The synthesis, characterization, removal of toxic metal ions and in vitro biological applications of a sulfanilamide–salicylic acid–formaldehyde terpolymer

Abstract

A new terpolymer was synthesized by the polymerization of sulfanilamide, salicylic acid and formaldehyde (SASF) in the presence of glacial acetic acid as a catalyst. The characterization of the synthesized SASF terpolymer was conducted using various techniques such as elemental analysis, gel permeation chromatography (GPC), Fourier transform infrared (FTIR), ultraviolet-visible (UV-Vis), ¹H nuclear magnetic resonance (NMR), ¹³C NMR, thermogravimetric analysis (TGA), X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) measurements. The synthesized SASF terpolymer was used for the removal of Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions from aqueous solution. The effect of factors affecting the metal ion adsorption on the SASF terpolymer was studied as a function of the electrolytes in different concentrations, and the influence of pH in different ranges was studied using the batch equilibrium technique. The metal ion removal efficiency increased with an increase in the pH and concentration of the electrolytes. The maximum removal percentage was achieved at pH 6–7. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) was employed for the determination of all metal ions. The in vitro antibacterial and anticancer activities of the SASF terpolymer were also investigated. The SASF terpolymer exhibited an effective antibacterial activity against Gram-positive methicillin-resistant Staphylococcus aureus, Bacillus subtilis and Gram-negative Salmonella typhi, Escherichia coli bacterial strains. The cytotoxicity studies indicated that the SASF terpolymer possesses much potential against the HeLa (mammalian cancer) cell line.

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1. Introduction

Environment pollution by heavy metals is a major threat to human and aquatic ecosystems. Toxic heavy metals and their ions are not only potential hazards to human health but also to other life forms.^1–3 From an eco-toxicological point of view, considerably dangerous metals are lead, cadmium, mercury, copper, nickel, arsenic, cobalt, zinc and vanadium, which are known to be toxic to living beings at any concentration. The most common sources of toxic heavy metals in water and/or waste water are the effluents from textile, leather, electroplating, pigment, dye, refining process, petrol refining, metallurgical and painting industry sectors containing large amounts of heavy metal ions.⁴ The removal of toxic heavy metals from these effluents is an important challenge to avoid water and soil pollution. Several approaches have been developed for the removal of metal ions such as precipitation, ion-exchange, electrowinning, electro-coagulation, reverse osmosis and solid–liquid extraction. Most of these methods suffer from some drawbacks such as high capital operational costs and the problem of disposal of residue metal sludge. However, metal ion removal by chelating ion-exchange terpolymers using the batch equilibrium method is a powerful eco-friendly technique for the separation and recovery of toxic heavy metal ions from industrial waste contaminated water.^5–8 Chelating ion-exchange terpolymers with substituents such as –OH, –COOH and –NH₂ are important groups of metal ion chelating agents.⁹

The eco-friendly metal ion removal and biological properties of terpolymers have further provoked research into the synthesis of new terpolymers. A survey of the literature reveals that terpolymers have attracted significant research by synthetic chemists due to their varied applications. Terpolymers find quite useful applications in the fields of fibers and fabrics, ion-exchange, semiconductors, biological applications, fire proofing agents, binders and molding materials.^10–14 Terpolymers involving 4-hydroxy acetophenone and biuret with formaldehyde in the presence of an acid catalyst proved to be an excellent ion exchanger.¹⁵ Similarly, the solution polymerization of three monomers involving 8-hydroxyquinoline, salicylic acid and formaldehyde was carried out to obtain a terpolymer. From the thermogravimetric analysis (TGA) results, the terpolymer was found to be thermally stable and the order of reaction for the thermal decomposition was determined.¹⁶ An ion-exchange and antimicrobial study of the poly[(2-hydroxy-4-methoxy benzophenone) ethylene] terpolymer and its polychelates with lanthanides(III) was reported.¹⁷ A series of acrylic terpolymers with arginylglycylaspartic acid (RGD) peptides was used for biomedical applications.¹³ Chemically crosslinked metal-complexed chitosans for the comparative adsorptions of Cu(II), Zn(II), Ni(II) and Pb(II) ions in aqueous medium were studied.¹⁸ The removal of cations using an ion-binding terpolymer involving 2-amino-6-nitro-benzothiazole and thiosemicarbazide with formaldehyde by a batch equilibrium technique and the effect of pH, contact time and electrolytes and the reusability of the terpolymer was also studied.¹¹ The anti-bacterial activities of a thiosemicarbazide–formaldehyde terpolymer and its polymer–metal complexes were reported due to the higher inhibition characteristics of the terpolymers against the chosen microbes.¹⁹ The protein antifouling properties of a poly(methyl methacrylate–acrylic acid–vinyl pyrrolidone) terpolymer modified with a polyethersulfone hollow fiber membrane with pH sensitivity were studied.²⁰ The kinetics of the thermal decomposition and antimicrobial screening of a terpolymer prepared by polycondensation of anthranilic acid and urea with formaldehyde have also been reported.²¹

In this article, we describe a new terpolymer synthesized from sulfanilamide, salicylic acid and formaldehyde (SASF). The synthesized SASF terpolymer has been characterized by basic spectral analysis and confirmed. The sorption properties of the SASF terpolymer towards selected toxic divalent metal ions were investigated in detail. The antibacterial activity of the terpolymer was studied against Gram-positive and Gram-negative bacterial strains. The cytotoxicity of the terpolymer was investigated against the HeLa (mammalian cancer) cell line.

