The Influence of pH/Salt Concentrations on Tuning Lower Critical Solution Temperature of Poly(NIPAAm-co-DMAA-co-DTBAVA) Multi-Environmentally Terpolymer

New series of thermo-pH multi-responsive terpolymers were fabricated. Cationic acrylate monomer was synthesized and named by [2-((ditert-butylamino)methyl)-4-formyl-6-methoxyphenyl acrylate] and abbreviated as (DTBAVA). 1H NMR and 13C NMR investigated the new compounds and FT IR. The new terpolymers were fabricated by the free radical polymerization of N-isopropylacrylamide NIPAAm, 10 mol% N, N-dimethylacrylamide and 5, 10, and 20 mol% DTBAVA. The investigation process involved the chemical method such as 1H NMR and FT IR; physical methods for the solid terpolymers as glass temperature by DSC, polymer degradation by TGA, and polymer crystallinity via XRD. GPC was performed for molecular weights and dispersity; contact angles for identifying the hydrophilic or hydrophobic terpolymers solutions. The lower critical solution temperatures Tc,s, and cloud points Cp,s of terpolymers were recorded considering the impact of pH solutions and the concentrations of different sodium salts of (SO4−2, Cl−1, and SCN−1) in the Hofmeister series; turbidity measurements were used via UV/vis spectroscopy and micro-DSC. In a new optimization study, we will use these terpolymers in the post-polymerization with biomolecules such as chitosan, protein, and amino acids via Schiff base.


Introduction
To define the environmental polymers, it is a material that can change their behavior according to the surrounding environment. In the last few decades, scientists have revealed several kinds of environmental materials; they are defined with different meanings, such as responsive, intelligent, smart, and stimuli-responsive [1][2][3][4][5][6]. This material has been rapidly developed according to modern technology and its applications [7][8][9]. The sensitivity of polymers to temperature is known as thermo-responsive polymeric material [10][11][12]. According to the phase separation temperature of the polymer solution, there are two significant kinds; first is the most popular and has exhibited separation (two phases) after heating over its transition temperature, and is known as lower critical solution temperature or (LCST) [13][14][15]. On the other hand, the separation process occurs before its separation temperature, one phase after heating, while two phases before heating and is defined as upper critical solution temperature (UCST) [16,17]. The main factor in both behaviors is the hydrogen bonding interaction between polymer chains and water molecules. Increasing the hydrogen bonding interaction raises the separation temperature to higher values and vice versa [13]. Massive studies have focused on preparing LCST copolymers using N-ispropylacrylamide (NIPAAm) [14][15][16][17][18][19][20][21][22][23]. The homo-polymerization of NIPAAm (PNIPAAm) shows the LCST at 32 °C [13,15]; moreover, the copolymerization with different monomers with hydrophilic or hydrophobic groups will change LCST significantly to higher or lower value [24][25][26][27]. Other significant environmental polymers are pH-sensitive; they can change their behavior in response to the pH solution [28,29]. They are polyelectrolytes with acidic or basic functional groups that can ionize in acidic or basic solutions [30,31]; Carboxylic functional polymers such as poly(acrylic acid) PAA is a significant example of anionic polyelectrolyte [32]. Introducing of tertiary amine group into the monomer molecule is responsible for the formation of cationic polyelectrolyte, e.g., poly((2-dimethylamino) ethyl methacrylate) PDMAEMA [33]. The pH-responsive polymers have been widely used in many applications, essentially in bioseparation and drug delivery [34,35]. Both thermo-responsive and pH-responsive monomers have been copolymerized to prepare dual-responsive thermo-pH responsive polymeric materials [36,37]. In special interests, several scientific kinds of research have been studying the copolymerization of NIPAAm with cationic or anionic monomer to optimize the general characterization of polymeric material with the advantage to the lower critical solution temperature polymer solutions [1,2,19,20]. Several research articles recently prepared vanillin-based monomers and its derivative due to their natural resource and nontoxic properties [38][39][40][41][42][43]. N, N-dimethylacrylamide (DMAA) is a hydrophilic monomer that has been copolymerized with N-isoprpylacrlamide to enhance the hydrophilic efficiency of copolymer that is reflected in the change of LCST to a higher value [9,42,44,45]. The Hofmeister series, also known as the lyotropic series, was unveiled by Franz Hofmeister [46]. They have discussed the influence of the anions and cations of salts on the solubility or coagulation of proteins [47]. Anions are the most familiar used than cations; they have two major types according to their arrangement in the Hofmeister series. Chaotropes anions are singly charged and have low charge density, e.g., Cl − , NO 3 − , SCN − ; they weakly interact with water molecules and order-making exhibiting the formation of hydrogen bonds and the ability of solubility. On the other hand, kosmotropes anions are small with highly charged density, e.g., CO 3 2− , SO 4 2− , S 2 O 3 2− ; they strongly interact with water molecules than the self-interaction of water, forming breaking in the hydrogen bonds order-breaking [48][49][50]. Recently, several studies focused on Hofmeister salts' effect on the LCST and the cloud point of PNIPAAm solutions and its copolymers [51,52]. All studies unveiled the lower critical solution temperature lowered by the addition of kosmotropes; however, the effect has been weaker by using chaotropic [53,54]. This work aims to prepare a series of thermo-pH-salt multi-responsive terpolymers by the copolymerization of NIPAAm, DMAA, and new tertiary pH monomer DTBAVA to optimize LCST of polymer solution as well as the formation of functional terpolymer. Next, this work will get many applications for the bioseparation of biological molecules by a one-pot reaction via Schiff's base post-polymerization.

