Optical devices for the detection of cyanide in water based on ethyl(hydroxyethyl)cellulose functionalized with perichromic dyes

Article abstract of DOI:10.1016/j.carbpol.2016.11.039

Films of three polymers, based on ethyl(hydroxyethyl)cellulose functionalized with protonated perichromic dyes, were used for anion sensing. The polymer functionalized with protonated Brooker's merocyanine acts as a chromogenic/fluorogenic system for the

Full text of DOI:10.1016/j.carbpol.2016.11.039

Accepted Manuscript
Title: Optical Devices for the Detection of Cyanide in Water
Based on Ethyl(hydroxyethyl)cellulose Functionalized With
Perichromic Dyes
Author: Leandro Guarezi Nandi Celso Rodrigo Nicoleti
´
Vanderleia Gava Marini Ismael Casagrande Bellettini Silvano
Rodrigo Valandro Carla Cristina Schmitt Cavalheiro Vanderlei
Gageiro Machado
PII:
S0144-8617(16)31304-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2016.11.039
CARP 11751
To appear in:
Received date:
Revised date:
Accepted date:
13-9-2016
4-11-2016
11-11-2016
Please cite this article as: Nandi, Leandro Guarezi., Nicoleti, Celso Rodrigo.,
´
Marini, Vanderleia Gava., Bellettini, Ismael Casagrande., Valandro, Silvano
Rodrigo., Cavalheiro, Carla Cristina Schmitt., Machado, Vanderlei
&
Gageiro., Optical Devices for the Detection of Cyanide in Water Based on
Ethyl(hydroxyethyl)cellulose Functionalized With Perichromic Dyes.Carbohydrate
Polymers http://dx.doi.org/10.1016/j.carbpol.2016.11.039
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1
Optical Devices for the Detection of Cyanide in Water Based
on Ethyl(hydroxyethyl)cellulose Functionalized With
Perichromic Dyes
Leandro Guarezi Nandia, Celso Rodrigo Nicoletia, Vanderléia Gava Marinia, Ismael
Casagrande Bellettinia†, Silvano Rodrigo Valandrob, Carla Cristina Schmitt Cavalheirob,
Vanderlei Gageiro Machadoa*
aDepartamento de Química, Universidade Federal de Santa Catarina, UFSC, CP 476,
Florianꢀpolis, SC, 88040−900, Brazil
bDepartamento de Físico−Química, Instituto de Química de São Carlos, Universidade
de São Paulo, USP, CP 780, São Carlos, SP, 13560−970, Brazil
* Corresponding author: vanderlei.machado@ufsc.br
† Current address: Universidade Federal de Santa Catarina, UFSC, Campus de
Blumenau, Blumenau, SC, 89065−300, Brazil

2
Research Highlights
 Ethyl(hydroxyethyl)cellulose was functionalized with protonated perichromic
dyes.
 The systems were designed to be used for chromogenic/fluorogenic anion
sensing.
 Films were utilized for the highly selective optical detection of CN− in water.
 The polymeric chain protects the sensing unit from the medium, improving the
detection limit of CN− in water.
 The films can be reused.
Abstract
Films of three polymers, based on ethyl(hydroxyethyl)cellulose functionalized with
protonated perichromic dyes, were used for anion sensing. The polymer functionalized
with protonated Brooker’s merocyanine acts as a chromogenic/fluorogenic system for
the selective detection of cyanide in water. An increase of >28 times was verified for the
fluorescence lifetime of the sensing units in the polymer in comparison with protonated
Brooker’s merocyanine in water. Moreover, an increase in the pKa values was verified
for the sensing units in the polymers. Data suggest that the hydrocarbonic polymeric
chains provide an adequate microenvironment to protect the sensing unit from bulk
water. The other polymer, functionalized with an iminophenol, also showed high

