Simple azo-based salicylaldimine as colorimetric and fluorescent probe for detecting anions in semi-aqueous medium

Article abstract of DOI:10.1002/jmr.2268

A series of novel, highly sensitive, and selective azo-based anion sensors 1-3 have been designed and synthesized from the condensation reaction between 4-amino azo benzene and three different aldehydes. The structure of the sensors 1-3 were confirmed by

Full text of DOI:10.1002/jmr.2268

Research Article
Received: 20 September 2012,
Revised: 31 December 2012,
Accepted: 29 January 2013,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2268
Simple azo-based salicylaldimine as
colorimetric and fluorescent probe for
detecting anions in semi-aqueous medium
Sivalingam Suganya and Sivan Velmathi*
A series of novel, highly sensitive, and selective azo-based anion sensors 1–3 have been designed and synthesized
from the condensation reaction between 4-amino azo benzene and three different aldehydes. The structure of the
sensors 1–3 were confirmed by IR, HRMS, 1H NMR, and 13C NMR spectroscopic methods. Colorimetric naked-eye
analysis revealed the anion detection by receptors 2 and 3 as color changes from yellow to pink and yellow to
orange, respectively. Anion sensing ability of all receptors was further investigated by 1H NMR titration, UV-vis
experiment, and fluorescence titration. UV-vis measurements highly indicate the selective recognition of fluoride
and acetate ions in 9:1 dimethyl sulfoxide–H2O (v/v) for receptors 2 and 3. Binding constant value showed among
all receptors, receptor 3 has strong affinity toward Fꢀ and AcOꢀ in semi-aqueous medium, which is due to the
presence of chromogenic signaling unit in it. The Fꢀ ion detection property of receptor 2 in organic medium was also
extended in the real sample like toothpaste. Copyright © 2013 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this paper
Keywords: chromogenic sensor; Azo derivative; Fꢀand AcOꢀ recognition; aqueous medium; fluorescence
anions and organic molecules in biological and environmental
systems will occur in aqueous medium. However, the difficulty
INTRODUCTION
in making the anion sensor in aqueous medium is that strong
hydration stops the sensors from detection of anions through
hydrogen bonding. Still now, only a few sensors have been
designed for anion recognition in the aqueous phase (Wang
et al., 2005; Gunnlaugsson et al., 2006; Kim et al., 2009; Chaicham
et al., 2010). This diversity made an interest to develop anion
sensors 2 and 3 that work in dimethyl sulfoxide (DMSO) and in
DMSO/H2O binary solutions by colorimetric titration and UV-vis
spectroscopy.
In this article, we described the synthesis and anion sensing
behavior of sensors 1–3 bearing phenol O–H and imine group.
Sensor 1 was reported by Peker and Serin (2004) for the
preparation of metal complex. To the best of our knowledge,
for the first time, we are reporting the synthesis of sensors 2
and 3 and anion sensing studies of sensors 1–3. Sensors 2 and
3 showed colorimetric sensitivity for Fꢀ and AcOꢀ in DMSO/
H2O (9:1 v/v) solutions. The detection of anions in aqueous
environment is the most interesting target in chemosensor field.
Water plays an important role in the analysis of real samples in
industries, and the detection of anions in biological system
needs an aqueous environment.
The construction of chemical sensor toward various anions
(Suksai and Tuntulani, 2003) is an extensive area in the field of
supra molecular chemistry (Gale et al., 2008; Gale and Quesada,
2006) due to their vital roles in biological system and environ-
mental and clinical applications. Anions play an important role
in a wide range of industrial (Lehn, 1985; Hijji et al., 2009;
Zuhair et al., 2006) and biological processes (Nguyen and
Kim, 2009; Mizuno et al., 2002; Yu et al., 2007; Beer and Gale,
2001), and considerable attention has been focused for the
recognition of anion species selectively through visible, elec-
trochemical, and optical responses. Among all anions, fluoride
and acetate are of particular interest because of their wide
range of biological and environmental application. For exam-
ple, Fꢀ ion is used for the prevention of dental problems
and treatment of osteoporosis, and excess fluoride causes
fluoride toxicity, namely, “fluorosis” (Xu and Tarr, 2004; Sole
and Gabbai, 2004; Lee et al., 2004; Cho et al., 2003). AcOꢀ have
abundant application in metabolic processes of enzymes and
antibodies through carboxylate ion, and AcOꢀ production has
been used as an indicator of organic decomposition (Ho
et al., 2002; Gupta et al., 2006; Nie et al., 2004). Therefore,
much attention has been paid to design anion sensor for the
detection of Fꢀ and AcOꢀ ions.
The common binding sites for hydrogen bonding formation
used in fluorogenic anion sensors are imines (Suganya et al.,
2011; Udhayakumari et al., 2011), urea (Cho et al., 2005), thiourea
(V´azquez et al., 2004), amide (Kang et al., 2006), phenol (Libra
and Scott, 2006), –OH (Lee et al., 2001), and pyrrole (NH) subunits
(Velmathi et al., 2012). The advantage of producing aqueous
sensing receptor is the determination of water content in a given
substrate (Zhang et al., 2009), and also the contact between
* Correspondence to: Sivan Velmathi, Organic and Polymer Synthesis
Laboratory, Department of Chemistry, National Institute of Technology,
Tiruchirappalli-620 015, India.
E-mail: velmathis@nitt.edu
S. Suganya, S. Velmathi
Organic and Polymer Synthesis Laboratory, Department of Chemistry,
National Institute of Technology, Tiruchirappalli 620 015, India
J. Mol. Recognit. 2013; 26: 259–267
Copyright © 2013 John Wiley & Sons, Ltd.

