Synthesis and polymerization of a new hydantoin monomer with three halogen binding sites for developing highly antibacterial surfaces

Article abstract of DOI:10.1039/c8nj02743a

N-Halamine-based antibacterial polymers play a significant role in controlling microbe-associated surface contamination. Hydantoin-containing polymers are usually synthesized by tethering polymerizable moieties to 5,5′-dialkyl hydantoin. However, such app

Full text of DOI:10.1039/c8nj02743a

View Article Online
View Journal
NJC
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: R. K. Rai and A.
Jayakrishnan, New J. Chem., 2018, DOI: 10.1039/C8NJ02743A.
This is an Accepted Manuscript, which has been through the
Volume 40 Number
1 January 2016 Pages 1–846
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
Accepted Manuscripts are published online shortly after
New Journal of Chemistry
www.rsc.org/njc
A journal for new directions in chemistry
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. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
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, outlined
in our author and reviewer resource centre, 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.
ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

Please do not adjust margins
New Journal of Chemistry
Page 1 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
NJC
ARTICLE
Synthesis and Polymerization of a New Hydantoin Monomer With
Three Halogen Binding Sites for Developing Highly Antibacterial
Surfaces
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Rajani Kant Rai a, A. Jayakrishnana*
www.rsc.org/
N-halamine-based antibacterial polymers play
a significant role in controlling microbe-associated surface
contamination. Hydantoin-containing polymers are usually synthesized by tethering polymerizable moieties to 5, 5’-
dialkyl hydantoin. However, such approaches sacrifice the imide hydrogen in the molecule, thereby restricting the
halogen capture only to the amide nitrogen. In this paper, we report the synthesis of a novel polymerizable hydantoin
monomer containing one more amide group in addition to the amide and imide groups already present in hydantoin to
maximize the halogen capture and hence its antibacterial activity. The synthesized monomer was copolymerized with
methyl methacrylate (MMA) and styrene. The monomer and its copolymers were characterized using 1H and 13C NMR,
HRMS, FTIR, XPS, TGA, DTA and GPC. The oxidative chlorine content of the copolymers ranged from 0.9% to 1.21%. The
polymers were spin coated onto glass surface to study their antimicrobial potential. Polymer-coated glass surfaces
challenged with both Gram positive S. aureus and Gram negative E. coli bacteria using standard protocols showed total
kill of the bacteria within 15 minutes of exposure. These new hydantoin polymers showed many superior antibacterial
activities compared to many such polymers reported so far in the literature.
effective against a broad range of pathogenic microbes. The
hydantoin chemistry has been extensively reviewed [31-33].
1. Introduction
Microorganisms-based surface contamination in water purification
systems, in hospital and dental equipments, in medical devices, in
surgical gowns and gloves, in food processing and storage and in
household sanitation is a major growing concern [1-5]. To minimize
the transmission of the microbes through contamination of the
surfaces, several approaches have been examined for developing
highly effective antibacterial surfaces. Inactivation of surface-bound
microbes by light, coating surfaces using polymers doped with
quaternary ammonium compounds, iodine, phenols, salts of silver
and tin has been reported [6-13]. However, leaching of the biocide
and the resulting toxicity pose problems. Polymers having biocidal
groups such as N-halamines covalently bound to them, known as
polymeric biocides have emerged as potential antibacterial agents
in recent years [14-29]. The cyclic N-halamines, known as
hydantoins received much attention as they are able to regenerate
their biocidal activity on reaction with hypochlorite to generate the
biocidal chloramide function [30]. These hydantoin-containing
polymers are stable, durable, and non-toxic to humans and are
Generally, polymerizable hydantoins are synthesized by
tethering vinyl moieties to the imide hydrogen in 5, 5’-dialkyl
hydantoin thereby restricting the halogen capture only to the amide
nitrogen (Scheme 1). The biocidal activity of such polymers directly
depend on the halogen content in them and if both amide and
imide nitrogen atoms are available for halogenation, such polymers
should exhibit better biocidal activity. N-halamine monomers
wherein both nitrogens are available for halogenation are few in
the literature [27, 34, 35].
In a recent report, we have shown that a hydantoin monomer
where both the amide and imide functionalities are available for
halogen capture can be synthesized from α-amino acids such as L-
tyrosine and can be copolymerized with other vinyl monomers such
as methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate to
exhibit better biocidal activity [36].
In this report, we demonstrate the synthesis of a new polymerizable
vinyl hydantoin monomer containing an additional amide group, in
addition to the amide and imide functions that exist in hydantoin
thereby increasing its halogenation capacity and thereby its
antimicrobial properties [(d) in Scheme 1]. The synthesized
monomer was copolymerized with commercially available monom-
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 2 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
ARTICLE
Journal Name
pressure and stored in amber coloured bottles in the refrigerator.
BPO was recrystallized from methanol/chloroform. Dry DMF and
dry DCM were prepared by drying the solvents overnight over
barium oxide and calcium hydride respectively, followed by
distillation
under
vacuum.
THF
was
dried
over
sodium/benzophenone and distilled prior to use. All other reagents
and chemicals were of analytical or equivalent grade. Gram positive
S. aureus (ATCC29, 213) and Gram negative E. coli (BL21) were
employed for anti-bacterial study. Milli
throughout.
Q water employed
2.2. Characterization
1H NMR and 13C NMR spectra were recorded using a Bruker AV
500 MHz spectrometer (Bruker, Germany) using DMSO-d6 as the
solvent. High resolution mass spectra (HRMS) were obtained by
using Thermo Orbitrap Elite mass spectrometer (Thermo Scientific,
U.S.A.). Fourier transform infrared (FTIR) spectra were recorded
using a Perkin Elmer (Spectrum1 FT-IR) instrument using KBr pellets.