3
selectivity for cyanide (detection limit = 9.36×10−6 mol L−1 and quantification limit =
3.12×10−5 mol L−1). The polymer functionalized with azophenol units is unable for the
detection of cyanide, due to the low pKa value verified for its chromogenic units.
Keywords: ethyl(hydroxyethyl)cellulose; cyanide; naked−eye detection; chromogenic
chemosensors; fluorogenic chemosensors; anion sensing.
1. Introduction
Research on the recognition and detection of anions has increased in recent years
due to the very important role played by these species in chemical and biological
processes (Anslyn, 2007; Cho & Sessler, 2009; Gai et al., 2015; Gale, 2010;
Gunnlaugsson, Glynn, Tocci, Kruger & Pfeffer, 2006; Huynh, Sharma, Sosnowska,
D'Souza & Kutner, 2015; Martínez-Máñez & Sancenón, 2003; Suksai & Tuntulani,
2003; Wenzel, Hiscock & Gale, 2012; Yang, Zhao, Feng & Li, 2013; Zimmermann-
Dimer & Machado, 2008). An important target analyte in terms of detection is CN− (
Nelson, 2006), which is lethal in very small concentrations because it binds strongly to
the active site of cytochrome−oxidase, causing a decrease in the oxidative metabolism
(Bhattacharya & Flora, 2015; Luque-Almagro, Moreno-Vivián & Roldán, 2016;
Nelson, 2006; Zelder & Männel-Croisé, 2009). CN− is a chemical species present in
several processes, being fundamental in various industrial activities, such as the
synthesis of polymers, metallurgy, and mining. In addition, this anion is released in the
hydrolysis of cyanohydrins, present in certain fruit seeds and roots, and also of some

4
neurotoxic warfare agents (Agatemor, 2009; Nelson, 2006; Zelder & Männel-Croisé,
2009).
The detection of analytes using chromogenic and fluorogenic chemosensors has
been extensively employed because of the sensitivity, selectivity, and low cost of this
technique. The wide variety of strategies which have been investigated has allowed the
detection of analytes in different situations (Anslyn, 2004; Buske, Nicoleti, Cavallaro &
Machado, 2015; Chhatwal, Kumar, Singh, Gupta & Awasthi, 2015; Heying, Nandi,
Bortoluzzi & Machado, 2015; Houk, Wallace, Hewage & Anslyn, 2008; Nicoleti,
Marini, Zimmermann & Machado, 2012; Nicoleti, Nandi & Machado, 2015; Pati, 2016;
Snowden & Anslyn, 1999).
One type of system that has been widely explored in the recent years involves the
use of polymeric materials with chromogenic or/and fluorogenic units anchored in their
macromolecular structure, in order to achieve the selective detection of a particular
analyte (Busschaert, Caltagirone, Van Rossom & Gale, 2015; Isaad & El Achari, 2011;
Isaad, El Achari & Malek, 2013; Ma, Wu & Zeng, 2010; Machado, Stock & Reichardt,
2014; Sharma, Kaur, Singh, Kuwar & Singh, 2016; Trupp et al., 2010; Wu, Tang, Li &
Li, 2015).
An attractive macromolecular material which can be employed as a support for
the development of analyte sensing materials is ethyl(hydroxyethyl)cellulose (EHEC,
Fig. 1) (Feller & Wilt, 1991; Kamide, 2005; Klemm, Heublein, Fink & Bohn, 2005).
This modified cellulose can be easily functionalized for use in different applications
(Isaad & El Achari, 2011; Karlberg, Thuresson & Lindman, 2005).
Recently, EHEC was functionalized with Brooker’s merocyanine (BM) and the
resulting polymer (1a), in a form of a film, was used in an acid–base strategy for anion
sensing (Nandi, Nicoleti, Bellettini & Machado, 2014). BM is a classical merocyanine

5
which has been extensively used as a perichromic dye (Cavalli, da Silva, Machado,
Machado & Soldi, 2006; Koopmans & Ritter, 2007; Zimmermann-Dimer, Reis,
Machado & Machado, 2009). System 1a acts as chromogenic/fluorogenic system for the
highly selective detection of CN− in water (Nandi, Nicoleti, Bellettini & Machado,
2014). It was demonstrated that the polymer matrix acts as a support for anchoring the
sensing units, resulting in a system with interesting features, such as the ability to detect
CN− in a highly selective fashion and in concentrations below the minimum established
by the World Health Organization (WHO). The results obtained motivated us for the
design of other related chromogenic polymeric systems for the detection of CN− and
even other anionic species in aqueous solution. Moreover, a comparison between related
polymeric systems using different experimental procedures should provide further
information about the microenvironment of the sensing units in the polymeric chains.
Thus, herein, we describe the preparation and characterization of two other
modified EHEC polymers, obtained by the functionalization of EHEC with two
merocyanine units with a molecular structure similar to BM, differing in terms of the
replacement of the carbon−carbon double bond with carbon−nitrogen or
nitrogen−nitrogen double bonds as conjugated bridges. The ability of the new systems
to detect CN– in water was compared with that of polymer 1a. In addition, this study
provided new evidence aimed at gaining a better understanding of the features
responsible for the ability of the EHEC polymers to detect CN– in water. The reuse of
the films prepared was also evaluated.
2. Experimental
2.1. Reagents

