1026 DOI: 10.17344/acsi.2019.5681 Acta Chim. Slov. 2019, 66, 1026-1037 ©amnions Scientific paper Temperature Dependent Behavior of Isotactic and Atactic Poly(Methacrylic Acid) in the Presence of MgCl2 and CaCl2 Patricija Hribersek1 and Ksenija Kogej1'* 1Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: ksenija.kogej@fkkt.uni-lj.si Tel.: 01-4798-538; Received: 11-06-2019 Abstract Temperature (T) induced nanoparticle formation of isotactic (iPMA) and atactic (aPMA) poly(methacrylic acid) chains is investigated in aqueous solutions in the presence of divalent cations (Mg2+ and Ca2+) by UV spectroscopy, visual observations and pyrene fluorimetry. In aPMA solutions, aggregation and phase separation take place with increasing T. The onset of aPMA aggregation (heating) and re-dissolution of the aggregates (cooling) is shifted to lower Ts with increasing ionic strength of the solutions. iPMA associates partly decompose upon heating, but stable nano-sized particles are present at all Ts. Structural characterization of the aggregates was carried out by dynamic and static light scattering measurements at 25 °C. Results revealed that aggregates of both PMAs with Mg2+ ions involve a lot of water and have a rather even mass distribution. This is attributed to strong hydration of Mg2+ ions and their preference towards mono-dentate binding to carboxylate groups. Differently, Ca2+ ions bind more strongly and in a bidentate manner, they lose the hydration water and form aggregates with several structural elements, depending on chain tacticity. Keywords: Temperature-responsive polymers; poly(methacrylic acid) isomers; atactic; isotactic; divalent cations; intermolecular association 1. Introduction Despite the ubiquitous presence of polyelectrolytes in chemistry, biology and physics, their interactions with numerous species, among those also with oppositely charged and multivalent metal ions, still present one of the most challenging aspects of macromolecule chemistry. These interactions have a significant effect on size, charge and structure of the polymer molecules in solutions, on dynamics of polymer chains, kinetics of eventual phase separation, and furthermore have implications to biological systems, since metal ions perform important functions in biological pro-cesses.1-3 Among the most common cations found in nature and in living systems are divalent Mg2+ and Ca2+. Since these two ions play a crucial role in stabilizing the structure of many proteins, many studies have been performed on their interactions with macromolecules.1,4-10 For example, Mg2+ is responsible for the DNA replication1,5 and Ca2+ has a stabilizing function in bones, teeth and shells.1,11 They are both so-called hard ions and prefer to bind to hard ligands containing oxygen, such as carboxylates, carbonyls, water, and hydroxyl groups.1,12 However, Mg2+ and Ca2+ each has specific properties that make them different from each other. Apart from small ionic (0.65 Ä13) and significantly larger hydrated radius (4.76 Ä13,14) , one of the main characteristics of Mg2+ is strong interaction with water molecules leading to a bulky hydration sphere with a hexa-coordinated geometry around this ion. The slow exchange rate of the hydrated water molecules is responsible that Mg2+ ions do not readily lose water molecules.1,4,6,12-18 These features of Mg2+ play a crucial role in stabilizing the monodentate binding of Mg2+ to carboxylate group (see Scheme S1 in Supporting Information (SI)).1,4,12,13,17 Ca2+ has quite different properties in comparison to Mg2+ and therefore participates in different biochemical processes.11,13 Its ionic radius (0.9913) is considerably larger in comparison to Mg2+ and this leads to significant flexibility in the coordination number of its complexes with various ligands. The water coordination numbers for Ca2+ range from 6 to 8, but also up to 12 is possible, which leads to irregular geometry of the ion's coordination sphere.1,11-13 Hydrated radius of Ca2+ thus depends on the coordination Hriberšek and Kogej: Temperature Dependent Behavior of Isotactic Acta Chim. Slov. 2019, 66, 1026-1037 1027 number, but is always smaller than that of Mg2+. For coordination numbers 6-8, the hydrated radius is 2.95.13 These properties cause that Ca2+ can easily lose water molecules from its hydration sphere.13,15,16 Its complexes, in which bidentate binding prevails,17 are therefore classified as 'dry'. There are two types of bidentate binding of Ca2+ to carbox-ylate groups: chelating bidentate and bridging bidentate binding (see Scheme S1). In chelating coordination, one central Ca2+ ion is bound to two oxygen atoms of one car-boxyl group, whereas in bridging coordination Ca2+ bridges two different carboxyl groups via their oxygen atoms.19 Interactions of macromolecules and counterions are important also in the (bio)technological, industrial, and pharmaceutical fields, where synthetic polymers are more commonly used.2,3 For example, poly(acrylic acid) is a promising candidate for controlling water hardness20 and poly(methacrylic acid) (PMA) is widely studied due to its pH- and temperature dependent behavior,21-24 which is very appropriate in targeted drug delivery.25 In the case of PMA, chain tacticity brings in qualitatively different solution behavior. For example, the atactic form (aPMA) is known to form intermolecular