The Influence of Amphoteric Terpolymerization of Poly (N-Isopropylacrylamide) in the Optimization of the Phase Separation Temperatures

The aim of this study is to fabricate an ampholyte thermo-responsive terpolymers and explore the influence of ampholyte on the phase separation temperatures in different pH solutions. The fabrication of the ampholyte thermo-responsive terpolymers was achieved by terpolymerization of N-isopropylacrylamide (NIPAAm), DIVA (5, 10, and 15 mol.%), and acrylic acid (AA) 10 mol.%. The preparation of the cationic monomer from vanillin was achieved in a facile reaction named 2-((diisopropylamino) methyl)-4-formyl-6-methoxyphenyl acrylate (DIVA). The chemical structures of the monomers and terpolymers were evaluated successfully by 1H, 13C NMR, and FTIR. The average molecular weight and dispersity of the terpolymers were characterized using gel permeation chromatography (GPC); the glass transition temperature, crystallinity, and morphology were characterized using DSC, XRD, and SEM, respectively. The hydrophilic/hydrophobic properties of the fabricated terpolymers, at different pH conditions, were evaluated using contact angle measurements. The lower critical solution temperature (LCST) for all samples was measured using UV–vis spectrophotometer. Moreover, the LCST was tested using different Hofmeister salts in kosmotropic and chaotropic conditions. The findings of this study can be utilized in the bio-separation of biological molecules, as well as drug delivery applications.


Introduction
In the last few decades, scientists have focused on studying smart materials, also known as environmentally responsive or intelligent materials, which are materials that respond to changes in their surrounding conditions [1][2][3][4][5][6].Among these materials is a class of polymers known as thermo-responsive polymers (TRPs) which change their phase from a coiled state to a globular one in response to temperature changes.TRPs have been popular in various biomedical applications, sensors, and actuators [7,8].The polymeric solutions of these materials have a unique attitude towards temperature [9].Two popular categories of TRPs have been studies.The first type, known as the lower critical solution temperature (LCST)-TRP, appears as an opaque solution (turbid or milky) when increasing the solution temperature above a specific limit, which is known as the cloud point at the mid-transmittance or the lower critical solution temperature [10][11][12].The second type is known as the upper critical solution temperature (UCST)-TRP, which behaves in contrast to the LCST-TRPs [13].
Po ly ( N -i s o p r o p yl a c r yl a m i d e ) s ( P N I PA M ) ((C6H11NO)n) and its derivatives are among the most popular thermo-responsive polymers.They display reversible changes in water solubility upon heating and cooling by undergoing a phase transition from a hydrophilic coiled state to a hydrophobic globule state with a lower critical solution temperature close to the human body temperature of − 32 °C [14][15][16].The tailoring of PNIPAAm structure to specific applications has been widely studied in several recent works [17][18][19][20][21][22].Copolymerization of PNIPAAm with other hydrophilic or hydrophobic monomers has been proposed to optimize the phase separation temperature (T c ) to higher, or lower, temperatures for various applications [23][24][25][26][27][28][29][30].
Another important class of stimuli-responsive materials is the pH-responsive polymers [31][32][33].The monomers and polymers of these materials have functional groups which facilitate the formation of ionic charges in pH solutions; hence, they are distinguished with their electrical charge as anionic or cationic in the pH solutions [34][35][36][37][38][39].The copolymerization of anionic monomer-like acrylic acid (AA) with NIPAAm has been demonstrated to increase the phase transition temperature (T c ) of PNI-PAAm in higher pH solutions due to the ionization of the carboxylic group [40].Whereas; cationic monomers with tertiary amine exhibit a change in their phase transition temperatures in lower pH solutions due to the high protonation in highly charged solutions [41][42][43][44][45].
Copolymerization and terpolymerization of PNIPAAm/ AA have been studied extensively with emphases on the fluctuations in LCST and the cloud points in low and high pH solutions [46].The copolymers exhibited higher transition temperatures in higher pH solutions and lower transition temperatures in lower pH solutions compared to homo-PNIPAAm.This was attributed to the effect of hydrophilic/hydrophobic interaction of both NIPAAm and acrylic acid groups, which increases/decreases the hydrogen bonds in water solutions; respectively [47].The polymerization of cationic and anionic monomers with the N-isopropylacryamide produced a thermo-responsive polyampholyte [46][47][48][49].A zwitterionic polymer was produced with anionic and cationic functionality, which exhibited special characteristics in polymer solutions [50].The phase separation temperature of the polymer solutions was influenced by the different pH levels in the case of dual pH-thermo-responsive polymers [51,52].Changes in the LCST due to the ionization of polymer solutions in both lower and higher pH solutions were observed; the adjustment of the phase separation temperature depends mainly on each hydrophilic/hydrophobic group in the thermoampholyte copolymer [1-3, 13, 14, 20-22].Applications of these polymers were demonstrated in various biological and biomedical scopes [49][50][51][52].
Monomers from vanillin derivatives have been studied in many recent publications due to the ability of vanillin compounds to be modified with hydroxyl and aldehyde groups [53,54].Franz Hofmeister is credited for revealing the lyotropic series [55] in which groups of anions and cation salts were arranged according to their solubility and coagulation properties in water [56,57].Anions were divided into two groups, one called chaotropic, which is distinguished with low charge density like Cl − , NO 3 − , SCN − ; their interaction with water molecules is weak.The other group is called kosmotropic, which is highly charged anions, like CO 3 2− , SO 4 2− , S 2 O 3 2− , and their interaction with water molecules is strong [58][59][60][61][62][63].Many recent studies focused on studying the effect of these anions on the phase separation temperature of PNI-PAAm and its copolymers; they demonstrated a strong decrease of the LCST with the kosmotropic anions; however, a weak effect has been noticed in the case of the chaotropic anions [64][65][66][67].
In this study, the preparation of a new pH-responsive monomer, which is used in the fabrication of thermo-ampholyte terpolymers, is demonstrated.The influence of ampholyte on the fluctuations and optimization of the phase separation temperature in different pH solutions is presented.The fabricated terpolymers can be used in bio-separation of biological molecules, as well as drug delivery systems.