2. Experimental

2.1. Chemicals and regents

Sulfanilamide, salicylic acid and formaldehyde (37%) were procured from Merck, India. Analytical reagent (AR) grade metal nitrates of the chosen metals were purchased from Merck, India. Multi-elemental standard solutions for inductively coupled plasma-optical emission spectroscopy (ICP-OES) were obtained from PerkinElmer (USA). All working metals and standard solutions were prepared immediately before use by stepwise dilutions of 1000 mg L⁻¹. All other chemicals and solvents were of AR grade and used as received. Milli-Q water was used for all the experiments.

2.2. Terpolymerization

The terpolymer was synthesized by the condensation polymerization of sulfanilamide (0.1 M), salicylic acid (0.1 M) and formaldehyde (0.2 M) using 2 M glacial acetic acid as a catalyst and refluxing this mixture with occasional shaking at 140 ± 2 °C in an oil bath for 6 h. The resultant product was cooled, poured into crushed ice with constant stirring and left overnight. The obtained dark orange colored product was separated and washed with warm water, ethanol and ether to remove the starting materials and acid monomers. Then the product was air-dried and further purified by dissolution in 10% aqueous sodium hydroxide solution. Finally it was filtered and regenerated by the gradual dropwise addition of ice-cold 1 : 1 (v/v) concentrated hydrochloric acid/distilled water with constant and rapid stirring to avoid the formation of lumps. This regeneration technique was repeated twice, then the product was filtered off and cured at 75 °C for 24 h in an air oven.^12,14 The yield of the terpolymer was found to be 82%. The dried terpolymer was finely ground and sieved to obtain uniform particles of 100 mesh size and stored in a polyethylene container. The sieved terpolymer was used for further characterization. The reaction sequence of the synthesis of the SASF terpolymer is shown in Scheme 1.

Scheme 1 Synthesis sequence of the SASF terpolymer.

2.3. Characterization of the SASF terpolymer

The percentage of elements such as C, H, N and S present in the SASF terpolymer was determined on an Elementar instrument (Model vario EL III, Germany). The number-average (M_n), weight-average molecular weight (M_w) and polydispersion index (PDI = M_w/M_n, M_w/M_z) of the terpolymer were determined using a gel permeation chromatography (GPC) system equipped with a Waters 2690D separations module. Waters millennium module software was used to calculate the molecular weight on the basis of a universal calibration curve generated by a polystyrene standard.

The Fourier transform infrared (FTIR) spectrum of the terpolymer was scanned using the KBr pellets technique on a FTIR spectrophotometer (Bruker Tensor 27) to identify the linkages and functional groups. The electronic absorption spectrum of the terpolymer was recorded on an ultraviolet-visible (UV-Vis) double beam spectrophotometer (Shimadzu 1601 PC) in pure DMSO in the region of 200–800 nm at a scanning rate of 100 nm min⁻¹ and a chart speed of 5 cm min⁻¹. The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of the terpolymer were recorded on a Bruker 400 MHz NMR spectrometer using dimethyl sulfoxide (DMSO)-d₆ as the solvent and tetramethylsilane (TMS) was used as the reference material. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of the terpolymer were carried out on a thermal analyzer (SDT Q600 V8.3 Build 101Universal V4.7A TA Instruments systems) at a heating rate of 10 °C min⁻¹ in a static nitrogen atmosphere in the range of 30–800 °C. X-ray diffraction (XRD) analysis (Rigaku Rotoflux) was performed to investigate the phase structure of the terpolymer. The surface morphology of the terpolymer was analysed by scanning electron microscopy (SEM, Zeiss18 Evaluation) and the chemical composition was characterized by energy dispersive X-ray spectroscopy (EDX, Oxford X-Act).

2.4. ICP-OES measurements

Determination of all metal ions was performed with ICP-OES using a Perkin Elmer Optima™ 7000 DV dual view series sequential spectrometer (Shelton, CT, USA) equipped with WinLab™ 32 for ICP Version 4.0 software. In order to avoid any carry-over effects, the ultrasonic nebulizer was washed out with 1% (v/v) HNO₃ for 60 s between each sample run. Argon gas (99.99%) was used as the ICP torch gas and nitrogen gas was used as the optical purge gas. A charge-coupled device (CCD)-array detector was used to collect both the analyte spectra and the nearby background spectra, which provided improved precision and analytical speed. The instrumental operating conditions and parameters are given in Table 1. The following spectral lines (wavelength in nm) were monitored for various analyte quantification: Ni-231.604, Cu-327.393, Pb-220.353, Cd-228.802, Hg-253.652 and Zn-206.200. All emission lines monitored are ionic (II). These results were obtained by the high sensitive detection of ultra-trace and trace analysis.^22,23