associates in dilute aqueous solutions26 and gels in concentrated solutions upon heat-ing,22-24,27,28 whereas the isotactic form (iPMA) associates upon cooling.26,28,30 These findings are attributed to different distribution of polar (carboxyl) and nonpolar (methyl) groups on aPMA and iPMA chains and consequently within the associates. The favorable isotactic arrangement of carboxyl groups on iPMA favors strong hydrogen bonding between neighboring chains and exposes the methyl groups towards the polar solvent (water), leading to insolubility of unionized iPMA in water. Increasing the temperature destroys the H-bonds, the associates decompose and consequently carboxyl groups become exposed to water. This is different when the distribution of functional groups is irregular, such as in aPMA. In this case, hydrogen bonding is much less advantageous. The mostly free carboxyl groups are exposed to water and the methyl groups can effectively hide from the polar aqueous environment by forming associates with a hydrophobic core. Increasing the temperature would lead to exposure of these methyl groups to water, which is clearly unfavorable. As a result, the polymer chains first form intermolecular associates and finally precipitate from solution at elevated temperatures. Our previous results show that the added simple salt, in particular the cation charge, affects the temperature at which association takes place, either by heating (aPMA) or by cooling (iPMA).28 The main focus of this paper is to compare the effect of biologically relevant divalent Mg2+ and Ca2+ cations on the temperature-dependent association of iPMA and aPMA chains in aqueous solutions. For this purpose, we first carry out UV spectroscopy measurements supported by visual observations in the temperature range between 0 and 95 °C to clarify the effect of polymer tacticity on the association process. To identify the role that the specific nature of Mg2+ and Ca2+ plays in the interaction with car-boxylate groups on PMA isomers, fluorescence measurements using pyrene as a polarity probe are used. Finally, the molecular characteristics of the associates are determined by dynamic (DLS) and static light scattering (SLS), which are further supported by pH measurements, both carried out at 25 °C. 2. Experimental 2. 1. Materials The aPMA sample (product number P2419-MAA) with the weight (Mw) and number average molar masses (Mn) of Mw = 159 900 g mol-1 and Mn = 123 000 g mol-1 and a polydispersity index (PDI) of PDI = 1.3 was purchased from Polymer Source Inc (Montreal, QC, Canada). iPMA was obtained by acidic hydrolysis of its ester form, isotactic poly(methylmetacrylate) (iPMMA), which was synthesized following the procedure reported in the literature29,30 at the Catholic University of Leuven. Detailed description of hydrolysis and further purification of iPMA was described previously.29,31 The molecular characteristics of the starting iPMMA were the following: the triad content or tacticity was 94% of isotactic, 4% of syndiotactic, and 2% of atactic triads, Mw = 138 000 g mol-1, Mn = 30 000 g mol-1, and PDI = 4.6. After hydrolysis and purification, the Mw and PDI (Mw = 69 500 g mol-1 and PDI = 3) were determined by size exclusion chromatography, and the degree of hydrolysis (> 98%) was determined from the 1H nuclear magnetic resonance spectrum of the sample in D2O. MgCl2 (> 98%; Sigma Aldrich, St. Louis, Missouri, USA) and CaCl2 (in the form CaCl2 x 2H2O; pro analysis, Merck KGaA, Darmstadt, Germany) were used to prepare stock salt solutions in water. The exact concentration of salts (cs) was determined by potentiometric titration using a standardized AgNO3 solution. Pyrene (optical grade) for fluorimetric measurements was purchased from Aldrich (Darmstadt, Germany). The saturated solutions of pyrene in aqueous MgCl2 and CaCl2 were prepared as reported previously.32,33 2. 2. Preparation of Solutions Stock solution of aPMA was prepared at a degree of neutralization (aN) aN = 0 by dissolving the dry polymer in water. After one day of stirring, the solution was filtered through 0.45 ^m Millex HV filter and the exact polymer concentration (cm, in g L-1) was determined by potentio-metric titration with a standardized NaOH solution. The iPMA does not dissolve in water at aN = 0.29,34,35 Therefore, the iPMA stock solution was prepared at a higher aN (> 0.8) to ensure that the polyacid was completely dissolved. A calculated volume of1 M NaOH was slowly added to the iPMA suspension in water under continuous stirring to obtain the desired aN value. The dissolution process at Hriberšek and Kogej: Temperature Dependent Behavior of Isotactic ... 