Adsorption Properties of Soft Hydrophobically Functionalized PSS/MA Polyelectrolytes

: We investigated the adsorption properties of the newly synthesized, hydrophobically functionalized polyelectrolyte (HF-PE), poly(4-styrenesulfonic-co-maleic acid) copolymer (PSS/MA). The hydrophobic alkyl side chains (C 12 or C 16 ) were incorporated into the polyelectrolyte backbone via the labile amid linker to obtain the soft HF-PE product with the assumed amount of 15% and 40% degree of grafting for every length of the alkyl chain, i.e., PSS/MA-g-C 12 NH 2 (15% or 40%) as well as PSS/MA-g-C 16 NH 2 (15% or 40%). In the present contribution, we determined both the effect of grafting density and the length of alkyl chain on adsorption at water/air and water/decane interfaces, as well as on top of the polyelectrolyte multilayer (PEM) deposited on a solid surface. The dependence of the interfacial tension on copolymer concentration was investigated by the pendant drop method, while the adsorption at solid surface coated by poly(diallyldimethylammonium chloride)/poly(styrene sulphonate) PEM by the quartz crystal microbalance with dissipation (QCM-D), attenuated total reﬂection Fourier transform infrared spectroscopy (FTIR-ATR) and contact angle analysis. We found that surface activity of the hydrophobized copolymer was practically independent of the grafting ratio for C 16 side chains, whereas, for C 12 , the copolymer with a lower grafting ratio seemed to be more surface active. The results of QCM-D and FTIR-ATR experiments conﬁrmed the adsorption of hydrophobized copolymer at PEM along with the modiﬁcation of water structure at the interface. Finally, it can be concluded that the hydrophobically modiﬁed PSS/MA can be successfully applied either as the efﬁcacious emulsiﬁer for the formation of (nano)emulsions for further active substances encapsulation using the sequential adsorption method or as one of the convenient building blocks for the surface modiﬁcation materials. We 40%) ﬁlms the surface of PEM with the top polycationic layer. The results of the quartz crystal microbalance with dissipation (QCM-D), Fourier transform infrared spectroscopy in the attenuated total reﬂectance mode (FTIR-ATR) experiments and contact angle analysis revealed the efﬁcacious ability of the studied PSS/MA-g-C n NH 2, to adsorb at the PEMs. In contrast to non-modiﬁed PSS/MA, coating of the ﬁlm with hydrophobically modiﬁed polyelectrolyte increased its stability for exposure to the mildly acidic and basic medium. Our FTIR-ATR experiments revealed the changes in water structure upon the adsorption of the layers of PEMs. Adsorption of polycation led to a uniform reduction of all water ν OH stretching bands, while when polyanion was adsorbed on top of the polyelectrolyte ﬁlm, the intensity of bands associated with strong hydrogen bonding was mostly decreased. Moreover, upon adsorption of hydrophobized polyanion, the intensity of water bands associated with weak hydrogen bonding becomes also reduced and the decrease was more pronounced for a higher degree of the alkyl chain grafting. Thus, the modiﬁcation polyelectrolyte ﬁlm’s surface, either by adsorption of polycation or hydrophobically modiﬁed polyanion, to a of water irrespective of its hydrogen bonding with the underlying polymer layer. In contrast, more hydrophilic polyanion preferably water with strong hydrogen bonds.


Introduction
Nowadays, polyelectrolytes as classical soft-matter materials are of particular interest because they can modify many substrates at any interface, functionalize them, and convert them into various compatible forms with many solvents. Particularly they exert profound effects on colloidal stability for many biomedical purposes and engineered surfaces and can be applied at biointerfaces for many pharmaceutical or cosmetic applications [1][2][3][4]. Moreover, they can be designed as responsive polymer materials, playing an increasingly important part in a diverse range of applications, such as drug delivery, diagnostics, tissue engineering, and 'smart' optical systems, as well as biosensors, microelectromechanical systems, coatings, textiles and much more [5]. The associative behavior of oppositely charged of grafting for every length of alkyl chain (for the synthesis procedure see the previous report [6]). While the literature concerning grafting polymers is abundant [2,32], the reports on hydrophobically modified grafted PSS/MA are limited [6]. The synthesized amphiphilic polyelectrolytes were found to reveal excellent aqueous solubility as well as structure and concentration dependent self-association behavior. Surface tension at aqueous polymer solution/air interface and interfacial tension at the solution/decane were determined using the pendant drop shape analysis method. The quartz crystal microbalance with dissipation analysis (QCM-D) technique provided mass and viscoelastic properties of films adsorbed on quartz crystal sensors' surface. Fourier transform infrared spectroscopy (FTIR) in the attenuated total reflectance (ATR) mode was applied for the in-situ observation of the polyelectrolyte multilayer (PEM) assembly and adsorption of hydrophobically modified PSS/MA-g-C n NH 2 (n = 12, 16 the grafting ratio = 15%, 40%). We correlated the observed changes in the interfacial water structure with the hydrophobicity of polymer deposited as a terminating PEM layer.