Chemical and Physical Analysis
Bruker Avance spectrometer 500 and 125 MHz were used for detecting 1 H and 13 C NMR, respectively.Monomer samples were dissolved in deuterium CDCl 3 (99.8,Sigma); whereas, polymer samples were dissolved in D 2 O (≥ 99.96%, Sigma).Fourier transform infrared FT-IR spectra of monomers and polymers have been carried out using Vertex 70; they were recorded in the range between 400 and 4000 cm −1 .The average molecular weights ‾M w and the number average molecular‾M n were measured using size exclusion chromatography (SEC).Dimethylacetamide (DMA) was the eluent of 14 mg/ml of polymer solution.The polystyrene molecular weight was carried out as standard.
Modulated Differential Scanning Calorimetry (MDSC) was used to record the polymer's glass-transition temperature (T g ) at a heating rate of 5 °C/min.The onset point was used as the glass-transition temperature.Bruker AXS D8 Advance-X-ray diffraction (2.2 kW Cu and Co, 40 kV, 40 mA) was used to measure the crystallinity of solid polymer samples.It was fixed with a microprocessorcontrolled Goebel mirror, goniometer, optical encoders, sample spinner, video camera, and thin-film reflectometry.The morphological features of polymers were investigated using a Zeiss NEON 40 scanning electron microscope (SEM) at 700X magnification.
The surface contact angle (ϴ) is commonly used to characterize the degree of hydrophilicity of solid surfaces by exposing the surface to an aqueous solution using a micropipette.A digital camera was used to take photographs of drops on the surface of polymer pellets.ImageJ software was used to magnify the images and determine the contact angles.
Perkin Elmer (Lambda 45) UV-vis spectrometer was used to measure the lower critical solution temperature via turbidity of polymer solution as the temperature is raised above its critical temperature.The relationship between the transmittance percent in the polymer solution versus its temperature was plotted, and the phase-transition temperature (T c ) was detected at the inflected point.The cloud point was determined at 50% transmittance.The solution temperature was increased from 5 to 80 °C, with a polymer concentration of 1 wt.% using different pH solutions.Micro-DSC III (Setaram) was used as an alternative method to measure LCST.The polymer was dissolved in deionized water DI (pH 6.9) and heated/cooled at 5 °C/min in deionized water.The thermograms were drawn and the LCST was recorded at the onset value.