X-ray photoelectron spectra (XPS) was performed on Kratos Ultra-2
(Shimadzu Axis Supra) with Al Mg Kα radiation
weight (MW) of copolymers was determined using Gel permeation
chromatography (GPC) on Shimadzu instrument (Shimadzu,
. The Molecular
Scheme 1 Preparation of hydantoin monomers (a), (b) and (c) as reported in
the literature. The new hydantoin monomer with improved chlorine binding
site (d).
a
Japan) equipped with 510 pump, SPD-20A, R401 refractive index
detector and 7725 Rheodyne injector. The column used was
Phenogel OOH-646-K0 (Phenomenex, Hyderabad, India). The
mobile phase was THF at a flow rate of 1 mL/min. Polystyrenes of
known MW were employed as the standard. Thermo gravimetric
analysis (TGA) and differential scanning calorimetry (DSC) were
performed using a Perkin Elmer (TGA7, Q500Hi-Res TGA) and Perkin
Elmer (DSC7, Q200MDSC) instruments respectively at a heating rate
of 10 °C/min under nitrogen atmosphere. The elemental
composition of copolymers was determined using CHNSO Perkin
Elmer 2400 series analyzer. Coating of surface was achieved using
spin coater (Spin NXG-P1, West Bengal, India).
ers such as, MMA and styrene by free radical polymerization using
benzyl peroxide (BPO) as the initiator. The monomer and its XPS,
TGA, DTA and GPC. The oxidative chlorine content of the
copolymers ranged from 0.9% to 1.21%. Glass surface was modified
with the copolymers by employing the spin-coating method. Coated
surface was washed with aqueous bleach to activate their
chloramide functions and exposed to Gram positive S. aureus and
Gram negative E. coli bacteria. Surfaces modified with copolymer
containing 5 mole % of hydantoin monomer and MMA or styrene
exhibited 6 log kill (total kill) of the bacteria within 20 min, whereas,
15 min was enough to sterilize the surface coated with copolymer
containing 7.5 % mole % of hydantoin monomer and MMA or
styrene.
2.3. Synthesis of tert-butyl (4-hydroxyphenyl) carbamate (TBHC)
4-aminophenol (10 g, 91.64 mmol) and triethylamine (23 mL,
137.46 mmol) were dissolved in 150 mL of methanol in a 500 mL RB
flask. Di-tert-butyldicarbonate (22 mL, 100.8 mmol) was added to
the reaction mixture and stirred magnetically for 4 h at room
temperature. After completion of reaction, solvent was evaporated,
and the residue was purified using column chromatography using
1:4 ratio of ethyl acetate/hexane (Yield 96%). 1H NMR (500 MHz,
DMSO-d6): ꢀ = 9.05 (s, 1H, -NH), 8.99 (s, 1H, -OH), 7.21 (d, 2H, J =
7.5 Hz, Aromatic H), 6.64 (d, 2H, J = 9 Hz, Aromatic H), 1.45 (s, 9H,
Boc) 13C NMR (125 MHz, DMSO-d6): ꢀ = 153.46, 152.95, 131.46,
120.41, 115.46, 78.88, 20.65. HRMS (ESI): Calculated mass
C11H15NO3 [M+ Na]+ = 232.0950 and observed mass = 232.0953.
FTIR: 3367 cm-1 (O-H stretch), 2979 cm-1, 1700 cm-1, 1526 cm-1,
1438 cm-1, 1235-1168 cm-1, 1053 cm-1, 830 cm-1, 767 cm-1 and 630
cm-1.
2. Experimental section
2.1. Materials
Hydantoin acetic acid, 4-aminophenol, di-tert-butyldicarbonate
(BoC), BPO, (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) (HBTU), N, N-diisopropylethylamine (DIPEA),
4-dimethylaminopyridine (DMAP), silica gel for column
chromatography (100-200 mesh), MMA and styrene were
purchased from Sigma Aldrich (Bangalore, India). Trifluroacetic acid
(TFA) and methacryloyl chloride were procured from Alfa Aesar
(Chennai, India). Solvents such as N,N'-dimethyl formamide (DMF),
dichloromethane (DCM), Tetrahydrofuran (THF), ethyl acetate,
toluene, methanol, hexane etc. were from S. D. Fine Chemicals,
Mumbai, India. MMA and styrene were washed with aqueous
sodium hydroxide to remove the inhibitor, followed by water, dried
over anhydrous sodium sulphate and distilled under reduced
2.4 Synthesis of 4-((tert-butoxycarbonyl)amino)phenyl) metha-
crylate (TBPM)
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 3 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
Journal Name
ARTICLE
ꢀ = 166.37, 146.97, 141.06, 136.08, 127.44, 122.29, 114.50, 18.60.
HRMS (ESI): Calculated mass C10H11NO2 [M+ H]+ = 178.0868 and
observed mass = 178.0857. FTIR: 3500-3300 cm-1, 2970 cm-1, 1714
cm-1, 1632 cm-1, 1509 cm-1, 1400 cm-1, 1190-1143 cm-1, 1035 cm-1,
830 cm-1, 760 cm-1 and 630 cm-1.
2.6 Synthesis of 4-(2-(2,5-dioxoimidazolidin-4yl-)acetamido)phenyl
2-oxoproanoate (DAPO)
Hydantoin acetic acid (3 g, 19 mmol), DIPEA (8.2 mL, 47.5 mmol)
and HBTU (8.65 g, 22.8 mmol) were added sequentially into 15 mL
dry DMF in
a 100 mL RB flask. After stirring the contents
magnetically for 10 min, APM (3 g, 16.95 mmol) was added to the
reaction mixture and stirred for 12 h. The reaction mixture was
taken up in ethyl acetate (100 mL) and washed with dilute NaHCO3
followed by water. The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent was evaporated in vacuum. The
crude product was then purified using column chromatography
using 1:1 ethyl acetate/hexane to obtain the hydantoin monomer in
75% yield. 1H NMR (500 MHz, DMSO-d6): ꢀ = 10.64 (s, 1H, imide H)
10.14 (s, 1H, amide H), 7.94 (s, 1H, amide H), 7.62 (s, 2H, Aromatic
H), 7.11 (d, 2H, J= 10 Hz, Aromatic H), 6.26 ( s, 1H, =CH), 5.88 (d, 1H,
J = 1 Hz, =CH2), 4.32 ( t, 1H, J = 10 Hz, -CH), 1.95 (s, 3H, -CH3). 13C
NMR (125 MHz, DMSO-d6): ꢀ = 176.18, 167.81, 165.92, 158.08,
146.27, 137.08, 135.76, 128.10, 122.42, 120.46, 55.10, 38.31, 18.53.
