High-Resolution Ultrasonic Spectroscopy: Looking at the Interpolyelectrolyte Neutralization from a Different Perspective

High-Resolution Ultrasonic Spectroscopy: Looking at the Interpolyelectrolyte Neutralization from a Different Perspective Tin Klacic,*Adam Jugl, Miloslav Pekar, and Davor Kovacevic* Cite This: Macromolecules 2023, 56, 1434−1445 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: In this study, the high-resolution ultrasonic spectroscopy (HR-US) technique was applied to examine interpolyelectrolyte neutralization. The mentioned method was tested on the example of complexation between poly(allylammonium) cations and poly(acrylate) anions in aqueous solutions at pH = 7. It was confirmed by HR-US that the type of titration (stepwise or abrupt), the direction of titration, and the type of background salt affect the outcome of interpolyelectrolyte neutralization. The obtained results were explained on the basis of ultrasonic velocity and attenuation changes in the context of suspension compressibility, a parameter that is extremely sensitive to molecular organization and intermolecular interactions. Moreover, the results of HR-US measurements proved to be consistent with previous results obtained by more traditional methods such as dynamic light scattering, microcalorimetry, and electrokinetics. This research demonstrates that HR-US is a convenient and reliable method that can be employed for the investigation of interpolyelectrolyte neutralization and polyelectrolyte-related processes. 1. INTRODUCTION Polyelectrolytes are macromolecules (e.g., synthetic polymers or biopolymers) with ionic or ionizable functional groups that can carry positive or negative charges in solution. Mixing of oppositely charged polyelectrolytes in aqueous solution may result in polyelectrolyte complex (PEC) formation. The occurrence of PECs often goes along with phase separation, resulting in either solid−liquid or liquid−liquid phase separation. In the former case, dense polyelectrolyte precipitates are formed, while the latter case results in the formation of polyelectrolyte complex coacervates with liquidlike properties. 1−4 As this self-assembly processes for many polyelectrolyte pairs are generally isoenthalpic, it is believed that PECs are formed due to an increase in entropy caused by the release of structured water molecules and counterions associated with polymer chains. 5−12 The morphology, structure, composition, and physicochemical properties of formed PECs will depend on numerous assembly parameters such as concentration and charge density of polyelectrolytes, 13 temperature, pH, and ionic strength of the medium. 5,12,14−16 One interesting factor that also affects the structure and properties of PECs is the type of used background salt. As has been recently shown by our and Schlenoff group, 7−10 the influence of supporting anions on PEC composition and energetics of interpolyelectrolyte neutralization can be reasonably well correlated with the position of the counteranion in the Hofmeister series. The Hofmeister series was discovered in protein precipitation experiments, 17,18 and this series classifies anions and cations according to their increasing precipitation power as follows: < < < < < < <SCN ClO I NO Br Cl F H PO 4 3 2 4 < < < < < < + + + + + + + Li Na K Rb Cs NH (CH ) N 4 3 4 This list of ions rank-ordered in terms of how strongly they modulate protein solubility provides a path for the modification of PEC properties simply by changing the type of salt during their preparation. Clearly, PECs are versatile materials that could possibly be used in a wide range of applications, such as matrices for enzyme immobilization, 19 biocompatible coatings for drug delivery, 20 and vectors for gene therapy, 21,22 to name a few. The process of interpolyelectrolyte neutralization is commonly studied by light scattering methods, turbidimetry, conductometry, potentiometry, spectrophotometry, and calorimetry. 5−13,23 Each of these experimental techniques gives valuable information about the examined system. Potentiometry, for example, provides information on the proportion of paired monomers by monitoring counterion activity in solution. However, it is limited to complexation reactions of “pure” polyelectrolytes (without excess counterions). Calorimetry provides valuable information on the energetics of interpolyelectrolyte neutralization. However, as mentioned earlier, interpolyelectrolyte neutralization processes are mostly isoenthalpic, 5−12 which limits the application of calorimetry in research. In addition to the above, measurable changes in Received: November 17, 2022 Revised: January 31, 2023 Published: February 14, 2023 Article pubs.acs.org/Macromolecules © 2023 American Chemical Society 1434 https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 Downloaded via BRNO UNIV OF TECHNOLOGY on March 27, 2023 at 12:26:25 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. enthalpy always indicate that a chemical process is taking place. The only problem is to determine which process it is. Methods that monitor the scattering of light and electrophoretic mobility of particles provide valuable information on the size and structure of PECs. However, they are limited to the molar ratios of titrant and titrand before the onset of flocculation. UV/vis spectrophotometry is suitable for determining the proportion of oppositely charged monomers in the reaction products. Spectrophotometry can also be used to investigate the conversion of metastable products to equilibria. 7 However, to perform a spectrophotometric experiment, it is necessary to use spectrophotometrically active polyelectrolytes such as poly(sodium 4-styrenesulfonate) (PSS) or at least label the polyelectrolytes with some kind of a marker. From all of the mentioned, it can be concluded that the processes of interpolyelectrolyte neutralization are best studied by a combination of complementary techniques, but even then, as shown by Philipp et al., 13 different methods often indicate different “titration endpoints” for the same pair of polymers. High-resolution ultrasonic spectroscopy (HR-US) is a relatively novel spectroscopic technique for material analysis. A detailed description of this method can be found in recently published review papers. 