Synthesis of 3-((Diisopropylamino) Methyl)-4-Hydroxy-5-Methoxybenzaldehyde (DIV)
In a two-neck flask, 4 g (0.09 mol) of vanillin (4-hydroxy-3-methoxy benzaldehyde) was added to 14 g (0.47 mol) 37% formaldehyde, and 14 g (0.14 mol) diisopropylamine, and then dissolved in 150 ml ethyl alcohol.The mixture was connected with a reflux condenser fixed with a water trap and heated in an oil bath at 130 °C for five hours.The solvent was evaporated to collect the product.The physical state appeared as a yellow solid.The yield of the final product was 96%.

Synthesis of 2-((Diisopropylamino) Methyl)-4-Formyl-6-Methoxyphenyl Acrylate (DIVA)
We prepared a mixture of 12.0 g (0.045 mol) of 1a 3-((diisopropylamino)methyl)-4-hydroxy-5-methoxybenzaldehyde (DIV), and 4.6 g, (0.045 mol) triethylamine in 100 ml dry dichloromethane CH 2 Cl 2 , then transported the mixture to a three-neck flask connected by nitrogen balloon, rubber stopper, and dropping funnel.The mixture was stirred in an ice bath (1-5 °C) during the dropping of 4.1 g (0.045 mol) acryloyl chloride in dichloromethane.After that, the mixture was stirred at room temperature for about 7 h.The solid salt of tertiary amine was disposed of by filtration, and the solvent was evaporated.The purification was carried out by the following steps: dissolving in dichloromethane; transporting to a separating funnel; washing in deionized water DI, 0.1 M sodium carbonate (Na 2 CO 3 ), 0.1 M hydrochloric acid (HCl), and neutralizing by washing in deionized water.The final product was dried by stirring in MgSO 4 overnight; then the solvent was evaporated.The TLC was used for the purification by dissolving in a mixture of methanol/n-hexane 3:1, R f = 0.44.The physical state appeared as an orange solid.The yield of the final product was 77%.

Monomer and Terpolymers Chemical Evaluations
This work is based on the preparation of a new acrylate monomer with functional and cationic groups as described in Scheme 1.

Monomer
The new acrylate monomer has functional groups that add unique properties to its general characterization.It was prepared in two steps; vanillin was used as the starting compound as shown in Scheme 1.The formation of a cationic tertiary amine group was implemented by the reaction of vanillin with formaldehyde and diisopropylamine to produce compound (1a) 3-((diisopropylamino) methyl)-4-hydroxy-5-methoxybenzaldehyde (DIV).The new compound was evaluated by chemical analysis using 1 H NMR, 13 C NMR, and FTIR; the protons NMR confirmed the chemical structures and the presence of all protons, through the presence of 2H of CH (CH 3 ) 2 at δ = 2.95 ppm, also 2H of (-NCH 2 ) at δ = 3.54 ppm and 12H due to the methyl groups of tertiary amine at δ = 1.03 ppm; the aldehyde proton was observed at δ = 9.98 ppm, as shown in Fig. 1.The corresponding 13 C has also detected the essential carbons in the chemical structure; The 1 H NMR in Fig. 3 illustrated the formation of the vinyl group through the appearance of protons at δ = 6.05, 6.37, and 6.62 ppm attributed to the three protons of -CH = CH 2 the spin-spin coupling and the integrations have also been observed and confirmed its chemical structure.Additionally, the 13 C NMR elucidated and confirmed the new compound via the presence of the two specific carbons of the vinyl group at δ = 127.14, and 134.50 ppm, the aldehyde group was noticed in both 1 H and 13 C NMR at δ = 9.98  6, compound (1a) illustrated the essential functional groups of (CN) at ν = 2345-2480 cm −1 that proves the formation of a tertiary amine group, otherwise, the appearance of a stretched peak at ν = 1653 cm −1 for the carbonyl aldehyde group.The final product emphasized the formation of vinyl acrylate group -C=C-at ν = 1538 cm −1 stretched peak, moreover; the tertiary amine group was confirmed by the presence of -CNgroup at 2345-2480 cm −1 , and the carbonyl aldehyde group at 1653 cm −1 (Fig. 4).

