Sulfonyl Amide Salicylic Acid

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: V. Raj, V. V, A.
Raja, A. V, S. Prakash, P. Priya and A. Saranya, RSC Adv., 2016, DOI: 10.1039/C6RA05115D.
This is an Accepted Manuscript, which has been through the

Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after

acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the

Information for Authors.
Please note that technical editing may introduce minor changes

to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances
Page 1 of 34 RSC Advances
View Article Online
DOI: 10.1039/C6RA05115D
1
Synthesis, Characterization, Removal of toxic metal ions and in-vitro biological

applications of sulfanilamide-salicylic acid-formaldehyde terpolymer
V. Vasanthakumar, A. Saranya, A. Raja, S. Prakash, V. Anbarasu, P. Priya, V. Raj*

Published on 23 May 2016. Downloaded by UNIVERSITY OF NEBRASKA on 24/05/2016 07:22:26.
Advanced Materials Research Laboratory, Department of Chemistry, Periyar University,
RSC Advances Accepted Manuscript

Salem-636 011, Tamil Nadu, India.
*Correspondence: V. Raj, Professor and Head, Department of Chemistry, Periyar University,

Salem- 636 011, Tamil Nadu.
Phone: Off: +91-427-2345271, Mobile: +91-9790694972, Fax: +91-427-2345565,
E-mail: alaguraj2@rediffmail.com, doctorvraaj@gmail.com
RSC Advances Page 2 of 34
View Article Online
DOI: 10.1039/C6RA05115D
2
Abstract: A new terpolymer was synthesized by polymerization of sulfanilamide, salicylic acid and
formaldehyde (SASF) in presence of glacial acetic acid as a catalyst. The characterization of the
synthesized SASF terpolymer was conducted by various techniques such as elemental analysis, GPC,
FTIR, UV-Visible, 1H NMR, 13C NMR, TGA, XRD, SEM and EDAX 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 ions adsorption on SASF terpolymer has
been studied as function of electrolytes in different concentrations and the influence of pH in different
ranges were studied by batch equilibrium technique. The metal ions removal efficiency is increases
with increase of pH and concentration of the electrolytes. The maximum removal percentage is
achieved at pH 6-7. Inductively coupled plasma-optical emission spectroscopy 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 effective antibacterial activity
against gram-positive Saphylococcus aureus, Bacillus subtilis and gram-negative Salmonella typhi,
Escherichia coli bacterial strains. The cytotoxicity studies indicate that SASF terpoymer possesses
much potential against Hela (mammalian cancer) cell line.

View Article Online
DOI: 10.1039/C6RA05115D
3
1. Introduction
The environment pollution by heavy metals is a major threat to human and aquatic ecosystem.
The toxic heavy metals and their ions are not only potential human health hazards but also to another
life forms [1-3]. From the eco-toxicological point of view, the considerable dangerous metals are lead,
cadmium, mercury, copper, nickel, arsenic, cobalt, zinc and vanadium which are known to be toxic for

living beings at any concentration. The most common sources for toxic heavy metals to water and/or
waste water are, effluent from textile, leather, electroplating, pigment and dyes, refining process, petrol
refining, metallurgical and painting industries 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, electro winning, electro-coagulation, reverse osmosis and solid-liquid
extraction. Most of these methods suffer from some drawbacks such as high capital operational cost
and problem of disposal of residue metal sludge. However, the metal ions removal by chelating ion-
exchange terpolymer using batch equilibrium method is a powerful eco-friendly technique for the
separation and recovery of toxic heavy metal ions from the industrial wastes contaminated water [5-8].
Chelating ion-exchange terpolymer with substituent like –OH, –COOH and –NH2 are important groups
of metal ion chelating agents [9].
The eco-friendly metal ions removal and biological properties of terpolymers have further
provoked researchers for the synthesis of new terpolymer. A survey of the literature reveals that the
terpolymers exert a pull on the concentration of synthetic chemists due to their varied applications.
Terpolymer 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].
Terpolymer involving 4-hydroxy acetophenone and biuret with formaldehyde in the presence of acid
catalyst was proved to be an excellent ion exchanger [15]. Similarly, solution polymerization of three
monomers involving 8-hydroxyquinoline, salicylic acid and formaldehyde was carried out to obtain a
View Article Online
DOI: 10.1039/C6RA05115D
4
terpolymer. From the TGA results, the terpolymer was found to be thermally stable and the order of
reaction for the thermal decomposition was found [16]. Ion-exchange and antimicrobial study of
poly[(2-hydroxy-4-methoxy benzophenone) ethylene] terpolymer and its polychelates with
lanthanides(III) was reported [17]. Series of acrylic terpolymer with RGD peptides were used for
biomedical applications [13]. The chemically crosslinked metal-complexed chitosans for comparative