6
All chemicals employed in this study were high−purity commercial reagents. The
deionized water used in the measurements was boiled and bubbled with nitrogen and
kept in a nitrogen atmosphere to avoid the presence of carbon dioxide.
Tetra−n−butylammonium hydroxide was purchased from Sigma−Aldrich. All anions
–
–
–
(HSO4 , H2PO4 , NO3 , CN–, CH3COO–, F–, Cl–, Br–, and I–) were used as
tetra−n−butylammonium salts with purity greater than 97−99% and were dried over
phosphorous pentoxide under vacuum before use. The anions were purchased from
–
–
Fluka (F–, > 97%; Cl–, > 98%; NO3 , > 97%; H2PO4 , > 97%), Vetec (Br–, > 99%; I–, >
–
99%; HSO4 , > 99%), and Sigma−Aldrich (CH3COO–, > 97%).
EHEC, Bermocoll E 230FQ lot no. 14770, was donated by Akzo Nobel Surface
Chemistry AB, Stenungsund, Sweden. The degree of substitution of ethyl groups
(DSethyl) in the polymer 0.9−1.0 per anhydroglucose unit and the molar substitution of
ethylene oxide groups (MSEO) is 1.9−2.2 per anhydroglucose unit. The molar weight of
EHEC, determined by light scattering, is 3.30×105 g mol−1. The polymer has a degree
of polydispersity (Mw/Mn) below 2 (Dal Bó, Schweitzer, Felippe, Zanette & Lindman,
2005; Dal-Bó, Laus, Felippe, Zanette & Minatti, 2011; Modolon, Dal Bó, Felippe,
Minatti & Zanette, 2009).
2.2. Instruments
UV−vis experiments were carried out on an HP 8452A spectrophotometer
equipped with a thermostated bath and all measurements were performed at 25 °C,
employing a 1 cm quartz cuvette. The films were fixed with adhesive tape on a quartz
plate with 1 cm width  4 cm height and subsequently introduced into the sample holder
of the spectrophotometer. The spectrum was collected and the maximum wavelengths
(max) in the UV−vis spectra were calculated from the first derivative of the absorption

7
spectrum. The max values thus obtained were transformed into ET(dye) values,
according to the expression ET(dye) = 28590/max (Machado, Stock & Reichardt, 2014),
given in kcal mol−1 with a precision of ±0.1 kcal mol−1. Melting points were obtained on
a Kofler hot stage and were uncorrected. The nuclear magnetic resonance (NMR)
spectra were recorded on a Varian AS−400 spectrometer. Chemical shifts were recorded
in ppm with the solvent resonance as the internal standard. Data are reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, and bs = broad singlet), integration,
and coupling constants (Hz). Infrared (IR) spectra were obtained on a Shimadzu model
Prestige−21 spectrophotometer, with KBr pellets or silicon plates. In the latter case, the
polymer was dropped in a 5% aqueous solution (wt/vol) onto the silicon plate and dried
in an oven for 12 h at 30 °C before the analysis.
The thermal degradation of the films was analyzed using a Shimadzu TGA−50
thermogravimetric analyzer. The experiments were performed in the temperature range
of 25−600 ºC at a heating rate of 5 ºC min−1. The nitrogen flow was kept at 50 cm3
min−1 and the analysis was performed in triplicate.
The time−resolved fluorescence experiment involving 1a was carried out on an
Easy life VTM lifetime fluorometer using the stroboscopic technique (Optical Building
Blocks Corp.). The fluorescence decay was obtained using a pulsed light from a diode
(LED) of 445 nm as the excitation source and a long pass filter of 550 nm in order to
isolate the emission of the film of polymer 1a from the scattered light. The instrument
response function (IRF) was measured using a Ludox scattering solution. Four scans
were averaged for the fluorescence decay experiments. For every scan, the number of
channels was 600, and the time of integration, over which the signal was averaged for
every point of each scan, was 4 s.

8
The fluorescence decay curve for BMH in water was obtained using
time−correlated single−photon counting (TCSPC). The pulsed excitation was at 400
nm, with a pulse duration of 150 fs, using a Ti:sapphire laser operating in locked mode,
tuned at 800 nm, Mira−Coherent. The Mira laser oscillator was pumped by a Coherent
Verdi 5W laser. The detection system used was a PMTMCP (Hamamatsu R3809U−50),
cooled by the Peltier effect. The adjustment of the photomultiplier in this experiment
was carried out employing a time increment of 48.83 ps, with the focal plane operating
at −20 °C. The typical IRF was 50 ps at FWHM. The monochromator was set at 480
nm, close to the emission peak of the sample. The typical IRF was 50 ps at FWHM. The
decays were recorded using TC900 and Edinburgh Instruments software and the curve
was fitted using monoexponential analysis.
2.3. Synthesis of the compounds and the functionalized polymers
BM was prepared as described previously (Minch & Shah, 1977)
2.3.1. 4−((Pyridin−4−ylmethylene)amino)phenol (8)
4−Pyridinecarboxaldehyde (0.56 mL, 4.6 mmol) and 4−aminophenol (0.5 g, 4.6
mmol) were mixed, with magnetic stirring, in ethanol (5 mL). A drop of acetic acid was
added and the reaction mixture was stirred for 4 h. A light green solid was formed,
which was filtered under vacuum and recrystallized from ethanol. 72% yield. m.p.
o
o