Instrumentation
Vertex 70 Fourier transform infrared instrument FT-IR. Bruker AV 500 spectrometer at 500 MHz and 125 MHz was used for recording 1 H and 13 C NMR, respectively, at 25 °C; samples were dissolved in CDCl 3 or DMSO-d 6 . The dry sample was milled with KBr and then pressed into pellets for the measurement process.
Differential Scanning Calorimeter (DSC) Perkin Elmer Pyris 1; this technique is used to record the glass transition temperature (T g ) of solid polymers at a heat and cool rate of 5 ℃/min. It was recorded at the onset value of the thermogram.
Thermogravimetric analysis (TGA); was used to investigate the thermal stability of the polymer sample related to its chemical decomposition by heating from 25 to 700 °C at and heating rate of 5 ℃/min. X-ray diffraction-(Bruker AXS D8 Advance); was used for recording the degree of crystallinity. The detector is Vantec-1; high precision microprocessor-controlled, Goebel mirror; two circle goniometer with independent stepper motors and optical encoders, reproducibility ± 0.0001°, smallest angular step 0.0001°, high-temperature MRI stage (RT-1400C), sample spinner, video camera, thin-film Reflectometry, sample plate fixed with 9-sample holders. The instrument was run as 2.2 kW Cu and Co, Running 40 kV, 40 mA. The stability of power is better than 0.01%.
Size exclusion chromatography (SEC) has been used for the determination of molecular weights (M n , M w ) and dispersity (Ð) in THF as eluent contaminated with 0.1 vol% triethylamine and a flow rate of 0.75 ml/min at 30 °C. The sample was dissolved at 15 mg/ml; an automatic injection ran it. PSS-SDV Columns were used with 5 µm gel. The molecular weight M w of polystyrene (PS) was used as standard.
To measure the contact angle (ϴ): the sample was converted into a pellet and allowed a 4 μl drop of pH solution over the sample surface using a micropipette. The photographs were taken for each drop using a digital camera. The contact angles were detected from the photographs by using the software ImageJ.

Measurements of the Phase Separation Temperature or Lower Critical Solution Temperature (LCST) (T c ) and Cloud Point for Terpolymer Solution
(1) It was performed by UV-vis spectrometer (Perkin Elmer Lambda 45); it is known by the turbidity method. The process depends on the relation of transmittance and temperature of the polymer solution. The instrument has been built by metal covet stands with the water cycle and fixed using a thermostat and cooling system. The internal heat of the polymer solution was measured by a manual thermostat inside the solution at 2 ℃/min over the range from 5 to 80 ℃. The polymer solution concentration was at 1 wt% in water or pH solution. (2) Another instrument was used for T c measurement of polymer solution; it is micro-DSC III from setaramto. The thermograms of the terpolymer solutions were recorded. The heating rate was at 5 °C/min; the concentration was 50 mg/ml in deionized water. The transition temperature T c was detected at the onset value.