adsorptions of Cu(II), Zn(II), Ni(II) and Pb(II) ions in aqueous medium was studied [18]. Removal of
cations using ion-binding terpolymer involving 2-amino-6-nitro-benzothiazole and thiosemicarbazide
with formaldehyde by batch equilibrium technique with effect of pH, contact time and electrolytes and
the reusability of the terpolymer was also studied [11]. Anti-bacterial activities of thiosemicarbazide–
formaldehyde terpolymer and its polymer–metal complexes were reported for their higher inhibition
characteristics against the chosen microbes [19]. Protein antifouling property of poly (methyl
methacrylate–acrylic acid–vinyl pyrrolidone) terpolymer modified with polyethersulfone hollow fiber
membrane with pH sensitivity was studied [20]. Kinetics of thermal decomposition and antimicrobial
screening of terpolymer prepared by polycondensation of anthranilic acid, 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 on Hela (mammalian cancer) cell line.
2. Experimental
2.1. Chemicals and regents
Sulfanilamide, salicylic acid and formaldehyde (37 %) were procured from Merck, India. Metal
nitrate of chosen metals were purchased from AR grade merck, India. Multi-elemental standard
View Article Online
DOI: 10.1039/C6RA05115D
5
solution for ICP-OES was obtained from PerkinElmer (USA). All working metal and standard solution
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
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 ºC in an oil bath for 6 h. The resultant product
was cooled, poured into crushed ice with constant stirring and left overnight. The dark orange colored
obtained product was separated, washed with warm water, ethanol and ether to remove 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 drop
wise addition of ice-cold 1:1 (v/v) concentrated hydrochloric acid/distilled water with constant and
rapid stirring to avoid lump formation. This regeneration technique was repeated twice, then filtered off
and cured at 75 ºC for 24 h in an air oven [12, 14]. The yield of the terpolymer was found to be 82%.
The dried terpolymer was finely grounded and sieved to obtain uniform particle 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 SASF terpolymer is shown in Scheme 1.
2.3. Characterization of SASF terpolymer
The percentage of elements such as C, H, N and S present in the SASF terpolymer was carried
out on elementar instrument (Model vario EL III, Germany). The number-average (Mn), weight-average
molecular weights (Mw) and polydispersion index (PDI = Mw/Mn, Mw/Mz) of the terpolymer were
determined by gel permeation chromatographic (GPC) system equipped with Waters 2690D
separations module. Waters millennium module software was used to calculate molecular weight, on
the basis of a universal calibration curve generated by polystyrene standard.

View Article Online
DOI: 10.1039/C6RA05115D
6
The FTIR spectrum of the terpolymer has been scanned by using the KBr pellets technique on a
(Bruker Tensor 27) FTIR spectrophotometer to identify the linkages and functional groups. The
electronic absorption spectrum of the terpolymer was recorded on (Shimadzu 1601PC) UV-Visible
double beam spectrophotometer in pure DMSO, in the region of 200-800 nm at the scanning rate 100
nm min-1 and chart speed of 5 cm min-1. 1H and 13C NMR spectra of terpolymer have been scanned on

Bruker 400 MHz NMR spectrometer using DMSO-d6 as a solvent and tetramethylsilane (TMS) was
used as reference material. Thermogravimetric analysis (TGA) and Differential thermal analysis (DTA)
of the terpolymer were recorded on (SDT Q600 V8.3 Build 101Universal V4.7A TA Instruments
systems) thermal analyzer at heating rate of 10 ºC/ min in static nitrogen atmosphere in the range of 30-
800 ºC. X-Ray Diffraction analysis (Rigaku Rotoflux) was performed to investigate phase structure of
the terpolymer. The surface morphology of the terpolymer was analysed by scanning electron
microscope (Zeiss18 Evaluation) and the chemical composition were characterized by energy
dispersive X-ray spectroscopy (EDAX Oxford X-Act).
2.4. ICP-OES Measurement
Determination of all metal ions was performed with inductively coupled plasma-optical
emission spectroscopy (ICP-OES) using a Perkin Elmer OptimaTM 7000 DV dual view series sequential
spectrometer (Shelton, CT, USA) an equipped with WinLab™ 32 for ICP Version 4.0 software. In
order to avoid any carry-over effect, 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. The CCD-array detector was used to collect both the analyte spectra and
the nearby background spectra, which provide improved precision and analytical speed. The
instrumental operating conditions and parameter 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 high sensitive detection of ultra-trace and trace analysis. [22,23].
View Article Online
DOI: 10.1039/C6RA05115D
7
2.5. Batch sorption experiments
The metal ions binding capacity of SASF terpolymer was studied as a function of various
electrolytes in different concentrations and pH in different ranges by batch equilibrium technique. A 50
mg of synthesized SASF terpolymer was suspended in 30 mL of electrolyte solutions such as NaCl,

Na2SO4 and NaNO3 in different concentrations viz. 0.1, 0.5, 1.0, 1.5 and 2.0 M. To each suspension,