Materials and Methods
The hydrophobized poly(4-styrenesulfonic-co-maleic acid) (PSS/MA) with side alkyl chains with twelve and sixteen carbon atoms attached to the backbone via secondary amide bonds were recently synthesized. The polymer precursor poly(4-styrenesulfonic-co-maleic acid) was purchased from Sigma-Aldrich (average M W~2 0,000 Da, 1:1 4-styrenesulfonic acid:maleic acid mole ratio, viscosity 30 cP (30% in H 2 O), density 0.94 g/mol at 25 • C). We obtained PSS/MA polyelectrolytes with 15% and 40% degree of grafting for every length of alkyl chain, i.e., PSS/MA-g-C 12 NH 2 (15%), PSS/MA-g-C 12 NH 2 (40%), PSS/MAg-C 16 NH 2 (15%) and PSS/MA-g-C 16 NH 2 (40%). The degree of grafting was calculated as the mol % content of C x side chains with respect to maleic acid groups, i.e., it indicates percent of carboxylic acid groups with chemically attached alkyl chain. The calculated grafting densities (the number of pendant C x chains per copolymer) were equal to 18.6 (for PSS/MAg-C 12 NH 2 (15%) and PSS/MA-g-C 16 NH 2 (15%)) and 49.6 (for PSS/MA-g-C 12 NH 2 (40%) and PSS/MA-g-C 16 NH 2 (40%)). The general procedure for the synthesis of those functionalized copolymers was described in our previous paper [6]. Figure 1 illustrates the structure of synthesized polymers. The detailed synthesis route as well as IF-IR and 1 H NMR spectra (Figures S1-S4) of resulting copolymers are given in ESI.
As a continuation of our studies on soft HF-PEs [6,20] in the present contribution, we report our findings on adsorption behavior of newly synthesized, hydrophobically functionalized polyelectrolyte (HF-PE), i.e., decorated along its backbone with alkyl chains (C12 or C16) via the labile amid linker with the assumed amount of 15% and 40% degree of grafting for every length of alkyl chain (for the synthesis procedure see the previous report [6]). While the literature concerning grafting polymers is abundant [2,32], the reports on hydrophobically modified grafted PSS/MA are limited [6]. The synthesized amphiphilic polyelectrolytes were found to reveal excellent aqueous solubility as well as structure and concentration dependent self-association behavior. Surface tension at aqueous polymer solution/air interface and interfacial tension at the solution/decane were determined using the pendant drop shape analysis method. The quartz crystal microbalance with dissipation analysis (QCM-D) technique provided mass and viscoelastic properties of films adsorbed on quartz crystal sensors' surface. Fourier transform infrared spectroscopy (FTIR) in the attenuated total reflectance (ATR) mode was applied for the in-situ observation of the polyelectrolyte multilayer (PEM) assembly and adsorption of hydrophobically modified PSS/MA-g-CnNH2 (n = 12, 16 the grafting ratio = 15%, 40%). We correlated the observed changes in the interfacial water structure with the hydrophobicity of polymer deposited as a terminating PEM layer.

Surface/Interfacial Tension
Surface tension at aqueous polymer solution/air interface or interfacial tension at the solution/decane were performed using the pendant drop shape analysis method based on fitting the solution of the Young-Laplace equation of capillarity to the shape of the pendant drop, recorded by a digital camera. The home-made experimental set-up and the fitting procedure were described in detail in [33]. The dynamic surface tension measurements were performed every 5 s. Measured equilibrium surface tension corresponded to the one at steady-state reached after adsorption time dependent on polymer concentration. Since the studied solutions of copolymers are polydisperse concerning the molecular weight, the kinetic curves for different drops of the same solution and various batches of modified PSS/MA did often not overlap. So the experimental values are the mean values from all performed experiments (at least three drops from two batches) and the error bars represent its standard deviation. All surface tension measurements were performed at 295 K.