Terpolymers
The random free radical polymerization in solution was used to fabricate a series of three terpolymers based on the N-isopropylacrylamide as the thermo-responsive monomer, while the amphoteric property was achieved by using the new cationic monomer DIVA with three different concentrations (5, 10, and 15 mol.%); the acrylic acid was used as the anionic monomer with 10 mol.%.These polymers were evaluated by the 1 H NMR and the FTIR.The multiple protons have appeared in the 1 H NMR chart as shown in Fig. 5.The protons for each monomer that indicate the formation of terpolymer are represented in the synthesis section.Three protons were used for the confirmation of each monomer concentration in the polymer chain; the proton of the isopropyl group (-CH) was used for NIPAAm at δ = 3.81-4.09ppm, and 1H of aldehyde -CHO was recorded at δ = 9.86-10.07ppm, 1H of the carboxylic acid of the acrylic acid was also detected δ = 10.96-11.13ppm.The absorptions of the functional groups in the polymer chain were detected via FTIR, indicating some carbonyl groups for ester at ν = 1767 cm −1 ; the aldehyde carbonyl of DIVA was observed at 1714 cm −1 , and amide carbonyl -CONH-at 1625 cm −1 , furthermore, many absorptions were detected as illustrated in Fig. 6.

Miscellaneous Characterizations of the New Terpolymers
This work emphasized the fabrication of a new series of ampholyte terpolymers via free radical polymerization, as discussed earlier.The new terpolymers have zwitterion properties due to the effect of the cationic and the anionic groups for each of the tertiary amine and the carboxylic group in the polymer main chain.The polymerization process was based on N-isopropylacryamide monomer as the main component in the terpolymer chain.Their sensitivities to temperature, pH, and salt led to diversity in characterization.The molecular weights and the dispersity were analyzed via gel permeation chromatography (GPC) in dimethylacetamide eluent and PS column, the number average molecular weight M n and dispersity Ð are summarized in Table 1 and illustrated in Fig. 7.The GPC data showed a significant decrease in both M n and Ð with the increase of DIVA content in the terpolymer chain.It demonstrated M n (64,500, 19,800, and 14,100), corresponding to Ð (3.06, 2.16, and 2.06) for 4a (5), 4b (10), and 4c (15), respectively.This is due to the stearic hindrances caused by DIVA, as the DIVA content increased the stearic hindrances increased and therefore the effect on the molecular chain length of the terpolymer to the shorter one [13,14].
The glass transition temperature (T g ) of the ampholyte terpolymers was analyzed using differential scanning calorimetry (DSC) at a heating rate of 5 °C/min.The dry terpolymers samples of 4a (5), 4b (10), and 4c (15) were measured by quenching them from the melted temperature to the liquid nitrogen temperature.The resulting thermogram was calibrated via the standards.The specific glass temperature for each terpolymer was taken at the inflected point of the thermogram.Figure 8 shows the thermograms for   terpolymers 4a (5), 4b (10), and 4c ( 15), with their associate date presented in Table 1.The glass transition temperatures for terpolymers 4a (5), 4b (10), and 4c (15) were measured to be 152, 130, and 125 °C; respectively.We observed a gradual descent of the glass transition temperatures with an increase in the concentration of DIVA.This was attributed to the highly stearic hindrances of DIVA as an aromatic compound [20][21][22].X-ray diffraction (XRD) was used to determine the crystallinity state of the new ampholyte terpolymers.Figure 9 shows the XRD plots for terpolymers 4a (5), 4b (10), and 4c (15); it illustrates three main peaks indicating three stages of 2ϴ, from 20° to 25°, 26° to 36°, and then 37° to 48° which indicate crystallinity enhancement in the polymeric material.The crystallinity percent was calculated according to reference [68] to be 58, 52, and 44% for 4a (5), 4b (10), and 4c (15), respectively.A decrease in the percent crystallinity with increasing the DIVA content in the terpolymer chain supports the results presented earlier.The absence of sharp peaks in the XRD plots indicates a semi-crystalline nature of these materials [66].
The surface morphology of the ampholyte terpolymers 4a (5), 4b (10), and 4c (15) was investigated using scanning electron microscopy (SEM) as shown in Fig. 10.The scanning was performed on a small disk of dry terpolymer samples at 700 × magnification.A mild aggregation of waxy substance was observed in the lowest content of DIVA terpolymer 4a (5) as shown in Fig. 10A, while a waxy substance with small holes was observed in the samples with medium content of DIVA 4b (10), and intensive aggregation of waxy substance was observed in the sample with the highest content of DIVA 4c (15).
The wettability of the polyampholyte terpolymers was tested using contact angle measurements to explore the tendency of these materials to be hydrophilic or hydrophobic.The tests were performed at different pH levels: Fig. 9 X-ray diffractions (XRD) of solid terpolymers Fig. 7 The molecular weight graphs of terpolymer recorded by GPC Fig. 8 DSC thermographs for the glass transition temperature of solid terpolymers 1.38, 3.6, 5.6, 6.9, 9.3, 11.2, and 13.4.The contact angles for hydrophilic, hydrophobic, and superhydrophobic behaviors were used as defined in a recent article [67].The contact angle values for 4a (5), 4b (10), and 4c (15) at pH 1.38 were measured to be 70.3°,67.6°, and 64.7°, respectively, which indicates higher hydrophilic moiety in the polymer chains with higher DIVA content due to the ionization and protonation of the tertiary amine group of the terpolymer solution under strongly acidic conditions.Higher contact angle values 73°, 76.4°, and 79.7° were observed at higher pH values in 4a (5), 4b (10), and 4c (15), respectively.Table 2 presents the fluctuations in the contact angles due to the change in the balance of the hydrophilic to the hydrophobic groups in the terpolymer main chain, which is attributed to the degree of the ionization of tertiary amine groups and carboxylic groups in the polymer main chains.Figure 11 shows the measured contact angles at various pH levels.It is evident that a hydrophilic terpolymer exhibiting lowest contact angle was observed under the strongest acidic solutions (pH 1.38), whereas a more hydrophobic terpolymer exhibiting the highest contact angle was observed in the neutral media (pH 6.9).