HRMS (ESI): Calculated mass C10H11NO2 [M+ Na]+ = 340.0909 and
observed mass = 340.0924. FTIR: 3550 cm-1, 3310 cm-1, 1742 cm-1,
1647 cm-1, 1145-1210 cm-1, 760 cm-1 and 700 cm-1. The synthesis of
the monomer is depicted in Scheme 2.
Scheme 2 The synthesis of novel hydantoin monomer
THBC (5 g, 23.9 mmol) was dissolved in 100 mL of dry DCM in a
double necked RB flask under nitrogen environment. DIPEA (8.24
mL, 47.8 mmol) and catalytic amount of DMAP were added to the
reaction mixture followed by drop-wise addition of methacryloyl
chloride (2.57 mL, 26.3 mmol) using a syringe at 0°C. The contents
were magnetically stirred for 3 h at room temperature after which
the mixture was filtered, the organic layer was taken up in ethyl
acetate (300 mL), washed with dilute NaHCO3 (200 mL) followed by
water, dried over anhydrous Na2SO4, filtered and the solvent
evaporated in vacuum. The residue was then purified by column
chromatography using 1:5 ethyl acetate/hexane to give the product
in 85% yield. 1H NMR (500 MHz, DMSO-d6): ꢀ = 9.43 (s, 1H, -NH),
7.47 (d, 2H, J= 10 Hz, Aromatic H), 7.04 (d, 2H, J = 10 Hz, Aromatic
H), 6.25 (s, 1H, =CH), 5.87 (s, 1H, =CH2), 1.98 (s, 3H, -CH3), 1.47 (s,
9H, Boc). 13C NMR (125 MHz, DMSO-d6): ꢀ = 166.0, 153.29, 145.53,
137.63, 135.78, 128.01, 122.31, 119.33, 79.61, 28.55, 18.52. HRMS
(ESI): Calculated mass C11H15NO3 [M+ Na]+ = 300.1212 and observed
mass = 300.1211. FTIR: 3367 cm-1, 2970 cm-1, 1714 cm-1, 1632 cm-1,
1509 cm-1, 1400 cm-1, 1190-1143 cm-1, 1035 cm-1, 830 cm-1, 760 cm-
1 and 630 cm-1.
2.7 Preparation of copolymers
DAPO was copolymerized with MMA and styrene. A pressure
tube was charged with 5 or 7.5 mole % of DAPO with MMA and
styrene in 20 mL dry THF. After adding BPO (0.1 mole %), the
contents were deoxygenated by flushing with dry nitrogen for 30
min. After sealing the tube, polymerization was performed by
heating the tube at 70 °C under continuous magnetic stirring for 24
h. The obtained copolymer was precipitated in cold methanol,
filtered, washed with methanol and dried under vacuum.
2.8 Coating of glass surface
2.5 Synthesis of 4-aminophenyl methacrylate (APM)
To develop the antibacterial surface, copolymer films were
coated on a glass cover slips (2.5 cm x 2.5 cm) using a spin coater
operating at 2500 rpm for 45 sec from a 10 % solution in toluene
[37]. The coated cover slips were dried in an air oven at 50 °C
overnight.
TBPM (5g, 18.03 mmol) was dissolved in 100 mL dry DCM,
followed by drop-wise addition of TFA (16.5 mL, 144 mmol) at 0°C
and stirred for 2 h to complete the reaction. 200 mL of saturated
solution NaHCO3 was added to the reaction mixture and stirred for
20 min. The contents were transferred to a separating funnel and
150 mL of DCM was added. The organic layer was washed twice
with 1% KOH followed by water respectively. After drying over
anhydrous Na2SO4 followed by evaporation of solvent in vacuum,
the compound was purified by column chromatography using 1:4 of
ethyl acetate/hexane to give the product in 60% yield. 1H NMR (500
MHz, DMSO-d6): ꢀ = 6.78 (d, 2H, J = 10 Hz, Aromatic H), 6.56 (d, 2H,
J= 9Hz, Aromatic H), 6.20 (s, 1H, =CH), 5.82 (t, 1H, J = 3 Hz, =CH2),
5.05 (s, 2H, -NH2), 1.97 (s, 3H, -CH3). 13C NMR (125 MHz, DMSO-d6):
2.9 Chlorination of copolymers
The spin coated glass cover slips were treated with 9-12%
sodium hypochlorite at pH 7 for 1.5 h, washed thoroughly with
water and dried at 45 °C for 1 h to remove free chlorine from the
surface [38].
2.10 Determination of oxidative chlorine content
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 4 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
ARTICLE
Journal Name
The oxidative chlorine content in copolymers was determined characterized using 1H and 13C NMR, HRMS and FTIR. In 1H NMR,
by the modified iodometric/thiosulfate titration procedure [18]. appearance of singlet at ꢀ = 1.55 ppm suggested the protection of
Chlorinated copolymers (100 mg) were dissolved in 20 mL DMF to NH2 group with Boc (See supplementary information, Figure S1). In
which 20 mL water containing 1.0 % acetic acid and 1 g potassium the 13C NMR spectrum, appearance of aliphatic and carbonyl
iodide were added. The mixture was stirred for 1 h at room carbons of Boc at ꢀ = 28.65 and 153.46 ppm respectively further
temperature under nitrogen atmosphere. The released iodine was confirmed the formation of TBHC (Figure S2). In the FTIR spectrum,
titrated against a 0.01 M aqueous sodium thiosulfate till the end appearance of peaks at 1700 cm-1 (C=O stretching) and at 1053 cm-1
point. Blank titrations were performed under the same conditions (C-O-C stretching) again confirmed the BoC protection of amine
(without polymer) to serve as controls. The percentage of the group (Figure S3, a).
