than that of spin coated film (12.9 ng/cm2/µM). The similar protocol was employed for sensing urea in milk. The sensitivity of urea in milk by LB film of G-COOH is found to be about 5 times better than that of spin coated film of G-COOH. This study clearly indicates the potential of LB film of G-COOH as a sensitive and low concentration detectable urea sensor.

Impact of polyphenol-rich green tea extracts on the protection of DOPC monolayer against damage caused by ozone induced lipid oxidation.

Effectiveness of green tea (compared to two single polyphenols) in removing ozone derived reactive oxygen species acting on dioleoylphosphatidylcholine monolayers was determined. Lipid oxidation was followed by changes in mechanical properties of the layer spread on the aqueous subphase containing various amounts of ozone in the presence and absence of polyphenolics. It was shown that the tea extract (containing 8.5 x 10-4 mg/cm3 polyphenols) is capable of inactivating 0.4 ppm ozone. The DPPH radical scavenging test set polyphenols in the order of increasing activity, consistent with their protective effect in relation to lipid oxidation, showing the highest efficacy of EGCG.

Mobile liquid-substrate for self-assembly of solution-processable poly (5-aminoindole) by Langmuir technique

Polymers based device performance greatly depends on the π-backbone chain orientation along the charge transportation direction. Therefore, rearrangement of polymer chains at molecular level and thin film fabrication via Langmuir technique is highly desirable for large area electronic device applications. Here, we have utilized air-water interface of Langmuir technique for proper molecular orientation of the interfacially (water-chloroform interface) synthesized, electroactive poly (5-aminoindole) (5-APIn). 5-APIn is assembled over aqueous (mobile) subphase and variation in film compactness with varying surface pressure has been plotted as surface-pressure per area (п-A) isotherm. The films fabricated via Langmuir-Schaefer (LS) technique at surface pressures 35, 45 and 55 mN/m are qualitatively investigated using scanning electron microscopy (SEM). Surface roughness of the 5-APIn LS film is also investigated via atomic force microscopy (AFM). Polymer ordered assembly obtained in LS film is compared to bulk via Raman spectroscopy. Further, we have explored its large area electronic application via Al/5-APIn LS film/ITO diode configuration. Our study illustrates the significant role of mobile liquid substrate in formation of large area, smooth and ordered LS film of 5-APIn that unlocks its way for utilization in low cost and large area display electronic applications.

Structure of a monolayer of molecular rotors on aqueous subphase from grazing-incidence X-ray diffraction

In situ grazing-incidence X-ray scattering shows that a monolayer of artificial rod-shaped dipolar molecular rotors produced on the surface of an aqueous subphase in a Langmuir trough has a structure conducive to a 2D ferroelectric phase. The axes of the rotors stand an average of 0.83 nm apart in a triangular grid, perpendicular to the surface within experimental error. They carry 2,3-dichlorophenylene rotators near rod centers, between two decks of interlocked triptycenes installed axially on the rotor axle. The analysis is based first on simultaneous fitting of observed Bragg rods and second on fitting the reflectivity curve with only three adjustable parameters and the calculated rotor electron density, which also revealed the presence of about seven molecules of water near each rotator. Dependent on preparation conditions, a minor and variable amount of a different crystal phase may also be present in the monolayer.

Pb arachidate Langmuir-Blodgett coatings of silicon wafers: relation between Pb particle density and subphase composition

The formation of lead arachidate (Pb(AA)2) coatings produced by Langmuir tensiometry (LT) was studied in dependence of lead nitrate (Pb(NO3)2) concentration in the aqueous subphase at pH 5.5. The stable Pb(AA)2 monolayers were transferred to Si wafers and TEM grids at 35 mN/m in the liquid condensed phase of the AA monolayer applying the Langmuir-Blodgett (LB) (ten monolayers) or the Langmuir-Schaefer (LS) technique, respectively. The most homogeneous monolayer was obtained at a Pb(NO3)2 concentration of 0.1 mM by using the LS method. Most homogeneous films (five bilayers) were obtained for a Pb(NO3)2 concentration of 0.01 mM by using the LB method. The formed films are investigated by transmission electron microscopy (monolayer produced by LS), X-ray reflectivity (XRR), and atomic force microscopy (films produced by LB). These methods revealed the formation of homogeneously distributed lead inclusions in the formed monolayers and films. Pb aggregates increase in number whereas their average size stays constant at ≈ 50 nm2 with increasing Pb(NO3)2 concentration. Thickness parameters were determined by XRR. With increasing Pb(NO3)2 concentration, the bilayer thickness was found to increase. Furthermore, the patterns (111) and (200) for a cubic lead structure were found. The experimental results are compared with simulations obtained from geometry optimization on a semi-empirical quantum level to discuss the lead-ion binding to the AA molecules at pH 5.5.