Table 1 ICP-OES: operational conditions and instrumental parameters

RF power	1450 watt
Plasma gas flow rate	15 L min^−1
Auxiliary gas flow rate	0.2 L min^−1
Nebulizer carrier gas flow rate	0.75 (pneumatic) and 0.70 (ultrasonic)
Sample pump rate	1.5 mL min^−1
Plasma view mode	Axial and radial
Resolution	High
Background correction	2 points per peak
Integration time (min–max)	2.5–5.0 s
Signal processing	Peak area (3–7 points per peak)
Replicates	3

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2.5. Batch sorption experiments

The metal ion binding capacity of the SASF terpolymer was studied as a function of various electrolytes in different concentrations and pH values by a batch equilibrium technique. 50 mg of the synthesized SASF terpolymer was suspended in 30 mL of electrolyte solutions such as NaCl, Na₂SO₄ and NaNO₃ at different concentrations viz. 0.1, 0.5, 1.0, 1.5 and 2.0 M. To each suspension, a 400 mg L⁻¹ (10 mL) mixture of Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions was added. Then, the mixture was shaken homogeneously with shaker rotating at a speed of 200 rpm for 12 h to facilitate the adsorption of metal ions into the SASF terpolymer. After the specified time, the SASF terpolymer was separated from the solution by filtration through the Whatman filter paper no. 42. The initial and final concentrations of Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions were determined by ICP-OES. A blank was analyzed to ensure that no metal ions were carried over from the previous sample. High purity deionized water was chosen as the blank solution. The blank values were subtracted from the values determined for the different metal ions to give the exact adsorbed metal ion concentrations.^24,25

The same procedure was repeated for the pH studies. The effect of pH on the sorption of metal ions by the terpolymer was studied in a range of 2–7. Before mixing the metal ions, the pH of the suspension was adjusted to the required value by adding a minimum amount of HCl (0.5 M) and/or NaOH. The amount of metal ions taken up by the SASF terpolymer during the series of batch investigations was determined using the following equation:

Removal (%) = [(C_o − C_f)/C_o] × 100

where: C_o and C_f are the initial and equilibrium concentrations (ppm) of the metal ions in solution, respectively.

2.6. In vitro biological studies of the SASF terpolymer

2.6.1. Antimicrobial properties of the terpolymer. The antimicrobial properties of the SASF terpolymer were investigated against Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis and Gram-negative Salmonella typhi, Escherichia coli bacterial strains by the agar disc diffusion method according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS, 1997) with slight modifications.²⁶ The inoculums of the Gram positive and Gram negative bacterial strains were prepared from fresh overnight broth cultures (0.6% yeast extract) that were incubated at 37 ± 1 °C with constant stirring and the resulting broth cultures were used for the diffusion test.

The agar disc diffusion test was performed by pouring agar into Petri dishes to form 4 mm thick layers and adding 2 mL dense inoculums of the test organisms of the strains in order to obtain semi confluent growth. Sterile circular Whatman filter paper discs of size 6 mm were placed on the agar and loaded with varying concentrations of the SASF terpolymer (such as 25, 50, 100, 250, 500 and 1000 μg) along with the standard reference drug (ciprofloxacin). Then, the Petri plates were incubated at 37 ± 1 °C for 24 h and were placed at equal distance. The experiment was repeated thrice. After the test, the antibacterial activity (bacteria-free area) was measured as the zone of inhibition (mm) around the disc, which was indicative of the ability of the antimicrobial test sample to kill the bacteria.

2.7. Anticancer study

2.7.1. Cell line and culture medium. A HeLa cell line was obtained from the National Centre for Cell Sciences Repository (NCCS, Pune, India). The minimum essential medium (MEM) was supplemented with 100 units of penicillin, streptomycin and 10% (v/v) of foetal bovine serum (FBS) in a humidified atmosphere of 5% CO₂ at 37 °C until confluent. The cells were seeded at 1 × 10⁴ cells per well in 96 well culture plates for 24 h.

2.7.2. MTT assay. The SASF terpolymer was dissolved in DMSO, diluted in the culture medium and used to treat HeLa cells with a concentration range of 25 to 100 μg mL⁻¹ for a period of 24 h. DMSO was used as the solvent control. A miniaturized viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was performed as per the International Organization for Standardization (ISO) 10993:5 method. The control and test sample in triplicates were added to the cell. After incubation at 37 ± 1 °C for 24 h, the monolayer was examined microscopically for the response around the test sample. As per Table 2, the reactivity was graded as 0, 1, 2, 3 and 4 based on the zone of lysis, vacuolization, detachment and disintegration of the membrane.

Table 2 Cytotoxic reactivity grade and their description

Grade	Activity	Description of reactivity zone
0	None	No detectable zone around or under specimen
1	Slight	Some malformed or degenerated cell under specimen
2	Mild	Zone limited to area under specimen
3	Moderate	Zone extending specimen size up to 1 cm
4	Severe	Zone extending farther than 1 cm beyond specimen

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3. Results and discussion

3.1. Analytical and elemental data

Elemental analysis was carried out on the synthesized SASF terpolymer, which was dark orange in color. The calculated and determined C, H, N and S elements values are given in Table 3. The yield of the SASF terpolymer was 82%. Based on the results, the empirical formula and the empirical weight of the single repeating unit of the terpolymer were found to be C₁₅H₁₄N₂O₅S and 334.35, respectively, which is in good agreement with the calculated values.