chlorine content was calculated according to the following
equation,
Percentage chlorine =
3.2 Synthesis of TBPM
ꢌꢍ
ꢃ
ꢉ
ꢁꢂ.ꢂ × ꢄꢅꢆ ꢇꢄꢈ ×ꢊꢋ
× ꢋ.ꢋꢊ × 100
TBPM was synthesized by esterification of THBC with
methacryloyl chloride. Disappearance of phenolic proton signal and
appearance of both olefinic proton (ꢀ = 5.87 and 6.25 ppm) as well
as –CH3 proton (ꢀ = 1.94 ppm) suggested the esterification with
methacryloyl chloride (Figure S4). Further, the 13C NMR spectrum
exhibited an additional aliphatic carbon signal, carbonyl carbon
(C=O) and olefinic carbon (C=C) at ꢀ =18.52, 166.0 and 135.78,
137.63 ppm respectively confirming the synthesis of TBPM (Figure
S5). Appearance of C=C stretching suggested the tethering of
polymerizable moiety via esterification (Figure S3, b).
ꢎ × ꢏꢐ
Where, VCl and Vo are the volumes (mL) of sodium thiosulphate
solution consumed in the titration of the polymer solution and the
control, respectively, and Wg is the weight of the polymer. Each test
was repeated three times, and the average was recorded.
2.11 Antibacterial assessment
E. coli (BL21) and S. aureus (ATCC29 213) were used as target
strains to evaluate the antibacterial activity of the chlorinated
copolymers. E. coli and S. aureus were grown in nutrient broth for
12 h at 37 °C and 180 rpm. The cells were harvested and washed
with phosphate buffered saline (pH 7.4). A “Sandwich assay”
method as reported recently [36] was adopted to study the
antibacterial potential of modified glass surface. Glass cover slips
coated with the copolymers were challenged using 50 µL of 6.0-6.5
log10 CFU/mL bacterial cells and the bacterial suspension was
covered with another polymer-coated glass cover slip. A small
sterilized beaker was placed on top of the “sandwich” to ensure the
contact of bacteria with the surface. After various contact times,
the cover slips were immersed in 5 mL of a 0.03 wt % aqueous
sodium thiosulfate to completely neutralize the active chlorine in
the films without affecting bacterial growth. After vigorously
shaking the solution for 5 min, an aliquot of the solution was serially
diluted and 100 μL of each dilution was plated onto nutrient agar.
Unchlorinated films were used as control. Viable bacterial colonies
on the agar plates were counted after incubation at 37 °C for 24 h.
The percentage reduction was calculated using the following
formula,
3.3 Synthesis of APM
TFA was employed for deprotection of Boc to synthesize APM
to generate amine group. Appearance of a singlet at ꢀ = 5.05 ppm
and disappearance of Boc signal at ꢀ = 1.47 ppm evidenced the
generation of amine group (Figure S6). Further, the disappearance
of the aliphatic carbon peak at ꢀ =28.65 ppm and carbonyl peak at
ꢀ=153.29 ppm suggested Boc deprotection (Figure S7). The
appearance of the characteristic two bands for primary amine (–
NH2) at 3400-3500 cm-1 in the FTIR spectrum confirmed the
generation of amine group (Figure S3, c).
3.4 Synthesis of DAPO
Hydantoin acetic acid was coupled to the amino group of APM
to obtain DAPO. In 1H NMR, the appearance of imide and amide
protons at ꢀ = 10.64 ppm and 10.14 ppm respectively as a singlet
and doublet and the peak at ꢀ = 4.37 ppm of –CH of hydantoin ring
confirmed the coupling of hydantoin acetic acid with NH2 group to
form polymerizable hydantoin monomer (Figure 1). In addition, in
the 13C NMR, amide and imide carbonyl carbons appeared at 176.18
and 158.08 ppm respectively (Figure S8). In FTIR, DAPO containing
benzene ring showed corrugated peaks at 700 cm-1 and aromatic C-
H bending at 760 cm-1. Moreover, functional groups such as C=O,
C=C, C-N and N-H gave their corresponding peaks at 1742 cm-1,
1647 cm-1, 1210 cm-1 and 3310 cm-1 respectively, whereas C-O-C
stretching appeared in the range of 1145-1210 cm-1 confirming the
structure of the monomer (Figure 2, b).
Percentage Reduction = ꢑꢇꢒ × 100
ꢑ
Where X is the number of CFU on control plate and Y is the number
of CFU on experimental plate. The log reduction was then
calculated as,
Log reduction = log(X – Y).
The plate counting was done in triplicate.
