Characterization of Monoolein Langmuir Monolayers Spread at The Salt Solution/Air Interface

The Langmuir monolayer is commonly described at interfaces for an insoluble homogenous single molecular layer. Langmuir monolayers have demonstrated various soft matters and complex fluids by forming ideal uniform two-dimensional structures over the air-water interface. The Langmuir monolayer has advantages for evaluating physicochemical properties at interfaces and, for the insoluble molecules, can be applied simultaneously to the different interaction occurrences at interfaces. In this regard, monoolein lipid was used as a spreading solvent to create a Langmuir monolayer. Five different types of salt subphases were applied for the physicochemical properties interaction studies. On the air-water interface, the surface properties of monoolein lipids were investigated for interfacial phase behaviors using the Wilhelmy plate pressure sensor technique compression isotherm (π -a). Data and analysis were also contributed to correspondent and precise verification of physical state behaviour with the surface pressure measurements on the interfaces through the compressibility modulus parameters on the surface. In the experiments, the interfacial activity of the monoolein lipids was found to be stable on the aqueous subphase. At the same time, the area per molecule over the interface did not affect much as a subphase with the change in various salts. The experiment's repeatability and reproducibility were affirmed by the difference in Langmuir monolayer's particular phase transition orientation behavior and the stability of colloidal lipid dispersion. However, Langmuir monolayer formation contributes to several special groups being restructured and found to be a more remarkable natural process for their attractive organic, dynamic structural properties over the interface. Still, the interfacial molecular dynamics have proved challenging to calculate.


Introduction
The rising popularity of the Langmuir monolayer technique from 1920 to 2020 has enormously increased. In the past, Irving Langmuir's discovery was awarded the Nobel Prize for remarkable achievement in chemistry in 1932 [1]. Afterward, many researchers have studied various applications based on science and technology interfaces [2]. These applications include protein-lipid macromolecules [3], molecular thin films [4], colloids [5], nanomaterials [6], and more recently, monolayers as vehicles for bioactive macromolecules membrane [7].
Nowadays, the advancements carried out in the Langmuir monolayer technique were successfully utilized in Alzheimer's disease and the surface chemistry interaction of peculiar conditions [6,8]. During the Langmuir monolayer formation, the uniform distribution occurs, due to which the colloidal film description became more comfortable for the particle size identification and structural properties characterization [2,9]. The Langmuir monolayer's interfacial properties have become an attractive source for its ability to control accurate measurements of the homogeneous monolayer at the interfaces [10,11]. The difficulty lies in the surface pressure measurement properties for performing the real-time characterization of Langmuir monolayer experimentation [4,8]. Monoolein forms monolayers in the liquid expanded phase only, and no solid phase could be observed by the values of compression modulus [12,13].
Natural lipids can carry a negative net charge or are neutral, depending on the head group structure interacting with the aqueous phase [14]. Lipids are widely used for the biomembrane process, and they are primarily heterogeneous in chain length, chain saturation, and head group structure [15]. It is commonly used in the food industry and widely used for low fat and instant food mix surfactants in traditional foods [16][17][18]. Langmuir lipid monolayer formation is a bit straightforward since the second most essential macromolecule is after the protein bio-macromolecules. In molecular species, the lipid macromolecules are highly surface-active amphiphilic materials and are purely hydrophobic. Many researchers studied the monolayer approach of Langmuir lipid using compression isotherm [4,19]. The lipid monolayer macromolecule assists in the understanding of the folding unfolding behaviour of interacting molecules along with the structural analysis of the formed monolayers [20]. The elastic energy measurement was recently carried out with the lipid macromolecules' topographical changes by the Langmuir monolayer approach [21][22][23][24]. Similarly, several other theoretical methods were developed and used for the various experiments to understand better the driving force for folding unfolding phase transition behaviour observations. In addition, the heterogeneity of the molecules plays a significant role in determining the film's 3D structure and stability [25,26].
A commonly known chemical name for monoolein lipid is 9-Octadecenoic acid (Z)monoester with 1,2,3-propanetriol and 1-Monoolein (1-(cis-9-Octadecenoyl)-rac-glycerol) is also familiar [27]. It is a viscous, pale yellow liquid with a characteristic odour in terms of physicochemical properties. From the point of view of the molecular structure shown below, it is composed of a hydrocarbon chain, which is attached by an ester bond to a glycerol backbone (see Figure 1). Macroscopically speaking, the moiety of glycerol gives polar characteristics to this part of the molecule for a hydrophobic head, which implies that the formation of hydrogen bonds with water in aqueous solutions by the hydrocarbon chain known as the hydrophilic tail, with a double bond position, is highly hydrophobic [39]. In water and cold alcohol, monoolein is insoluble but is regularly soluble in oil, petroleum ether, chloroform, and even hot alcohol.
Since it is a non-toxic, biodegradable, and biocompatible material, monoolein lipids could have very desirable properties as in a food emulsion [28].  [27] The significant interest in evaluating the lipid Langmuir monolayer technique on the air-water interface has increased to understand the lipid macromolecules interaction effect with the change in subphase containing liquids. The surface properties of monoolein lipid macromolecules were investigated in this paper by using the Wilhelmy plate surface pressure sensor at the air-water interface. For the phase behavioural orientation of the lipid molecules and their interaction with various ionic salt subphase molecules on an interface, compression isotherm (π-a) was observed. The shift in the phase transmission of the lipid macromolecules was expressed by surface pressure-area measurements per molecule (π-a) for aqueous salt subphases. We took advantage of the subphase surface and interface head groups in this experiment to examine interaction at the interface. Monoolein lipid spread from 0.01 M to 0.1 M across various salt sub-phases and observed their phase transition in molar concentration.
NaCl, KCl, KH2PO4, (NH4)2S2O8, and (NH4)2SO4 were the subphase salts used for lipid interactions. Extension of π-a isotherms mean a molecular area was used to find the compressibility parameters for the miscibility of lipid-salt interaction at the interface.