24,25 Briefly, this technique is based on the measurements of velocity and attenuation (energy losses that include absorption and scattering contributions) of ultrasonic compression waves propagating through the analyzed sample. These ultrasonic waves probe the elastic properties of materials determined by intermolecular interactions and molecular organization. Thus, HR-US offers a number of advantages over traditional methods such as UV/vis spectrophotometry. For example, it is a nondestructive technique that does not require optical markers and can be used on concentrated and opaque samples. Another advantage is that it is relatively easy to change the wavelength of the ultrasonic wave. Unlike spectrophotometry where the electromagnetic wave originates in a light source and therefore needs special effort to obtain the required spectral purity, ultrasonic waves are generated electronically. Therefore, a typical ultrasonic spectrometer can cover a broad range of frequencies (from 1 to 20 MHz). In addition, HR-US measurements can be performed with resolution down to 10−5% for ultrasonic velocity in small sample volumes (typically 1 mL and down to 0.03 mL) at ambient or elevated pressures over a broad temperature range (−40 to 130 °C) and in various measuring regimes such as automatic titrations and measurements in flow. Also, HR-US measurements can be performed in different media ranging from dilute solutions to semi-solid materials such as waxes and gels. Therefore, HR-US is a very versatile method that can be used to analyze a broad range of molecular processes. Current applications of this technique include analysis of aggregation, micellization, and gelation phenomena, 26−30 phase diagrams of microemulsions, 31 conformational transitions in polymers and biopolymers, 32 interactions between polyelectrolytes and surfactants or amino acids, 33−35 stability of emulsions and suspensions, 36−38 polymer adsorption onto surfaces, 39 enzymatic hydrolysis of proteins and carbohydrates, 25,40−43 and many others. Despite being employed for the characterization of numerous chemical and physical processes, there is a lack in the literature regarding the potential use of HR-US for studying the formation of polyelectrolyte complexes. The aim of this work is to use, for the first time, highresolution ultrasound spectrometry in a titration regime to gain a more detailed insight into interpolyelectrolyte neutralization of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) in different salt media, namely, NaF, NaCl, NaClO4, LiCl, and (CH3)4NCl. To the best of our knowledge, there is no report published yet on using this method to investigate the formation of polyelectrolyte complexes. In our previous study, 9 complexation between the same pair of macroions was examined by means of electrokinetics, microcalorimetry, and dynamic light scattering (DLS). It was found that the presence of different electrolytes affected the PAH/ PAA interpolyelectrolyte neutralization considerably, leading to ion-specific aggregation of polyelectrolyte particles. Here, we expand the research to HR-US measurements, which are discussed also with respect to the earlier study. 9 2. EXPERIMENTAL SECTION 2.1. Materials. Poly(allylamine hydrochloride) (Mw≈17.5 kDa) and poly(acrylic acid) (Mw≈1000 kDa) were purchased from SigmaAldrich. The monomer functionalization degrees (f), also known as degrees of substitution, 44 were determined by potentiometric titrations with standardized NaOH (Merck) and AgNO3(Kemika) solutions. The values obtained were 0.88 ±0.01 for PAH and 0.97 ± 0.02 for PAA (for more details about the procedure of determining f, see the Supporting Information). All polyelectrolyte solution concentrations were corrected accordingly to f-values and are expressed with respect to the monomer repeating unit (denoted cm in the text). The polyelectrolyte solutions used for HR-US and density investigations were prepared by dissolving a certain amount of the polyelectrolyte, 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (Sigma-Aldrich), and a background salt in deionized water. Before dissolving them, PAH and PAA were dried at 60 °C, and salts were dried at 110 °C for about 200 min. To ensure the maximum charge density of both macroions, 45 the pH of polyelectrolyte solutions was adjusted to 7.0 ±0.1 with a 1.0 M NaOH solution (Penta). For that purpose, a pH meter (888 Titrando, Metrohm) equipped with a combined glass microelectrode (6.0262.100, Metrohm), precalibrated with standard buffers (Hamilton) with pH values of 4.01, 7.00, and 9.21, was used. NaCl, NaClO4, NaF, (CH3)4NCl, and LiCl salts of analytical purity were purchased from Sigma-Aldrich and were used without further purification except for (CH3)4NCl. Tetramethylammonium chloride (hereafter referred to as Me4NCl), a salt of a tetramethylammonium cation Me4N+and a chloride anion, was purified by recrystallization from a mixture of methanol (Honeywell Riedel-de-Haen) and acetone (Honeywell Riedel-de-Haen) as described in the literature. 46 The ethanol used for cleaning the tube of the densitometer was purchased from Penta. The deionized water used in all experiments was prepared in a PURELAB Classic purification system (ELGA) and had an initial conductivity lower than 0.056 μS/cm. 2.2. High-Resolution Ultrasonic Spectroscopy Measurements. High-resolution ultrasonic spectroscopy measurements were performed in a titration regime on an HR-US spectrometer (model 102T) equipped with a titration accessory produced by Ultrasonic Scientific (Ireland). All experiments were carried out at six selected frequencies (2.8, 5.3, 8.0, 11.7, 12.3, and 14.9 MHz) and 25.00 °C. Temperature control was achieved by a Haake PC300/A28 water bath (Thermo Scientific), which provided a temperature stability of 0.01 °C. Before the measurements, each solution was degassed by a 5452 miniSpin centrifuge (Eppendorf) for 5 min at 3500 rpm. After that, the reference cell was filled with 1 mL of deionized water and the sample cell with 1 mL of polycation or polyanion solution. Then, the sample cell was closed with a stopper, which incorporated a line for the injection of titrant solution. The titration accessory was equipped with a 50 μL Hamilton syringe and the titrant solution was injected into the sample cell automatically every 13 min in steps controlled by Pump Monitor software (Ultrasonic Scientific). After each injection, the titrand solution was vigorously stirred for 3 min using a double stirring system that provided effective mixing. Afterward, the Macromolecules pubs.acs.org/Macromolecules Article https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 1435 polyelectrolyte mixture was not stirred until the next addition of titrant. The differences in ultrasound velocity and attenuation between the cells were monitored continuously throughout the titration. Data are therefore reported here in terms of velocity difference (Δu) and attenuation difference (ΔN) between the sample and reference cells. =u u u(sample) (water) (1) =N N N(sample) (water) (2) The measured data were processed