obtained: 207 C (literature (Demir, Kaya & Saçak, 2006): 210 C). IR (KBr,
max/cm−1): 3447 (ν, OH), 3063−2996 (ν, C−H), 1620, 1460 (ν, C=C), 1577 (ν, C=N),
1
1250 (ν, C−O). H NMR (400 MHz, DMSO−d6) δ/ppm: 9.69 (1H, s), 8.70 (2H, d, J =
6.1 Hz), 8.67 (1H, s), 7.80 (2H, d, J = 6.1 Hz), 7.30 (2H, d, J = 9.0 Hz), 6.83 (2H, d, J =
9.0 Hz). TGA: 231.75, 345.93, 470.73 ºC.

9
2.3.2. 4−(Pyridin−4−yldiazenyl)phenol (9)
4−Aminopyridine (1.5 g, 0.0159 mol) was dissolved in 7.5 mol L−1 hydrochloric
acid (12 mL). A mixture containing 10% NaOH (10 mL), phenol (1.25 g, 0.0132 mol),
and NaNO2 (1 g, 0.014 mol) was then prepared and added dropwise at 8 °C to the
4−aminopyridine solution. The crude product was isolated by filtration and purified by
crystallization from acetone/ethanol to give an orange powder (Agostini, Milani,
o
Martínez-Máñez, Licchelli, Soto & Sancenón, 2012). 70% yield. m.p. obtained: 253 C
(literature (Buncel & Keum, 1983): 256 oC). IR (KBr,

max/cm−1): 3550−3414 (ν, OH),
1
3043 (ν, C−H), 1640, 1580 (ν, C=C), 1468−1403 (ν, N=N), 1295 (ν, C−O). H NMR
(400 MHz, DMSO−d6 + 2 drops of D2O) δ/ppm: 8.92 (2H, d, J = 5.6 Hz), 8.14 (2H, d, J
= 5.6 Hz), 7.96 (2H, d, J = 8.4 Hz), 7.01 (2H, d, J = 8.4 Hz). TGA: 262.96, 373.59 oC.
2.3.3. 4−(((4−Hydroxyphenyl)imino)methyl)−1−methylpyridin−1−ium iodide (4a)
Compound 8 (0.1 g, 0.5 mmol), iodomethane (0.03 mL, 0.5 mmol), and dry
acetone (10 mL) were refluxed for 4 h and the flask was cooled in an ice bath. The
brown solid formed was washed, filtered in cold acetone, and then recrystallized in
o
ethanol. 80% yield. m.p. obtained: 223−225 C (literature (Machado, Nascimento &
o
Rezende, 1994): 225−227 C). IR (KBr,

max/cm−1): 3424 (ν, OH), 3153−3017 (ν,
1
C−H), 1640, 1475 (ν, C=C), 1581 (ν, C=N), 1258 (ν, C−O), 1163 (δ, C−N+). H NMR
(400 MHz, DMSO−d6) δ/ppm: 10.00 (1H, s), 9.04 (2H, d, J = 6.6 Hz), 8.95 (1H, s), 8.43
(2H, d, J = 6.6 Hz), 7.46 (2H, d, J = 8.6 Hz), 6.88 (2H, d, J = 8.6 Hz), 4.35 (3H, s).
TGA: 244.91, 373.90, 522.41 oC.
2.3.4. 4−((4−Hydroxyphenyl)diazenyl)−1−methylpyridin−1−ium iodide (5a)
Compound 5a was prepared following the procedure described in the literature
(Buncel & Keum, 1983; Garcia-Amoros, Nonell & Velasco, 2012). Compound 9 (0.5 g,