Quartz Crystal Microbalance with Dissipation (QCM-D) Measurements of the Mass of the Adsorbed Polymer
The QCM-D technique allows analyzing mass and viscoelastic properties of films adsorbed at the surface of quartz crystal sensors. During the adsorption process, a decrease in the frequency of crystal resonance and an increase in oscillations energy dissipation is observed. An observed negative frequency shift is an indication of the mass increase due to the adsorption process. The dissipation of the crystal oscillations energy, ∆D, is related to the viscous losses in the film. A high value of ∆D and spread for the oscillation over-tones indicates a soft film structure. If the adsorbed film is rigid, i.e., the measured dissipation is less than 1 × 10 −6 per 10 Hz of the frequency shift, the Sauerbrey equation describes the linear relation between the frequency shift (∆f ) and the areal mass density of the adsorbed layer (∆m) [34]: where C is the constant of the crystal (C = 17.7 ng/cm 2 /Hz based on the physical properties of quartz crystal) and n is the oscillation overtone number. This linear relation is not obeyed for soft films, and the analysis based on viscoelastic models of an adsorbed film is needed. The formation of polyelectrolyte multilayers (PDADMAC/PSS) using the sequential adsorption method and adsorption of copolymers on top of them was observed using the QCM-D QSense E4 system (Biolin Scientific, Gothenburg, Sweden). The AT-cut, piezoelectric, gold-coated quartz sensors (14-mm diameter, 4.95-MHz fundamental resonance frequency) (Q-Sense, Biolin Scientific, Gothenburg, Sweden) were washed with piranha solution (96% H 2 SO 4 : 30% H 2 O 2 , 2:1 (v/v); 5 min) and then boiled in distilled water in 80 • C, thoroughly rinsed with distilled water and stream-dried with air. They were then immersed in the solution of polycation (PDADMAC) for 15 min and rinsed in a 0.15 M NaCl solution to remove weakly adsorbed chains of polyelectrolyte from the substrate surface. Next, the substrate was immersed in the negatively charged solution (for 15 min) of polyanion (PSS) and rinsed (0.15 M NaCl), which led to the formation of the polyelectrolyte bilayer. By repeating this deposition cycle, one can obtain multilayer coatings with the desired structure and thickness. In this case, that was nine layers' (PDADMAC/PSS) 4  until again steady-state values of the frequency shift and dissipation were attained. Then the salt solution with pH = 3 was introduced to the cell for the time needed to attain steady frequency and dissipation shifts, followed by rinsing with the solution with pH = 7. In the second set of experiments, after rinsing with the salt solution with pH = 7, the cell was filled with the solution at pH = 11, steady frequency and dissipation shifts were attained, and the cell was rinsed again at pH = 7. The example of the crystal frequency changes during those experiments is illustrated in Figure S5 given in the Supplementary Materials. Since adsorbed layers were relatively rigid (measured dissipation was less than 1 × 10 −6 per 10 Hz of the frequency shift), the mass of adsorbed film was calculated with the Sauerbrey equation. The calculations were performed with Qtools 3 software (QSense, Biolin Scientific, Gothenburg, Sweden). The determined mass was the average of the results from four QCM-D cells. The experimental error was less than 10% of the measured mass. In the separate experiment, the formation of PDADMAC/PSS film was followed in the QCM-D cell to confirm its buildup. The experiment was performed according to the methodology described in [35].

Contact Angle Analysis
The contact angle of water droplets was determined using the Drop Shape Analyzer Krüss DSA 100 M. Water droplets with a volume of 12 µL were placed at the surface of silicon wafers cleaned by washing in piranha solution and by submerging in water at 70 • C for 30 min. The wafers were covered by the polyelectrolyte multilayer using the sequential adsorption technique described above. The adsorption of (PDADMAC/PSS) 4 PDADMAC film eliminated the influence of the substrate on the contact angle of the outer layer of hydrophobically modified PSS/MA. Each deposition step took 10 min, and after the deposition of a subsequent layer of the polymer, the sample was rinsed three times with NaCl solution for 1 min. Samples terminated with hydrophobized polymers and PSS/MA were also exposed to basic conditions for 15 min by submerging in NaCl solution adjusted to pH = 11 by the addition of NaOH. To prevent NaCl crystals from forming on the samples, they were rinsed three times in water for 1 min after the films were deposited. Then the samples were left to dry for 16 h in ambient air. Each measurement was the average of two runs with at least five drops. Contact angle measurements were performed at 295 K.
Fourier transform infrared spectroscopy (FTIR) in the attenuated total reflectance (ATR) mode was for the in-situ observation of the polyelectrolyte multilayers assembly and adsorption of hydrophobically modified PSS/MA. The experiments were performed using the Thermo Scientific Nicolet iS10 (Waltham, MA, USA) FTIR spectrometer equipped with Specac 25 Reflection Variable Angle ATR accessory with Liquid Holder P/N GS 11003 flow cell. The cell was equipped with Ge crystal (25 reflections, trapezoid dimensions 52 × 20 × 2 mm), which together with Teflon gaskets on both sides, formed two closed compartments, where adsorption of polyelectrolytes on the germanium surface took place. The polymer solution was introduced through the flow system into the liquid holder, where it was left for 15 min for the polymer to adsorb onto the crystal surface. The system was washed with NaCl solution, and then the FTIR spectrum was recorded. The multilayer polyelectrolyte films were formed on the crystal surface by the layer-bylayer technique by alternating adsorption of PDADMAC and PSS, and the spectrum was recorded after each deposition and rinsing step. The PSS/MA copolymer without and with grafted hydrophobic chains was adsorbed on the top of (PDADMAC/PSS) 4 PDADMAC multilayer. Since the method is very sensitive to swelling and shrinking of the polymer layers, the constant conditions were maintained during experiments-salt concentration 0.15M NaCl and ambient temperature (22 • C). The spectra were acquired in the range 800-4000 cm −1 with the resolution 4 cm −1 ; scans were repeated 256 times and the resulting spectra were processed with PeakFit software (SeaSolve Software, San Jose, CA, USA). The background spectrum for the cell filled with 0.15 M NaCl solution was first subtracted from the ones received after the deposition of the consecutive layers of the multilayer film, the baseline established and the characteristic bands of the polyelectrolytes PDADMAC, PSS, and hydrophobized PSS/MA copolymer were identified. Bands were deconvoluted and the second derivative of the spectra was used for the decomposition of spectra into a sum of Gaussian and Lorentzian peaks. The differential spectra of the adsorbed polymer on top of polyelectrolyte films were determined according to the method described previously [36].

