Preparation and Characterization of Environmental Functional Poly(Styrene-<i>Co</i>-2-[(Diethylamino)Methyl]- 4-Formyl-6-Methoxy-Phenyl Acrylate) Copolymers for Amino Acid Post Polymerization

Vanillin was used as renewable resource for preparing new monomer in two stops. The monomer has tertiary amine group which facilitates the pH change and functional aldehyde group that encourages the formation of Schiff base. It was abbreviated by DEAMVA and evaluated using chemical analysis e.g. 1H NMR, 13C NMR and FT IR all data were in logic case. Copolymerization of Styrene with 5 and 15 mol% of DEAMVA has been done by free radical polymerization and AIBN as initiator. The copolymers have been chemically and physically characterized e.g. 1H NMR, FT IR, GPC, and DSC. Post polymerization of poly (styrene-Co-DEAMVA) with 15 mol% (III b) was prepared for immobilization of tryptophan and investigated by the same methods used lately. Moreover, the sensitivity of the posted copolymer to pH has also studied by UV-vis. Spectroscopy. Scanning electron microscopy (SEM) was used to study the morphological feature of polymer surface after immobilization of tryptophan.


Introduction
Environmental pH-responsive polymers are polyelectrolytes that have weak panded state has been explained by changes in the osmotic pressure exerted by mobile counter ions neutralizing the network charges [2]. Several applications for pH-responsive polymers and hydrogel are especially in the field of biomedicine e.g. Drug delivery systems and Gene carriers [5]- [11].
Several scientists were looking for new alternatives of styrene monomer due to its disadvantages such as, hazardous air pollutants and emitting during metering mixing process and curing; additionally the unreacted styrene continues to be released from composites during life cycle [12]. Renewable resources like (cellulose, starch, natural oil… etc.) have been used as alternative to produce bio-based monomers [13]. The lignin produced from Vanillin becoming relatively easily accessible; still there are only a handful of reports on attempts to utilize vanillin as monomers for bio-based polymer synthesis [14] [15]. Several chemical modifications on vanillin have occurred due to the presence of both aldehyde and hydroxyl active groups [2] [16]. The dimerization and the Polymerization of vanillin through electrochemical reductive with horseradish peroxidase have recently been discussed [17].
Schiff's base has also been played a vital role in the coordination chemistry and was found to be stable under oxidative and reductive conditions [18]. Vanillin was used to prepare Schiff bases [19]. The biological activity of Schiff's base polymers have been widely reported by several authors [20] [21] [22] [23] [24]. The most interested articles used Schiff base as a stimuli-responsive linker in the polymer chain which has been published by Yuan et al. [25]. The imine linkage is very sensitive to pH value which is responsible on the responsive feature of the polymer molecule [26] [27]. The formation of complexes with the imine can also be used to demonstrate the pH responsiveness of the linker [28]- [33]. Several publications have reported ''click chemistry'' as linkers for many bio-based molecules [34] [35] [36]. Here, we synthetized new functional polystyrene polymers by copolymerization with pH-responsive monomer from vanillin as renewable resource. The aldehyde group in the copolymer chain was used for grafting biomolecule by Schiff base click reaction. In future our work will focus on the applications of these kinds of polymers and their gel in the bio-separation for many kinds of biological macromolecule.
Other chemicals were used as received.

Instruments
Bruker AV 500 spectrometer was used to record 1 H and 13 C NMR spectra in DMSO d 6