10
2.5 mmol) was refluxed with iodomethane (12.5 mmol) for 4 h in a mixture of
acetone/tetrahydrofuran (7:3 v/v). The orange solid formed was washed and filtered in
cold acetone. 80% yield. m.p. obtained: 217−220 ºC (literature (Buncel & Keum, 1983):
220−222 C). IR (KBr,

max/cm−1): 3550−3414 (ν, OH), 3124−3055 (ν, C−H), 1633,
o
1582 (ν, C=C), 1411 (ν, N=N), 1295 (ν, C−O), 1146 (δ, C−N+).1H NMR (400 MHz,
DMSO−d6) δ/ppm: 11.38 (1H, s), 9.10 (2H, d, J= 4.5 Hz), 8.27 (2H, d, J = 4.5 Hz), 7.97
(2H, d, J = 7.0 Hz), 7.06 (2H, d, J = 7.0 Hz), 4.36 (3H, s).
2.3.5. Preparation of the polymers functionalized with the sensing units
Polymers 1a−3a were prepared using a tosylated EHEC (6) as the intermediate,
for which the synthesis was previously described together with that of polymer 1a
(Nandi, Nicoleti, Bellettini & Machado, 2014).
The film formed from 6 was refluxed in 250 mL of ethanol with 0.3 g (1.55
mmol) of compound 8 for 12 h to give 2a. The resulting film was then washed five
times with 100 mL of ethanol resulting in a yellow colored film. IR (silicon wafer,

max/cm−1): 3433 (ν, OH), 2972−2874 (ν, C−H), 1640−1470 (ν, C=C), 1634 (δ, OH),
1157 (δ, C−N+). TGA: 239.44, 299.29 ºC. The same procedure was used for the
synthesis of 3a, by means of the reflux of 6 with compound 9. IR (silicon wafer,

max/cm−1): 3430 (ν, OH), 2972−2871 (ν, C−H), 1099 (δ, C−N+). TGA: 317.63, 365.91,
571.09 ºC.
2.4. pKa determinations
A solution of 4a or 5a was prepared in a concentration of 4×10–3 mol L–1 in
acetone, and stored in glass flasks closed with rubber stoppers to avoid the evaporation

11
of the solvent. An aliquot of the solution, sufficient to give a concentration of the
compounds of 2×10–5 mol L–1, was collected with a microsyringe and placed in a flask.
After the evaporation of the solvent, distilled water was added, the pH was adjusted
with the addition of small aliquots of HCl (0.1 mol L−1) or KOH (0.1 mol L−1), and the
UV−vis spectrum of the compound was recorded at 25 ºC. The pH of the solution was
measured and the UV−vis spectrum was obtained. Data were fitted using a sigmoid
equation to give the pKa value.
The pKa values were also determined for the films of EHEC with the anchored
chemosensors. In these experiments, the film was immersed in 10 mL of distilled water
in vials, with the pH previously adjusted by the addition of small aliquots of HCl (0.1
mol L−1) or KOH (0.1 mol L−1). The UV−vis spectrum of the film was recorded at 25
°C after 3 min of immersion. Data were fitted using a sigmoid equation to give the pKa
value.
2.5. Influence of pH on the detection of cyanide
To determine the pH dependence on the CN− detection, using films of 1a and 2a,
various CN− aqueous solutions (6.0×10−4 mol L−1) were prepared at different pH values
in vials containing 10 mL of distilled water with the pH previously adjusted with HCl
solutions (0.1 mol L−1) or KOH (0.1 mol L−1). The films were immersed into these
solutions and after 5 min the UV−vis spectra were collected and compared with the
spectrum of the film in an aqueous solution of tetra−n−butylammonium hydroxide
(6×10−4 mol L−1). A bar graph of the concentration percentage in relation to the real CN−
value as a function of pH was constructed.
2.6. Determination of the apparent concentration of the sensing units in the films