S. SUGANYA AND S. VELMATHI
a
R
EXPERIMENTS
N
N
N
Materials
OH
All solvents such as dimethyl sulphoxide (DMSO), acetonitrile (ACN),
ethanol (EtOH), and reagents used for spectroscopic experiments
and synthesis were received commercially and used as such without
any further purification. All anions used for titration experiment
were added in the form of tetrabutylammonium (TBA) salts,
which were purchased from Sigma-Aldrich Chemical, stored in a
vacuum desiccator containing self-indicating silica, and dried fully
before using.
fluorophore unit
binding unit
chromophore unit
S1: R= H
S2: R= Cl
S3: R= NO2
b
Apparatus
1H NMR spectra were obtained on a BRUKER AV III 500-MHz
spectrometer using DMSO-d6 as solvent. IR spectra were recorded
on NICOLET IS5 instrument using KBr plates. UV-vis spectra were
recorded on a Shimadzu UV-2600 spectrophotometer with a
quartz cuvette (path length = 1 cm) at room temperature (r.t).
Fluorescence spectra were recorded on Shimadzu RF-5301 PC
spectrophotometer. A 2.5ꢁ 10ꢀ5 M solution of receptors 1–3 and
1.5 ꢁ 10ꢀ3 M TBA salts of the respective anions in DMSO and
ACN were prepared and used for all spectroscopic studies.
General experimental procedure for the synthesis of the
receptors
One millimole of 4-amino azobenzene (0.197 g) and 1 mmol of
salicylaldehyde (0.1221 g) were dissolved in 10 ml of EtOH, and
the resulting mixture was allowed to reflux for 2–3 h at 70 ꢂC.
After cooling to room temperature, the precipitate formed was
filtered, washed with cold EtOH, recrystallized from EtOH, and
dried in a vacuum oven (receptor 1). Following the previous
procedure, receptors 2 and 3 were synthesized (Figure 1). The
results were as follows:
Figure 1. (a) Structure of receptors 1, 2, and 3; (b) ORTEP of the receptor 2.
ꢂ
are a = 12.2215(3) A ꢂ, b = 4.5174(9) A ꢂ, c = 27.775(2) A
along with R value of 0.0374. The intra molecular hydrogen
bonding (2.593 Aꢂ) present within the receptor 2 was confirmed
from the ORTEP diagram of receptor 2 (Fig.1b). Intra and
intermolecular hydrogen bonding and other parameters present in
receptor c are given in (Suppo. info Figure. 11).
Receptor 1. Yield, 80%; mp, 160 ꢂC. 1H NMR (500 MHz,
DMSO-d6 d ppm): 12.83 (s, 1H, OH), 9.04 (s, 1H, CH = N), 8.00
(d, 2H, Ar), 7.98 (d, 2H, Ar), 7.70 (dd, 1H, Ar), 7.58–7.59 (m, 5H, Ar),
7.40–7.45 (m, 1H, Ar), and 6.9–7.0 (m, 2H, Ar).13C NMR
(125 MHz, DMSO-d6 d ppm): 164.7, 160.8, 152.4, 151.3, 150.8,
134.2, 133.2, 132, 129.9, 124.4, 123, 119.9, 119.8, 117.2. IR
(KBr plates, cmꢀ1): 3449, 3057, 1619, 1560.
Colorimetric naked-eye analysis
The binding event of anions by receptor molecules 1–3 was
monitored by “naked eye” through color changes. The variation
in color changes can affect the electronic properties of the
receptor molecule and lead to the formation of the complex
between anion and receptor molecule through hydrogen