Synthesis of Monomer, Copolymers and Grafted Copolymers
Scheme 1 describes the chemical procedure for synthetizing monomer, copolymers and grafted copolymers. Monomer (II) or 2-[(diethylamino)methyl]-4-formyl-6-methoxyphenyl acrylate and abbreviated by (DEAMVA), it was fabricated in two steps reaction. The first step Is the formation of (3-[(diethylamino)methyl)-4-hydroxy-5-methoxy-benzaldehyde) and has been done by the reaction of vanillin with diethylamine and formaldehyde according to Mannich reaction mechanism. In this reaction we did not use any catalysis especially acid catalysis which famous to use in Mannich reaction. The second Step Is the formation of 2-[(diethylamino)methyl]-4-formyl-6-methoxyphenyl acrylate (DEMAVA). This was achieved by reaction of compound (I) with acryloyl chloride in the presence TEA to form (II). They have chemically evaluated by 1 H NMR and 13 C and FT IR in Figure 1, Figure 2 and Figure 5. All data was in logic state and proved the presence of active aldehyde group at 9.97 ppm and 196 ppm.
To improve the functionality of styrene free radical polymerization with 5 and 20 mol% of DEAMVA has been done in the presence of AIBN as initiator as described in Scheme 1. The chemical structure of each polymer was evaluated    Functionality with the aldehyde group in the polymer main chain was an interested to make grafting with any amino compound to produce Schiff's base which is familiar by click reaction Scheme 1. The grafting process has been done at room temperature in THF at different pH (pH4, pH7, pH9, pH10, pH12).
Grafted copolymers were elucidated by 1 H NMR and FT IR as shown in Figure 4 and Figure 5. The 1 H NMR showed the disappearance of aldehyde signal at 9.74 ppm and formation of imino (HC=N) signal at about 8.5 ppm as shown Figure   4. Figure 5 showed FT IR spectra and proved the presence of (C=N stretch) imine at about 1563 cm −1 .

Molecular Weight
Size exclusion chromatography was used for determination of molecular weight (Mw), number average molecular weight (Mn) and polydispersity (Đ) of polymers using Polystyrene (PS) as standard in CHCl 3 . Table 1 has summarized all recorded data for all copolymers. Figure 6 shows the relation between molecular weight and log M as recoded by GPC and demonstrates one peak, proofing the disappearance of low molecular weight like monomers or impurities.

Glass Transition Temperature
The glass transition temperature (T g ) is very important parameters for solid material. It has been recorded by Differential Scanning Calorimeter of dried samples at heating rate 5˚C/min as described in experimental part. The T g was taken as the midpoint inflection. The (T g ' s) values have been tabulated in Table 1 for copolymers and grafted copolymers. Figure 9 showed a single T g for each Open Journal of Polymer Chemistry  sample, which indicating the formation of random copolymers [36]. The homo-polystyrene (PS) showed T g at 100˚C [37]. Incorporation of DEAMVA moieties in the copolymers chain with hydrophobic and hydrophilic groups demonstrated in aromatic and tertiary amine respectively resulted in increased T g , which might be attributed to decrease in the spacing and hence greater interaction between polymer chains leading to lesser flexibility and T g of the polymer increased [36]. Introducing grafting molecule in copolymer (IV) main chain has directly influenced on raising the T g due to the steric hindrance of aromatic molecule.   . DSC shows the T g of copolymers and grafted copolymer. Figure 10 is the Scanning Electron Microscopy (SEM) image obtained at a magnification of 1000× for grafted copolymer (IV). After grafting the porosity of the grafted polymer surface increases the whole surface looks like waxy with cross-linking referring to the imine linkage and grafting of tryptophan.

Conclusion
Here we synthetized new functional polystyrene copolymers. New pH responsive monomer with tertiary amine and aldehyde functional groups were prepared in two steps. Free radical polymerization of styrene with two different mole ratios of DEAMVA was used for synthetizing copolymers. The presence of aldehyde group facilitated the formation of Schiff base with primary amine.
Tryptophan was used as biological molecule for immobilization. UV-vis. Spectroscopy was used to detect the immobilization and formation of Schiff base at different pH. We observed the highest absorption at pH12 and lowest at pH4. In Open Journal of Polymer Chemistry Figure 10. SEM of copolymer IV (grafting) copolymer with tryptophan 1000× magnification.
future, we are looking for using this kind of polymer and its gel in the separation of biomolecules.