12
To determine the concentration of compound 4a anchored in the film of 2a, a
calibration curve was obtained using 4a as the standard in DMSO (Fig. S19). A linear
fit of the experimental data gave the equation: Absorbance = (1.55±0.01)×104 c (4a),
with R2 = 0.9999 and S.D. = 1.0×103. Subsequently, a film of 2a (1.5 ×10−4 g) was
dissolved in DMSO (5 mL) and the maximum absorbance value (at 455 nm) was
collected, this being 0.30, allowing the apparent concentration of 4a covalently bonded
to the film to be estimated with the use of the calibration curve, which was calculated as
c (4a) = 1.93×10−5 mol L−1 or 2.18×10−3 g of 4a per g of EHEC. This methodology was
also used in the determination of the apparent concentration of compound 5a (Fig. S20)
in the film of 3a (1.5×10−4 g dissolved in 5 mL of DMSO). A linear fit of the
experimental data gave the equation: Absorbance = (1.56±0.018)×104 c(dye), with R2 =
0.994 and S.D. = 4.8×102. The value of the absorbance found at 409 nm was 0.78,
providing c (5a) = 1.56×10−5 mol L−1 or 5.7×10−3 g of 5a per g of EHEC.
2.7. Titration experiments using the film of 2a with cyanide and determination of limits
of detection and quantification
Titration experiments were performed in water (adjusted to pH = 8.0) through the
immersion of the film of 2a (5 min) in flasks with different concentrations of
tetra−n−butylammonium cyanide. UV−vis spectra were obtained and the absorbance
values were collected at 498 nm.
The linear segment of the titration curve was used to estimate the optical detection
limit (DL) and the optical quantification limit (QL). Based on the linear fit of the
experimental data, the DL and QL were obtained from equations (1) and (2) according
to a procedure reported in the literature (Isaad, El Achari & Malek, 2013; Skoog &
West, 1982), where Sb1 is the standard deviation of the blank solution and S is the slope
of the calibration curve. The B values are 3 for DL and 10 for QL.

13
DL = B Sb1 / S
QL = B Sb1 / S
(1)
(2)
3. Results and Discussion
3.1. Functionalization of EHEC
The decorated polymers 1a−3a were prepared as shown in Scheme 1. The
tosylation of EHEC, in the first step, was carried out using a previously described
methodology (Nandi, Nicoleti, Bellettini & Machado, 2014). In the second step, the film
of the tosylated intermediate 6 was reacted with compound 8 or 9 to yield the
functionalized EHEC products. The films have different colors depending on whether
the anchored sensing units are protonated or deprotonated. The deprotonation was easily
accomplished by dipping the films into aqueous solutions of tetra−n−butylammonium
hydroxide.
3.2. Characterization
The synthetically−modified polymers were characterized using the IR technique.
The characterization of 6 and polymer 1a has been previously reported (Nandi, Nicoleti,
Bellettini & Machado, 2014). With the reaction of 6 with 8, the bands relative to the

14
tosyl group disappeared in the IR spectrum of the resulting polymer 2a (Fig. S5). In
addition, the appearance of the bands relative to the anchored 4a unit (see Fig. S3 for
comparison) could be observed at 1643−1470 cm−1 (ν, C=Carom) and 1157 cm−1 (δ,
C−N+). These data are consistent with values reported in the literature (Alsaygh, Al-
Humaidi & Al-Najjar, 2014; Demir, Kaya & Saçak, 2006; Xu, Ling & He, 2008).
Polymer 3a was similarly characterized and the bands relative to the anchored 5a unit
(Fig. S10) could be observed at 1099 cm−1 (δ, C−N+).
Data obtained from the thermogravimetric (TG) curves (mass versus degradation
temperature) and the corresponding derivative thermogravimetric (DTG) curves (mass
loss rate versus temperature) were used to assess the effect of the functionalization on
the degradation pattern of the EHEC film. The TG/DTG curves are provided in the
Supporting Information. For the EHEC film the maximum degradation temperature
(Tmax) is 375.3 ºC, while for the film of tosylated EHEC (6) the Tmax value is 213.0 ºC
(Nandi, Nicoleti, Bellettini & Machado, 2014). For the film obtained from 2a values of
239.44 and 299.20 °C were obtained (Fig. S13). For comparison, for a film prepared
with a physical mixture of EHEC and 4a (which corresponds to the same sensing unit
in functionalized 2a), Tmax value is 367.4 ºC (Fig. S14), which differs only slightly from
that obtained for the EHEC film. The same pattern was found for the polymer 3a, with
Tmax values of 317.63 and 365.91 ºC (Fig. S17), while a film obtained from the physical
mixture of EHEC and the corresponding azophenol 5a exhibits Tmax = 372.4 ºC (Fig.
S18), which is very similar to the EHEC film value (Nandi, Nicoleti, Bellettini &
Machado, 2014). These studies showed that significant changes in Tmax only occur when
the EHEC film is chemically modified to generate the functionalized polymers 2a and
3a.