using Titration Analysis software (version 4.50.27.35, Ultrasonic Scientific) in a way that means values of ultrasonic parameters were calculated based on data measured in the last 100 s before the next titrant addition. Each measurement was made at least in triplicate, and in the text and tables, average values of measured quantities with their standard error of the mean are reported. However, for clarification, only average values of ultrasound velocity and attenuation are reported in the figures. The highest standard errors of velocity and attenuation were 0.08 m/s and 0.36 m−1, and the highest relative errors were 1.1% and 78%, respectively (for examples of titration profiles with standard errors, see Figure S5). Therefore, reproducibility was much better in the case of ultrasound velocity than that in attenuation. However, this did not affect the conclusions of this research because attenuation was used only as an auxiliary qualitative parameter and only the quite reproducible shapes of the measured attenuation curves were relevant. The ultrasound frequency had no effect on the measured data (Figure S6); consequently, only results for 14.9 MHz are reported here. Also, the effect of dilution of the titrand solution on measured ultrasound parameters was negligible (Figure S7). It should also be mentioned that visual observation of the titrated systems was also performed outside the HR-US spectrometer in a test tube with a bottom stirrer, using the same steps of the titration procedure performed in the spectrometer to obtain a better insight into the titration process. 2.3. Density Measurements. The density of polyelectrolyte solutions was measured at 25.000 °C using a densitometer DSA 5000 M (Anton Paar). The densitometer calibration was performed at 25.000 °C using air as a standard. Before the measurements, the samples were degassed as described earlier, and then they were carefully injected into a U-shaped borosilicate glass cell so that no gas bubbles were present. To perform the measurements, the U-cell was electronically excited to vibrate at its characteristic frequency. After the measurements, the U-cell was thoroughly cleaned first with 10 mL of a 5.0 M NaClO4solution to remove any residues of adsorbed polyelectrolytes and then with a large amount of deionized water and ethanol. Density measurements were made on three samples of the same composition in duplicates, and the densities of polyelectrolyte solutions presented here are average values of six measurements ± standard errors of the mean. 3. RESULTS AND DISCUSSION 3.1. Complexation of PAH and PAA without the Added Supporting Electrolyte: Stepwise Titration. Ultrasonic velocity (u) is one of the main outputs of HR-US measurement. For liquids, this parameter strongly depends on the density (ρ) and adiabatic compressibility (βS) of the sample according to the well-known Newton−Laplace equation. =u 1 S (3) The compressibility is mainly determined by the molecular organization and intermolecular forces in the medium. Therefore, ultrasonic measurements are usually accompanied by measurements of density to enable calculations of compressibility from ultrasonic velocity and density of the sample. Here, in Table 1, we present the results of density and velocity measurements carried out with solutions of poly- (allylamine hydrochloride) and poly(acrylic acid) in the presence of MOPS buffer. The density and ultrasonic velocity of both polyelectrolyte solutions are similar, which means that the studied systems have much the same compressibility. These results suggest that the conformation of PAH and PAA polymer chains in solution is almost identical, as well as the structure of the ionic atmosphere and the hydration shell around charged monomer units. However, it should be pointed out that the buffer concentration was 10 times higher than that of polyelectrolytes to maintain the pH of the solutions constant. As at pH = 7.0, approximately half of MOPS molecules are present in zwitterionic and another half in the anionic form, 48 it is to be expected that MOPS molecules are associated with both PAH and PAA charged segments. This means that MOPS molecules bound to polyelectrolyte chains can effectively eliminate the electrostatic repulsions between groups of the same charge so that macroions adopt globular shape conformation. Furthermore, the compressibility of prepared solutions is probably most affected by the state of free buffer molecules because MOPS is in a large surplus. As will be evident from the results of HR-US measurements made on polyelectrolyte solutions in the presence of different background salts, the compressibility of the solution is often determined by the most abundant species. To further expand our research, the formation of PEC was monitored by the HR-US technique. For this purpose, the interpolyelectrolyte neutralization of PAH and PAA in aqueous MOPS buffer solutions was carried out at pH = 7.0. The ultrasonic velocity and attenuation of PAH/PAA complexes, prepared by stepwise additions of PAH solution to PAA solution and vice versa, are shown in Figure 1. As can be seen in Figure 1, three regions can be identifiable for the titration profiles measured in both directions. In the first region, velocity and attenuation differences linearly increase as the titrant-to-titrand molar ratio enlarges. Visually, the macroion mixture in this part changed from clear to milky clouded after the first titrant addition, and then the turbidity evolved with new titrant additions (Figure 2). In region II, which expands very narrowly around the equivalence point, the velocity and attenuation differences suddenly decrease with the addition of titrant solution. In this part, the formation of flaky white precipitates can be observed, which sediment over time (Figure 2). Chollakup and co-workers 49,50 also reported that polyelectrolyte complexation between PAH and PAA in the absence of salt results in a precipitate (not the coacervate), independent of the mixing ratio, pH (7.0 and 8.6), and the Table 1. Density, Ultrasonic Velocity, and Compressibility of 0.005 M Polyelectrolyte Solutions at pH = 7.0 (0.05 M MOPS Buffer) and 25.0 °C a PAH PAA ρ(kg/m3) 1001.07 ±0.02 1001.22 ±0.06 u(m/s) 1502.77 ±0.06 1502.61 ±0.11 βS(10−13 Pa−1) 4423.3 ±0.3 4423.8 ±0.9 a The ultrasonic velocities were calculated from the measured difference in velocity between the sample and reference cells at 14.9 MHz using the velocity of pure water at 25.0 °C (u= 1496.687 m/s) from the literature. 