Table 3 Elemental data of the SASF terpolymer

Empirical formula of repeating unit	Formula mass	Elemental analysis (%) found (calc.)				Color	Yield (%)
		C	H	N	S
C15H14N2O5S	334.35	51.80 (53.88)	4.69 (4.22)	7.87 (8.38)	8.41 (9.59)	Dark orange	82%

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The average molecular weight of the SASF terpolymer determined by the GPC technique is given in Table 4. The M_n and M_w of the terpolymer were found to be 2137 and 2281 g mol⁻¹, respectively. The size average molecular weight M_z of the SASF terpolymer was found to be 2213 g mol⁻¹. The polydispersity index (M_w/M_n) and (M_w/M_z) found to be 1.0673 and 1.0307 respectively. The results of the polydispersity index values indicate that the SASF terpolymer has a narrow molecular weight distribution.

Table 4 GPC data of the SASF terpolymer

Sample	Formula mass of the repeating unit	(Mn)	(Mw)	(Mz)	Polydispersity (Mw/Mn)	Polydispersity (Mw/Mz)
SASF	334.35	2137	2281	2213	1.0673	1.0307

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3.2. Spectral analysis

3.2.1. FTIR spectral analysis. The FTIR spectrum of the SASF polymer is depicted in Fig. 1. The spectrum shows a broad band at 3480 cm⁻¹, which is due to the stretching vibration of the hydroxyl group of –COOH present in the aromatic ring, which is involved in intermolecular hydrogen bonding.^27,28 A broad and strong band at 3259 cm⁻¹ is assigned to the –NH stretching vibration.²⁹ The 1,2,3,5 tetra substitution in the aromatic benzene ring is confirmed by the bands between 1200 cm⁻¹ and 800 cm⁻¹.^30,31 The band observed at 1661 cm⁻¹ is assigned to the –C=O (carboxylic ketone) stretching vibration of the –COOH group in the terpolymer.^31,32 A band at 2945 cm⁻¹ is attributed to the –CH₂ linkage present in the terpolymer.^33,34 A strong band in the range of 1376–1228 and 1163–1136 cm⁻¹ is assigned to the asymmetric and symmetric stretching of the O=S=O present in the terpolymer.³¹ The characteristic stretching frequencies confirm the structure of the SASF terpolymer as given in Scheme 1.

Fig. 1 FTIR spectrum of the SASF terpolymer.

3.2.2. UV-Vis spectral analysis. The electronic spectrum of the SASF terpolymer is shown in Fig. 2. The UV-Vis spectrum of the SASF terpolymer exhibits two characteristic bands at 266 nm and 308 nm. The observed positions of the absorption bands with different intensities indicate that the more intense band at 266 nm is due to the π → π* transition of the carbonyl group (ketonic) in conjugation with the aromatic nucleus, whereas the later (less intense) band at 308 nm is due to a n → π* electronic transition. The presence of a phenolic hydroxyl group (auxochromes) is responsible for the hyperchromic shift. This observation is in good agreement with the proposed structure for the SASF terpolymer.^9,29,35

Fig. 2 UV-Vis spectrum of the SASF terpolymer.

3.2.3. ¹H NMR and ¹³C NMR spectrum. The ¹H NMR spectrum of the SASF terpolymer recorded in DMSO-d₆ solvent is presented in Fig. 3. The obtained signals for the terpolymer were interpreted on the basis of the literature.^{21,31,36–38} The signal at 6.5–7.3 ppm is due to the aromatic protons (Ar–H). The signal at 7.6 ppm is assigned to the –OH proton of the Ar–COOH group and this downfield shift is due to the intramolecular hydrogen bonding between the Ar–COOH and Ar–OH present in the terpolymer. The signal appearing in the region of 3.1–3.3 is due to the Ar–CH₂ moiety and a signal around 4.4–4.6 ppm may be due to the Ar–CH₂–N moiety. A signal typically appearing around 5.8 and 9.1 ppm is due to the –NH proton of the terpolymer.

Fig. 3 ¹H NMR spectrum of the SASF terpolymer.

The ¹³C NMR spectrum of the SASF terpolymer is shown in Fig. 4. The observed peak positions are assigned according to the basis of the literature.^29,31 The ¹³C NMR spectrum shows peaks at 127.39, 120.07, 117.89, 117.03, 131.24 and 152.03, corresponding to the aromatic ring of the sulfanilamide present in the terpolymer. The signals at 112.89, 161.09, 128.19, 137.64, 127.41 and 128.64 ppm may be attributed to the C1–C6 of the aromatic ring of the salicylic group. A peak around 172.08 ppm is due to the C=O group of the carboxylic acid present in the aromatic ring of the terpolymer. A peak at 45.01 ppm may be assigned to the –CH₂ linkage of the terpolymer.