Monomer and Polymer Chemical Evaluations
The new monomer has been synthesized according to Scheme 1. The procedure was implemented in two steps. The cationic monomer from vanillin was first prepared via the reaction of vanillin, formaldehyde, and di-tert-butylamine producing compound II [3-((di-tert-butylamino) methyl)-4-hydroxy-5-methoxy-benzaldehyde] (DTBAV). The evaluation has been done using the 1 H, 13  H of the methyl groups and 2 H methylene -NCH 2 ; they have also been shown in 13C NMR at δ = 30.83, and 43.48 ppm; additionally, they keeping the aldehyde group at δ = 9.76 ppm, and 190.65 ppm for 1 H and 13 C. Through the next step and formation of the final product, compound II was reacted in an alkaline solution such as triethylamine in an inert atmosphere via purging nitrogen entailed the dropping of acid chloride (acryloyl chloride) for compound III (DTBAVA), all in equivalent molar concentrations. At first, the reaction appears exothermic and requires cooling; after that, the reaction proceeds at room temperature. The evaluation process was performed by the same tools discussed lately. Both of the 1 H and 13 C NMR appeared the identical protons, and 13C demonstrated a successful reaction and achieved the chemical structure; the essential protons for the formation of the vinyl group were proofed 3H, at δ = 6.09, 6.39, and 6.63 ppm, and their analog 13C 2C, at 127.16, and 133.24 ppm. Other detected protons and 13 C affirmed the chemical structure, as shown in Figs. 3 and 4. Fortier infrared spectroscopy FT IR has been selected for detecting the functional groups. The vibrations were observed for the essential functional groups. Compound II exhibited ν = 2645-2525, 1745-1733, and 1660-1658 cm −1 for -C-N, C=O carbonyl, and C-OH; however, compound III demonstrated the presence of vinyl group -C=C-group at ν = 1660-1658 cm −1 , as shown in Fig. 6.
Eventually, the work has been accomplished by fabricating thermo-pH environmentally terpolymers via the free radical polymerization of N-isopropylacrylamide and N, N-dimethylacrylamide, and three different molar concentrations of (DTBAVA) (5, 10, and 20 mol%) in solution and initiated by AIBN. One notable was taken in the yield percentage of polymer; they decreased with increasing the molar concentration of DTBAVA in the polymer chain. The investigations of terpolymers have been performed via 1 H NMR and FT IR. Figure 5 illustrates the 1 H NMR of terpolymers in CDCl 3 ; the essential 1H peaks and integrations were evaluated and used in the estimation of the actual molar concentration of each monomer in the chain of the terpolymer, as apparent in Table 1; they are δ = 2.63-3.27 ppm multiple peaks of dimethyl groups of N, N-dimethylacrylamide, at δ = 3.84-4.15 ppm 1H for -CH-isopropyl of N-isopropylacrylamide, the last peak is related to 1H of the aldehyde of DTBAVA at δ = 9.89-10.01. The functional groups have also been tested via FT IR, indicating different vibrations related to -C=O ester at ν = 1705 cm −1 , ν = 1670 cm −1 , -C=O, aldehyde group, ν = 1576 cm −1 , -C=O, the amide of -CONH-NIPAAm, as shown in Fig. 6.

Physical Properties of Solid Polymers
Solid terpolymers of poly(NIPAAm-co-DMAA-co-DTBAVA) with various DTBAVA molar concentrations starting with 5 mol%, 10, and finally 20 mol% have been investigated. These different molar concentrations have defiantly impacted their general characterizations.

The Glass Transition Temperature T g Via Differential Scanning Calorimetry (DSC)
The glass transition temperature T g of polymer is a specific character like the figure print and has been influenced by other characterizations of the polymeric material. Differential scanning calorimetry DSC was used to record the glass temperature T g at a heating rate of 5 °C/min. All terpolymers were quenching from melting to liquid nitrogen temperature. The diffractogram was calibrated with standards; the glass temperature T g was measured at the inflected point. Figure 7 shows the diffractogram curves of terpolymer samples indicating the glass transition temperatures at 141, 135, and 119 °C for VIa, VIb, and VIc, respectively, as mentioned in Table 1. The regular decrease of the T g , s inflected the effect of the molar concentration of DTBAVA in the polymer main chain; the reason is the addition of aromatic monomer, which restricts the flexible rotation of repeating unit in the polymer chain [55].