15
Spectrophotometric measurements involving a comparison of dyes 4a and 5a in
DMSO solution with the polymers 2a and 3a allowed the apparent concentration of
each sensing unit anchored in EHEC to be calculated. For 2a, c (4a) was 1.93×10−5 mol
L−1 or 2.18×10−3 g of dye/g of EHEC, while the value determined for 5a in the film of
3a was c (5a) = 1.56×10−5 mol L−1 or 5.7×10−3 g of dye/g of the polymer.
The films of 2a and 3a were immersed in an aqueous solution of
tetra−n−butylammonium cyanide and it was verified that the sensing units chemically
anchored in the polymer matrix were not released into the aqueous solution (Figs. S21A
and S22A). In contrast, when a film prepared from a physical mixture of 4a and EHEC
(or 5a and EHEC) was immersed in an aqueous solution of tetra−n−butylammonium
cyanide the solution quickly became colored, indicating that the dyes are lixiviated to
the aqueous solutions (Figs. S21B and S22B).
3.3. Studies on pKa of the functionalized polymers
Plots of the maximum absorbance as a function of the pH for compound 4a in
water and for the film of 2a immersed in water were used for the determination of the
pKa values for the phenols at 25 oC (Fig. S23 a, b). Similar experiments were performed
for compound 5a and for the film of 3a (Fig. S23 c, d). The pKa values for BMH and 1a
have been previously reported (Davidson & Jencks, 1969; Nandi, Nicoleti, Bellettini &
Machado, 2014). All pKa values are given in Table 1. The pKa value for the sensing
units in 2a was 9.75±0.04. This value is higher than that for free 4a in aqueous solution
(pKa = 9.00±0.03). These trends are very similar to those reported for BM and polymer
1a and for 5a in comparison with the film of 3a. Thus, all data show that the acidity of
the phenols is decreased if their microenvironment is changed from water to the
polymeric chains. It is well known that the more acid the phenol, the lesser the basicity

16
of its conjugated base, being this decrease associated with stereoelectronic effects
and/or with the ability of the medium in stabilizing the conjugated bases by solvation.
The higher pKa values verified for the sensing units anchored in the polymers, in
comparison with the values for the corresponding compounds in water, suggest that the
microenvironment of the sensing units in the films are protected from the bulk water.
.
3.4. Perichromic studies on the polymer films
Table 2 shows the molar transition energy (ET) values for the vis perichromic
bands of BM, 4b, and 5b in various solvents, as well as for the films of 1b–3b
immersed in aqueous solution. The deprotonation of 4a in aqueous solution leads to the
appearance of the solvatochromic band at λmax= 476.0 nm. Interestingly, the
deprotonation of the phenol units in the film of 2a led to the appearance of the
perichromic band corresponding to the dye units at λmax = 498.0 nm. This bathochromic
shift of Δλmax= +22 nm suggests that the dye is in a less polar environment in the film
(Machado, Stock & Reichardt, 2014). Fig. 2 shows solutions of BM, 4b, and 5b in
some solvents, as well as the films of 1b−3b after immersion in water. The film of 2b in
aqueous medium has a similar color to the dye 4b in methanol, a less polar solvent than
water, i.e., the environment of the dye in the film has a polarity very similar to that of
methanol. For the film of 3b, the dye units in the film are responsible for the
perichromic band at λmax = 540.0 nm. The corresponding dye 5b in aqueous solution has
a solvatochromic band with λmax= 530.0 nm and a bathochromic shift of Δλmax= +10 nm.
The film of 1b has a similar behavior, as previously reported (Nandi, Nicoleti, Bellettini
& Machado, 2014).

17
3.5. Fluorescence decay curve for 1a
Fluorescence decay curves for the film of 1a and for BM (Fig. S24) were fitted
using single−exponential analysis. The fluorescence lifetime of BMH in water was
determined as 0.030 ns (χ2 = 1.351), while the lifetime observed for the film of 1a was
0.85±0.01 ns (χ2 = 0.9). The increase (by a factor of >28) in the lifetime value suggests
that the hydrocarbonic chains in 1a provide hydrophobic microenvironments and
increase the rigidity of the system, which leads to a decrease in the non−radiative
transitions of the BMH units.
3.6. UV−vis study of 2a and 3a with the anions
Fig. 3A shows films of 2a before and after immersion in aqueous solutions of
various anions. The film of 2a is yellow and that of 2b (obtained after immersion in an
aqueous HO− solution) is red. Of the several anion solutions studied, only CN− is able to
change the color of the film to red, which is sufficiently basic to deprotonate the dye in
aqueous solution, forming 2b. Fig. 3B shows films of 3a before and after immersion in
aqueous solutions of various anions. The film of 3a is yellow and that of 3b (obtained
after immersion in an aqueous HO− solution) is pink. The very low pKa value of 5a
influences the selectivity and precludes the use of this compound in the detection of
CN− in aqueous media.
Fig. 4 shows the UV−vis spectrum for the film of 2a, which exhibits a band with
maximum wavelength λmax = 408 nm. This band disappears with the immersion of the
film in an aqueous solution of CN− simultaneously with the appearance of another band
with λmax = 498 nm, while the other anions studied did not cause any spectral alteration.