Adsorption at Fluid Interfaces
Measurements of surface tension at air/polymer solution and interfacial tension at decane/polymer solution interfaces using the pendant drop shape analysis technique were performed to determine the adsorption properties of the hydrophobized PSS/MA copolymers (i.e., PSS/MA-g-C 12 NH 2 and PSS/MA-g-C 16 NH 2 ) at fluid interfaces. The dependence of surface/interfacial tension on the mass concentration of copolymers with grafted C 12 and C 16 alkyl chains via amide bond for 15% and 40% grafting ratio is shown in Figure 2. The results demonstrate that unmodified PSS/MA polyelectrolyte has no surface activity up to high concentration (5000 ppm). For hydrophobized copolymers, we can observe surface activity for concentrations above 200 ppm. Interestingly, for the PSS/MA-g-C 12 NH 2 we observed slightly higher surface activity (decrease of surface/interfacial tension at a lower concentration) for a lower grafting ratio (15%), whereas, for PSS/MA-g-C 16 NH 2 it seemed not to depend on the grafting ratio.  Triangles denote surface/interfacial tension for unmodified PSS/MA copolymer at air/water (black symbols) and decane/water (white symbols) interface, respectively.
To account for the effect of molecular weight difference, we evaluated the average Mw of the synthesized copolymers taking into account the reactant used for the synthesis and the grafting degree as: 23,000 Da-PSS/MA-g-C12NH2 (15%), 28,000 Da PSS/MA-g-C12NH2 (40%), 24,000 Da-PSS/MA-g-C16NH2 (15%) and 31,000 Da-PSS/MA-g-C16NH2 (40%). We used the average Mw to express surface/interfacial tension isotherms as molar concentration dependence. The results are given in Figure 3. Contrary to the expectation, the surface activity for the PSS/MA-g-C12NH2 copolymer was again slightly higher for the lower grafting ratio while for the PSS/MA-g-C16NH2 no differences in surface activity were observed. To account for the effect of molecular weight difference, we evaluated the average M w of the synthesized copolymers taking into account the reactant used for the synthesis and the grafting degree as: 23,000 Da-PSS/MA-g-C 12 NH 2 (15%), 28,000 Da PSS/MA-g-C 12 NH 2 (40%), 24,000 Da-PSS/MA-g-C 16 NH 2 (15%) and 31,000 Da-PSS/MA-g-C 16 NH 2 (40%). We used the average M w to express surface/interfacial tension isotherms as molar concentration dependence. The results are given in Figure 3. Contrary to the expectation, the surface activity for the PSS/MA-g-C 12 NH 2 copolymer was again slightly higher for the lower grafting ratio while for the PSS/MA-g-C 16 NH 2 no differences in surface activity were observed.
(40%). We used the average Mw to express surface/interfacial tension isotherms as molar concentration dependence. The results are given in Figure 3. Contrary to the expectation, the surface activity for the PSS/MA-g-C12NH2 copolymer was again slightly higher for the lower grafting ratio while for the PSS/MA-g-C16NH2 no differences in surface activity were observed.  In our previous papers [6,20], we demonstrated that the hydrophobic domains are formed consisting of two to three hydrophobic chains collapsed onto the polyelectrolyte backbone. With increasing grafting density, these domains can merge and the polyelectrolyte molecule can assume the form of a pseudo-micelle. For the polymer to be adsorbed at the air/water or oil/water interface, those micelles need to re-conform with the alkyl chains exposed to the hydrophobic phase. That re-conformation can be less favorable for In our previous papers [6,20], we demonstrated that the hydrophobic domains are formed consisting of two to three hydrophobic chains collapsed onto the polyelectrolyte backbone. With increasing grafting density, these domains can merge and the polyelectrolyte molecule can assume the form of a pseudo-micelle. For the polymer to be adsorbed at the air/water or oil/water interface, those micelles need to re-conform with the alkyl chains exposed to the hydrophobic phase. That re-conformation can be less favorable for PSS/MA-g-C 12 NH 2 (40%) than for PSS/MA-g-C 12 NH 2 (15%). On the other hand, the re-conformation tendency is the same for PSS/MA-g-C 16 NH 2 disregarding the alkyl chain grafting density, however, lower than for PSS/MA-g-C 12 NH 2 taking into account smaller surface activity (c.f. Figure 3a,b). That result is consistent with one obtained by Théodoly et al., who studied the adsorption of hydrophobic polyelectrolyte, polystyrene with various degrees of sulphonation, at the air/water interface [37]. They found that such polyelectrolyte's surface activity first increased with the charged fraction, reached an optimum at 55%, and then decreased. They explained the presence of optimum of adsorption with the degree of sulfonation by a conformational barrier effect, more hydrophobic chains assuming the compact shape of a charged colloid that prevented adsorption. Similarly, Barazza et al. studied the adsorption of hydrophobically modified polyelectrolytes at the n-octane/water interface. They concluded that the free energy of adsorption could be split into contributions arising from two different processes, namely, the distribution of the polymer between the bulk of the solution and the arrangement of the macromolecule at the interface [38].