Studying the Molecular Mechanisms of Interaction of Nanoaerosol Particles with a Model Membrane

An X-ray study of the interaction of Langmuir monolayers with nanoparticles under the conditions when nanoparticles arrive at the monolayer from the side of the air medium has been performed for the first time. Measurements by the X-ray standing-wave method and two-dimensional diffraction have made it possible to investigate the transport of nanoaerosol particles through a lipid monolayer and observe the structural changes occurring in it. Nanoaerosol particles were formed by electrohydrodynamic sputtering of desalted solutions of ferritin. It was shown that nanoaerosol particles pass through the monolayer without disrupting the two-dimensional packing of lipid molecules. The ferritin molecules penetrating the aqueous subphase after sputtering are found to be adsorbed under the monolayer.

Effects of Aggregate Charge and Subphase Ionic Strength on the Properties of Spread Polyelectrolyte/Surfactant Films at the Air/Water Interface under Static and Dynamic Conditions.

We demonstrate the ability to tune the formation of extended structures in films of poly(sodium styrenesulfonate)/dodecyltrimethylammonium bromide at the air/water interface through control over the charge/structure of aggregates as well as the ionic strength of the subphase. Our methodology to prepare loaded polyelectrolyte/surfactant films from self-assembled liquid crystalline aggregates exploits their fast dissociation and Marangoni spreading of material upon contact with an aqueous subphase. This process is proposed as a potential new route to prepare cheap biocompatible films for transfer applications. We show that films spread on water from swollen aggregates of low/negative charge have 1:1 charge binding and can be compressed only to a monolayer, beyond which material is lost to the bulk. For films spread on water from compact aggregates of positive charge, however, extended structures of the two components are created upon spreading or upon compression of the film beyond a monolayer. The application of ellipsometry, Brewster angle microscopy, and neutron reflectometry as well as measurements of surface pressure isotherms allow us to reason that formation of extended structures is activated by aggregates embedded in the film. The situation upon spreading on 0.1 M NaCl is different as there is a high concentration of small ions that stabilize loops of the polyelectrolyte upon film compression, yet extended structures of both components are only transient. Analogy of the controlled formation of extended structures in fluid monolayers is made to reservoir dynamics in lung surfactant. The work opens up the possibility to control such film dynamics in related systems through the rational design of particles in the future.

Surface Activity of Poly(ethylene glycol)-Coated Silver Nanoparticles in the Presence of a Lipid Monolayer.

We have investigated the surface activity of poly(ethylene glycol) (PEG)-coated silver nanoparticles (Ag-PEG) in the presence or absence of lipid monolayers comprised of monounsaturated dioleoylphosphocholine and dioleoylphosphoglycerol (DOPC/DOPG; 1:1 mol ratio). Dynamic measurements of surface pressure demonstrated that Ag-PEG were surface-active at the air/water interface. Surface excess concentrations suggested that at high Ag-PEG subphase concentrations, Ag-PEG assembled as densely packed monolayers in the presence and absence of a lipid monolayer. The presence of a lipid monolayer led to only a slight decrease in the excess surface concentration of Ag-PEG. Surface pressure-area isotherms showed that in the absence of lipids Ag-PEG increased the surface pressure up to 45 mN m-1 upon compression before the Ag-PEG surface layer collapsed. Our results suggest that surface activity of Ag-PEG was due to hydrophobic interactions imparted by a combination of the amphiphilic polymer coating and the hydrophobic dodecanethiol ligands bound to the Ag-PEG surface. With lipid present, Ag-PEG + lipid surface pressure-area (π-A) isotherms reflected Ag-PEG incorporation within the lipid monolayers. At high Ag-PEG concentrations, the π-A isotherms of the Ag-PEG + lipid films closely resembled that of Ag-PEG alone with a minimal contribution from the lipids present. Analysis of the subphase silver (Ag) and phosphorus (P) concentrations revealed that most of the adsorbed material remained at the air/lipid/water interface and was not forced into the aqueous subphase upon compression, confirming the presence of a composite Ag-PEG + lipid film. While interactions between "water-soluble" nanoparticles and lipids are often considered to be dominated by electrostatic interactions, these results provide further evidence that the amphiphilic character of a nanoparticle coating can also play a significant role.