Materials
Monoolein lipid purchased from the TCI Chemicals Pvt. Ltd., Chennai, India. The molecular weight of monoolein lipid with an empirical formula of C21H40O4 was 356.55 g/mol in molecular weight, and the boiling point was 238-240 0 C with a density of 0.94 g cm -3 density [27]. Hexane and chloroform were used as spreading spectroscopic grade lipid solvent,

Surface pressure measurements
The surface pressure measurement was carried out on a fully automatically customized Langmuir Blodgett trough (APEX Instruments Co. Pvt. Ltd., India). The total trough area is 26 cm × 15 cm. The surface pressure v/s area per molecule isotherms was obtained for different subphases by a Wilhelmy plate pressure sensor [5,29]. Teflon trough was thoroughly cleaned with acetone twice before each experimental run, followed by rinsing with Milli-Q water. The purity of the subphase has been verified by ensuring no increase in the surface pressure during

Molecular stability of monolayer phases
Langmuir monolayer approach analysis to investigate the surface properties of lipid macromolecules was conducted at the air-water interface. Langmuir monolayer formation involves the molecule spreading across the surface of the subphase using a volatile organic solvent. As the organic solvent evaporates within 30 min waiting time, molecules at the interfaces achieve a system equilibrium state. The stock solution was prepared by 10 percent chloroform:hexane (v/v) and macromolecules spreading carried using the traditional Trurnit method [30,31]. A gas-tight Hamilton micro-syringe was used to carry out the spreading of molecules. During preliminary trials, the required optimal spreading volumes and spreading concentrations were standardized. The two barriers, which were moving symmetrically, were operated at 1 mm/min. A standard monolayer experiment is appropriate to spread a few micrograms of spreading solution compounds on a subphase. The resulting compact monolayer film is stable for an extended period if the surface pressure is not too high.
The monolayer will provide the information on two-dimensional surface behaviours and immediate access to the disorder to an organized, ordered layer of confirmations of physical state transmission under compression isotherm. The phase transition sequence, under compression, follows the order of a gas-like, liquid-like, and solid-like monolayer formation at the air-water interface, in phase from right to left, respectively. In addition, during the Langmuir isotherm process, the compact macromolecule ensures the hydrophobic-head and hydrophilic-tails of present molecules. Performing the same tests confirmed repeatability and reproducibility. Macromolecules need to be an experiment in the form of a single thin layer for the interaction with external media shown in the schematic diagram below Figure 3. The spreading will continue until the surface pressure has elevated to an equilibrium value until the colliding surface pressure. The colliding surface pressure is defined as when the formed Langmuir monolayer breaks and indicates the sudden shift in surface pressure value towards the reduction. The spreading pressure of the equilibrium is defined as that produced spontaneously when the solid bulk is put in contact with a surface of the water. Again, the vapor pressure of a bulk solid will disburse compression. In the presence of its vapor, an equilibrium vapor pressure occurs for the solid. If this vapor pressure reaches, as the vapor is over-saturated, a deposit on the solid surface will occur. In specific experiments, however, relatively stable surface pressure can be calculated up to higher values than the spreading pressure of the equilibrium [32].