The addition of electrolyte increased surface activity of hydrophobized PSS/MA polyelectrolyte; however, that effect was much more pronounced at the decane/solution interface, as it is demonstrated in Figure 4, where the surface/interfacial tension isotherms in water and 0.15 M NaCl for PSS/MA-g-C 16 NH 2 (15%) and PSS/MA-g-C 16 NH 2 (40%) are compared. Similar results were obtained for PSS/MA-g-C 12 NH 2 (15%) and PSS/MA-g-C 12 NH 2 (40%) (not shown). The addition of 0.1 M NaOH to the copolymer solution had a small effect on surface activity (except for small concentration) due to the compensation of two effects, screening of ionic charges due to the higher ionic strength of the solution and higher charge of the polymer caused by the deprotonation of carboxylic groups at high pH.

Adsorption at Solid Surface Covered with Polyelectrolyte Multilayer
To analyze the adsorption properties of the hydrophobized PSS/MA copolymers, PSS/MA-g-C 12 NH 2 (15%), PSS/MA-g-C 12 NH 2 (40%), PSS/MA-g-C 16 NH 2 (15%), and PSS/MA-g-C 16  the deposited film and its stability while exposed to acidic (pH = 3) and basic (pH = 11) conditions. The results are collected in Table 1. interface, as it is demonstrated in Figure 4, where the surface/interfacial tension isotherms in water and 0.15 M NaCl for PSS/MA-g-C16NH2 (15%) and PSS/MA-g-C16NH2 (40%) are compared. Similar results were obtained for PSS/MA-g-C12NH2 (15%) and PSS/MA-g-C12NH2 (40%) (not shown). The addition of 0.1 M NaOH to the copolymer solution had a small effect on surface activity (except for small concentration) due to the compensation of two effects, screening of ionic charges due to the higher ionic strength of the solution and higher charge of the polymer caused by the deprotonation of carboxylic groups at high pH.

Adsorption at Solid Surface Covered with Polyelectrolyte Multilayer
To analyze the adsorption properties of the hydrophobized PSS/MA copolymers, PSS/MA-g-C12NH2 (15%), PSS/MA-g-C12NH2 (40%), PSS/MA-g-C16NH2 (15%), and PSS/MA-g-C16NH2 (40%), we determined the mass of adsorbed polymer on top of the (PDADMAC/PSS)4PDADMAC multilayer, deposited on top of the QCM sensor as described in the "Materials and Methods" section. Then we determined the response of the deposited film and its stability while exposed to acidic (pH = 3) and basic (pH = 11) conditions. The results are collected in Table 1.  Adsorption of the PSS/MA-g-C n NH 2 on positively charged polyelectrolyte film terminated by the PDADMAC layer appeared to be slightly less effective than that of the non-modified copolymer. Upon exposure to acidic conditions, pH = 3, the mass of the films largely decreased, but for films covered with the hydrophobically modified copolymer, it was regained. Since the process is mostly reversible, it can be attributed to a decrease in film hydration. Carboxylic groups of maleic acid become protonated, the negative charge decreases and the excess of compensating counterions are repelled from the film together with hydrating water. After exposure to basic conditions, pH = 11, the opposite phenomenon could be observed, carboxylic groups become fully deprotonated, negative charge increases, and the film swells. Measured film mass increased and then grew upon rinsing at pH = 7. The coating of the polyelectrolyte film with hydrophobically modified PSS/MA seemed to stabilize the whole multilayer structure.
We determined the effect of the external layer of a polyelectrolyte multilayer film formed with the hydrophobically modified PSS/MA copolymer on its wettability. For that purpose, we deposited (PDADMAC/PSS) 4 PDADMAC on a silicon wafer and then adsorbed the copolymer by deep coating in 200 ppm aqueous solution in 0.15 NaCl. We determined the equilibrium contact angle by the sessile drop method. The results are collected in Table 2.