Lead bromide-based layered perovskite Langmuir–Blodgett films having π-conjugated molecules as organic layer prepared by using squeezed out technique

Using the squeezed out technique, we successfully prepared PbBr-based layered perovskite Langmuir–Blodgett (LB) films, which have π-conjugated materials as an organic layer (i.e., a phenylenevinylene oligomer, a dithienylethene derivative, and a π-conjugated polyfluorene derivative). The mixed monolayers of π-conjugated materials and octadecylammonium bromide were spread on an aqueous subphase containing saturated PbBr2. During pressing, octadecylammonium molecules were squeezed from the mixed monolayer, and the squeezed ammonium molecules formed the PbBr-based layered perovskite structure at the air-aqueous subphase interface. The monolayers with the PbBr-based layered perovskite structure could be deposited on fused quartz substrates by the LB technique. In addition to the preparation procedure, the structural and optical properties of the layered perovskite LB films and their formation mechanism are reported in this paper.

Study of aggregation behavior of water insoluble metalloporphyrin (Zn) in LB film

In the present communication, a water insoluble metalloporphyrin, 5,10,15,20-Tetraphenyl-21H,23H-porphine zinc (abbreviated as ZnTPP) has been used to prepare stable Langmuir monolayer at the air–water interface by allowing it to adsorb from the aqueous subphase, into the preformed Langmuir monolayer of stearic acid (SA). Surface pressure versus area per molecule (π-A) isotherm studies gave the thermodynamical behavior of the ZnTPP-SA mixed Langmuir monolayer formed at the air water interface. Access area per molecule (AE) vs molefraction curve indicated the repulsive interaction between the two binary components in the mixed Langmuir monolayer. BAM images gave the visual evidence of the aggregation pattern of the mixed molecules at the air-water interface. UV-vis spectroscopic studies confirmed the formation of prominent J-band in the ultra-thin films.

Graphene oxide supported filtration of cesium from aqueous systems

Graphene Oxide-Cesium (GO-Cs) composites, in the form of dispersions as well as solid state thin films, were prepared by introducing different concentrations of Cs into aqueous GO dispersions or subphase. Interaction mechanism of Cs with various functional groups of GO and the extent of Cs uptake on GO sheets under different ambient conditions were investigated using FT-IR, Raman, PL and XPS techniques. Cs was noticed to exhibit preferential interaction with hydroxyl groups of GO and furthermore, incorporation of Cs causes significant change in relative ratios of functional groups, inferring a major structural reorganization in the composite sheets. FT-IR and PL studies showed that aged GO samples can accommodate relatively larger quantities of Cs compared to fresh GO sheets, owing to largely available –COOH and free/associated hydroxyl groups in aged GO sheets. Intercalation studies on GO multilayers indicated that extent of Cs uptake on GO layers increases with duration of intercalation as well as quantity of GO available for interaction. Membrane filters of GO were prepared by controlled coating of GO sheets on filter papers and their adsorption and filtration abilities were tested by filtration of aqueous Cs solutions. Aged GO coated membrane filter displayed superior adsorption abilities compared to those coated with fresh GO sheets, which corroborate well with the inferences drawn from the spectroscopic investigations. In the present work, the prepared GO membrane filters showed a maximum Cs adsorption capacity of ∼148 mg/g.

Effect of pH and Salt on Surface pKa of Phosphatidic Acid Monolayers.

The pH-induced surface speciation of organic surfactants such as fatty acids and phospholipids in monolayers and coatings is considered to be an important factor controlling their interfacial organization and properties. Yet, correctly predicting the surface speciation requires the determination of the surface dissociation constants (surface pKa) of the protic functional group(s) present. Here, we use three independent methods-compression isotherms, surface tension pH titration, and infrared reflection-absorption spectroscopy (IRRAS)-to study the protonation state of dipalmitoylphosphatidic acid (DPPA) monolayers on water and NaCl solutions. By examining the molecular area expansion at basic pH, the pKa to remove the second proton of DPPA (surface pKa2) at the aqueous interface is estimated. In addition, utilizing IRRAS combined with density functional theory calculations, the vibrational modes of the phosphate headgroup were directly probed and assigned to understand DPPA charge speciation with increasing pH. We find that all three experimental techniques give consistent surface pKa2 values in good agreement with each other. Results show that a condensed DPPA monolayer has a surface pKa2 of 11.5, a value higher than previously reported (∼7.9-8.5). This surface pKa2 was further altered by the presence of Na+ cations in the aqueous subphase, which reduced the surface pKa2 from 11.5 to 10.5. It was also found that the surface pKa2 value of DPPA is modulated by the packing density (i.e., the surface charge density) of the monolayer, with a surface pKa2 as low as 9.2 for DPPA monolayers in the two-dimensional gaseous phase over NaCl solutions. The experimentally determined surface pKa2 values are also found to be in agreement with those predicted by Gouy-Chapman theory, validating these methods and proving that surface charge density is the driving factor behind changes to the surface pKa2.