1028 Acta Chim. Slov. 2019, 66, 1026-1037 room temperature was slow. The iPMA suspension was stirred for several days with occasional heating to ~50 °C, which accelerated the dissolution. When the polymer was visually dissolved, the solution was filtered through 0.45 ^m Millex HV filter and the exact aN and cm values were determined by potentiometric titration, first with 0.1 M NaOH in the direction of increasing aN (to determine the concentration of the still protonated COOH groups) and then with 0.1 M HCl in the direction of decreasing aN (to determine the total concentration of carboxyl groups). Due to differences in solubility, different procedures were used also for the preparation of aPMA and iPMA solutions with added MgCl2 and CaCl2. Solutions of aPMA with a desired salt concentration (cs) were prepared by adding a calculated volume of concentrated salt solutions to the aPMA stock solution and diluting the solution with triple distilled water to adjust the concentration of aPMA and the ionic strength (I) to the desired values. For all the measurements with aPMA, aN = 0, cm = 2 g L-1 (or in moles of COOH groups per volume, designated as cp, cp = 0.023 mol L-1), and the concentration of MgCl2 and CaCl2 was cs = 0.0333 and 0.0667 mol L-1, which corresponds to I = 0.1 and 0.2 mol L-1, respectively (these data are collected in Table S1). UV and pH measurements with aPMA were performed also at higher I (or cs) values, i.e. in the range I = 0.1-0.5 mol L-1. For the preparation of iPMA solutions with the lowest possible aN, at which the polyacid was still soluble, a calculated amount of 0.1 M HCl was gradually added to the stock solution with aN > 0.8, which resulted in some NaCl in solution. The NaCl was removed by dialysis using dialysis membranes Float-A-Lyzer G2 with a molecular weight cut-off MWCO = 3.5-5 kDa. Finally, calculated amounts of concentrated MgCl2 or CaCl2 solutions and water were added slowly and under continuous stirring to adjust I and cp to the desired final values, so as in the case of aPMA. The final I values were 10 times lower in comparison to aPMA, because iPMA precipitated from solutions with I = 0.1 mol L-1 in the presence of the studied divalent cations. For both, MgCl2 and CaCl2, I was 0.01 (or cs = 0.0033 mol L-1) and 0.02 mol L-1 (or cs = 0.0067 mol L-1) and aN was 0.2. In the case of MgCl2, the iPMA solution with aN = 0.2 and I = 0.02 mol L-1 was stable (no phase separation occurred). However, in the case of CaCl2, aN in iPMA solution with I = 0.02 mol L-1 could not be reduced to aN = 0.2, because the solution became opaque when aN was decreased from aN > 0.8 to aN = 0.63 and remained cloudy even after prolonged stirring. This solution was therefore studied at aN = 0.63. All I, cs, and aN values for iPMA solutions are reported in Table S1. 2. 3. Methods 2. 3. 1. Visual Observations In order to obtain visual information about temperature-dependent aggregation of a- and iPMA chains in aqueous solutions, samples were kept in cuvettes and equilibrated at different temperatures (T) (T = 15, 25, 45, 55, 65, 75, 85 and 95 °C) in a Julabo circulator. aPMA in MgCl2 and CaCl2 precipitated on a macroscopic scale during heating. The samples were photographed and the photographs are presented in Tables S2 and S3. For iPMA, only pictures at 25 °C are shown (Table S4), because the solutions were transparent in the whole temperature range. Photographs of iPMA in CaCl2 with I = 0.02 mol L-1 and aN = 0.63 (not shown) resembled the case of aPMA at 75 °C. 2. 3. 2. UV Measurements The UV absorbance (A) was recorded with a UV-Vis spectrophotometer Cary BIO 100 (Varian, Australia) at a wavelength X = 280 nm in the temperature range between 0 and 95 °C. To avoid condensation on the cuvette walls, measurements were carried out by blowing dry nitrogen around the cuvette. Samples were degassed prior to measurements. The heating and cooling rate programs were different for a- and iPMA solutions and are reported in Table S5. 2. 3. 3. Fluorimetric Measurements Fluorimetric measurements were performed by using pyrene as the external fluorophore. The fluorescence emission spectra of pyrene were recorded on a Perkin-Elmer model LS-100 luminescence spectrometer in the direction of increasing temperature (from 15 to 95 °C) with a step of 10 °C, also under nitrogen flow similar to UV measurements. The excitation light wavelength was 330 nm, the recording speed was 100 nm min-1, and 20 emission spectra were collected in the wavelength region 350-450 nm and averaged. From the averaged spectra, the ratio of intensities of the first (I1) and the third (I3) vibra-tional peak in the emission spectrum of pyrene or so-called pyrene polarity ratio (I1/I3) was calculated. 2. 3. 4. Light Scattering In order to obtain the information on size and structure of aPMA and iPMA associates in MgCl2 and CaCl2 solutions, dynamic (DLS) and static light scattering (SLS) measurements were performed with the 3D-DLS-SLS cross-correlation spectrometer from LS Instruments GmbH (Fribourg, Switzerland). The measurements were carried out only at 25 °C. The source of incident light was the He-Ne laser with a wavelength X0 = 632.8 nm. Intensity of scattered light was collected in the angular range between 40° and 150° with a step of 10° after keeping the samples at 25 °C for 30 min. At each angle, five correlation functions were collected and averaged. For processing the measured correlation functions, CONTIN analysis was used. Prior to light scattering (LS) measurements, all aPMA and iPMA solutions were filtered through hydro- Hribersek and Kogej: Temperature Dependent Behavior of Isotactic Acta Chim. Slov. 2019, 66, 1026-1037 1029 philic and low protein binding Millex-HV filters (diameter 13 mm, pore size 0.45 ^m) directly into the sealable dustfree cylindrical quartz LS sample cells. LS measurements were performed 3 days after filtration. Due to opacity, iPMA solution in 0.0067 mol L-1 CaCl2 (I = 0.02 mol L-1 and aN = 0.63; see above) was studied without being filtered. Note that the 3D cross-correlation technique was