3. Results and discussion
3.1 Synthesis of TBHC
3.5 Synthesis of poly(DAPO-co-MMA)
TBHC was synthesized by protecting the amine group in 4-
aminophenol using Boc anhydride. The compound was
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 5 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
Journal Name
ARTICLE
Fig.1 1H NMR spectrum of hydantoin monomer (DAPO)
Fig. 3 1H NMR spectrum of copolymer Poly(DAPO-co-MMA)
Fig. 2 FTIR spectrum PMMA (a), DAPO (b), Poly(DAPO-co-MMA) (c) and
Chlorinated Poly(DAPO-co-MMA) (d)
Fig. 4 XPS analysis of PMMA (a), Poly(DAPO-co-MMA) (b) and Chlorinated
Poly(DAPO-co-MMA) (c)
suggesting the incorporation of DAPO in PMMA. Moreover,
disappearance of vinylic protons from DAPO (ꢀ = 6.26 and 5.88
ppm) in the copolymer confirmed the formation of the poly(DAPO-
co-MMA The elemental composition of the copolymers is shown in
Table 1. Oxidative chlorine content was quantified through
iodometric titration. The oxidative chlorine content of the
copolymers increased on increasing the amount of hydantoin
monomer in the feed. XPS analysis was performed to examine the
composition of polymer surfaces. The XPS spectrum of PMMA,
showed peaks of C 1s and O 1s (Figure 4a). Poly(DAPO-co-MMA)
spectrum gave additional peak of N 1s at 400 eV along with O 1s
and C 1s. Presence of N 1s and O 1s signal in the spectrum is proof
for copolymerization of hydantoin monomer with PMMA (Figure
4b). The advent of additional peak for Cl 2p at 200 eV in chlorinated
polymer sample evidenced the N-H to N-Cl transformation (Figure
4c). Identical C 1s and O 1s spectrum of chlorinated and non-
chlorinated sample suggest that the chlorination treatment had no
damaging effect on the polymeric carbon skeleton. All the spectra
The formation of the copolymer was confirmed by FTIR and NMR
spectroscopy). In the FTIR spectra, PMMA exhibits a characteristic
peak at 1740 cm-1 corresponding to the stretching vibrations of C=O
and peaks at 1450 and 2990 cm-1 due to the bending and stretching
vibrations of C-H. The O=C-O-C ester stretching peak appears in the
range of 1150-1200 cm-1 (Figure 2, a). The spectrum of DAPO
showed characteristic peaks at 1742 cm-1, 1647 cm-1, 1210 cm-1 and
3310 cm-1 for C=O, C=C, C-N and N-H functional groups. (Figure 2,
b). The introduction of N-H stretching
vibration at 3310 cm-1 and diminishing of the stretching vibration
of C=C at 1647 cm-1 suggested the formation of the copolymer
poly(DAPO-co-MMA) (Figure 2, c). When the copolymer film was
chlorinated, the shrinkage of the vibrational peak of N-H in the FTIR
spectrum suggested the successful transformation of N-H to N-Cl
(Figure 2, d). The 1H NMR spectrum of copolymer (Figure 3)
showed the presence of amide and imide proton of hydantoin ring
at ꢀ = 10.21 and 10.69 ppm respectively and the resonance peaks of
O-CH3, CH3 and CH2 at ꢀ= 3.6, 2.15 - 1.9 and 0.99-0.81 ppm of MMA
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 6 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
ARTICLE
Journal Name
Fig. 6 XPS analysis of Polystyrene (a), Poly(DAPO-co-styrene) (b) and
Chlorinated Poly(DAPO-co-styrene) (c)
Fig. 5 FTIR spectrum Polystyrene (a), DAPO (b), Poly(DAPO-co-styrene (c)
and Chlorinated Poly(DAPO-co-styrene) (d)
poly(DAPO-co-styrene) spectrum gave additional peak of N 1s and O
1s at 400 eV and 532 eV respectively along with C 1s to provide the
important evidence for copolymerization of hydantoin monomer
with polystyrene (Figure 6, b).The appearance of additional peak for
Cl 2p at 200 eV in chlorinated polymer sample revealed the N-H to
N-Cl transformation (Figure 6, c). Identical C 1s and O 1s spectrum
of chlorinated and non-chlorinated sample suggested that the
chlorination treatment had no adverse effect on the polystyrene
backbone.
exhibited Si 2s and Si 2p signal at 154 eV and 103 eV respectively
from Si support (glass) used for immobilization of the sample.
3.6 Synthesis of poly(DAPO-co-styrene)
The FTIR spectrum of polystyrene showed (Figure 5, a) the
characteristic peaks at 1601, 1493 and 1451 cm-1 for C=C stretching
and at 756 cm-1 for C-H bending of benzene ring, while corrugated
vibration of benzene ring was observed at 698 cm-1. Peaks at 2921
and 3024 cm-1 can be attributed to the symmetric bending vibration
of –CH bond. Appearance of additional stretching vibrational peak
for C=O and N-H at 1726 and 3319 cm-1 in poly(DAPO-co-styrene)
spectrum confirmed the successful synthesis of the copolymer
(Figure 5, c). Diminishing of NH stretching vibration in chlorinated
copolymer suggested the generation of N-Cl (Figure 5, d). Further,
XPS analysis of the polymers was performed to confirm again the
incorporation of hydantoin moiety in polystyrene backbone and
chlorination of amide and imide function. XPS spectrum of
polystyrene displayed peak at 285 eV for C 1s (Figure 6, a).
3.7 Molecular weight of copolymers
GPC analysis was performed to determine the MW and
polydispersity index (PDI) of the copolymers. The weight average
molar mass Mw and PDI for poly(DAPO-co-MMA) were 49,000 Da
and 2.71 respectively, whereas poly(DAPO-co-styrene) copolymer
exhibited Mw of 30,000 and PDI of 2.5. The Mw of the copolymers
was good enough to coat the glass surface forming thin films that
were stable during the antibacterial and durability studies
Table 1. Elemental analysis of polymers using CHNSO analyser and iodometric titration
Sample
Mole % of DAPO in Polymers
C (%)a
H (%)a
N (%)a
Cl (%)b
PMMA
0%
57.2
14.28
0
0
5%
59.09
58.03
13.83
14.19
4.72
5.83
0.91 ± 0.05
1.21 ± 0.02
7.5%
P(DAPO-co-MMA)
Polystyrene
0%
5%
92.58
84.40
83.30
7.40
7.61
7.32
0
0
4.30
5.45
0.85 ± 0.07
1.18 ± 0. 04
P(DAPO-co-styrene)
7.5%
a Obtained from CHNSO analysis. b Obtained from iodometric/ thiosulfate titration
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 7 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
Journal Name
ARTICLE
3.8 Elemental analysis
Elemental composition of the polymers was determined using
CHNOS analyzer in CHN mode. Oxidative chlorine content was
quantified through iodometric/thiosulfate titration. Quantitative
analysis of elements in the copolymers is shown in the Table 1.
Iodometric/thiosulfate titration method based quantitative analysis
showed the expected increment in oxidative chlorine content on
increasing the feed ratio of hydantoin monomer in both
copolymers.