Results and Discussion
In this contribution, the surface properties of monoolein lipid macromolecules, the single molecules Langmuir monolayer, were developed using the Wilhelmy plate pressure sensor on the air-water interface for the interaction of different salt subphases. The Langmuir monolayer confirms different transition phase positioning, such as gas-like, liquid-like, and solid-like behavior on compression isotherm. It is recognized by Langmuir monolayer that the force, which causes the spreading of lipid on the surface of water subphase, was due to the attraction or repulsion of interacting lipid molecules and also surface activity of monoolein lipid. Using the equilibrium surface pressure compression isotherm results, the compressibility calculation was carried out with the mean molecular area of the isotherm. Therefore, an active group of lipid molecules has a marked affinity for spreading the uniform single layer molecule on the subphase.

Effect of subphase on monoolein lipid Langmuir monolayer
Compression isotherm [surface pressure (π) -area per molecule (a)] (π-a) study allows us to determine the surface pressure increase as a function of decreasing surface area with compression, illustrating the monolayer packing at the interface shown below Figure 4(a). The pure monoolein lipid isotherm was observed to undergo a sharp transition at a molecular area of ∼ 0.88-0.17 nm 2 , consistent with previous studies [1,27,33]. Due to the compactness of the molecules on barrier compression and also the phenomenon of liftoff surface pressure observed for monoolein lipid monolayer has been shown in Figure 4(a). On the experimental side, the values of salt-free monoolein experimental studies for the area per lipid for monoolein monolayers were recorded for various temperature ranges [33][34][35]. To validate, our observed value for the area per lipid is therefore in good agreement with the available experimental data at room temperature [34].
In the gaseous phase, lipid molecules' initial compression implies that molecules don't interact with each other. The lipid molecules come closer at further compression, and an increase in surface pressure was observed in Figure 4 Using different salt subphases in monolayer formation observed the effect of interaction on monoolein lipids at the air-water interface and to identify the effect of lipid monolayer on mono valiant and divalent changes in the distribution of solvent at interfaces. In addition, lipids are weakly interacting with monovalent salts and have a strong interaction with divalent salts [36]. Effects from observations shown in Figure 4 In the monolayer, the interactions between components can be investigated through the excess area from the miscibility between components in the subphase. These results suggest that higher surface pressures are dominated by salt solution. We represented in Figure 4(a) hypothetical π-a isotherms for compression monolayer calculated for lipid molecules on pure water subphase is present at the air-water interface. Likewise, other salt's compression isotherm plots were shown in Figure 4   modulus were computed using the following eq. 1. The mean molecular area obtained by means of compression isotherm is respectively. Many researchers conduct the compressibility modulus recognition based on eq.1 to select parameters over the lipid monolayer's physical state, in their extension analysis towards the Langmuir isotherm [38,39]. The information provided by the -A isotherm, the compressibility , was determined to be evaluated in more detail.