designed especially for the characterization of such strongly turbid samples.36 After 2 weeks two phases were observed in the cuvette in this case. The one at the bottom was more turbid than the upper one. By changing the height of the cuvette in the decalin bath, we were able to perform the LS characterization of both phases separately. LS experiments identified two (in some cases three) populations of particles in iPMA and aPMA solutions (see examples of calculated hydrodynamic radii (Rh) distributions in Figure S1). The Rh values of smaller particles (Rh < 10 nm, assigned to individual chains, and 10 nm < Rh < 20 nm, assigned to small pre-associates; not reported) did not depend on the angle. We were interested in the Rh values of larger particles (70-200 nm), which were assigned to inter-chain associates and designated as Rh,ass. Their values depended considerably on the angle of observation. For further calculations, the Rh,ass values at 9 = 0° were obtained by extrapolation of the measured Rh,ass to zero angle. Due to large size of the associates, their radius of gyration (Rg,ass) could be determined from the angular dependency of the LS intensity following the procedures reported previously.37-39 From Rh,ass at 9 = 0° and Rg,ass, the shape parameter (p) was calculated as p = Rg,ass/Rh,ass. This parameter is often used to characterize mass distribution within the scattering particles.37,38 All further details of LS data evaluation were reported previously.37-40 2. 3. 5. pH Measurements The pH values of the samples were measured by using the combined glass micro-electrode from Mettler Toledo (type InLab® 423; Schwerzenbach, Switzerland) and the Iskra pH meter model MA 5740 (Ljubljana, Slovenia). 3. Results and Discussion 3. 1. Temperature Dependent Behavior of iPMA and aPMA in the Presence of MgCl2 and CaCl2 3. 1. 1. UV Spectroscopy and Visible Observations In Figure 1, A is presented as a function of temperature for aPMA (aN = 0) solutions with I = 0.1 mol L-1 and for iPMA (aN = 0.2) solutions with I = 0.01 mol L-1 in the presence of both, MgCl2 and CaCl2. In addition, measurements for aPMA at higher I (= 0.2-0.5 mol L-1) are shown in Figure 2. The data demonstrate that aqueous aPMA solutions with added MgCl2 and CaCl2 behave as polymer mixtures with so-called lower critical solution temperature (LCST) behavior. The LCST behavior (phase separation induced by heating) is inferred from the fact that UV absor-bance is close to zero at low temperatures (below 70 and 80 °C in CaCl2 and MgCl2, respectively; Figure 1b) and steeply increases at high temperatures. Strong increase in A indicates the formation of larger particles in solution, which is ultimately followed by phase separation of the polymer-rich phase (see visual observations reported below). Clearly, the increase in A is more pronounced in the presence of Ca2+ ions. For the aPMA-CaCl2 solution, a plateau in A is reached between 80 and 90 °C, followed by a fall as T approaches 95 °C. We anticipate that this final drop in A is a consequence of the fact that the aPMA precipitate in the presence of CaCl2 is effectively removed from the solution by settling down to the bottom of the cuvette, whereas it still fills the whole cuvette in the MgCl2 solution at this I (= 0.1 mol L-1; for more details see photographs in Table S2). The LCST for aPMA-CaCl2 solution clearly appears at lower Ts in comparison with that in aPMA-MgCl2 solution (see the LCST values reported in Table S6 and the method for their determination shown in Figure S2). 1 35 - 1.0- o. 2.5 2.0- 0.0 a) iPMA heating cooling MgCI2 ---- CaCI2 ---- b) aPMA heating cooling MgCI2 ---- ,—^ CaCI2 ---- 1 A -r V / i * / / X / / 1 / 1 / 10 20 30 40 50 60 70 80 90 100 T / °C Figure 1. UV absorbance at 280 nm as a function of temperature for a) iPMA (cp = 0.022 mol L-1, aN = 0.20) in MgCl2 and CaCl2 with I = 0.01 mol L-1, and for b) aPMA (cp = 0.023 mol L-1, aN = 0) in MgCl2 and CaCl2 with I = 0.1 mol L-1. Heating (solid lines) and cooling (dashed lines) were performed according to the temperature programs reported in Table S5. Hriberšek and Kogej: Temperature Dependent Behavior of Isotactic ... 1030 Acta Chim. Slov. 2019, 66, 1026-1037 During cooling the aPMA-MgCl2 solution back to the initial temperature, the absorbance first decreases gradually and then drops steeply to 0 at lower temperatures (below approximately 60 °C) as compared to the heating direction. Meanwhile, the initial drop in A upon cooling the aPMA-CaCl2 solution is steeper, but reaches zero at approximately the same temperature (at around 58 °C) as in MgCl2 solution. The drop in A to zero indicates that aPMA aggregates, which form in the presence of MgCl2 and CaCl2 upon heating, disintegrate upon cooling. We conclude that Mg2+ and Ca2+ cations induce reversible association of aPMA chains at elevated temperatures. These findings are in agreement with our recent report on intermolecular association of aPMA chains in the presence of cations with increasing charge.28 The occurrence of the aPMA rich phase (the turbid part that eventually forms a precipitate) in the CaCl2 solution is similar to that in LaCl3.28 This is not surprising, since both cations (Ca2+ and La3+) are known to bind to carboxylate groups in a biden-tate manner.19 The La3+ cations are also recognized as Ca2+ analogs in living systems and can thus