400 mgL-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
adsorption of the 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 inductively
coupled plasma-optical emission spectroscopy (ICP-OES). A blank was analyzed to ensure that no
metal ions were carried over from the previous sample. Highly pure 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 ions concentration [24].
The same procedure was repeated for pH studies. The effect of pH on sorption of metal ions by
terpolymer was studied in the range of 2-7. Before mixing the metal ions, the pH of the suspension was
adjusted to required value by adding minimum amount of (0.5 M) HCl and/or NaOH. The amount of
metal ions uptake by the SASF terpolymer during the series of batch investigations were determined
using the following equation:
where: Co and Cf are the initial and equilibrium concentration (ppm) of metal ions in solution,
respectively.
2.6. In vitro biological studies of the SASF terpolymer
2.6.1. Antimicrobial properties of terpolymer
The antimicrobial properties of the SASF terpolymer has been investigated against gram-
positive Saphylococcus aureus (MRSA), Bacillus subtilis and gram-negative Salmonella typhi,
View Article Online
DOI: 10.1039/C6RA05115D
8
Escherichia coli bacterial strains by the agar disc diffusion method according to the guidelines of
National Committee for Clinical Laboratory Standards (NCCLS, 1997) with slight modification [25].
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 ºC 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 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 concentration of SASF terpolymer (such as 25, 50, 100, 250, 500 and 1000 µgs)
along with standard reference drug (ciprofloxacin). Then the petri plates were then incubated at 37±1
ºC 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 zone of inhibition (mm) around the disc, that
was indicative of the ability of the antimicrobial test sample to kill bacteria.
2.7. Anticancer study

2.7.1. Cell line and culture medium
HeLa (mammalian cancer) cell line was obtained from National Centre for Cell Sciences
Repository (NCCS- Pune, India). Minimum Essential Medium (MEM) 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 ºC until confluent. The cells were seeded at 1×104 cells/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 (mammalian cancer) cells with 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 ISO 10993:5 method. The
View Article Online
DOI: 10.1039/C6RA05115D
9
control and test sample in triplicates were added on the cell. After incubation at 37±1 ºC for 24 h. the
monolayer was examined microscopically for the response around the test sample. As per the table 2
the reactivity were graded as 0, 1, 2, 3 and 4 based on zone of lysis, cacuolization, detachment and
disintegration of membrane.
3. Results and discussion

3.1. Analytical and elemental data
The elemental analysis of synthesized SASF terpolymer was brown in color. The calculated and
founded C, H, N and S elements value 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.
The average molecular weight of SASF terpolymer determined by gel permeation
chromatography technique is given in Table 4. The number average (Mn) and weight average (Mw)
molecular weight of terpolymer were found to be 2137 and 2281 respectively. The size average
molecular weight Mz of the SASF terpolymer was found to be 2213. The polydispersity index (Mw/Mn)
and (Mw/Mz) found to be 1.0673 and 1.0307 respectively. The results of polydispersity index values
indicate that the SASF terpolymer has narrow molecular weight distribution.
3.2. Spectral analysis
3.2.1. FTIR spectral analysis
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 hydroxyl group of –COOH present in the aromatic
ring which is involving intermolecular hydrogen bonding [27,28]. A broad and strong band at 3259 cm-
1
is assigned to –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 –C=O (carboxylic ketone) stretching vibration of the –COOH group in the
View Article Online
DOI: 10.1039/C6RA05115D
10
terpolymer [31,32]. A band at 2945 cm-1 is attributed to –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 asymmetric and symmetric
stretching of O=S=O present in the terpolymer [31]. The characteristic stretching frequencies confirm
the structure of SASF terpolymer as given in Scheme 1.

3.2.2. UV-Visible spectral analysis

The electronic spectrum of the SASF terpolymer is shown in Fig. 2. The UV-visible spectrum
of SASF 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 (π→
π*) transition of carbonyl group (ketonic) in conjugation with the aromatic nucleus, whereas the later
(less intense) band at 308 nm is due to (n → π*) electronic transition. The presence of phenolic
hydroxyl group (auxochromes) is responsible for hyperchromic shift. This observation is in good
agreement with the proposed structure for the SASF terpolymer [9,29,35].
3.2.3. 1H NMR and 13C NMR Spectrum
The 1H NMR spectrum of SASF terpolymer recorded in DMSO-d6 solvent is presented in Fig. 3.
The obtained signals for the terpolymer were interpreted on the basis of 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 Ar–COOH group and this downfield shift is due to the intramolecular hydrogen bonding
between Ar–COOH and Ar–OH present in the terpolymer. The signal appeared in the region at 3.1-3.3
is due to Ar-CH2 moiety and a signal around 4.4-4.6 ppm may due to Ar-CH2-N moiety. A signal
typically appears around 5.8 and 9.1 ppm is due to–NH proton of the terpolymer.
13
The C 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]. The13C NMR spectrum shows
peaks at 127.39, 120.07, 117.89, 117.03, 131.24 and 152.03 corresponding to 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 C1-C6 of the aromatic ring of the salicylic group. A peak around
View Article Online
DOI: 10.1039/C6RA05115D
11
172.08 ppm is due to (C=O) group of 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.
3.3. Thermal behavior of the terpolymer
Thermogravimetric analysis (TGA) and Differential thermal analysis (DTA) curve of the SASF
terpolymer are shown in Fig. 5 and its percentage of weight loss at various temperatures is presented in