Determination of surface compressibility modulus
Whereas, A denotes the mean molecular area, πsurface pressure, is at room temperature T (approx 20 0 C). From the experimental observation, shown the compressibility modulus is consistent with the results previously published [22]. Also, their LE state did not start from the zero surface pressure, and then it decreased, as we observed in Figure 5. With the shift in slope values, the physical state transitions behaviour can be discovered from the − curve study.
In addition, , according to the Davies and Rideal description, offers information on the monolayer phases and phase transitions physical state [40].
However, lipid surfactants have attracted a lot of attention at the air-water interface to create a Langmuir monolayer to monitor the molecular structure interface. In addition, combinations of various subphase solutions are employed to understand both the fundamental knowledge of lipid interaction and the monolayer formation influence, as well as the role of net loading, etc. [39,40]. A derived compressibility equation of the modulus is defined using the average mean molecular area of the monolayer at the air-water interface to deduce the physical state and orientation of the formed monolayer Langmuir. For monooline lipid, the compression of the lipid spreading isotherm (π-A), which occurs at the lowest level of the −1 surface pressure isotherms, which occurs at the lowest level of the −1 surface pressure isotherm, was observed on the plateau of transitional changes in gas-like, fluid-like and solidlike behaors. The obtained −1 values were below 50 mN/m, indicating a liquid expanded phase, and a change in the phase-state of the lipid on the air-water interface, was not due to any change of the π-A isotherms [41]. Even at low surface pressures, the compressibility module is not directly linked with its true rheological and shear properties [42]. indicates that a more liquid extended surface layer is developed [43]. The Golden rule says that the less the compression module value is, the more the monolayer is compressible [44].
Furthermore, the overall compressibility values can help to improve lipid monolayer stability.
When adding salt to the subphase of lipid monolayers causes a reduction in the values of −1 , it means that the salt and the monolayer components may be partly miscible.
The well-formed physical state displays the order of the gas, liquid, liquid-expanded to liquid-condensed, and in the end solid phase transition state in sequential order. But sometimes, the distribution of homogeneous layer, penetration time, and size of the molecule is deformed so that they are no longer in a circular shape [45]. These superficial structural characteristics might cause our monoolein lipid, which shows only one transition phase, the acceptable version of a phase transition asymmetric peak consisting of one single step at least, as shown in Figure   5. However, the effect is different for subphase conditions used on interfacial interactions of a monoolein lipid monolayer. More data on the dynamic and elastic properties and the application of −1 study on membrane lipids and their mixtures can be found in several studies [4,[46][47][48]. The presence of interacting forces is almost the same in the compression equation as surface pressure α-a isotherms measurements. The slit complement to the interactions between van der Waals and the hydrocarbon chain of the lipid and hydrogen bond links at the interface form the network with the lipid polar head group. This enhances the attraction or repulsion of the uniform distributive single-molecule lipid layer, particularly the affinity to change the subphase of salt in the bulk aqueous process [35,49]. It may also be associated in the bulk aqueous phase with monolayer molecular loosening by collapse and dissolution, as seen for the same lipid propagation to the aqueous solution [50].
With salt surface groups and lipid heads being loaded, electrostatic interactions seem highly likely to be the driving force, but interactions with the lipid tail might justify all this.
This could allow the salt to penetrate the lipid layer deeper and, therefore, significantly higher surface pressure changes than other molar concentrated subphases in 0.1 M salt forms. Figure   5(a-f) shows that higher concentrations of 0.1M salt have resulted in monoolein interaction, while monoolein monolayer binding did not affect this. Interestingly, the anionic salt subphase demonstrated substantially greater interaction with both monolayers at high concentrations than the cationic salt subphase. Since Na ions are almost not bound, the high concentration of sodium does not directly affect the density of the surface. Still, a high level of Na + ion will indirectly affect the density of surface charges. Thus, their impact on the surface concentration of lipids, in particular by an increase in Na + ion concentration [14]. However, this study can help develop finished products and determine the physicochemical properties of a stable system. The main goal is to connect different possible subphase interaction behaviours to understand the fundamental research behind the interactive lipid interface affected by other molecules at the air-water interface.

Conclusion
Using the Langmuir monolayer technique on air-water interfaces, we observed the surface properties of various salt subphases and their change in concentration for the spreading of monoolein lipid macromolecules at interfaces. Characterizes the effect of sub-phase ions on the monoolein lipid monolayer 's phase behaviour, conformation, and head group structure. Langmuir lipid monolayer must be established and monitored in multiple food systems to ensure improved structural stability, its physicochemical properties, macrostructure, and the resulting rheological surface characteristics.