We noted the typical dependence of the contact angle observed for the polyelectrolyte multilayers, i.e., the structure with a polycation (PDADMAC) in the top layer being more hydrophobic than one with a polyanion (PSS or PSS/MA) [39,40]. Surfaces covered with hydrophobically modified PSS/MA were slightly more hydrophobic than ones with unmodified PSS/MA at the top layer and the contact angle increased with the length of the alkyl chain grafted at the polyelectrolyte backbone. That limited increase of hydrophobicity and lower adsorption of PSS/MA-g-C n NH 2 can be the evidence that the modified polyelectrolyte is adsorbed preferably in the form of pseudo-micelles; thus, alkyl chains can be less exposed to the aqueous phase. We observed an increase of the contact angle when the films were exposed to basic conditions (pH = 11) for 15 min (c.f. Table 2) except for the PEM with PSS/MA-g-C 16 NH 2 as a top layer. As it was demonstrated in Table 1, the film swells due to hydration, polycation chains become more mobile and can penetrate the top layer of the film, which increases its hydrophobicity. Since the swelling is the lowest for PSS/MA-g-C 16 NH 2 the increase of hydrophobicity was not observed. The decrease of the contact angle at the film terminated by the PDADMAC layer upon exposure to pH = 11 seems to confirm that hypothesis as the top layer can be penetrated by more mobile polyanions. We applied the FTIR-ATR technique to investigate the formation of the polyelectrolyte multilayer and adsorption of the hydrophobically modified PSS/MA as its external layer. Application of that technique allowed to monitor changes of IR spectra of films and interfacial water due to the increased hydrophobicity of the copolymers. The example of the IR spectra obtained at the consecutive steps of the formation of (PDADMAC/PSS) 4 PDADMAC multilayer film on the surface of Ge crystal, followed by the adsorption of unmodified PSS/MA, is illustrated in Figure 5a. lowest for PSS/MA-g-C16NH2 the increase of hydrophobicity was not observed. The decrease of the contact angle at the film terminated by the PDADMAC layer upon exposure to pH = 11 seems to confirm that hypothesis as the top layer can be penetrated by more mobile polyanions. We applied the FTIR-ATR technique to investigate the formation of the polyelectrolyte multilayer and adsorption of the hydrophobically modified PSS/MA as its external layer. Application of that technique allowed to monitor changes of IR spectra of films and interfacial water due to the increased hydrophobicity of the copolymers. The example of the IR spectra obtained at the consecutive steps of the formation of (PDADMAC/PSS)4PDADMAC multilayer film on the surface of Ge crystal, followed by the adsorption of unmodified PSS/MA, is illustrated in Figure 5a. Several fingerprint bands for PSS and PDADMAC can be clearly identified. At 1008, 1035, 1125, and 1190 cm −1 , respectively, the in-plane bending vibrations of the para-substituted benzene ring, symmetric and asymmetric stretching vibration of the SO3 − group for PSS, and 1475 cm −1 the vibration of the amine group of PDADMAC [41]. Upon adsorption of consecutive layers of the film, the intensity of those fingerprint bands increases with the characteristic odd-even effect (odd layers-PDADMAC, even layers-PSS), but the overall growth of the film is linear, as it is illustrated in Figure 5b. That correlates well with the results of previous studies of PDADMAC/PSS multilayer film formation by the QCM-D [35] (see also Figure S6). The adsorption of PSS/MA was confirmed by an increase in PSS characteristic bands' intensity, while the intensity characteristic bands for the car- Several fingerprint bands for PSS and PDADMAC can be clearly identified. At 1008, 1035, 1125, and 1190 cm −1 , respectively, the in-plane bending vibrations of the parasubstituted benzene ring, symmetric and asymmetric stretching vibration of the SO 3 − group for PSS, and 1475 cm −1 the vibration of the amine group of PDADMAC [41]. Upon adsorption of consecutive layers of the film, the intensity of those fingerprint bands in-creases with the characteristic odd-even effect (odd layers-PDADMAC, even layers-PSS), but the overall growth of the film is linear, as it is illustrated in Figure 5b. That correlates well with the results of previous studies of PDADMAC/PSS multilayer film formation by the QCM-D [35] (see also Figure S6). The adsorption of PSS/MA was confirmed by an increase in PSS characteristic bands' intensity, while the intensity characteristic bands for the carboxylic groups were too weak to be identified and partly overlap with the band at 1475 cm −1 . Table 3 presents the intensity differences of these bands upon adsorption of PSS/MA copolymers (non-modified or hydrophobically modified) on top of (PDADMAC/PSS) 4 PDADMAC multilayer film (see Figure S7). These results confirm that the adsorption is not dependent on the hydrophobicity of the copolymer. The ATR analysis of the changes of IR spectra of the water ν OH stretching band in the region between 3000 and 3800 cm −1 allows determining a variation of water structure at the interface upon adsorption of the consecutive polyelectrolyte layers [42,43]. The infrared spectrum of water in that region contains four components that can be ranked by hydrogen bonding strength. These components at approximately 3280, 3440, 3550, and 3630 cm −1 correspond to water from that which is most hydrogen-bonded with its surroundings to water that is least hydrogen-bonded [42,44]. Figure 6 demonstrates the differential spectra of the multilayer film after adsorption of top polycation layer (PDADMAC/PSS) 2 PDADMAC, (PDADMAC/PSS) 3 PDADMAC, (PDADMAC/PSS) 4 PDADMAC, adsorption of the polyanion (PDADMAC/PSS) 3 , (PDADMAC/PSS) 4 and non-functionalized and hydrophobized PSS/MA. These results suggest that upon adsorption of polycation (PDADMAC), the intensity of all water ν OH stretching bands is uniformly reduced. On the other hand, when polyanion is adsorbed on top of the polyelectrolyte film, the intensity of bands associated with strong hydrogen bonding is mostly decreased. Moreover, the hydrophobization degree of PSS/MA copolymer has a strong effect on the water structure. Upon adsorption of hydrophobized polyanion, the intensity of water bands associated with weak hydrogen bonding becomes reduced stronger with a higher degree of the alkyl chain grafting. Correlating those findings with the contact angle measurements we can conclude that hydrophobization of the surface of polyelectrolyte film, either by adsorption of polycation or hydrophobized polyanion, leads to reduction of water irrespective of its hydrogen bonds with the underlying polymer layer, while adsorption of more hydrophilic polyanion mostly eliminates water with strong hydrogen bonds. That result may correlate with the finding of Ho et al., who applied ATR-FTIR and showed that water in the multilayer is subject to noticeably less hydrogen bonding than water in the bulk [43].