47 Macromolecules pubs.acs.org/Macromolecules Article https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 1436 molecular weight of the employed PAA. Finally, the third region of titration profiles is characterized by a reincrease in ultrasound velocity and attenuation upon the addition of titrant solution. This part is somewhat similar to the first region of the titration profiles, but the slope of velocity and attenuation dependence on the polymer molar ratio is slightly smaller than that in the first region. Visually, this stage of the titration profile corresponds to a clear solution with flocs that have been sedimented (Figure 2). Obtained velocity titration profile (Figure 1a) can be explained reasonably well within the framework of the Newton−Laplace relationship (eq 3) by considering the sequence of events that occur upon stepwise addition of one polyelectrolyte into the solution of the other, as proposed by Fuoss and Sadek. 51 For simplicity, this sequence of events is illustratively shown for the case of interpolyelectrolyte neutralization of PAH with PAA in Figure 2. When the first drop of PAA is added to PAH solution, the polyelectrolytes interact, forming a stable colloidal dispersion. The resulting polymer aggregates, denoted here as primary complexes (often called nonstoichiometric 52,53 or quasisoluble 54 complexes), are compact clusters cross-linked by electrostatic interactions. Since PAH is initially in excess, the interior of these primary complexes is mostly composed of PAA molecules, while the exterior contains positively charged PAH chains. Overall, as reported earlier, 9 PAH/PAA primary complexes are positively charged, and their hydrodynamic diameter is approximately constant over a wide range of polyion molar ratios (r< 0.8). Therefore, each subsequent addition of titrant primarily causes the formation of new electrically charged primary complexes. Consequently, in this part of the titration profile (region I in Figure 1a), the ultrasonic velocity, as well as turbidity (Figure 2), of the colloid solution increases. As mentioned before, ultrasonic velocity is determined by the compressibility and the density of the medium. Generally, the compressibility response, which is extremely sensitive to molecular organization and intermolecular interactions, is dominating. 24 When a polyelectrolyte molecule encounters a macroion of opposite charge, the strong attractive field between them causes electrostatic binding of their monomer segments, and many of the counterions are displaced. Around displaced ions, new hydration shells composed of less compressible water molecules are formed, and thus, the compressibility of the system decreases. Figure 1. Velocity (a) and attenuation (b) difference obtained by stepwise titrations of PAH solution with PAA solution (blue squares) and vice versa (red circles) in MOPS buffer (c= 0.05 M) at pH = 7.0, 25.0 °C, and 14.9 MHz frequency. The titrant-to-titrand molar ratio of repeating units was calculated on the basis of the added volume of titrant solution (cm= 0.05 M) to titrand solution (cm= 0.005 M, V0= 1.0 mL). Dashed lines have no physical meaning and were added as guides to the eye. Figure 2. Illustration of the sequence of events that occur upon stepwise addition of PAA solution into PAH solution accompanied with images of the mixture at different PAA-to-PAH molar ratios denoted r. Macromolecules pubs.acs.org/Macromolecules Article https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 1437 Therefore, the linear increase in ultrasonic velocity in region I of the titration profile (Figure 1a) is an outcome of the formation of compact and stiff PAH/PAA primary complexes, the release of water and associated counterions (in our case MOPS ions) from charged polyelectrolyte monomers, and the formation of new hydration shells around PECs and counterions. As the ratio of titrant to titrand monomers further increases and approaches equimolar value, the primary complex corona charge is compensated by titrant molecules. Consequently, oppositely charged secondary complexes bearing the excess of PAA monomers at the surface are formed. Their interactions with the primary complexes lead to macroscopic flocculation and over a short period to sedimentation of flocs (Figure 2). The flocculation is accompanied by the reduction of total solid surface area and by the expulsion of stiff hydration water from the surface of PECs, which increases the compressibility and decreases the ultrasonic velocity of the polyelectrolyte mixture. This sharp decrease in the velocity is visible in Figure 1a (region II), regardless of the polyelectrolyte addition order. However, it is interesting to note that the steep velocity and attenuation decrease in region II occurs at different molar ratios depending on the direction of titration. In the case of the PAH to PAA titration direction, it appears at a molar ratio of 0.80, and in the case of the opposite direction, it appears at a higher molar ratio of 0.95. Moreover, this step decrease is larger for the case of PAA addition to PAH solution than that for PAH addition to PAA solution. The observed dependency of the flocculation point on the direction of titration is in good agreement with the results of DLS measurements reported earlier, 9 and it was also found for PECs prepared by other polyelectrolytes. 55−57 Mainly, this mixing order effect can be explained by mismatches in kinetically controlled monomer pairing. After all, nonstoichiometric complexes, especially those that depend on the order of mixing, are a signature of kinetic control in polyelectrolyte association. 8,58,59 Other reasons for observed titration endpoint discrepancy could be associated with the degree of ionization, degree of functionalization, and molecular weight of polyelectrolytes used in this research (Table 2). According to the literature, 45 under conditions of the performed experiments (pH = 7.0), PAH has a higher degree of ionization than PAA. It means that fewer polycation molecules are needed to overcharge primary complexes that contain an excess of the polyanion, and thus, phase separation takes place at lower molar ratios. It also means that the molar ratio presented in Figure 1 is not equal to the charge ratio of polyelectrolytes. However, Li et al. 15 have recently demonstrated that polyelectrolyte pairing in PAH/ PAA complexes is not only achieved by electrostatic interactions but also by the hydrophobic interactions of the aliphatic polymer backbone and the interpolymer hydrogen bonding of nonionized acrylic monomer units. The former two interactions are pronounced especially at acidic pH. Therefore, taking hydrogen bonding and hydrophobic interactions into account makes it difficult to rely only on the charge ratio for expressing the titration endpoint. Furthermore, the real monomer ratios can also