Table 5. The SASF terpolymer exhibits three major stages of degradation in temperature range 33.5 to
603.7 °C. The first stage of degradation takes place at 236 ºC with the weight loss 11 % which is
corresponding to loss of carboxylic and alcoholic group attached to the aromatic ring. The second stage
degradation takes place from 237 ºC and ends at 345 ºC, involving 27 % of weight loss, which may
attributed to amine and CH2 moiety of the terpolymer. The final and highest weight lose attaining about
79 % takes place at 603.7 ºC, which may be due to degradation of aromatic nucleus of the terpolymer. The
melting temperature Tm of SASF terpolymer as evident from the (DTA) curve is found to be around 458
ºC. The results indicate that the SASF terpolymer possesses better thermal stability.
3.4. Binding capacity of SASF terpolymer with the metal ions
3.4.1. Effect of Electrolytes and pH
The electrolyte solution is one of the most important factors in the adsorption process of heavy
metal ions. A 50 mg of 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 has been studied at room temperature for 12 h and the
results are shown in Figs. 6-8 respectively. From the figure it is observed that the metal ions 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 is 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 electrolytes ligand and the metal ions. Generally, if the electrolyte ligand–metal ion
View Article Online
DOI: 10.1039/C6RA05115D
12
complex is weaker than the polymer–metal ion complex, the terpolymer can break easily the electrolyte
ligand–metal complex and the free metal ions easily form complex with terpolymer and hence removal
of metal ion is more. But, if electrolyte ligand–metal ion complex is stronger than polymer–metal ion
chelates, then the more metal ions form strong complexes with electrolyte ligand itself, which make
lower metal ion removal capacity of terpolymer. From Fig. 8 it can be seen, at higher concentration of

Na2SO4, the sulfate (SO42-) ions form strong complex with metal ions and so adsorption of metal ions
on terpolymer is reduced.
The effect of pH plays a unique role in the removal of metal ions. The reaction between 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 value ranging form 2 to 7. The metal ions
removal efficiency of terpolymer is shown in Fig. 9. It can be seen from the figure that adsorption
quantity is increased with the increasing aqueous solution of pH. At below pH 3.0, the removal
efficiency of terpolymer is low, This may be due to the fact that in acidic environment, the functional
groups of the terpolymer are highly protonated with hydrogen ion (H+) and lose the coordinate ability
with divalent metal ions because of the loss of negative charge. As the pH is increased in the range of
3.0 to 7.0, the functional groups of the terpoymer become less protonated, thus providing greater
coordination with heavy metal ions and hence the percentage of metal ions removal was increased [40].
The removal efficiencies of 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 best results for Ni2+(76), Cu2+(87), Pb2+(83), Cd2+(57), Hg2+(69)
and Zn2+(74%) . On increasing the pH above 7, the precipitation of metal ion may occur. Fig.10. shows
that the color of SASF terpolymer after metal ions adsorption is changed from dark orange to dark
brown which may due to more availability of metal ions on the surface of the terpolymer. The study of
adsorption mechanism indicates that the hydroxyl and amino groups on the terpolymer plays an
View Article Online
DOI: 10.1039/C6RA05115D
13
important role and the metal ions removal capacity of the terpolymer in weak acidic or neutral solutions
is better than in strong acidic solutions.
3.5. X-ray diffraction studies
The X-ray diffraction analysis was conducted to elucidate phase structure of the SASF terpolymer,
as shown in Fig. 11(a). The spectrum shows a broad diffraction peak between at 23 and 32 (2 theta)