Tuning in the Phase Transition Temperature of the Ampholyte Terpolymers
The recent studies of the lower critical solution temperature or the phase separation temperature of poly(Nisopropylacrylamide) solution exhibited 32 °C due to the hydrophilic/hydrophobic groups in the polymer chain.This temperature is imperfect for many applications of thermo-responsive polymers.Here, the new ampholyte thermo-responsive terpolymers were synthesized with the incorporation of DIVA and AA to demonstrate a new behavior of the phase transition temperature (T c ) that reflected the ionization of the zwitterion in both of the tertiary amine group of DIVA and the carboxylic group as well.The tuning in the phase separation temperatures with a variation in pH solutions (1.35, 5.6, 6.9, 9.5, 11.2, and 13.4) for all terpolymers (4a (5), 4b (10), and 4c (15)) have been studied in detail.
In the first run, the terpolymers were dissolved in a pH 1.35 solution.The change of the transmittance with the temperature was drawn and the LCST was taken at the inflected point, while the cloud point (C p , s ) was taken at 50% of the transmittance as shown in Fig. 12A.The data are recorded in Table 1.The LCST (T c , s ) of the ampholyte, thermo-responsive terpolymers exhibited 39.4, 42.5, and 48.5 °C with the corresponding cloud points of 40.6, 43.9, and 49 °C for 4a (5), 4b (10), and 4c (15), respectively.The significant increase in the LCST with increasing DIVA content indicates high ionization and protonation of the tertiary amine group in the DIVA.This is also the reason for increasing the hydrophilicity in the terpolymer solution, and increasing the hydrogen bonding in the polymer solution which delays the phase separation to a higher temperature than homo-PNIPAAm [10,16].
In the next run, we used a pH 3.6 solution, as illustrated in Fig. 12B (5), 4b (10), and 4c (15).The lower values were attributed to the lower ionization and protonation at pH 3.6 than pH 1.35.Furthermore, lesser hydrophilicity and lower LCST were recorded for the polymer solution in weakly acidic media at pH 5.6 as shown in Fig. 12C.By increasing the pH level to a neural media, a decrease in the LCST and the cloud point were observed.The test of samples 4a (5), 4b (10), and 4c (15)  By changing the solution into weak alkaline at pH 9.5, the ionization of the carboxylic acid started to form the carboxylate ion and release the protons in the terpolymer solution which increased the hydrophilic interaction and further spiking in the LCST but with a lower rate due to lower ionization of the carboxylic group of terpolymer solutions.In addition, decreasing the content of DIVA in the polymer chain reflected the opposite effect of DIVA in the alkaline solution by showing a hydrophobic character.The T c , s were recorded at 34.6, 33.8, and 31.5 °C; whereas, the C p , s were recorded at 35.4, 34.9, and 32.3 °C for 4a (5), 4b (10), and 4c (15), respectively, as shown in Fig. 12E.