be different from those given in Figure 1 due to experimental errors in the standardization of polyelectrolytes. On the other hand, even if the functionalization degrees of both polymers are correctly determined, the unfunctionalized monomers are randomly distributed among the PAH and PAA chains. This affects the value of the corrected molar ratio at which precipitation occurs, especially in the case of low functionalization degrees. However, the functionalization degree of the polycation and polyanion used in the herein investigated case is large enough (Table 2) that its deviation from unity probably does not affect the pairing of charged groups. One should also keep in mind that the reversed mixing order cannot be expected to yield PECs of the same structure, since PAH used in the frame of our investigation has a much smaller molecular mass (shorter chains) than PAA, which must induce somewhat different opportunities for assembly during the PEC formation process. The recent investigation of PAH/PAA interpolyelectrolyte neutralization in solution conducted by Gardlund and coworkers 60 supports this statement. Returning to the results in Figure 1a, one can see that after the flocs are formed, ultrasonic velocity increases upon further addition of titrant solution (region III of titration profile). Although subsequent incorporation of titrant chains into the flocs may contribute to the solution compressibility and thus to the velocity, we believe that velocity increases with new titrant additions due to the increasing number of free titrant molecules with hydration shells composed of less compressible water molecules. In support of this conclusion, we state the following: First, the slope of the linear dependence of ultrasonic velocity on the molar ratio after the flocculation point (region III) was not the same as before flocculation (region I) when the titrant molecules were incorporated into the polyelectrolyte complex. Furthermore, in the earlier study, 9 the corresponding reaction was investigated calorimetrically, and no measurable heat effects above equivalence were observed. This finding indicates that titrant molecules do not participate in further build-up of PECs, as the results of calorimetric measurements were corrected for the heats of titrant dilution. In the last few paragraphs, it was demonstrated how measuring ultrasonic velocity during the course of polyelectrolyte complexation by the HR-US spectrometer can provide a better insight into this process based on the compressibility changes of the system. Like ultrasonic velocity, measuring attenuation during interpolyelectrolyte neutralization can be of great importance for a better understanding of PEC formation. Ultrasonic attenuation represents the energy losses in compressions and decompressions of the medium in ultrasonic waves, and it is mainly affected by the heterogeneity of the medium (in this case, this means the formation of colloidal particles in a continuous liquid medium). 25 In a few sentences Table 2. Degree of Ionization (α), Degree of Functionalization (f), Weight-Average Molecular Weight (Mw), Degree of Polymerization (DP), and Polydispersity Index (PDI) of Polyelectrolytes Used in This Research a PAH PAA α(%) ≈85 ≈65 f(%) 88 ±1 97 ±2 Mw(kDa) ≈17.5 ≈1000 DP 187 13 877 PDI 4.3 a Values of αcorrespond to pH = 7.0 and were taken from data published by Choi and Rubner. 45 Degrees of functionalization were determined by potentiometric titrations as explained in Section 2. Molecular weights of polymers, degrees of polymerization, and polydispersity indexes (except for PAH) were provided by the supplier. Macromolecules pubs.acs.org/Macromolecules Article https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 1438 that follow, we will explain the results of attenuation measurements obtained by the HR-US device, which are shown in Figure 1b. The increased attenuation in region I of the attenuation titration profile reflects the formation of new heterogeneities, i.e., stable nonstoichiometric PAH/PAA nanoassemblies. Then, close to the equivalence point, attenuation decreases steeply (region II). The step decrease corresponds to the coalescence of primary and secondary complexes that sediment in the absence of stirring. As a result, the mixture clears up (region II in Figure 2), and there are only several unsettled particles on the way of ultrasonic wave propagation that account for the energy losses. This behavior is consistent with the results of turbidity measurements carried out by Chen et al. 56 They noted a drop in turbidity near the equivalence point during titration of poly- (diallyldimethylammonium chloride) (PDADMAC) solution with potassium salt of poly(vinylsulfonic acid) (PVSK) and explained it by aggregation and sedimentation of polymer particles. After the sharp decrease in attenuation in the vicinity of the equivalence point (region II), the attenuation again moderately increases with new additions of the titrant. An increase in attenuation in region III indicates increasing heterogeneity of the system, as the concentration of free titrant molecules that scatter the ultrasonic wave increases. 3.2. Complexation of PAH and PAA without the Added Supporting Electrolyte: Abrupt Titration. At the beginning of the stepwise titration, the monomers of titrand are in considerable excess compared to the added monomers of titrant, i.e., the primary complexes contain excess monomers of titrand. As the titration progresses, the free chains of titrand run out, which leads to the formation of secondary complexes that instantly flocculate with the primary ones. If the charge inversion is linked exclusively to the excess of one of the polyelectrolytes, only secondary complexes should form from the primary complexes without macroscopic phase separation when a large excess of titrant molecules is suddenly added to the corresponding suspension. To test this hypothesis by HRUS, positive PAH/PAA primary complexes were prepared in MOPS buffer (pH = 7.0) by stepwise titrant to titrand additions up to a 0.6 monomer molar ratio. This procedure was followed by the addition of the equivalent amount of titrant to achieve a molar ratio of 1.6. The results of the abrupt titrant addition in excess following the stepwise addition procedure are depicted in Figure 3. As can be seen in Figure 3, the outcome of the abrupt type of PAA to PAH titration was different from the outcome of the stepwise titration experiment. Although the ultrasonic velocity and attenuation increased equally up to a molar ratio of 0.6 in both types of titration due to the primary complexes formation, no drop in ultrasonic parameters near equivalence was observed in