Fig. 8 (B) DSC curve of Polystyrene (a) and Poly(DAPO-co-styrene) (b)
3.9. Thermal analysis
TGA was carried out to study the thermal stability of the
synthesized copolymers (Figure 7). Poly(DAPO-co-MMA) exhibited
degradation at three different stages. Degradation of the copolymer
started at 150
only 5% of the polymer was left behind at 980
styrene) weight loss process started at approximately 310
°
C, 50 % degradation was observed at 400
C. In poly(DAPO-co-
C, after
°C and
°
°
Fig. 7 TGA curve of Poly(DAPO-co-styrene) (a) and Poly(DAPO-co-MMA) (b)
that continuous weight loss was observed in response to increase in
temperature. 50% degradation was observed at approximately 400
°
C. Only 1.7 % of the material was left at 450 °C. DSC study of the
polymers and its corresponding copolymers were performed. The
glass transition temperature (Tg) for poly(DAPO-co-MMA) was
observed at 135
was 150 C (Figure 8 A and B). The dramatic increase in the Tg of the
copolymers compared to that of PMMA (105 C) and polystyrene
(95 C) could be attributed to bulky aromatic and hydantoin rings of
°C while in the case of poly(DAPO-co-styrene) it
°
°
°
DAPO in the copolymer.
3.10 Antibacterial assessment
Transfer of oxidative halogen from amide or imide nitrogen to
bacterial cells results in suppressing the metabolic process and
eventually expiration of cells. Therefore, amount of oxidative
chlorine attached to chloramide function is directly related to
antibacterial activity. Antibacterial sandwich assay was performed
by time dependent plating experiments to analyze the antimicrobial
properties of the copolymers. Control experiments with modified
Fig. 8 (A) DSC curve of PMMA (a) and Poly(DAPO-co-MMA) (b)
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 8 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
ARTICLE
Journal Name
Fig. 9 Optical photomicrographs showing the bacterial culture plates of S. aureus and E.coli at different time intervals. Control (a), chlorinated
poly(DAPO-co-MMA) containing 5 mole % of DAPO at 5 min (b), 10 min (c) and 15 min (d).
Fig. 10 Log reductions in bacterial growth on poly(DAPO-co-MMA) with 5 mole % (A) and 7.5 mole % (B) of DAPO against S. aureus and E. coli
and non-chlorinated surfaces showed dense growth of bacterial poly(DAPO-co-MMA) and poly(DAPO-co-styrene) exhibited almost
colony (Figure 9 a). However, bacterial strains treated with modified similar antimicrobial activity against both S. aureus and E. coli as the
and chlorinated surfaces showed reduction in growth with time, chlorine content in both the copolymers were almost same. Surface
suggesting their excellent bactericidal action (Figures 9 b, c and d). modified with the copolymer having 7.50 mole % of hydantoin
Copolymers with two different feed ratios of hydantoin monomer monomer exhibited improved bactericidal action over the
exhibited time dependent log reductions against S. aureus and E. copolymer with 5 mole % of hydantoin monomer in PMMA or
coli as shown in Figures 10 A and B. Glass surfaces coated with
polystyrene. Surfaces with 7.5 mole % of hydantoin in PMMA or
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 9 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
Journal Name
ARTICLE
Table 2 Durability and regenerability study of P(DAPO-co-MMA)
polystyrene copolymer exhibited 0.9 log reduction in 5 min. of
contact time, which increased to 3.1 log reduction in 10 min and
eventually total kill (6 log reduction) after 15 min of exposure
against S. aureus. In case of E. coli decreased killing was observed at
5 min and 10 min of exposure, but the reduction was less compared
to S. aureus due to presence of an extra layer of lipopolysaccharide
outer membrane, but total kill (6 log reduction) was observed at 15
min. Surfaces with 7.5 mole % of DAPO displayed the total kill in 20
min of contact time against both S. aureus and E. coli. The above
results again suggest that oxidative chlorine content in the
copolymer is directly related to the biocidal action. Choi et al [39]
reported that 5, 5’-dimethyl hydantoin attached to poly(ethylene
glycol) containing only amide hydrogen for halogen capture
exhibited a 5 log reduction in 60 min of contact time against E. coli,
when the polymer had 0.3% oxidative chlorine content. Xiu et al,
reported that 3-(4’-vinylbenzeneyl)-5,5 dimethyl hydantoin
(VBDMH )/polyurethane semi-interpenetrating polymer exhibited
total kill in 30 min of contact time against staphylococcus species
[40]. Incorporation of VBDMH in polyurethane resulted in a weight
increase from 32% to 56% but oxidative chlorine content was found
to be between 4.5 % and 5.5% due to single chloramide generating
group. Another report from same group observed total kill in 4 h
against staphylococcus when single amine based halogen binding
site containing monomer (N-chloro-2,2,6,6-tetramethyl-4-(piperidyl
methacrylate) was incorporated in to polyurethane [41]. The huge
difference in weight % of halamine (7.5 %) and chlorine content
(0.55 %) in above report is due to single halogen binding site, which
eventually resulted in reduced biocidal action. Sun and Sun [16]
reported that a vinyl benzyl derivative of 5,5′-dimethylhydantoin
copolymerized with MMA exhibited 2 log reduction in 30 min
against E. coli. At the same time, hydantoin monomer
copolymerized with vinyl acetate exhibited a 4 log reduction. Dong
et al [42] reported that 11% of 3-allyl-5,5’-dimethylhydantoin
copolymerized with MMA coated on magnetic silica nanoparticle
exhibited total kill in 30 min of contact time against S. aureus. In a
recent study, Bai et al [29] reported a 60 min contact time for total
kill of E. coli when exposed to electrospun polymer fibres of PMMA
containing 10% 1,3-dichloro-5,5′-dimethyl hydantoin. In another
study using hydantoin-containing polyurethanes, total kill was
observed in 30 min contact time for S. aureus but not for E. coli
when the polymer contained 0.8% of biocide [24]. In a recent report
it was shown that when the N-halamine monomer had three
halogen binding sites, the polymer exhibited improved antibacterial
activity with a 6 log reduction in bacterial growth against E. coli and
S. aureus in 15 min [43]. The above results support the observation
that increasing the halogen binding sites in the polymer results in
decreased contact time for biocidal activity. In our previous report,
we have shown that when both amide and imide hydrogens of
hydantoin were available for halogen capture, the polymers having
chlorine content in the range of 0.6 to 0.9 % displayed total kill in 30
min of contact time for both S. aureus and E. coli [36]. In
comparison to the previous reports, the present study showed
much improved antimicrobial activity against both Gram positive
No. of
washes
Antibacterial
evaluation
Exposure
time
(min)
Chlorine
content
(wt%)
0
No colony formation
No colony formation
No colony formation
No colony formation
No colony formation
Colony formation
15
15
15
15
15
15
1.21
1.07
0.9
5
15
25
35
38
0.6
0.4
0.2
and Gram negative strains because the hydantoin moiety in the
copolymers possessed more halogen binding sites.