Fig. 4 ¹³C NMR spectrum of the SASF terpolymer.

3.3. Thermal behavior of the terpolymer

The TGA and DTA curves of the SASF terpolymer are shown in Fig. 5 and its percentage weight loss at various temperatures is presented in Table 5. The SASF terpolymer exhibits three major stages of degradation in the temperature range 33.5 to 603.7 °C. The first stage of degradation takes place at 236 °C with a weight loss of 11%, which corresponds to the loss of the carboxylic and alcoholic group attached to the aromatic ring. The second stage of degradation takes place from 237 °C and ends at 345 °C, involving 27% of weight loss, which may be attributed to the amine and CH₂ moiety of the terpolymer. The final and highest weight loss attaining about 79% takes place at 603.7 °C, which may be due to the degradation of the aromatic nucleus of the terpolymer. The melting temperature T_m of the SASF terpolymer evident from the (DTA) curve is found to be around 458 °C. The results indicate that the SASF terpolymer possesses an improved thermal stability.

Fig. 5 TGA and DTA curves of the SASF terpolymer.

Table 5 TGA data of the SASF terpolymer

Sample	Temperature (°C)							Degradation temperature range (°C)	Tmax^a (°C)	T50^b (°C)
	150	250		350	450	550
	Weight loss of terpolymer (%)
a Maximum decomposition temperature.b Temperature at 50% weight loss.
SASF	7.2	12.1	27.0		51.6		74.2	33.5 to 603.7	603.7	491

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3.4. Binding capacity of the SASF terpolymer with the metal ions

3.4.1. Effect of the electrolytes and pH. The electrolyte solution is one of the most important factors in the adsorption process of heavy metal ions. 50 mg of the SASF terpolymer involving a mixture of 400 mg L⁻¹ of Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions in various electrolytes such as NaCl, Na₂SO₄ and NaNO₃ with different concentrations viz. 0.1, 0.5, 1.0, 1.5 and 2.0 M was studied at room temperature for 12 h and the results are shown in Fig. 6–8, respectively. From the figures, it can be observed that the metal ion removal capacity of the terpolymer with Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions significantly increased with increasing concentration of the electrolyte and the maximum adsorption capacities of the terpolymer were obtained in 2.0 M of NaNO₃ compared to other two electrolytes. The variation of the removal percentage between the three electrolytes may be due to the strong and weak complex formation between the electrolyte ligand and the metal ions. Generally, if the electrolyte ligand–metal ion complex is weaker than the polymer–metal ion complex, the terpolymer can easily break the electrolyte ligand–metal complex and the free metal ions can easily form a complex with the terpolymer and hence the removal of metal ion increases. However, if the electrolyte ligand–metal ion complex is stronger than the polymer–metal ion chelate, then more metal ions will form strong complexes with the electrolyte ligand itself, which lowers the metal ion removal capacity of the terpolymer. From Fig. 8, it can be seen that at higher concentration of Na₂SO₄, the sulfate (SO₄²⁻) ions form a strong complex with metal ions and so adsorption of the metal ions on the terpolymer is reduced.

Fig. 6 Adsorption capacity of Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions on the SASF terpolymer with different concentrations of NaCl.

Fig. 7 Adsorption capacity of Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions on the SASF terpolymer with different concentrations of Na₂SO_4.

Fig. 8 Adsorption capacity of Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions on the SASF terpolymer with different concentrations of NaNO₃.

The effect of pH plays a unique role in the removal of metal ions. The reaction between a fixed concentration (400 mg L⁻¹) of Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions mixture and 50 mg of the SASF terpolymer can be influenced by the changes in pH values ranging from 2 to 7. The metal ion removal efficiency of the terpolymer is shown in Fig. 9. It can be seen from the figure that adsorption quantity increases with the increasing pH of the aqueous solution. At a pH of below 3.0, the removal efficiency of the terpolymer is low. This may be due to the fact that in an acidic environment, the functional groups of the terpolymer are highly protonated with hydrogen ions (H⁺) and lose the coordination ability with divalent metal ions because of the loss of negative charge. As the pH increases in the range of 3.0 to 7.0, the functional groups of the terpolymer become less protonated, thus providing greater coordination with heavy metal ions and hence the percentage of metal ions removal was increased.³⁹ The removal efficiencies of the terpolymer for the selected metal ions at pH 4 are found to be Ni²⁺(76%), Cu²⁺(85%), Pb²⁺(76%), Cd²⁺(54%), Hg²⁺(69%) and Zn²⁺(74%). The maximum adsorption capacities are obtained at pH 6.0–7.0, which delivered the best results for Ni²⁺(76%), Cu²⁺(87%), Pb²⁺(83%), Cd²⁺(57%), Hg²⁺(69%) and Zn²⁺(74%). On increasing the pH above 7, precipitation of the metal ion may occur. Fig. 10 shows that the color of the SASF terpolymer after metal ion adsorption changed from dark orange to dark brown, which may be due to more availability of metal ions on the surface of the terpolymer. The study of the adsorption mechanism indicates that the hydroxyl and amino groups on the terpolymer play an important role and the metal ion removal capacity of the terpolymer in weakly acidic or neutral solutions is better than that in strong acidic solutions.