Thermal Stability Via Thermogravimetric Analysis TGA
Thermogravimetric analysis has performed the thermal degradation of terpolymers at 10 °C/min from 25 to 600 °C. Figure 8A, B illustrates the thermal decompositions and their 1st derivatives of synthetic terpolymers. The significant degradation of terpolymer VIa with 5 mol% of DTBAVA has occurred in three steps the first from 200 to 250 °C for evaporator materials water and ammonia, the principle degradation was detected from 390 to   415 °C, this is due to the decomposition of repeating unit in the polymer chain, eventually full decomposing from 495 to 550 °C for hydrocarbon. For terpolymer VIb 10 mol% DTBAVA has also exhibited three main degradable stages; the first has occurred from 130 to 170 °C, the next one from 365 to 385 °C, refereeing to the principal decomposition of the polymer chain, and finally 520-555 °C. The higher molar concentration of 20 mol% of DTBAVA VIc demonstrated the first degradation from 135 to 180 ℃; however, the principle degradation of polymer chains showed different features; it exhibited two distinct degradations as shown in Fig. 8B, from 325 to 360 , and from 365 to 390 ℃, this might be attributed to the decomposition of the polymer chain in two stages at the lowest temperature for DTBAVA repeating unit, while the highest to NPAAm and DMAA. The complete decompose has been detected from the range like lately of VIb terpolymer.

Degree of Crystallinity Via X-Ray Diffraction XRD
The crystallization process and the degree of crystallinity differentiate among the state of the material is crystalline, semi-crystalline, or amorphous. Figure 9 illustrates the crystallographic analysis of XRD of new synthetic terpolymers VIa-c. Diffraction peaks at 2θ = 25° to 37° and 38° to 47° are attributed to the enhancement of the crystallization. The crystallinity percent has been calculated for each terpolymer via the crystallographic by (area of crystalline peaks/area of all peaks × 100) [56]. They demonstrated crystallinity% of 53.4, 48.3, and 42% for VIa, VIb, and VIc, respectively. The essential feature of the crystallographic is the absence of the sharpest peak indicating the semi-crystalline state. One other note is that the correlation of crystallinity on the molar concentration of DTBAVA demonstrated a reversible process, increasing steric hindrance and restricted rotation sparked by increasing DTBAVA.

Molecular Weight/Dispersity
Gel permeation chromatography measures the molecular weight (weight average molecular weight M w and number average molecular weight M n ) and dispersity M w /M n , Ð for all polymeric samples dissolving in THF and using PS as standard. The chromatogram analysis has shown a good responsibility of the detector with the retention time. It has also demonstrated an essential feature as the formation of one peak for each sample, indicating monomers' disappearance [1,2]. Figure 10 Table 1, the number average molecular weight M n and dispersity Ð for all terpolymers; was observed an opposite relationship of M n and dispersity Ð with a polymer content of DTBAVA monomer in the chain that attributed to the aromaticity steric hindrances caused a reduction in the flexibility of polymer solution [5] (Table 2).

Terpolymers Contact Angles
The effect of incorporating hydrophilic and hydrophobic groups in the terpolymers has been studied by measuring the contact angles, which is the key to recognizing the tendency of polymeric material to hydrophilicity or hydrophobicity [57]. A recent study has been interpreted the difference among hydrophilic, hydrophobic, and superhydrophobic polymeric material via the determination of their contact angles; it has shown that hydrophilic polymers should be at ϴ < 90° and hydrophobic at ϴ ˃ 90°; however, superhydrophobic always measured at ϴ ≥ 145° [57]. The measurements of the contact angles of terpolymers VIa-05, VIb-10, and VIc-20 with 5, 10, and 20 mol% of DTBAVA showed the influence of the contact angles Table 1 Yield %, composition, molecular weight, dispersity, glass temperature, crystalinity, and transition temperature/cloud point of poly(NIPAAm-co-DMAA-co-DTBAVA)  the hydrophilicity decreased by increasing the pH from strong acidic solution to strong alkaline solution. Increasing the molar concentration of DTBAVA in the polymer chain VIb-10; showed the hydrophilic property in strong acid at pH 1.68 and lower contact angle ϴ ~ 82° than VIa-05 attributed to the effect of ionization of tertiary amine group in acidic solution. It is increased regularly in higher pH solutions. The highest hydrophilicity was detected for terpolymer VIc-20 at the strongest acidic solution pH 1.68 and the lowest contact angle at ϴ ~ 80.8°; the reason has been interpreted as the highly charged DTBAVA in the polymer chain reflected the highly hydrophilic character of the polymer solution. These changes in the contact angle measurements proved the influence of DTBAVA via pH solution producing hydrophilic or hydrophobic chains.