Much higher pH solution led to a higher LCST and cloud point for terpolymers as shown in Fig. 12F (5), 4b (10), and 4c (15), respectively.The reason for the highest value of 4a (5) and the lowest value of 4c (15) was attributed to the same content of AA which is responsible for the ionization in the alkaline solution and increasing the hydrophilicity and hydrogen bonding in the terpolymer solution, which lowers the value of DIVA (source of hydrophobicity); thus resulting in an overall effect as shown in Fig. 12G.The overall results are summarized in Fig. 12H, indicating the highest and the lowest values of T c and C p for the terpolymers solutions 4c (15) at pH 1.35, and pH 6.9, respectively.Another technique was used to measure the lower critical solution temperature or the phase separation temperature using the micro-differential calorimetry (Micro-DSC); it's more accurate than the turbidity (transmittance/temperature) method.The onset point of the thermogram was taken as the T c of the terpolymers solution.The measurements were recorded for all samples only in pH 6.9 (neutral media); they represented the T c , s at 33.4, 30.8, and 29.5 °C for 4a (5), 4b (10), and 4c (15), respectively.The decrease in T c , s has been interpreted as discussed earlier.The small differences in the values of T c , s measured using the earlier method (turbidity) compared with the micro-DSC values is attributed to the process for recording T c , s .In the first method, T c , s were detected at the inflected point of the graph, whereas, in the second method T c , s were detected at the onset of the graph, as shown in Fig. 13 [3,6].Finally, Fig. 14 represents the overall process of contact angle solution and the lower critical solution temperature at different pH solutions for the ampholyte terpolymers for 4a (5), 4b (10), and 4c (15).

Salting of Ampholyte Terpolymers
The lower critical solution temperatures or the phase separation temperatures (LCST, T c ) and the corresponding cloud points (C p ) of the ampholyte terpolymers were tested in three salt solutions: sodium sulfate (Na 2 SO 4 ), sodium chloride (NaCl), and sodium thiocyanate (NaSCN); these have been chosen to achieve both of kosmotropic and chaotropic anions.The salt concentrations in deionized water were varied from 0.1 to 0.5 wt.%.Turbidity tests by UV-vis spectrophotometer were performed for all samples 4a (5), 4b (10), and 4c (15) to measure the change in transmittances with temperatures, as shown in Fig. 15, with the data summarized in Table 3.
Figure 15A, shows the transmittance vs. temperature for ampholyte terpolymers samples 4a (5), 4b (10), and 4c (15) 15) at the highest concentration (0.5 wt.%).These results were attributed to the effect of sulfate groups in breaking the hydrogen bonds that is formed from the amide groups of NIPAAm and the tertiary amine of DIVA.Furthermore, the cationic sodium ions affect the carboxylic groups of AA and increase the hydrophilicity of the terpolymer solution in sample 4a (5), and vice versa in sample 4c (15).
A weak chaotropic salt solution like sodium chloride has significantly influenced the T c , s and C p , s to be spiky more than the lower critical solution temperature of PNI-PAAm.The ionization of AA by sodium ions increased  than the limit of the measurement.This was attributed to the higher building of the hydrogen bonds that increased the hydrophilicity of the terpolymers and further delay the phase separation process of the polymer solution to much higher temperatures, as shown in Fig. 15C and Table 3.A summary of the relationship between the concentrations of salts and the LCST is presented in Fig. 15D.