act as substitutes of Ca2+ in many proteins and cell membranes.41-43 However, in the case of aPMA the presence of trivalent La3+ ions at I = 0.1 mol L-1 induces irreversible precipitation from solution (no re-dissolution takes place upon cooling), whereas Ca2+ precipitates re-dissolve at the same I. Visual observations for aPMA (aN = 0) solutions are in good agreement with the UV results (see photographs presented in Table S2 for the heating and in Table S3 for the cooling direction). When temperature increases above 75 °C, aPMA solutions in MgCl2 and CaCl2 first become turbid. Fine flakes of precipitated aPMA are formed in the presence of MgCl2, which do not readily settle down (the solution remains turbid). In CaCl2 solutions, on the other hand, the initial turbidity is considerably more pronounced (see for example the photograph taken at 85 °C, Table S2). The precipitate, which subsequently forms in the aPMA-CaCl2 system with further heating to 95 °C, finally settles to the bottom of the cuvette or is partially stuck to the glass wall. This explains the plateau in A values in aPMA-CaCl2 solution at 80-90 °C (maximum turbidity) and their steep drop towards 95 °C (partial removal of the precipitate from solution). The LCST values for aPMA in the presence of MgCl2 and CaCl2 were determined in a broader range of I values (up to 0.5 mol L-1). Heating of aPMA-MgCl2 solution at higher I (> 0.1 mol L-1) leads to a similar plateau and a drop in A values as highlighted above for CaCl2 solution for all I (c.f. Figure 2). With increasing I, the rise in A shifts to lower LCSTs, whereas the plateau shifts to higher A due to higher turbidity and more extensive precipitation of aPMA from solutions at higher MgCl2 concentration. An- Figure 3. The dependences of LCST and Tde-ass on ionic strength (I) for aPMA (cp = 0.023 mol L-1, aN = 0) in MgCl2 and in CaCl2 solutions. Figure 0.1, 0.2: 2. UV absorbance at 280 nm as a function of temperature for aPMA (i^ = 0.023 mol L aN = 0) in a) MgCl2 and b) CaCl2 solutions with I = :, 0.3, 0.4 and 0.5 mol L-1. The heating (solid lines) and cooling (dashed lines) were performed with a rate of 1 °C min-1. Hribersek and Kogej: Temperature Dependent Behavior of Isotactic Acta Chim. Slov. 2019, 66, 1026-1037 54 other important observation is that upon cooling the plateau, and consequently the associated critical temperature of de-association, designated as Tde-ass,28 also shift to lower temperatures with increasing I. A strong hysteresis is clearly seen in the plots in Figure 2, indicating that Tde-ass is considerably lower than LCST (see Tde-ass and LCST values reported in Table S6 and plotted in Figure 3). Similar behavior is observed also for aPMA in the presence of CaCl2. Again, a strong hysteresis is detected along the heating and cooling paths. Nevertheless, some key differences between MgCl2 and CaCl2 solutions can be extracted from these plots. During heating, the plateau in A reaches higher values when Ca2+ cations are present, which means that these divalent cations induce more extensive association and consequently more extensive precipitation of aPMA from the solution in comparison to Mg2+ cations. This can be inferred also from a much steeper drop in A towards the end of the heating cycle in CaCl2 solutions (compare Figure 2b with Figure 2a). In agreement with this, the Tde-ass and in particular the LCST values, are lower for aPMA in CaCl2, suggesting a more effective intermolecular association of aPMA induced by Ca2+ ions. However, in the case of both divalent chlorides, the LCST and Tde-ass decrease with increasing I. Interestingly, Tde-ass approaches body temperature at the highest ionic strength of 0.5 mol L-1 (Tje-ass = 35 and 37 °C in CaCl2 and MgCl2, respectively). From the obtained results we can conclude that changing I of the solutions strongly affects the LCST and Tde-ass values for aPMA in aqueous MgCl2 and CaCl2 solutions, in spite of the fact that the aPMA chain is almost uncharged at aN = 0. The opposite effect of temperature is found for the iPMA isomer as demonstrated by the plot of UV absor-bance in Figure 1a. It should be emphasized right from the start that aN (= 0.2) is not negligible in the iPMA case, which puts electrostatic interactions at the forefront in this case. In contrast to aPMA (aN = 0), the UV absorbance is high in iPMA (aN = 0.2) solutions at low temperatures and starts to decrease at higher temperatures, but never drops to zero, so as is the case with aPMA. This is most clearly seen for the iPMA solution with added MgCl2: A is roughly constant up to 40 °C and starts to decrease more steeply above 50 °C, but does not drop below A ~ 0.95 even when approaching 95 °C. In the presence of CaCl2, the measured A values are even higher (A ~ 1.3-1.35), suggesting that the concentration and/or size of the iPMA associates are larger than in MgCl2 solution. In addition, no significant drop in A values can be identified from the curves for iPMA in CaCl2 and the hysteresis is almost absent, which suggests stronger interactions and different kinetics of the association process as compared to MgCl2. However, by inspecting the enlarged plot for iPMA-CaCl2 solution (Figure S3) we see that the trend in A values is similar to the one in MgCl2 or in other chlorides.28 This type of behavior of aqueous iPMA solutions was previously ascribed