angles. This spectral peak arises due to the formation of amorphous structure of the terpolymer.
However secondary peaks are observed with decrease in intensity around at 37, 43 and 50 (2 theta),
which is may be due to formation of slightly crystalline in nature. Therefore the obtained results
indicate that the terpolymer exist in the transition state between amorphous and crystalline nature. XRD
pattern of metal ions treated terolymer shown in Fig. 11(b). The broad diffraction characteristic peaks
appear at two theta angles between 24 and 35 for amorphous region, the secondary peaks are observed
at 40, 47 and 52 (2 theta). These observed peaks almost match with XRD pattern of untreated
terpolymer, which indicates unaffected morphological feature of the treated SASF terpolymer.
However after treatment, the characteristic peaks slightly shifted to higher 2 theta region due to the
conversion of some portion of SASF from amorphous to crystalline state. This obtained result is in
good agreement with structure of the terpolymer obtained from the SEM analysis earlier.
3.6. Morphological Study and EDAX Analysis
Surface morphologies of the SASF terpolymer before and after loading metal ions were
investigated by scanning electron microscopy. Fig. 12 represents the surface morphology of untreated
SASF terpolymer which has high porosity of honey bees hive (nest) like structure, hence it can be
easily accommodate metal ions into the its cavities and act as better adsorbent, which is evident form
different magnification of SEM images in Fig. 12(a-d). The SEM images of metal ions treated SASF
terpolymer is 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 different magnification of SEM images, as
shown in Fig. 13(a-d). After treatment, the porous morphology features remain unaffected.
View Article Online
DOI: 10.1039/C6RA05115D
14
The chemical composition of SASF terpolymer was characterized by energy dispersive X-ray
spectroscopy (EDAX) and the spectrum is shown in Fig. 14. The spectrum process shows around 67.24
% of C, 3.58 % of N, 22.35% of O and 6.83 % of S. Moreover, elements of C, N, O and S were
detected from the EDAX spectrum of SASF terpolymer which further confirms the presence of
sulfanilamide, salicylic acid and formaldehyde (SASF) in the terpolymer. Fig. 15 represents the SEM

images and their EDAX spectra of metal ions treated SASF terpolymer, which shows that the binding
of Ni2+, Cu2+, Pb2+, Cd2+, Hg2+ and Zn2+ metal ions and the uptake process happened on the membrane
throughout the SASF terpolymer. The SEM and EDAX results indicate that SASF terpolymer has an
extensive surface area, hence the porosity is suggested to bind the metal ions effectively.
3.7. In vitro biological studies of the SASF terpolymer
3.7.1. Antibacterial screening activity
The newly synthesized SASF terpolymer was screened for their antibacterial activity against
gram-positive Saphylococcus aureus (MRSA), Bacillus subtilis and gram-negative Salmonella typhi,
Escherichia coli bacterial strains by 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 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 inhibition zone (in mm) on agar plates are tabulated in Table 6.
The results obtained in this study clearly indicate that diameter of inhibition zone increases with
increasing concentration of the terpolymer. Obviously, high concentration the terpolymer exhibited
good antibacterial activity against the selected bacterial strains. For the SASF terpolymer sample, the
highest inhibition zone for Bacillus subtilis were 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 Saphylococcus aureus (MRSA) shows
View Article Online
DOI: 10.1039/C6RA05115D
15
lower inhibition at 25-250 µg, this may due to the multi peptidoglycan layer present in the bacterial
strain. The test results indicate the SASF terpolymer is efficient inhibitor of bacterial growth.
3.8. Cytotoxicity studies (in-vitro)
The cytotoxicity of the SASF terpolymer was tested against HeLa (mammalian cancer) cell line
using MTT assay. We examined the SASF terpolymer at four final concentrations such as 25, 50, 75

and 100 µg at 37 ± 1 ºC for 24 h. Suitable positive control was run and this experiment was repeated
thrice. Fluorescence morphology investigation of cell viability of control cells and treated HeLa cancer
cell are 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-e) shows that higher concentrations (75 and
100 µg) the most of cells zone were extending father than 1 cm beyond specimen, which indicate
severe cytotoxic activity of the terpolymer. Fig. 17 (b-c) shows that concentration of 25 and 50 µg, the
cells were zone round-shaped and loosely attaching degenerated cells under the specimen are observed,
which indicats slight cytotoxic activity against the cancer cells. The control gave none cytotoxic
activity as expected. Based on response around the test sample, the cytotoxic activity was graded, as
shown in Table 7. The achievement of numerical more than 2 are considered as cytotoxic effect. Since
the test sample achieved a numerical grade greater than 2 in 75 and 100 µg, reveals that SASF
terpolymer is considered as best cytotoxic agent at higher concentration.
4. Conclusion
We have successfully synthesized a novel sulfanilamide-salicylic acid-formaldehyde (SASF)
terpolymer via the condensation polymerization and its structure was confirmed by various spectral
studies. From thermal analysis the SASF terpolymer was found to possess higher degradation stability.
The batch equilibrium studies reveal 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
ions binding capacity of SASF terpolymer was confirmed by the ICP-OES, SEM and EDAX studies.
The antibacterial and cytotoxicity activity of SASF terpolymer were investigated. The antibacterial
View Article Online
DOI: 10.1039/C6RA05115D
16
studies showed that the SASF terpolymer possessed effective antibacterial properties against both
gram-positive Saphylococcus aureus, Bacillus subtilis and gram-negative Salmonella typhi,
Escherichia coli bacterial strains. The cytotoxicity studies revealed that the SASF terpolymer showed
strong cytotoxic activity against Hela (mammalian cancer) cell line. Since SASF terpolymer contains
alcoholic and amino groups, it plays a key role in the ions exchange phenomenon and the sulfa group