Conclusions
This work focused on the fabrication of new thermoresponsive ampholyte terpolymers by the terpolymerization of NIPAAm, AA (anionic monomer), and a new monomer synthesized from vanillin DIVA (cationic monomer) via free radical polymerization.The chemical content of each monomer was determined from the 1 H NMR of terpolymers.The terpolymers demonstrated a decrease in the number average molecular weight, dispersity, glass transition temperature, and degree of crystallinity with increasing the DIVA molar concentration.Contact angle measurements revealed the influence of the amphoteric properties of anionic and cationic groups, and the dominance of the hydrophilicity properties of the terpolymers solutions at the higher and lower pH solutions.The phase transition temperatures of the terpolymers solutions were measured by the turbidity and micro-DSC methods; fluctuations in the LCST affected by anionic and cationic groups in the pH solutions were observed.Additionally, the terpolymers with the highest concentration of DIVA exhibited the lowest LCST at pH 6.9, and the highest one at pH 1.35.The terpolymers were tested at salt concentrations of kosmotropic and chaotropic Hofmeister anions; the highest LCST was observed for the highest DIVA concentration (0.3 wt.%) in sodium thiocyanate solution (chaotropic anion).The lowest LCST was observed for the highest DIVA in the highest sodium sulfate solution (kosmotropic anion).The study will be applied in biological separation and drug delivery as well.

Fig. 2
shows the 1C, CH 2 N at δ = 45.56 ppm 2 C, -(CHN)2 at δ = 49.86 ppm, indicating the successful formation of the tertiary amine group.Moreover, the carbon of the aldehyde group was observed at δ = 191.57ppm.The final step in the preparation of the new monomer for the production of 2-((diisopropylamino) methyl)-4-formyl-6-methoxyphenyl acrylate (DIVA) (1b) was achieved by the reaction of compound (1a) with acryloyl chloride in an alkaline solution such as triethylamine; the compound was distinguished by the vinyl group that impressed in its chemical analysis.

Fig. 10 2
Fig. 10 SEM images for the surface morphology of solid terpolymers at 700 × magnification . The T c , s and C p , s showed lower values than those recorded at pH 1.35; they were 37.3, 40.8, and 46.5 °C for T c , s and 38, 41.4, and 47 °C for the C p , s for all terpolymers 4a resulted in 35.3, 34.6, and 33.7 °C for T c , s and 35.8, 35.6, and 34.4 °C for the C p , s respectively.At pH 6.9, Fig. 12D, the terpolymers solutions did not exhibit any ionization and the zwitterion effect was negligible.T c , s values appeared very close to the homo-PNIPAAm at 33.8, 31.7, and 30.6 °C, while the cloud points were 34.2, 32.3, and 31 °C for samples 4a (5), 4b (10), and 4c (15), respectively.

Fig. 11
Fig.11The contact angles with pH changes of terpolymers solutions in strong kosmotropic anions using sodium sulfate in different concentrations.The measurements demonstrate a decrease in transition temperature T c , s and cloud point C p , s values by increasing the molar concentration of DIVA in the terpolymer chain.From Table 3, the highest values for T c , s and C p , s were recorded for terpolymer 4a (5) at the lowest concentration of Na 2 SO 4 (0.1 wt.%) to be 34 and 34.5 °C, respectively.However, the lowest values for T c , s and C p , s were measured to be 20 and 21 °C, respectively, for 4c (