to so-called upper critical solution temperature (UCST) behavior, denoting polymer mixtures where phase separation (or merely association) is achieved upon cooling.28 The high A values in iPMA (aN = 0.2) solutions with added MgCl2 and CaCl2 are attributed to the presence of intermolecular associates that are abundant in these solutions in the whole temperature range (A stays high even at 95 °C). Similar to these observations, it was found previously that iPMA associates are stable in the whole temperature range in aqueous solutions with added trivalent La3+ ions, however they largely decomposed upon heating in the presence of monovalent Na+ ions.28 This again indicates the important role of electrostatics (counterion charge) in intermolecular association of iPMA (aN = 0.2) chains. Visual observations of iPMA solutions show that, in spite of high absorbance, no macroscopic phase separation occurs in iPMA (aN = 0.2) solutions at I = 0.01 mol L-1 (therefore only the photograph at 25 °C is presented in Table S4), indicating that iPMA associates are formed on a mesoscopic scale, as already established for iPMA solutions with mono- and trivalent cations under the same conditions (i.e. at I = 0.01 mol L-1).28 The iPMA (aN = 0.2) system is, however, very sensitive to salt concentration as the polymer precipitates from solution when I exceeds 0.01 mol L-1 (see Experimental and discussion below). Again, the relatively high charge of the iPMA chain plays the key role here. 3. 1. 2. Fluorescence Measurements Pyrene fluorescence measurements were used to follow the micropolarity of the aggregates' interior in aPMA and iPMA solutions. It is expected that pyrene, as a very hydrophobic molecule, would solubilize inside the aggregates if they are composed of a non-polar core. It is well-known that aPMA chains at low aN adopt a compact con- □ --—---5 u □ 5 — m/ iPMA aPMA MgCI2 □ ■ CaCI2 O • 0 20 40 60 80 100 T / °C Figure 4. Temperature dependence of the pyrene polarity ratio (I j/ I3) for iPMA (cp = 0.022 mol L-1, aN = 0.20) in MgCl2 and CaCl2 solutions with I = 0.01 mol L-1 and for aPMA (cp = 0.023 mol L-1, aN = 0) in MgCl2 and CaCl2 solutions with I = 0.1 mol L-1. Hriberšek and Kogej: Temperature Dependent Behavior of Isotactic ... 1032 Acta Chim. Slov. 2019, 66, 1026-1037 formation with hydrophobic methyl groups forming the interior and offering a suitable solubilization site for pyrene. We were interested in how the tacticity and added divalent metal ions affect the polarity of microenvironment in aPMA (aN = 0) and iPMA (aN = 0.2) solutions. The emission spectra of pyrene for both PMA isomers in MgCl2 and CaCl2 solutions are presented in Figure S4. From these spectra, the pyrene polarity ratio I1/I3 was calculated. The obtained values are reported in Table S8 and plotted as a function of temperature in Figure 4. The immediately evident feature of the spectra in Figure S4 is the decrease in fluorescence intensity with increasing temperature, which significantly distorts the spectrum for aPMA in CaCl2 solution at 95 °C. This is attributed to extensive precipitation of aPMA from this solution, as discussed above. No I1/I3 value is therefore reported for aPMA in CaCl2 at the highest temperature. The most obvious result of Figure 4 is that I1/I3 values are higher in iPMA (above 1.4 in iPMA-MgCl2 and above 1.1 in iPMA-CaCl2 solutions) than in aPMA solutions (here I1/I3 values start at around 0.85 at low Ts) and only weakly increase with temperature with no evident change of slope in the iPMA case. The value I1/I3 ~ 1.5 in iPMA-MgCl2 solutions points to a rather high polarity of microenvironment where pyrene molecules reside in these solutions. For comparison, I1/I3 is 1.3 in methanol, 1.35 in ethyl acetate, and around 1.7 (depending on the presence of eventual ions) in water.33 These high I1/I3 values are attributed to strong interactions of Mg2+ ions with water molecules, due to which these cations bring a lot of water into the associates with iPMA. At the same time the monodentate binding of Mg2+ is weaker in comparison with cations that release the hydration water (like Ca2+).37 However, the iPMA (aN = 0.2) chains must be held rather strongly in these associates, considering that they are charged and thus also repel each other. As proposed previ-ously28 we attribute the stability of the iPMA associates to strong cooperative intermolecular H-bonds between unionized COOH groups on different iPMA chains and in case of Mg2+ ions also to H-bonds between COOH groups and the hydration water molecules around the ion. The presence of large amounts of water in the iPMA-MgCl2 associates contributes to the observed high I1/I3 values. The I1/I3 values for iPMA in CaCl2 are somewhat lower (I1/I3 ~ 1.1-1.3) than in MgCl2 solutions, which can be explained by bidentate Ca2+ binding to carboxylate groups.11,17-19 This type of binding is accompanied by elimination of water from cation's hydration sphere and thus also from the associates. Consequently, the polarity of these associates is determined mainly by H-bonded COOH groups and is thus expected to be lower. Altogether, fluorescence measurements show that the interior of the iPMA associates is actually rather polar (in contrast to aPMA; see below) and stems from polar COOH groups and/or water in these associates. Strong H-bonding between COOH groups is facilitated