the case of abrupt titration. Instead, after the addition of an equivalent amount of PAA into a suspension of primary PECs at a molar ratio of 0.6, the ultrasound velocity and attenuation increased almost linearly compared to the values before this addition. Visual inspection of PEC suspension at a molar ratio of 1.6 revealed no macroscopic phase separation, just a milky clouded appearance (inset in Figure 3a). Judging by the obtained results, it can be stated that the abrupt titrant addition in excess led to the primary PEC overcharging and thus to the formation of PAH/PAA secondary complexes. Negatively charged secondary complexes did not transform into sparsely soluble precipitates due to electrostatic stabilization through repulsions between particles. Therefore, after the abrupt titrant addition, the ultrasonic velocity increased due to the coexistence of rigid secondary complexes and free PAA molecules in stable colloidal suspension, and the attenuation increases as a consequence of the increase in heterogeneity. In addition to the PAH/PAA pair, the overcharging of primary PECs achieved by abrupt titrant addition in excess was previously observed by DLS and electrophoretic methods in the case of PDADMAC-PSS and PAH-PSS pairs. 9 It, therefore, seems that the phenomenon of charge inversion of primary polyelectrolyte complexes is universal, just like the surface charge inversion that occurs during the deposition of each polyelectrolyte layer in the process of preparing polyelectrolyte multilayers (PEMs). 61−64 The described results are the additional strong evidence of the already observed 65 correlation between the process of interpolyelectrolyte complexation in solution and the formation of PEMs on the surface. 3.3. Complexation of PAH and PAA in Various Supporting Electrolytes. The complexation of PAH and PAA was also studied by HR-US in NaF, NaCl, NaClO4, LiCl, and Me4NCl aqueous solutions. The selection of these salts was based on remarkable anionand cation-specific effects observed earlier. 9 As in salt-free experiments, titrations were Figure 3. Velocity (a) and attenuation (b) difference obtained by abrupt titration of PAH solution (cm= 0.005 M, V0= 1.0 mL) with PAA solution (cm= 0.05 M) in MOPS buffer (c= 0.05 M) at pH = 7.0, 25.0 °C, and 14.9 MHz frequency (red circles). The results of corresponding stepwise titration are given for comparison (blue squares). The PAA-to-PAH molar ratio of repeating units was calculated on the basis of added volume of PAA solution to PAH solution. Dashed lines have no physical meaning and were added as guides to the eye. Image in (a) shows the stable colloidal solution of PAH/PAA secondary complexes obtained at a molar ratio of 1.6. Macromolecules pubs.acs.org/Macromolecules Article https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 1439 carried out in MOPS buffer solutions at pH = 7.0 with titrand and titrant monomer concentrations of 0.005 and 0.05 M, respectively. To ensure complete substitution of PAH and PAA counterions, which counterbalanced the macroion charge in corresponding buffer solutions with the ions of the introduced supporting electrolyte, a considerable excess of salt with respect to the buffer was added to polyelectrolyte solutions. Solutions of both polyions in all cases contained equal concentrations of the background salt (c= 0.1 M). The salt concentration was also kept high, since the influence of counterion type on the course of interpolyelectrolyte neutralization can be observed solely in concentrated salt solutions. 10 Before presenting the results of titration experiments, the adiabatic compressibility of prepared PAH and PAA titrand solutions in different supporting electrolytes will be considered (Figure 4). As before, corresponding compressibility values were calculated from eq 3 based on the measured ultrasonic velocity and density data (see Table S1). Several important conclusions can be drawn from the obtained results. First, the compressibility of polyelectrolyte solutions in the presence of all investigated background salts is lower than the compressibility of the same solutions without salts (Figure 4). This means that the presence of ions in polyelectrolyte solutions causes changes in molecular organization and intermolecular forces in the solution. These structural changes are primarily associated with the action of ions’ electric field on water and changes in hydrogen bonding among water molecules. 66−68 Moreover, the observed compressibility values are practically the same for PAH and PAA solutions prepared in the same salt. This fact indicates that the ions of the supporting electrolyte have the greatest influence on the compressibility of solutions. That is not so unexpected considering that there is a 20 times higher concentration of salt in solutions than the concentration of polymer monomer units. Therefore, the contribution of the dissolved macromolecules to the compressibility can be neglected in further consideration. Also, the type of background salt affects the compressibility of polyelectrolyte solutions. Considering the sodium salts used, the influence of anions on the compressibility of solutions increases in the order of F−< Cl−< ClO4 −. This rank order of ions is in accordance with the position of these ions in the Hofmeister series. 17,18 The influence of cations on the compressibility of solutions also follows the position of the ions in the Hofmeister series, so the compressibility increases in the sequence Me4N+< Na+< Li+. The adiabatic compressibility of the electrolyte solution can be usefully interpreted in terms of ions’ contributions to the structure of the water network. In this manner, ion−water and water−water interactions should be distinguished. 