plays a major role for biological properties. Thus, SASF terpolymer could be used for removal of toxic
metal ions in industrial waste water treatment and immense 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, J. Kaiser, India’s Spreading Health Crisis Draws Global Arsenic Experts, Science, 1996,
274, 174–17.
[2] Clayton GD, Clayton FE Patty’s, industrial hygiene and toxicology, Wiley-Interscience, New York,
2C, 1994.
[3] European Commission DG ENV, E3, Project ENV.E3/ETU/2000/0058, Heavy metal in waste,
Final report, 2002.
[4] S. S. Ahluwalia, 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, third ed., Wiley, New York, 1958.
[7] Treavor H. Boyer, Philip C. Singer, Water Res. 2006, 40, 2865-2876.
[8] D.Prabhakaran, M.S. Subramanian, Talanta, 2003, 59, 1227.
[9] Manjusha M. Jadhaoa, L.J. Paliwalb, N.S.Bhavea, Desalination, 2009, 247, 456–465.
[10] Amith K. Naskar, Robert A. Walker, Sarah Proulx, Dan D. Edie, Amod A.Ogale, Carbon, 2005,
43, 1065-1072.
View Article Online
DOI: 10.1039/C6RA05115D
17
[11] Mohamed A. Riswan Ahameda, Duraisamy Jeyakumar, Abdul R. Burkanudeen, J. Hazard.
Mater., 2013, 248-249, 59-68.
[12] R.N. Singru, W.B. Gurnule, V.A. Khati, A.B. Zade, J.R. Dontulwar, Desalination, 2010, 263, 200-
210.
[13] Garland W. Fussell, Stuart L. Cooper, Biomaterials, 2004, 25, 2971-2978.

[14] Narendra P.S. Chauhan, Suresh C. Ameta, Polym. Degrad. Stab., 2011, 96, 1420-1429.
[15] W.B. Gurnule, P.K.Rahangdale, L.J. Paliwal, R.B. Kharat, React. Funct. Polym., 2003, 55, 255-
265.
[16] Raja S. Azarudeen, Mohamed A. Riswan Ahamed, Abdul R. Burkanudeen, Desalination, 2011,
268, 90-96.
[17] M.M. Patel, M.A. Kapadia, G.P. Patel, J.D. Joshi, React. funct. polym., 2007, 67, 746-757.
[18] Arh-Hwang Chen, Cheng-Yu Yang, Chia-Yun Chen, Chia-Yuan Chen, Chia-Wen Chen, J.
Hazard. Mater., 2009, 165, 1068-1075.
[19] Shadma Parveen, Tansir Ahamad, Nahid Nishat, Appl. Organomet. Chem., 2008, 22, 70–77.
[20] Wen Zou, Yun Huang, Jie Luo, Jia Liu, Changsheng Zhao, J. Membr. Sci., 2010, 358, 76-84.
[21] Abdul R. Burkanudeen, Raja S. Azarudeen, Mohamed A. Riwan Ahamed, Wasudeo B. Gurnule,
Polym. Bull., 2011, 67, 1553-1568.
[22] C.B. Boss, K.J. Fredeen, Concept, Instrumentation and Techniques in Inductively Coupled Plasma
Optical Emission Spectrometry, 2nd ed., Perkin-Elmer, Norwalk, CT, 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.
[24] (a) Qun He, Zheng Hu, Yin Jiang, Xijun Chang, Zhifeng Tu, Lina Zhang, J. Hazard. Mater., 2010,
175, 710-714.
[25] P. A. Wayne, National Committee for Clinical Laboratory Standards, NCCLS Approved standard
M27-A, USA, 1997.

View Article Online
DOI: 10.1039/C6RA05115D
18
[26] Ru Gao, Zheng Hu, Xijun Chang, Qun He, Lijun Zhang, Zhifeng Tu, Jianping Shi, J. Hazard.
Mater., 2009, 172, 324–329.
[27] R.S. Azarudeen, M.A.R. Ahamed, D. Jeyakumar, A.R. Burkanudeen, Iran. Polym. J., 2009, 18,
821–832.
[28] Bhavna A. Shah, Ajay V. Shah, Riddhish R. Bhatt, Iran. Polym. J., 2007, 16, 173-184.