The Impact of pH Solutions on the Phase Separation Temperature and the Cloud Points
Two methods were used to identify and measure the lower critical solution temperature and the cloud point. The first method via turbidity test was UV/vis spectroscopy; the relation between transmittance percent to temperature, as shown in Fig. 13 A-D, represents the transition temperature T c at the inflected point and the cloud point C p at the mid transmittance to temperature (50%). The hydrophilic/hydrophobic balance in the terpolymers inflected the presence of hydrophilic groups besides the hydrophobic in NIPAAm, DMAA, Fig. 11 The contact angle photographs of polymer VIa-10 at different pH solutions Fig. 12 The relationship of contact angles (ϴ°) with pH value for terpolymer VIa, VIb, and VIc   [49][50][51][52]. The change of polymer solution from kosmotropes to chaotropic has influenced the transition temperature and the cloud points of terpolymers. The solubility of terpolymers in NaCl with different weight percent 0.1-0.5 wt.% was implemented, and the transmittances vs. temperatures were recorded. In this state, lower spiky was observed in the transition temperature and the cloud points than with Na2SO4 solutions, as shown in Fig. 15B; the T c /C p has fluctuated to record the highest value for 0.1 wt.% NaCl (VIa-05) at 35/35.7 °C, and the lowest at 17/17.9 °C for 0.5 wt.% NaCl (VIc-20); the interpretation of results has been discussed lately. Conversely, the solubility of terpolymers in sodium thiocyanate solutions has been seen in Fig. 15C. The strongest spiky for the T c , s /C p , s was detected at 52/53 °C for 0.5 wt.% NaSCN (VIc-20). The much higher hydrophilic groups based on the interaction of the thiocyanates anions with the amide group (NIPAAm) as well as the aldehyde group (DTBAVA), producing structure-making and increased the solubility of terpolymers and therefore slowing the phase separation process to higher temperature [55,57]. All data has been summarized in Table 2. Figure 15D illustrated the relationship between the LCST (T c , s ) and weight percent concentrations of terpolymer solutions with Na 2 SO 4 , NaCl, and NaSCN showing the highest T c , s /C p , s terpolymer VIc-20 dissolved in 0.5 wt.% NaSCN, while the lowest one for terpolymer VIc-20 dissolved in 0.5 wt.% Na 2 SO 4 .

Conclusion
A new monomer DTBAVA was prepared from vanillin in two facile methods; the chemical structure was confirmed by suitable instruments that achieved good results. Three thermo-pH responsive terpolymers were designed from NIPAAm, DMAAm, and DTBAVA. The terpolymerization process was made by free radical polymerization using different molar concentrations of DTBAVA 5, 10, and 20 mol%. The general physical characterizations were implemented; the glass transition temperature T g , molecular weight, and dispersity demonstrated their reversible relation with the content of DTBAVA in the polymer chain. Furthermore, the crystallinity by XRD has also represented a lower crystallinity percent with increasing DTBAVA. The degradation steps have illustrated two steps with VIa-05, VIb-10, and three steps with VIc-20. The lowest contact angle and the highest hydrophilic were also seen for VIc-20 at pH 1.68. The phase separation temperature of terpolymers was tested in various pH solutions and different Hofmeister salt via UV/ vis spectroscopy. They have been demonstrated the highest T c in pH 1.68 for VIc-20; however, the lowest value has also seen at pH 10.4 for the same terpolymer. The salt effect was tested using three solutions of salts from kosmotropic as Na 2 SO 4 , chaotropic NaCl, and NaSCN. The study illustrated the structure-breaking of Na 2 SO 4 terpolymer solution by recording the lowest transition temperature with the highest concentration of salt. Otherwise, the lowest T c value has been detected for terpolymer in chaotropic NaSCN solution with the highest salt concentration and the highest DTBAVA molar concentration. Future research will be interested in applying these terpolymers in the separation process of biological molecules.