66−68 Charged ions influence adjacent water molecule dipoles by causing strong electrostatic contraction. This phenomenon, usually called electrostriction, 69 causes a partial loss in the mobility of water molecules located near ions. As a result, hydration shells around ions are formed. Generally, hydration water is less compressible than bulk water. Therefore, solutions of ions with less hydration water, i.e., thinner hydration shells, will have higher values of compressibility. However, one should not neglect the effect of hydrogen bond reorganization induced by the presence of ions on the overall compressibility of the solution. According to Marcus, 70,71 the extent of hydrogen bonding in water can be satisfactorily described by a numerical value of ΔGHB. That physical parameter represents the ratio of standard molar Gibbs free energy of transfer of the solute from light (H2O) to heavy (D2O) water and the molar difference in the hydrogen bonding energies of light and heavy water. Ions with positive ΔGHB values are known as water structure makers, and ions with negative ΔGHB values are known as water structure breakers. In principle, solutions that contain large ions, which have a thin hydration shell and negative ΔGHB values, will be more compressible than solutions that contain small ions, which have a thick hydration shell and positive ΔGHB values. In Table 3,ΔGHB values of ions present in PAH and PAA solutions and the thickness of their hydration shells are listed. By inspection of Table 3, one can see that the relative relationship of observed anions compressibilities (F−< Cl−< ClO4 −) is in accordance with the rationale explained above. However, that is not the case for cations. Solutions of wellhydrated Li+ion have the highest compressibility values of all examined salts (Figure 4), but they should have the lowest compressibility according to Marcus formalism. 70−72 One reasonable explanation of this unexpected result (also observed by others 73,74 ) could be the formation of clusters in LiCl solution. As Thomas and Elcock 75 noticed in molecular dynamics (MD) simulations, Li+and Cl−ions form ionic linear clusters (strings). The formation of these clusters is accompanied by the expulsion of stiff hydration water molecules to the bulk space. Consequently, solution’s compressibility should increase. Interestingly, the same authors found that the presence of bulky Me4N+cations in solution increases the average number of hydrogen bonds per water Figure 4. Influence of different background salts (c= 0.1 M) on adiabatic compressibility of poly(allylamine hydrochloride) and poly(acrylic acid) solutions (cm= 0.005 M) at pH = 7.0 (0.05 M MOPS buffer) and 25.0 °C. Table 3. Thickness of Hydration Shells (Δr) and ΔGHB Values of Selected Ions a ion Δr(pm) ΔGHB F−79 0.08 Cl−43 −0.61 ClO4 −19 −1.01 Li+172 0.28 Na+116 −0.03 Me4N+14 −0.47 a Ion parameters were taken from data published by Marcus. 70,72 Macromolecules pubs.acs.org/Macromolecules Article https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 1440 molecule compared to the average number of hydrogen bonds in pure water. This structure-making behavior of Me4N+ions is not in accordance with the Marcus classification of ions 70,71 and could be an explanation for the observed lower compressibilities of Me4NCl solutions than expected. To truly demonstrate that the compressibility of LiCl and Me4NCl solutions is the exception rather than the rule, it would be necessary to determine βSvalues of a series of solutions containing different cations. Unfortunately, this is out of the scope of this paper because the aim was to explore how different background salts affect the formation of PAH/PAA complexes by HR-US. In Figure 5 one can see velocity profiles obtained by titrations of PAH solution with PAA and vice versa in the presence of examined salts. For the sake of easy visual comparison, the curves have been shifted vertically along the yaxis by adding a constant so that all curves start at the same point. As can be seen for all salts, the ultrasonic velocity changes with the molar ratio of polyelectrolytes in the same fashion as in salt-free experiments. In the beginning, velocity increases linearly with additions of titrant, then it suddenly decreases around the equivalence point, and finally again increases. The similarity between titration profiles obtained in experiments performed with and without supporting electrolytes indicates that formed PECs are more likely present in the form of a precipitate than in the form of a coacervate (probably due to the low concentration of salt in solutions). This conclusion is well supported by the literature 49,50 and by additional optical microscopy investigation. For example, in the optical micrographs, we obtained, for stoichiometric PECs prepared in NaCl solution, a distinguishable form of precipitate (Figure S8). Although the curves presented in Figure 5 look very similar, there are some important differences between them. For example, in some cases, the sharp drop in ultrasonic velocity occurs at lower molar ratios as compared to the “salt-free” curve. In the upper panel of Figure 5, the anion influence on the course of interpolyelectrolyte neutralization is depicted. In the case of PAA to PAH titration, a sudden decrease in the ultrasonic velocity of positively charged PAH/PAA complexes at a molar ratio of 0.6 (NaClO4) and a molar ratio of 0.9 (NaCl and NaF) can be observed. However, this kind of anionspecific effect is not observed in the case of titration of negatively primary complexes (PAH to PAA titration). The observed effect of anions on the formation of the PECs can be understood as follows. Normally, a charge of polyelectrolyte repeating units is balanced either by the complementary polyelectrolyte (intrinsic charge compensation) or by counterions (extrinsic charge compensation). 76 The increase in the ionic strength of polyelectrolyte solutions results in a more pronounced counterion-polyelectrolyte type of binding. 10 The counterion-polyelectrolyte binding constant is the one that exhibits ion specificity, i.e., polyelectrolytes and different salt ions are expected to interact with different strengths. Recently, it was found that PAH prefers the binding of weakly hydrated oxyanions such as perchlorates over strongly hydrated halide anions (e.g., Cl−and F−). 9 Therefore, it is to be expected that positively charged PAH/PAA primary complexes prepared in the presence of NaClO4will have a higher content of compensated charges in the corona than the same PECs prepared in the presence of NaCl or NaF. This Figure 5. Influence of different background anions (a, b) and cations (c, d) of 0.1 M concentration on the velocity difference obtained by stepwise titrations of PAH solution with PAA solution (a, c) and vice versa (b, d) in MOPS buffer (c= 0.05 M) at pH = 7.0, 25.0 °C, and 14.9 MHz frequency. The titrant-to-titrand molar ratio of repeating units was calculated on the basis of added volume of titrant solution (cm= 0.05 M) to titrand solution (cm= 0.005 M, V0= 1.0 mL). Dashed lines have no physical meaning and were added as guides to the eye. The results of corresponding salt-free experiments are given for comparison (dotted line). Macromolecules pubs.acs.org/Macromolecules Article https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 1441 leads to the secondary aggregation and finally to the flocculation of positive PEC particles at lower molar ratios. On the contrary, when negative PEC particles are prepared by titration of PAA with PAH, there is no such effect because the surface charge of this type of complex is not affected by the anions. Instead, the excess of PAA monomers at their surface is screened by Na+ions that are common to all investigated solutions. The herein-described HR-US results confirm the previously reported anion-specific aggregation of PAH/PAA complexes observed by DLS measurements. 