We investigated the IR spectra of PEMs with the hydrophobically modified PSS/MA as their external layer when exposed to alkaline conditions, pH = 11. The example of the spectrum changes for PSS/MA-g-C 12 NH 2 (15%) is illustrated in Figure 7. Immediately after the film was exposed to the alkaline environment, bands of carboxylic ion appeared at 1580-1565 cm −1 and around 1410 cm −1 and the weak band at 1710 cm −1 attributed to a protonated carboxylic group diminished [45]. Similar results were obtained for unmodified PSS/MA and PSS/MA-g-C 12 NH 2 (40%), as illustrated in Figure S8. We did not observe any significant changes in the spectra obtained after exposure to pH = 11 for up to two hours and flushing back with the solution with pH = 7. That indicates that within that time frame films were stable, and we did not observe base-catalyzed hydrolysis of the amide bond; however, that aspect requires further investigation, including more extended exposure to alkaline conditions and taking spectra in D 2 O to avoid overlapping of the amid and water bands.
anion, the intensity of water bands associated with weak hydrogen bonding becomes reduced stronger with a higher degree of the alkyl chain grafting. Correlating those findings with the contact angle measurements we can conclude that hydrophobization of the surface of polyelectrolyte film, either by adsorption of polycation or hydrophobized polyanion, leads to reduction of water irrespective of its hydrogen bonds with the underlying polymer layer, while adsorption of more hydrophilic polyanion mostly eliminates water with strong hydrogen bonds. That result may correlate with the finding of Ho et al., who applied ATR-FTIR and showed that water in the multilayer is subject to noticeably less hydrogen bonding than water in the bulk [43]. We investigated the IR spectra of PEMs with the hydrophobically modified PSS/MA as their external layer when exposed to alkaline conditions, pH = 11. The example of the spectrum changes for PSS/MA-g-C12NH2 (15%) is illustrated in Figure 7. Immediately after the film was exposed to the alkaline environment, bands of carboxylic ion appeared at 1580-1565 cm −1 and around 1410 cm −1 and the weak band at 1710 cm −1 attributed to a protonated carboxylic group diminished [45]. Similar results were obtained for unmodified PSS/MA and PSS/MA-g-C12NH2 (40%), as illustrated in Figure S8. We did not observe any significant changes in the spectra obtained after exposure to pH = 11 for up to two hours and flushing back with the solution with pH = 7. That indicates that within that time frame films were stable, and we did not observe base-catalyzed hydrolysis of the amide bond; however, that aspect requires further investigation, including more extended exposure to alkaline conditions and taking spectra in D2O to avoid overlapping of the amid and water bands.

Conclusions
The rationally designed hydrophobically functionalized poly(4-styrenesulfonic-comaleic acid) derivatives (i.e., PSS/MA-g-CnNH2; n = 12, 16)-with both strongly (sulfonic) and weakly (carboxylate) acidic moieties as well as having the side C12 and C16 chains attached to the PE backbone via the amide linker-made it possible to achieve a distinct group of amphiphilic pH-responsible PEs. Considering their soft (chemodegradable, hydrolyzable) structure, they can be treated as one of the custom-designed products. Our present studies made it possible to receive some crucial information about their structure influence, mainly of the grafting density and the hydrophobic chain length, on adsorption phenomena at the fluid interfaces and solid surface covered with polyelectrolyte multilayers. All newly synthesized HF-PEs were surface active in contrast with non-functionalized PSS/MA polyanion; however, the tendency to self-aggregation can affect surface activity at fluid interfaces. Therefore, some optimal grafting density can be expected.