[29] R.M. Silverstein, G.C. Bassler, Spectrometric Identification of Organic Compounds, second ed.,
John Wiley, New York, 1967.
[30] K. Nakanishi, Infrared Absorption Spectroscopy Practical, Nolden Day, INC and Nankodo Co.,
Ltd., Tokyo, 1967.
[31] E. Pretsch, P. Buhlmann, C. Afflolter, Structure Determination of Organic Compounds, Springer,
New York, 2000.
[32] M.A.R. Ahamed, R.S. Azarudeen, D. Jeyakumar, A.R. Burkanudeen, Int. J. Polym. Mater. 2011,
60, 124–143.
[33] J.A. Raj, C. Vedhi, A. Burkanudeen, P. Arumugam, P. Manisankar, Ionics, 2010, 16, 171-175.
[34] F. Cotton, G. Wilkinson, C. Murillo, M. Bochmann, Advanced Inorganic Chemistry, sixth ed.,
Wiley Interscience, New York, 1999.
[35] H. Dudley, I. Fleming, Spectroscopic Methods in Organic Chemistry, McGraw-Hill: UK, 1975.
[36] M. Riswan Ahamed, R. Azarudeen, M. Karunakaran, T. Karikalan, R. Manikandan, A.
Burkanudeen, Int. J. Chem. Eniviron. Eng., 2010, 1, 7.
[37] Xian-Long Wang, Kum Wan, Cheng-He Zhou, Eur. J. Med. Chem., 2010, 45, 4631-4639.
[38] Deepti B. Patle, Wasudeo B. Gurnule, Arabian J.Chem., (2011).
[39] Zahra Sekhavat Pour, Mousa Ghaemy, RSC Adv., 2015, 5, 64106-64118.
View Article Online
DOI: 10.1039/C6RA05115D
19
Scheme Caption:
Scheme 1. Synthesis sequence of SASF terpolymer.


View Article Online
DOI: 10.1039/C6RA05115D
20
Figure Captions:

Fig. 1. FT-IR Spectrum of SASF terpolymer.
Fig. 2. UV-Spectrum of SASF terpolymer.

Page 21 of 34
RSC Advances
Fig. 3. 1H NMR Spectrum of SASF terpolymer.

DOI: 10.1039/C6RA05115D
View Article Online
21

RSC Advances
Fig. 4. 13C NMR Spectrum of SASF terpolymer.

DOI: 10.1039/C6RA05115D
View Article Online
22

Page 22 of 34
View Article Online
DOI: 10.1039/C6RA05115D
23

Fig. 5. TGA and DTA Curves of SASF terpolymer.
Fig. 6. Adsorption capacity of Ni2+, Cu2+, Pb2+, Cd2+, Hg2+ and Zn2+ metal ions on SASF
terpolymer with different concentrations of NaCl.
View Article Online
DOI: 10.1039/C6RA05115D
24

terpolymer with different concentrations of Na2SO4.
terpolymer with different concentrations of NaNO3.
View Article Online
DOI: 10.1039/C6RA05115D
25

Fig. 9. pH dependence of metal ions uptake by SASF terpolymer.
Fig. 10. Images of (a) before and (b) after metal ions treated SASF terpolymer
View Article Online
DOI: 10.1039/C6RA05115D
26

Fig. 11. X-ray diffraction (XRD) patterns of (a) before and (b) after treated SASF terpolymer
View Article Online
DOI: 10.1039/C6RA05115D
27

Fig. 12. SEM images for metal ions unloaded SASF terpolymer with different magnification (a) 1000x,
(b) 2000x, (c) 2000x and (d) 3000x.
View Article Online
DOI: 10.1039/C6RA05115D
28

Fig. 13. SEM images for metal ions loaded SASF terpolymer with different magnification (a) 1000x,
(b) 2000x, (c) 3000x and (d) 5000x.
View Article Online
DOI: 10.1039/C6RA05115D
29

Fig. 14. EDAX Analysis and their spectrum process of SASF terpolymer.
View Article Online
DOI: 10.1039/C6RA05115D
30

Fig. 15. EDAX Analysis of metal ions loaded SASF terpolymer.
View Article Online
DOI: 10.1039/C6RA05115D
31
Fig. 16. Antibacterial activity of SASF terpolymer. Clear inhibition zone formed against the growth of
(a) B. subtilis, (b) S. typhi, (c) E. coli and (d) S. aureus (MRSA).
View Article Online
DOI: 10.1039/C6RA05115D
32
Fig. 17. Fluorescence images showing the viability of control cells and different concentration of
treated HeLa cancer cells on SASF terpolymer (after 24 hr treatment).
View Article Online
DOI: 10.1039/C6RA05115D
33
Table Captions:
Table 1. ICP OES: Operational conditions and instrumental parameters.