9 In that study, aggregation of positive complexes was detected by a large increase in the particle size at molar ratios of 0.6 and 0.8 in the presence of NaClO4and NaCl, respectively (Figure 6a in ref 9). Moreover, the anion-specific aggregation of PAH/PAA complexes was further explored by titration of positive primary complexes with simple electrolyte solutions. Compared to NaCl, the observed value of critical electrolyte concentrations needed for the onset of positive PAH/PAA complex aggregation was significantly lower when the suspension was titrated with NaClO4. The analogous behavior was noticed in the case of positive PAH/PSS complexes. 7 Undoubtedly, both PAH/PAA and PAH/PSS complexes bear an excess of polycation monomers at their surface, and ClO4 −ions more effectively screen this charge than Cl−ions, which results in lower values of flocculation endpoints. Opposite to the influence of anions, the effect of countercation type on the PAH/PAA interpolyelectrolyte neutralization is not so pronounced, i.e., the observed results for all investigated supporting electrolytes are relatively similar. As can be seen in Figure 5c,d, the sudden decrease in ultrasonic velocity for measurements performed in NaCl, LiCl, and Me4NCl occurs at a similar molar ratio as in the case of salt-free experiments, irrespective of titration direction. The observed flocculation at a relatively high titrant-to-titrand ratio (≈0.8) indicates that monomer pairing in these supporting electrolytes proceeds as efficiently as in MOPS buffer solutions. However, by closer examination of the presented data, one can see that there is a little shift in the flocculation onset toward lower monomer molar ratios in the case when the polycation was added to the polyanion solution in the presence of LiCl and Me4NCl. This finding can be interpreted as electrolyteinduced negative complex aggregation. Namely, Li+and Me4N+ions more efficiently screen the excess of negative charge at the PEC surface than Na+ions and thus induce aggregation of particles. To summarize, PAH/PAA interpolyelectrolyte neutralization in salt solutions seems to result in both positive and negative primary complex aggregation due to the masking effect of ions. This effect leads to asymmetric interpolyelectrolyte neutralization, resulting in the excess of titrand monomers in the obtained metastable precipitates. PAH seems to bind the examined ions more specifically than PAA. The reason for the observed differences in polycation and polyanion ion-binding abilities is still not fully understood. However, the fact that anions alter more strongly the formation of both PAH/PAA and PAH/PSS complexes than cations suggests that ion-specific effects are mainly related to the type of ion and not to such an extent to the type of polyelectrolyte. This statement is well supported by a previous study on PDADMAC/PSS multilayers. 77 While anion effects for this system become important above an ionic strength of 0.1 M, the cation effects become important at a much higher ionic strength of 0.25 M. Obviously, the greater specificity of polycations toward anions than that of polyanions toward cations can therefore be expected irrespective of the polyion type. 4. CONCLUSIONS To obtain a better insight into interpolyelectrolyte neutralization processes between poly(allylammonium) cations and poly(acrylate) anions, a relatively novel HR-US technique has been applied. The obtained results were compared with the previous study where more traditional methods (such as DLS and calorimetry) were used to examine PEC formation. 9 The results of HR-US measurements have confirmed that PAH/ PAA interpolyelectrolyte neutralization in the low salt regime undergoes solid−liquid phase separation and proceeds as described in the pioneering work of Fuoss and Sadek. 51 First, positively or negatively charged primary complexes are formed with the sign of surface charge that depends on the titration direction. Then, if the titration is performed stepwise, oppositely charged secondary complexes are generated, and they react with the primary complexes. This leads to macroscopic flocculation that is accompanied by the expulsion of stiff hydration water from the surface of PECs. However, if the titrant is added to the suspension of primary complexes in great excess, flocculation will not occur, but rather primary PECs will overcharge. In addition, the results of HR-US measurements made on PAH/PAA polyelectrolyte complexes in the different salt media have emphasized the important role of ions in PEC formation. Counterions present in the solution are electrostatically attracted by the charged polyelectrolyte units. Consequently, this charge screening causes asymmetric polymer neutralization (one of the polyelectrolytes in the formed complexes is present in considerable excess) and aggregation of primary PECs. 7,9,12,16,78 Interestingly, the polyion molar ratio at which flocculation occurs markedly depends on the type of supporting salt, and that effect is more pronounced in the case of anions. In the end, it can be established that the results obtained here and in the previous study 9 are in good agreement and mostly in line with expected values. Moreover, HR-US complements the methods normally used to study PECs with additional information. For instance, HR-US is not limited to the polyelectrolyte molar ratios before the onset of flocculation like DLS so we can make further conclusions about the process beyond the flocculation point. Also, many interpolyelectrolyte neutralizations are isoenthalpic 5−12 or do not include UV−vis active polymers (like in this research), which limits the application of calorimetry and spectrophotometry. In such cases, HR-US can be used as an appropriate alternative. Therefore, it can be concluded that HR-US is a convenient and reliable new method that can be used for monitoring interpolyelectrolyte neutralization. Overall, the capabilities of this technique could make it a valuable tool for further investigations of processes related to polyelectrolyte complexes such as precipitate/coacervate/solution phase transitions, 1,49,50 interpolyelectrolyte exchanges, 79 or glass transitions. 80,81 ■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.macromol.2c02349. Macromolecules pubs.acs.org/Macromolecules Article https://doi.org/10.1021/acs.macromol.2c02349 Macromolecules 2023, 56, 1434−1445 1442