by their suitable isotac- tic orientation on the iPMA chain. Meanwhile, the charged COO- groups are positioned on the surface and grant the solubility of the associates in water. The pyrene polarity ratio in aPMA solutions at temperatures below 45 °C is I1/I3 ~ 0.85-0.95 (irrespective of the cation) and demonstrates a considerably less polar microenvironment of the aPMA aggregates. This can be explained by so-called hydrophobic interactions that force the methyl groups to hide inside the cores of the aPMA aggregates, which is well known. The irregular orientation of COOH groups on aPMA actually facilitates the formation of such hydrophobic cores as a result of higher flexibility of the aPMA chain and at the same time does not allow extensive H-bonding (as in the iPMA case). The hy-drophilic COOH groups are positioned mostly on the surface and protect the core against the water solvent. Above 45 °C, an increase is observed in I1/I3 values for aPMA solutions in both, MgCl2 and CaCl2, which is attributed to precipitation of aPMA aggregates from solutions. Some of the pyrene molecules remain accumulated inside these precipitates and are removed from water, whereas the remaining ones detect the polarity of water without aPMA. The above conclusions apply also to higher I values, however the increasing in I has no significant effect on the I1/I3 ratio. Its value depends primarily on chain tacticity (see the I1/I3 values measured at 25 °C in Table S9). 3. 2. LS Characterization of aPMA and iPMA Associates at 25 °C Further structural information about the associates was obtained by LS measurements at 25 °C. In Table 1, the Rh,ass, Rg,ass and p values are reported for aPMA (aN = 0) and iPMA (aN = 0.2) associates in MgCl2 and CaCl2 solutions. The Rh,ass values (the dynamic LS data) are shown in Figures 5a and 5b as a function of q2 (q is the scattering vector given by the relationship q = (4nn0/A0)sin(9/2), with n0 the refractive index of the medium and A0 the wavelength of the incident light). From these dependencies, the zero angle values were obtained by using either linear function or second order polynomial for extrapolation to 9 = 0°. In Figures 5c and 5d, the static LS data are presented for both PMA isomers in the form of a Kratky plot (i.e. the dependence of (qRg)2P(9) on qRg) and compared with the calculated curves for some well-known particle topologies. The DLS results show that aPMA particles in the presence of both divalent salts have more or less comparable hydrodynamic radii (160-190 nm). However, the radii of gyration (the SLS result) of these particles are considerably smaller in the presence of Ca2+ ions, leading to lower p values in this case. The value p ~ 0.70 (aPMA-CaCl2 with I = 0.1 mol L-1) indicates micro-gel like structure of particles with mass concentrated in the core surrounded by a water swollen corona, whereas p ~ 0.90 in both aPMA-Mg-Cl2 solutions points to a more even mass distribution. For comparison, a homogeneous mass distribution, such as in Hribersek and Kogej: Temperature Dependent Behavior of Isotactic Acta Chim. Slov. 2019, 66, 1026-1037 1033 Table 1. Hydrodynamic radii (Rh,ass)> radii of gyration (Rgass) and parameter p (= Rg>ass/Rh,ass) for associates of iPMA (Cp = 0.022 mol L aN = 0.20) and aPMA (Cp = 0.023 mol L-1, aN = 0) in the presence of MgCl2 and CaCl2 at different I and at 25 °C. iPMA aPMA Added salt I /mol L1 ßh,ass / nm Rgass / nm P I /mol L1 Rh,ass / nm Rgass / nm P MgCl2 0.01 75 73 0.97 0.1 178 164 0.92 0.02 177 180 1.02 0.2 191 160 0.84 CaCl2 0.01 65 76 1.16 0.1 161 109 0.68 0.02a) 111b) 153b) 1.37b) 0.2b) 178b) 125b) 0.70b) 184c) 127c) 0.69c) a)aN = 0.63; b)upper phase; c)lower phase a) iPMA I / mol L"1 0.01 0.02 MgCI2 ■ □ CaCI2 • O upper phase O lower phase b) aPMA □ 1 / mol L 0.1 0.2 MgCI2 ■ □ CaCI2 • O q / 1CT q2/10~1 c) iPMA I / mol L"1 0.01 0.02 MgCL ■ CaCL □ o upper O lower d) aPMA I/mol L"1 0.1 MgCI2 a CaCL • Zimm hard sphere —i-1-1-1-1— 1 2 qRg qRg Figure 5. Hydrodynamic radii (Rh,ass) as a function of q2 for a) iPMA (Cp = 0.022 mol L aN = 0.20) in MgCl2 and CaCl2 with I = 0.01 mol L 1 (full squares) and I = 0.02 mol L-1 (open symbols) (note that aN = 0.63 for iPMA in CaCl2 with I = 0.02 mol L-1) and for b) aPMA (cp = 0.023 mol L-1, aN = 0) in the corresponding salt solutions with I = 0.1 and 0.2 mol L-1. The Kratky plot (the dependence of (qRg)2P(8) on qRg) for selected topologies (solid lines; for details on these scattering functions see44) and the experimental data for c) iPMA ( aN = 0.20 for all salt solutions, except for CaCl2 with I = 0.02 mol L-1 where aN = 0.63) and d) aPMA associates (aN = 0) in aqueous solutions of MgCl2 and CaCl2 salt solutions with I = 0.1 and 0.2 mol L-1. a (monodisperse) hard sphere, would result in p = 0.778. By considering that polydyspersity that is virtually always present in real samples tends to increase p, a particle with a measured p of 0.70 definitely cannot be a hard sphere. Lower p values in the presence of CaCl2 are actually expected due to the preference of strong bidentate binding of Ca2+ to carboxylate groups as discussed above. Ca2+ ions release coordinated water molecules upon binding to car- Hriberšek and Kogej: Temperature Dependent Behavior of Isotactic ... 