3.10 Durability and rechargeability
On treatment with sodium hypochlorite, the hydantoin based
copolymers of both MMA and styrene showed excellent
regeneration of biocidal chloramide function. The stability and
regenerability studies were conducted using poly(DPAO-co-MMA)
that has been stored in the dark condition at 4°C for 6 months
against E. coli. Durability of the copolymer surface against repeated
washing is summarized in Table 2. Up to 35 washes, no colony
formation was observed although a gradual decrease in the
percentage of chlorine content was observed. The chlorine content
of the polymer drops from 1.21% to 0.4% (66% drop) only after 35
washes and even with 0.4%, it was still bactericidal as there was no
colony formation. The total chlorine content is due to the
halogenation of the 3 chlorine binding sites in the copolymer. There
is a possibility that HCl elimination can take place between the H
atom on the carbon at position 7 and the chlorine at position 8 on
chlorination (See structure in Fig. 3) leading to loss in chlorine
content. If this elimination took place, the chlorine content should
drop down by 33% of the initial value (1.21 to 0.81%). This happens
after around 15 washes suggesting that the elimination is not
happening rapidly. In order to examine this, the ATR-FTIR spectrum
of the copolymer was recorded after different washes. As expected,
there was change in the intensity of the -NH vibrational band
pointing to HCl elimination (Fig. S9). The continued decrease in
chlorine content however, could not be explained by HCl
elimination alone. There is a possibility that the HCl elimination
further catalyses the hydrolysis of the adjacent amide bond
resulting in further loss of chlorine. In order to examine this, we
soaked the copolymer films in 0.1 M HCl for 24 h and recorded the
ATR-FTIR spectra of the films before and after soaking. There was a
decrease in the intensity of the -NH vibrational band in the
spectrum suggesting the hydrolysis of the amide bond (Fig S10). The
ester bond in the copolymer is less susceptible to hydrolysis as
PMMA is known to resist hydrolysis under mild acid or alkaline
conditions because of its hydrophobicity. Therefore, the fall in
chlorine content up on repeated washing and recharging could be
attributed to the elimination of HCl and the liberated HCl catalysing
the hydrolysis of the amide bond resulting in further decrease in
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 10 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
ARTICLE
Journal Name
6
7
8
9
L. Caballero, K. Whitehead, N. Allen and J. Verran, Dyes and
Pigments, 2010, 86, 56-62.
T. Zuccheri, M. Colonna, I. Stefanini, C. Santini and D.
Gioia, Materials, 2013, 6, 3270-3283.
G. Cheng, H. Xue, G. Li and S. Jiang, Langmuir, 2010, 26,
10425-10428.
J. Tiller, C. Sprich and L. Hartmann, Journal of Controlled
Release, 2005, 103, 355-367.
chlorine content. However, it is noteworthy that the copolymer
retains its antibacterial activity even up to 35 washes. The issue for
HCl elimination can be overcome for instance, by substitution of the
H atom on the carbon at position 7 with a methyl group. However,
it is important to point out that the more number of halogen sites
incorporated in the copolymer as demonstrated here, the higher its
antibacterial activity.
10 H. Tsuchiya, T. Shirai, H. Nishida, H. Murakami, T. Kabata, N.
Yamamoto, K. Watanabe and J. Nakase, Journal of
Orthopaedic Science, 2012, 17, 595-604.
11 N. Xu and D. Ding, RSC Advances, 2015, 5, 79820-79828
12 I. Perelshtein, G. Applerot, N. Perkas, E. Wehrschetz-Sigl, A.
Hasmann, G. Guebitz and A. Gedanken, ACS Applied
Materials & Interfaces, 2009, 1, 361-366.
13 R. Ortí-Lucas and J. Muñoz-Miguel, Antimicrobial Resistance
& Infection Control, 2017, 6.
14 Y. Sun and G. Sun, Journal of Applied Polymer Science, 2001,
80, 2460-2467.
15 L. Bastarrachea, L. McLandsborough, M. Peleg and J.
Goddard, Journal of Food Science, 2014, 79, E887-E897
16 Y. Sun and G. Sun, Journal of Polymer Science Part A: Polymer
Chemistry, 2001, 39, 3348-3355.
17 Y. Liu, Q. He, R. Li, D. Huang, X. Ren and T. Huang, Fibers and
Polymers, 2016, 17, 2035-2040
4. Conclusions
In this report, a novel strategy to synthesize a polymerizable
hydantoin monomer having three halogen binding sites, without
utilizing the imide position of hydantoin ring, is demonstrated. The
monomer was copolymerized with MMA and styrene to yield
copolymers having a halogenation capacity of 0.9 to 1.21%. Glass
surfaces modified with the copolymers containing higher feed of
the hydantoin monomer exhibited total kill in 15 min, whereas, the
copolymer with lesser feed displayed total kill in 20 min of contact
time. These results suggested that the bactericidal action is
dependent on the load of oxidative chlorine in copolymer.