Fig. 9 pH dependence of the metal ions uptake by the SASF terpolymer.

Fig. 10 Images of (a) before and (b) after metal ions treatment of the SASF terpolymer.

3.5. XRD studies

XRD analysis was conducted to elucidate the phase structure of the SASF terpolymer, as shown in Fig. 11(a). The spectrum shows a broad diffraction peak between 23 and 32 (2 theta) angles. This spectral peak arises due to the formation of the amorphous structure of the terpolymer. However, secondary peaks are observed with a decrease in intensity around 37, 43 and 50 (2 theta), which may be due to the formation of a slightly crystalline nature. Therefore, the obtained results indicate that the terpolymer exists in a transition state between an amorphous and crystalline nature. The XRD pattern of the metal ion treated terpolymer is shown in Fig. 11(b). Broad diffraction characteristic peaks appear at two theta angles between 24 and 35 for the amorphous region and secondary peaks are observed at 40, 47 and 52 (2 theta). These observed peaks almost match with the XRD pattern of the untreated terpolymer, which indicates the unaffected morphological feature of the treated SASF terpolymer. However, after treatment, the characteristic peaks slightly shifted to the higher 2 theta region due to the conversion of some portions of the SASF terpolymer from the amorphous to crystalline state. This obtained result is in good agreement with the structure of the terpolymer obtained from the SEM analysis earlier.

Fig. 11 X-ray diffraction (XRD) patterns of (a) before and (b) after treatment of the SASF terpolymer.

3.6. Morphological study and EDX analysis

The surface morphologies of the SASF terpolymer before and after loading of the metal ions were investigated by SEM. Fig. 12 represents the surface morphology of the untreated SASF terpolymer, which has a high porosity with a honeycomb-like structure, hence it can easily accommodate metal ions into its cavities and act as a better adsorbent, which is evident from the SEM images with different magnifications in Fig. 12(a–d). The SEM images of the metal ion treated SASF terpolymer are shown in Fig. 13. The presence of divalent Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions inside the pores is clearly seen in their respective SEM images with different magnification, as shown in Fig. 13(a–d). After treatment, the porous morphology features remained unaffected.

Fig. 12 SEM images of the metal ion unloaded SASF terpolymer with different magnifications: (a) 1000×, (b) 2000×, (c) 2000× and (d) 3000×.

Fig. 13 SEM images of the metal ion loaded SASF terpolymer with different magnifications: (a) 1000×, (b) 2000×, (c) 3000× and (d) 5000×.

The chemical composition of the SASF terpolymer was characterized by EDX and the spectrum is shown in Fig. 14. The spectral analysis shows approximately 67.24% of C, 3.58% of N, 22.35% of O and 6.83% of S. Moreover, the elements C, N, O and S were detected from the EDX spectrum of the SASF terpolymer, which further confirms the presence of SASF in the terpolymer. Fig. 15 represents the SEM images and the EDX spectra of the metal ion treated SASF terpolymer, which shows that the binding of the Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ metal ions and the uptake process happened on the membrane throughout the SASF terpolymer. The SEM and EDX results indicate that the SASF terpolymer has an extensive surface area, hence the porosity is suggested to facilitate the binding of the metal ions effectively.

Fig. 14 EDX analysis and the spectral analysis of the SASF terpolymer.

Fig. 15 EDX analysis of the metal ion loaded SASF terpolymer.

3.7. In vitro biological studies of the SASF terpolymer

3.7.1. Antibacterial screening activity. The newly synthesized SASF terpolymer was screened for its antibacterial activity against Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis and Gram-negative Salmonella typhi, Escherichia coli bacterial strains by the inhibition zone (disc-diffusion) method at five different concentrations such as 25, 50, 100, 250, 500 and 1000 μg. Fig. 16(a–d) shows the zone of inhibition formed around each disc loaded with the SASF terpolymer against Gram-positive and Gram-negative bacterial strains. The inhibitory effect was measured based on the clear zone surrounding the circular-shaped terpolymer and the diameters of the inhibition zone (in mm) on the agar plates are tabulated in Table 6. The results obtained in this study clearly indicate that the diameter of the inhibition zone increases with increasing concentration of the terpolymer. Clearly, a high concentration of the terpolymer exhibited a good antibacterial activity against the selected bacterial strains. For the SASF terpolymer sample, the highest inhibition zone for Bacillus subtilis was found to be 01.4 ± 0.38 mm for 25 μg, 04.3 ± 0.15 mm for 50 μg, 07.1 ± 0.86 mm for 100 μg, 08.2 ± 1.08 mm for 250 μg, 12.3 ± 0.47 mm for 500 μg and 15.8 ± 0.82 mm for 1000 μg, respectively. The terpolymer against MRSA shows a lower inhibition at 25–250 μg. This may be due to the multi peptidoglycan layer present in the bacterial strain. The test results indicate that the SASF terpolymer is an efficient inhibitor of bacterial growth.