RF power 1450 Watts
Plasma gas flow rate 15 L/min
Auxiliary gas flow rate 0.2 L/min

Nebulizer carrier gas flow rate 0.75 (pneumatic) & 0.70 (ultrasonic)

Sample pump rate 1.5 mL/min
Plasma view mode axial and radial
Resolution high
Background correction 2 points per peak
Integration time (min-max) 2.5-5.0 Sec
Signal processing peak area (3-7 points per peak)
Replicates 3
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 upto 1cm
4 Severe Zone extending farther than 1 cm beyond specimen
Table 3. Elemental data of the SASF terpolymer.
Empirical formula Formula Elemental analysis (%) Color Yield (%)

of Repeating unit Mass Found (Calc.)
C H N S
C15H14N2O5S 334.35 Dark 82 %
51.80 4.69 7.87 8.41 orange
(53.88) (4.22) (8.38) (9.59)
View Article Online
DOI: 10.1039/C6RA05115D
34
Table 4. GPC data of the SASF terpolymer.
Formula mass of Polydispersity Polydispersity

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


Table 5. TGA data of the SASF terpolymer.
Temperature (ºC)
Degradation
Sample 150 250 350 450 550 Tmaxa (ºC) T50 b (ºC)
Temperature
Range (ºC)
Weight loss of terpolymer (%)
SASF 7.2 12.1 27.0 51.6 74.2 33.5 to 603.7 603.7 491
a
Maximum decomposition temperature.
b
Temperature at 50% weight loss
Table 6. Antibacterial effect of SASF terpolymer at different concentrations

Zone of inhibition (in mm) SASF terpolymer
Bacterial strain (Average of three measurements). Standard
used 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
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

Sulfonyl Amide Salicylic Acid

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Sulfonyl Amide Salicylic Acid

Uploaded by

Copyright:

Available Formats

View Article Online

This is an Accepted Manuscript, which has been through the

Accepted Manuscripts are published online shortly after

You can find more information about Accepted Manuscripts in the

Please note that technical editing may introduce minor changes

Synthesis, Characterization, Removal of toxic metal ions and in-vitro biological

V. Vasanthakumar, A. Saranya, A. Raja, S. Prakash, V. Anbarasu, P. Priya, V. Raj*

Advanced Materials Research Laboratory, Department of Chemistry, Periyar University,

RSC Advances Accepted Manuscript

*Correspondence: V. Raj, Professor and Head, Department of Chemistry, Periyar University,

RSC Advances Accepted Manuscript

much potential against Hela (mammalian cancer) cell line.

RSC Advances Accepted Manuscript

precipitation, ion-exchange, electro winning, electro-coagulation, reverse osmosis and solid-liquid

of metal ion chelating agents [9].

poly[(2-hydroxy-4-methoxy benzophenone) ethylene] terpolymer and its polychelates with

RSC Advances Accepted Manuscript

cations using ion-binding terpolymer involving 2-amino-6-nitro-benzothiazole and thiosemicarbazide

methacrylate–acrylic acid–vinyl pyrrolidone) terpolymer modified with polyethersulfone hollow fiber

screening of terpolymer prepared by polycondensation of anthranilic acid, urea with formaldehyde

have also been reported [21].

against on Hela (mammalian cancer) cell line.

2.1. Chemicals and regents

Terpolymer was synthesized by the condensation polymerization of sulfanilamide (0.1 M),

RSC Advances Accepted Manuscript

reaction sequence of the synthesis of SASF terpolymer is shown in Scheme 1.

2.3. Characterization of SASF terpolymer

the basis of a universal calibration curve generated by polystyrene standard.

RSC Advances Accepted Manuscript

dispersive X-ray spectroscopy (EDAX Oxford X-Act).

2.4. ICP-OES Measurement

2.5. Batch sorption experiments

electrolytes in different concentrations and pH in different ranges by batch equilibrium technique. A 50

mg of synthesized SASF terpolymer was suspended in 30 mL of electrolyte solutions such as NaCl,

RSC Advances Accepted Manuscript

ions to give the exact adsorbed metal ions concentration [24].

using the following equation:

2.6. In vitro biological studies of the SASF terpolymer

2.6.1. Antimicrobial properties of terpolymer

RSC Advances Accepted Manuscript

2.7. Anticancer study

2.7.2. MTT assay

3. Results and discussion

RSC Advances Accepted Manuscript

with the calculated values.

The average molecular weight of SASF terpolymer determined by gel permeation

3.2. Spectral analysis

3.2.1. FTIR spectral analysis

the structure of SASF terpolymer as given in Scheme 1.

3.2.2. UV-Visible spectral analysis

RSC Advances Accepted Manuscript

3.2.3. 1H NMR and 13C NMR Spectrum

3.3. Thermal behavior of the terpolymer

RSC Advances Accepted Manuscript

3.4. Binding capacity of SASF terpolymer with the metal ions

3.4.1. Effect of Electrolytes and pH

RSC Advances Accepted Manuscript

is better than in strong acidic solutions.

3.5. X-ray diffraction studies

RSC Advances Accepted Manuscript

3.6. Morphological Study and EDAX Analysis

% of C, 3.58 % of N, 22.35% of O and 6.83 % of S. Moreover, elements of C, N, O and S were

RSC Advances Accepted Manuscript