We found that PSS/MA-g-CnNH2 (n = 12, 16, the grafting ratio = 15%, 40%) form satisfactorily stable films at the surface of PEM with the top polycationic layer. The results of the quartz crystal microbalance with dissipation (QCM-D), Fourier transform infrared

Conclusions
The rationally designed hydrophobically functionalized poly(4-styrenesulfonic-comaleic acid) derivatives (i.e., PSS/MA-g-C n NH 2 ; n = 12, 16)-with both strongly (sulfonic) and weakly (carboxylate) acidic moieties as well as having the side C 12 and C 16 chains attached to the PE backbone via the amide linker-made it possible to achieve a distinct group of amphiphilic pH-responsible PEs. Considering their soft (chemodegradable, hydrolyzable) structure, they can be treated as one of the custom-designed products. Our present studies made it possible to receive some crucial information about their structure influence, mainly of the grafting density and the hydrophobic chain length, on adsorption phenomena at the fluid interfaces and solid surface covered with polyelectrolyte multilayers. All newly synthesized HF-PEs were surface active in contrast with non-functionalized PSS/MA polyanion; however, the tendency to self-aggregation can affect surface activity at fluid interfaces. Therefore, some optimal grafting density can be expected.
We found that PSS/MA-g-C n NH 2 (n = 12, 16, the grafting ratio = 15%, 40%) form satisfactorily stable films at the surface of PEM with the top polycationic layer. The results of the quartz crystal microbalance with dissipation (QCM-D), Fourier transform infrared spectroscopy in the attenuated total reflectance mode (FTIR-ATR) experiments and contact angle analysis revealed the efficacious ability of the studied PSS/MA-g-C n NH 2, to adsorb at the PEMs. In contrast to non-modified PSS/MA, coating of the film with hydrophobically modified polyelectrolyte increased its stability for exposure to the mildly acidic and basic medium. Our FTIR-ATR experiments revealed the changes in water structure upon the adsorption of the layers of PEMs. Adsorption of polycation led to a uniform reduction of all water ν OH stretching bands, while when polyanion was adsorbed on top of the polyelectrolyte film, the intensity of bands associated with strong hydrogen bonding was mostly decreased. Moreover, upon adsorption of hydrophobized polyanion, the intensity of water bands associated with weak hydrogen bonding becomes also reduced and the decrease was more pronounced for a higher degree of the alkyl chain grafting. Thus, the modification of polyelectrolyte film's surface, either by adsorption of polycation or hydrophobically modified polyanion, leads to a removal of water irrespective of its hydrogen bonding with the underlying polymer layer. In contrast, adsorption of more hydrophilic polyanion mostly eliminates preferably water with strong hydrogen bonds.
Our findings allow concluding that the unique characteristics of hydrophobically modified PSS/MA may open the possibility of understanding its further performance properties, especially toward pH triggering structures for drug delivery. They can be successfully applied either as efficacious emulsifiers for the formation of (nano)emulsions for further active substance encapsulation using the sequential adsorption method or as one of the convenient building blocks for the surface modification materials.
Supplementary Materials: The following are available online at https://www.mdpi.com/2504-5 377/5/1/3/s1, Details of synthesis of hydrophobically functionalized poly(4-styrenosulfonic-comaleic acid), Figure S1: Synthetic routes for hydrophobically functionalized poly(4-styrenosulfonicco-maleic acid); Figure S2. 1H NMR spectra of the non-functionalized PSS/MA (0-8 ppm range); Figure S3. 1 H NMR spectra of the synthesized hydrophobically functionalized poly(4-styrenosulfonicco-maleic acid) with amide linking group (0-8 ppm range); Figure S4. 1H NMR spectra of the synthesized hydrophobically functionalized poly(4-styrenosulfonic-co-maleic acid) with amide linking group (0.5-3.5 ppm and 5.5-8.0 ppm ranges); Figure S5: The example of the QCM-D data obtained by monitoring the deposition of the copolymer (PSSMA-g-C 12 NH 2 (40%)) on top of the multilayer film. A-changes in the frequency during copolymer deposition on top of the (PDADMAC/PSS) 4 PDADMAC films and rinsing solution of 0.15 M NaCl in pH = 7 and pH = 11. B-changes in the frequency during copolymer deposition on top of the (PDADMAC/PSS) 4,5 films and rinsing solution of 0.15 M NaCl in pH = 7 and pH = 3. Spikes observed at c.a. 850 s are artifacts, disregarded in the analysis; Figure S6: The example of the QCM-D data obtained by monitoring the formation of the (PDADMAC/PSS) 4 PDADMAC multilayer film and the respective increase of the film's mass; Figure S7: The FTiR spectra in the PSS fingerprint region for (PDADMAC/PSS) 4 PDADMAC film without (dashed line) and with adsorbed copolymer layer (solid line): A. PSS/MA; B. PSS/MA-g-C 12 NH 2 (15%); C. PSS/MA-g-C 12 NH 2 (40%); Figure S8. The FTIR spectra of (PDADMAC/PSS) 4 PDADMAC multilayer film with adsorbed copolymer as the last layer during the exposure to the alkaline environment pH = 11. The dashed line illustrates the spectrum obtained after exposure for 30 min at pH = 11 and flushing with 0.15 M NaCl solution pH = 7. Top-PSS/MA, bottom-PSS/MA-g-C 12 NH 2 (40%). Funding: This work has been supported by the National Science Center Poland within a framework of the OPUS programme (2017/25/B/ST4/02450).

Data Availability Statement:
The data if not directly presented in this study are available on request from the corresponding author.