Durability and regenerability studies showed that the copolymer
retained its antibacterial activity up to 35 washes although there
18 L. Li, K. Ma, Y. Liu, Z. Xie, T. Huang and X. Ren, Polymers for
Advanced Technologies, 2014, 25, 963-968.
was continued decrease in content. This continued decrease in 19 Y. Sun and G. Sun, Macromolecules, 2002, 35, 8909-8912.
20 Y. Chen and Q. Han, Applied Surface Science, 2011, 257,
chlorine content on repeated recharging is attributed to the
6034-6039
possibility of HCl elimination involving the chlorine on the
21 L. Wu, A. Liu and Z. Li, Fibers and Polymers, 2015, 16, 550-
hydantoin ring and the hydrogen atom on the adjacent carbon and
the eliminated HCl further catalysing the hydrolysis of the amide
bond in the copolymer.
559
22 Y. Chen, S. Worley, J. Kim, C. Wei, T. Chen, J. Santiago, J.
Williams and G. Sun, Industrial & Engineering Chemistry
Research, 2003, 42, 280-284.
23 S. Worley, F. Li, R. Wu, J. Kim, C. Wei, J. Williams, J. Owens, J.
Wander, A. Bargmeyer and M. Shirtliff, Surface Coatings
International Part B: Coatings Transactions, 2003, 86, 273-
277.
24 U. Makal, L. Wood, D. Ohman and K. Wynne, Biomaterials,
2006, 27, 1316-1326.
Conflicts of interest
There are no conflicts to declare.
25 H. Kocer, I. Cerkez, S. Worley, R. Broughton and T.
Huang, ACS Applied Materials & Interfaces, 2011, 3, 3189-
3194.
26 A. Dutta, M. Egusa, H. Kaminaka, H. Izawa, M. Morimoto, H.
Saimoto and S. Ifuku, Carbohydrate Polymers, 2015, 115,
342-347.
27 I. Cerkez, H. Kocer, S. Worley, R. Broughton and T.
Huang, Reactive and Functional Polymers, 2012, 72, 673-679.
28 H. kocer, S. Worley, R. Broughton and T. Huang, Reactive and
Functional Polymers, 2012, 71, 561-568
29 R. Bai, Q. Zhang, L. Li, P. Li, Y. Wang, O. Simalou, Y. Zhang, G.
Gao and A. Dong, ACS Applied Materials & Interfaces, 2016,
8, 31530-31540.
Acknowledgements
RKR thanks the Department of Biotechnology for a graduate
research Fellowship. Thanks are due to for the XPS spectra analysis
in University of Miyazaki and Sophisticated Instrumentation Facility
(SAIF) at IIT Madras for the NMR spectra. The authors are grateful
to one of the reviewers for pointing out the possibility of HCl
elimination in the copolymer which helped us to explain the loss of
chlorine content on repeated recharging.
30 L. Timofeeva and N. Kleshcheva, Applied Microbiology and
Biotechnology, 2010, 89, 475-492.
Notes and references
31 E. Kenawy, S. Worley and R. Broughton, Biomacromolecules,
2007, 8, 1359-1384.
32 F. Hui and C. Debiemme-Chouvy, Biomacromolecules, 2013,
14, 585-601.
33 A. Dong, Y. Wang, Y. Gao, T. Gao and G. Gao, Chemical
Reviews, 2017, 117, 4806-4862.
34 G. Sun, W. Wheatley and S. Worley, Industrial & Engineering
Chemistry Research, 1994, 33, 168-170.
1
2
3
V.
Russotto,
A.
Cortegiani,
S.Raineri
Giarratano, Journal of Intensive Care, 2015, 3, 54
and
A.
R. Redulla, International Journal of Nursing Studies, 2016, 55,
131
S. Sattar, S. Springthorpe, S. Mani, M. Gallant, R. Nair, E.
Scott and J. Kain, Journal of Applied Microbiology, 2001, 90,
962-970.
4
5
E. Scott and S. Bloomfield, Journal of Applied Bacteriology,
35 G. Sun, L. Allen, E. Luckie, W. Wheatley and S.
Worley, Industrial & Engineering Chemistry Research, 1995,
34, 4106-4109.
1990, 68, 271-278
A. N. Neely, M. P Maley, Journal of Clinical Microbiology,
2000, 38, 724–726
.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 11 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
Journal Name
ARTICLE
36 V. Ravichandran, R. Rai, V. Kesavan and A.
Jayakrishnan, Journal of Biomaterials Science, Polymer
Edition, 2017, 28, 2131-2142.
37 D. Hall, P. Underhill and J. Torkelson, Polymer Engineering &
Science, 1998, 38, 2039-2045.
38 C. Li, L. Xue, Q. Cai, S. Bao, T. Zhao, L. Xiao, G. Gao, C.
Harnoode and A. Dong, RSC Advances., 2014, 4, 47853-
47864.
39 K. Choi, M. Nam, J. Kim, J. Yoon and J. Lee, Macromolecular
Research, 2011, 19, 1227-1232.
40 K. Xiu, J. Wen, J. Liu, C. He and Y. Sun, Industrial &
Engineering Chemistry Research, 2017, 56, 12032-12037.
41 K. Xiu, J. Wen, N. Porteous and Y. Sun, Journal of Bioactive
and Compatible Polymers, 2017, 32, 542-554.
42 A. Dong, S. Lan, J. Huang, T. Wang, T. Zhao, W. Wang, L. Xiao,
X. Zheng, F. Liu, G. Gao and Y. Chen, Journal of Colloid and
Interfacial Science, 2011, 364, 333–340.
43 B. Demir, R. M. Broughton, T. S. Huang, M. J. Bozack, and S.
D. Worley, Industrial & Engineering Chemistry Research,
2017, 56, 11773-11781.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

New Journal of Chemistry
Page 12 of 12
View Article Online
DOI: 10.1039/C8NJ02743A
335x173mm (96 x 96 DPI)