18
The results obtained in the experiments allowed us to verify that the films of 1a
are applicable for the detection of CN− in water within a pH range of 7.4−8.8 (Fig. 5A)
while the film of 2a detects CN− at pH 8.0−8.5 (Fig. 5B). Optical chemosensors based
on acid-base processes require attention with the results obtained since these strategies
are sensitive to changes in pH, which could lead to false positives (Schramm, Nicoleti,
Stock, Heying, Bortoluzzi, & Machado, 2017).
The recyclability of the systems was verified by immersing the films alternately in
CN− and in acid aqueous solutions. The film of 1a was reused several times after
appropriate treatment (Fig. 5C), but the film of 2a was only reused twice after being
washed with a slightly acidic aqueous solution (Fig. 5D). The limitation in the reuse of
the film of 2a is probably due to the hydrolysis of the double carbon−nitrogen bond
(Heying, Nandi, Bortoluzzi & Machado, 2015) of the sensing unit.
3.7. Titration of 2a with cyanide
Fig. 6A shows a plot of absorbance values at λmax = 498.0 nm for the film of 2a
as a function of c(CN−), which suggests a behavior typical of a 1:1 stoichiometry,
considering the anion and the dye units anchored in the polymer. The experimental data
were fitted with the use of equation (3) (Buske, Nicoleti, Cavallaro & Machado, 2015;
Nicoleti, Marini, Zimmermann & Machado, 2012; Valeur, Pouget, Bourson, Kaschke &
Ernsting, 1992), which is related to the following situation according to a 1:1
chemosensor:anion stoichiometry.
Abs = [Abso + Abs11K11CA- ] / [1 + K11CA- ]
(3)

19
In this equation, Abs is the absorbance value after each addition of an anion, Abs0
is the initial absorbance without an anion added, Abs11 is the maximum absorbance
value obtained with the addition of an anion considering a 1:1 1a:anion stoichiometry,
CA- is the anion concentration for each addition, and K11 is the equilibrium constant. A
fitting of the experimental data provided an equilibrium constant of (3.72±0.55)×103 L
mol−1. An analysis of the linear segment of the titration curve (Fig. 6B) gave DL =
9.36×10−6 mol L−1 and QL = 3.12×10−5 mol L−1 for the detection of CN− in aqueous
solution.
4. Conclusions
The functionalization of EHEC with the compounds 7–9 allowed the obtainment
of polymers 1a–3a. Films of 1a and 2a can be used for the highly selective
chromogenic (and in the case of 1a also fluorogenic) detection of CN− in water. The
role of the polymeric chain, protecting the sensing unit from the medium, explains the
ability of this ensemble to sense very small concentrations of CN− in water, which is not
possible if the corresponding protonated dyes are used in water.
The fluorescence lifetime obtained for the film of 1a suggests that the fluorophore
is protected from the bulk water, being located in hydrophobic microenvironments in
the polymer, leading to an increase in the rigidity and to a lowering of the non-radiative
transitions.
For the film of 2a the stoichiometry between the anion and dye anchored to the
film was determined by UV−vis techniques as being a 1:1 ratio, i.e., the CN− interacts
with the phenol group of the dye molecule, with deprotonation occurring and a
subsequent change in the color of the film.

20
Studies have shown that 1a can be used several times, while the film of 2a has a
limited number of uses (two cycles), this limitation being due to the hydrolysis of the
imine bond (Heying, Nandi, Bortoluzzi & Machado, 2015) in aqueous medium. The
low pKa value obtained for the film of 3a hinders its use in the detection of CN− in
water.
Polymeric films 1a and 2a are promising for the development of optical devices
for the detection CN− in water with low detection limits. Such films can also be used as
inspiration models for the development of new detection systems with various potential
analytical applications.
Acknowledgements
The authors are grateful to UFSC and for the financial support provided by the
Brazilian governmental agencies Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq; process number: 306400/2014-6), Fundação de Amparo à Pesquisa
e Inovação do Estado de Santa Catarina (FAPESC; process number: 468/2015), and
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; process
number: 23038.007802/2011-80). The authors are also grateful to Prof. Marcelo H.
Gehlen for assistance with determining the BMH lifetime in water by single photon
counting.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/

21
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28
Fig. 1. Structures of BM, EHEC, and 1a.

29
BM
4b
1b
2b
5b
3b
Fig. 2. Solutions of BM, 4b, and 5b in water, methanol, ethanol, and ethyl acetate. The films of
1b−3b after immersion in aqueous solution of tetra−n−butylammonium hydroxide are also
presented.