1034 Acta Chim. Slov. 2019, 66, 1026-1037 boxylate groups, the aggregates' interior is thus 'drier' (leaving only the outer layers hydrated) and therefore more compact. On the other hand, Mg2+ ions remain strongly hydrated due to their pronounced inability to dehydrate and thus the aPMA chains in MgCl2 solutions form rather loose and more water swollen aggregates. Such environment offers more mobility to the chains and the structure is more permeable for the solvent, which altogether leads to higher p values. Still, p ~ 0.90 for aP-MA-MgCl2 aggregates is far from values known for random coils of single chains in a 9-solvent (p ~ 1.5). The Kratky plots for aPMA aggregates in both metal chlorides (Figure 5d) confirm the suggested structures. The data points coincide with the Debye-Bueche scattering function, which is valid for micro-gel like particles with a core-shell structure and at the same time demonstrate that the increase in I does not greatly affect the structure, which is reasonable in case of aPMA (aN = 0) with charge close to zero. The experimental points for aPMA in CaCl2 solutions show a noticeable upturn from the Debye-Bueche towards the Debye scattering function at high qRg values. This kind of upturns were previously attributed to the ex-cluded-volume interactions,37,40 which may be more pronounced in case of Ca2+ ions in view of their stronger interaction with carboxyl groups. We actually demonstrate in the following (see results of pH measurements below), that Ca2+ ions replace some H+ ions from COOH groups and thus increase the ionization of aPMA. Different from aPMA, Rh,ass and Rg,ass values for iPMA associates in MgCl2 and CaCl2 with lower I (= 0.01 mol L-1) are significantly smaller in comparison to those in solutions with higher I (= 0.02 mol L-1). This result is reasonable, because salt concentration should play a more important role in the case of partly charged iPMA (aN = 0.2) chains: higher salt concentration (higher I) contributes to stronger electrostatic screening and enables more extensive intermolecular association and formation of larger particles. However, p values for iPMA-Mg2+ associates are not much affected by I (p ~ 1), whereas those for iPMA-Ca2+ associates range from 0.69 (the lower phase formed in the CaCl2 solution with I = 0.02 mol L-1 and aN = 0.63; see Experimental Section) to 1.37 (the upper phase in the same solution). In the Kratky plot we see that the experimental data for larger particles in iPMA solutions show a systematic positive shift from the Debye-Bueche function at high qRg values. We attribute these observations to a possibility that different structural elements are present in the iPMA-Ca2+ associates. Let's assume that the bidentate binding of Ca2+ proceeds predominately via bridging interaction with carboxyl groups on neighboring iPMA chains (i.e. intermolecularly), whereas the chelating (intramolecular) binding is less likely (see a scheme of both binding modes in Figure 6). The bridging bidentate binding with a concomitant pronounced dehydration of Ca2+ ions may induce structural elements of higher rigidity within these complexes, in particular considering also the isotacticty of the chain, and contributes to shifting of the points in the Kratky plot toward more rod-like structures. On the other hand, portions of the iPMA chains that are not involved in this complexation (presumably the more ionized parts) form coil-like outer regions with higher p (c.f. p = 1.16 and 1.37 for the homogeneous sample at I = 0.01 mol L-1 and for the upper phase at I = 0.02 mol L-1, respectively) around the compacted parts. Figure 6. Cartoon depicting proposed ways of bidentate binding (chelate and bridging) of Ca2+ to COO- groups on iPMA chains. At the end we should shortly comment also the result for the lower (polymer rich) phase in iPMA-CaCl2 solution with I = 0.02 mol L-1 and aN = 0.63 (note that these are average values for the whole sample). Here, p (= 0.69) is again very low and the points in the Kratky plot fit the Debye-Bueche function, showing that associates in this phase are much more compact as compared to the upper more solvated and dilute phase. We also presume, that aN in the bottom phase may be lower than the average value («N = 0.63). We conclude the discussion of LS results by proposing that PMA-Ca2+ associates could also include interactions in terms of so-called egg-box model, commonly used in interpretation of gelation phenomena of alginates and pectates, which belong to a class of polysaccharides with carboxylate groups.7,9,10 According to the egg-box model, interaction between COO- groups on the polysaccharide and divalent cations is cooperative if the ion has an appropriate size to fit into the space in the egg-box, which is formed by the polyion backbone. Ca2+ fulfils this criterion, while the hydrated Mg2+ is too big.7 By proposing the existence of egg-box-like structural elements into which Ca2+ ions fit due to their size, one can understand both, the for- Hribersek and Kogej: Temperature Dependent Behavior of Isotactic Acta Chim. Slov. 2019, 66, 1026-1037 58 mation of more gel-like polymer-rich phase in aPMA-Ca-Cl2 and more complex structural observations for iP-MA-CaCl2 solutions. 3. 2. 1. pH and Degree of Ionization In support of the above interpretation of differences between MgCl2 and CaCl2 solutions, we report the pH values for both PMA solutions, from which the degree of ionization (ai) of COOH groups was calculated from ai —