Fig. 16 Antibacterial activity of the SASF terpolymer. The clear inhibition zones formed against the growth of (a) B. subtilis, (b) S. typhi, (c) E. coli and (d) S. aureus (MRSA).

Table 6 Antibacterial effect of the SASF terpolymer at different concentrations

Bacterial strain used	Zone of inhibition (in mm) SASF terpolymer (average of three measurements)						Standard 1000 μg
	25 μg	50 μg	100 μg	250 μg	500 μg	1000 μg
B. subtilis	01.4 ± 0.38	04.3 ± 0.15	07.1 ± 0.86	08.2 ± 1.08	12.3 ± 0.47	15.8 ± 0.82	33.7 ± 0.33
S. typhi	01.5 ± 0.86	02.6 ± 0.35	03.8 ± 0.51	05.5 ± 0.13	06.1 ± 0.54	08.3 ± 0.65	37.4 ± 1.25
E. coli	01.3 ± 0.74	02.4 ± 0.31	07.2 ± 1.06	09.7 ± 0.87	10.5 ± 0.35	11.8 ± 0.81	29.3 ± 0.26
S. aureus	00.00 ± 0.00	0.00 ± 0.00	00.00 ± 0.00	01.3 ± 0.41	09.2 ± 0.64	10.6 ± 0.36	31.8 ± 0.63

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3.8. Cytotoxicity studies (in vitro)

The cytotoxicity of the SASF terpolymer was tested against the HeLa cell line using a MTT assay. We examined the SASF terpolymer at four final concentrations namely 25, 50, 75 and 100 μg at 37 ± 1 °C for 24 h. A suitable positive control was run and this experiment was repeated thrice. A fluorescence morphology investigation of the cell viability of control cells and treated HeLa cancer cells is depicted in Fig. 17(a–d). From the results, a significant change in cell viability is observed with changing the concentration of the terpolymer. Fig. 17(d and e) shows that at higher concentrations (75 and 100 μg), most of the cells zone extended father than 1 cm beyond the specimen, which indicated the severe cytotoxic activity of the terpolymer. Fig. 17(b and c) shows that at concentrations of 25 and 50 μg, the cells in the zone were round-shaped and loosely attaching degenerated cells under the specimen were observed, which indicates a slight cytotoxic activity against the cancer cells. The control showed no cytotoxic activity, as expected. Based on the response around the test sample, the cytotoxic activity was graded, as shown in Table 7. The achievement of numbers greater than 2 are considered to be a cytotoxic effect. Since the test sample achieved a numerical grade greater than 2 in 75 and 100 μg, this reveals that the SASF terpolymer is considered to be the best cytotoxic agent at higher concentrations.

Fig. 17 Fluorescence images showing the viability of control cells and different concentrations of the treated HeLa cancer cells on the SASF terpolymer (after 24 h treatment).

Table 7 Cytotoxic reactivity grade for the SASF terpolymer

Sample	Grade	Activity
Control	0	None
SASF 25 μg	1	None
SASF 50 μg	1	Slight
SASF 75 μg	4	Severe
SASF 100 μg	4	Severe

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4. Conclusions

We have successfully synthesized a novel SASF terpolymer via condensation polymerization and its structure was confirmed by various spectral studies. From thermal analysis, the SASF terpolymer was found to possess a higher degradation stability. The batch equilibrium studies revealed that the SASF terpolymer can act as an effective adsorption material for various divalent metal ions such as Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Zn²⁺ ions. The metal ion binding capacity of the SASF terpolymer was confirmed by ICP-OES, SEM and EDX studies. The antibacterial and cytotoxicity activity of the SASF terpolymer were investigated. The antibacterial studies showed that the SASF terpolymer possessed effective antibacterial properties against both Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis and Gram-negative Salmonella typhi, Escherichia coli bacterial strains. The cytotoxicity studies revealed that the SASF terpolymer showed strong cytotoxic activity against the HeLa cell line. Since the SASF terpolymer contains alcoholic and amino groups, it plays a key role in the ion exchange phenomenon and the sulfur group plays a major role in its biological properties. Thus, the SASF terpolymer could be used for the removal of toxic metal ions in industrial waste water treatment and has immense potential for various biological applications.

Acknowledgements

The authors are thankful to M. Suresh and S. Ragurm, Senior Chemist, TUV Rheinland Pvt. Ltd. Bangalore, India, for providing technical support and necessary facilities.

References

1. P. Bagla and J. Kaiser, India's Spreading Health Crisis Draws Global Arsenic Experts, Science, 1996, 274, 174–175.
2. G. D. Clayton, Clayton FE Patty's, industrial hygiene and toxicology, Wiley-Interscience, New York, 1994, vol. 2C.
3. European Commission DG ENV, E3, Project ENV.E3/ETU/2000/0058, Heavy metal in waste, Final report, 2002.
4. S. S. Ahluwalia and D. Goyal, Bioresour. Technol., 2007, 53, 1201–1210.
5. F. G. Helfferich, Ion Exchange, McGraw-Hill, New York, 1962.
6. R. Kunin, Ion Exchange Resins, Wiley, New York, third edn, 1958.
7. T. H. Boyer and P. C. Singer, Water Res., 2006, 40, 2865–2876.
8. D. Prabhakaran and M. S. Subramanian, Talanta, 2003, 59, 1227.
9. M. M. Jadhaoa, L. J. Paliwalb and N. S. Bhavea, Desalination, 2009, 247, 456–465.
10. A. K. Naskar, R. A. Walker, S. Proulx, D. D. Edie and A. A. Ogale, Carbon, 2005, 43, 1065–1072.
11. M. A. R. Ahameda, D. Jeyakumar and A. R. Burkanudeen, J. Hazard. Mater., 2013, 248–249, 59–68.
12. R. N. Singru, W. B. Gurnule, V. A. Khati, A. B. Zade and J. R. Dontulwar, Desalination, 2010, 263, 200–210.
13. G. W. Fussell and S. L. Cooper, Biomaterials, 2004, 25, 2971–2978.
14. N. P. S. Chauhan and S. C. Ameta, Polym. Degrad. Stab., 2011, 96, 1420–1429.
15. W. B. Gurnule, P. K. Rahangdale, L. J. Paliwal and R. B. Kharat, React. Funct. Polym., 2003, 55, 255–265.
16. R. S. Azarudeen, M. A. R. Ahamed and A. R. Burkanudeen, Desalination, 2011, 268, 90–96.
17. M. M. Patel, M. A. Kapadia, G. P. Patel and J. D. Joshi, React. Funct. Polym., 2007, 67, 746–757.
18. A.-H. Chen, C.-Y. Yang, C.-Y. Chen, C.-Y. Chen and C.-W. Chen, J. Hazard. Mater., 2009, 165, 1068–1075.
19. S. Parveen, T. Ahamad and N. Nishat, Appl. Organomet. Chem., 2008, 22, 70–77.
20. W. Zou, Y. Huang, J. Luo, J. Liu and C. Zhao, J. Membr. Sci., 2010, 358, 76–84.
21. A. R. Burkanudeen, R. S. Azarudeen, M. A. R. Ahamed and W. B. Gurnule, Polym. Bull., 2011, 67, 1553–1568.
22. C. B. Boss and K. J. Fredeen, Concept, Instrumentation and Techniques in Inductively Coupled Plasma Optical Emission Spectrometry, Perkin-Elmer, Norwalk, CT, 2nd edn, 1997.
23. U.S. EPA Method 200.7, Determination of metals and trace elements in water and wastes by Inductively Coupled Plasma Optical Emission Spectrometry, Cincinnati, Ohio, U.S, 1994.
24. Q. He, Z. Hu, Y. Jiang, X. Chang, Z. Tu and L. Zhang, J. Hazard. Mater., 2010, 175, 710–714.
25. R. Gao, Z. Hu, X. Chang, Q. He, L. Zhang, Z. Tu and J. Shi, J. Hazard. Mater., 2009, 172, 324–329.
26. P. A. Wayne, NCCLS Approved standard M27-A, National Committee for Clinical Laboratory Standards, USA, 1997.
27. R. S. Azarudeen, M. A. R. Ahamed, D. Jeyakumar and A. R. Burkanudeen, Iran. Polym. J., 2009, 18, 821–832.
28. B. A. Shah, A. V. Shah and R. R. Bhatt, Iran. Polym. J., 2007, 16, 173–184.
29. R. M. Silverstein and G. C. Bassler, Spectrometric Identification of Organic Compounds, John Wiley, New York, second edn, 1967.
30. K. Nakanishi, Infrared Absorption Spectroscopy Practical, Nolden Day, INC and Nankodo Co., Ltd., Tokyo, 1967.
31. E. Pretsch, P. Buhlmann and C. Afflolter, Structure Determination of Organic Compounds, Springer, New York, 2000.
32. M. A. R. Ahamed, R. S. Azarudeen, D. Jeyakumar and A. R. Burkanudeen, Int. J. Polym. Mater., 2011, 60, 124–143.
33. J. A. Raj, C. Vedhi, A. Burkanudeen, P. Arumugam and P. Manisankar, Ionics, 2010, 16, 171–175.
34. F. Cotton, G. Wilkinson, C. Murillo and M. Bochmann, Advanced Inorganic Chemistry, Wiley Interscience, New York, sixth edn, 1999.
35. H. Dudley and I. Fleming, Spectroscopic Methods in Organic Chemistry, McGraw-Hill, UK, 1975.
36. M. Riswan Ahamed, R. Azarudeen, M. Karunakaran, T. Karikalan, R. Manikandan and A. Burkanudeen, Int. J. Chem. Environ. Eng., 2010, 1, 7.
37. X.-L. Wang, K. Wan and C.-H. Zhou, Eur. J. Med. Chem., 2010, 45, 4631–4639.
38. D. B. Patle and W. B. Gurnule, Arabian J. Chem., 2011 DOI:10.1016/j.arabjc.2011.07.013.
39. Z. S. Pour and M. Ghaemy, RSC Adv., 2015 10.1039/c5ra08025h.

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