bonding. All the three receptors are expected to be in transform,
so that they can bind with anions effectively through charge
transfer mechanism. Also, the cis form of azo-based receptors
is inactive for anion sensing (Thangadurai et al., 2011). The
sensing action of receptors 1–3 were precisely investigated by
colorimetric studies, UV-vis, and fluorescence spectroscopy via
the addition of 4 Eq of different anions Fꢀ, Clꢀ, Brꢀ, AcOꢀ,
H2PO4ꢀ, and OHꢀ (1.5ꢁ 10ꢀ3 M) to sensors 1–3 (2.5ꢁ 10ꢀ5 M),
respectively. When 4 Eq of anions (1.5ꢁ 10ꢀ3 M in DMSO) was
introduced to the (2.5 ꢁ 10ꢀ5 M in DMSO) solution of sensors
1–2, sensor 1 responded with slight color changes from yellow
to light pink only for Fꢀ ion (Figure 2a) and sensor 2 showed
dramatic color change from yellow to pink for Fꢀ and AcOꢀ ions
(Figure 2b). The visible color changes are mainly because of the
detection of anions by the receptor molecules through hydrogen
bonding. The same titrations were repeated with other anions
under same conditions. But no color changes were observed
Receptor 2. Yield, 84%; mp, 204 ꢂC. 1H NMR (500 MHz,
DMSO-d6 d ppm): d 12.69 (s, 1H, OH), 9.04 (s, 1H, CH= N), 8.01–
8.10 (m, 2H, Ar), 7.91–7.92 (m, 2H, Ar), 7.82 (d, 1H, Ar), 7.58–7.60
(dd, 4H, Ar), 7.43–7.44 (m, 1H, Ar), 7.33–7.35 (m, 1H, Ar), and 7.05
(d, 1H, Ar). IR (KBr plates, cmꢀ1): 3440, 3059, 1610, and 1560. Mass:
calculated, 335.7944; found, 335.9604
Receptor 3. Yield, 86%; mp, 230 ꢂC. 1H NMR (500 MHz,
DMSO-d6 d ppm): d 13.91 (s, 1H, OH), 9.22 (s, 1H, CH = N),
8.72 (s, 1H, Ar), 8.29–8.30 (m, 2H, Ar), 7.97 (dd, 4H, Ar), 7.62–7.65
(m, 2H, Ar), 7.17 (d, 1H, Ar), 6.66 (d, 1H, Ar), and 6.08 (s, 1H, Ar). IR
(KBr plates, (cmꢀ1): 3493, 3096, 1627, and 1584. mass: calculated:
346.342, found: 347.1144.
RESULTS AND DISCUSSION
Single crystal of receptor 2 was obtained by slow evaporation
method in chloroform and its structure was analyzed by X-ray
diffraction (Enraf Nonius CAD4-MV31 Bruker Kappa APEXII
instrument). The crystal system of receptor 2 was found
as orthorhombic with Pca21 space group. Cell parameters
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2013; 26: 259–267

COLORIMETRIC CHEMO SENSORS
with other anions. To verify the sensing ability of receptor 2 and
Fꢀ/AcOꢀ in aqueous medium, the anions were prepared in
DMSO/H2O (v/v) (9:1, 7.5:2.5, and 5:5) with different ratios. When the
content of H2O was increased, the pink color of the anion–receptor
complex turned into orange (Supplementary Figure 1a) and then
yellow, indicating the regeneration of the receptor molecule.
Similarly, the same colorimetric titration was extended to
receptor 3 in ACN medium with same concentration of both
receptor and anions. The reason for choosing ACN as solvent is
because of the presence of highly acidic hydroxyl proton. If polar
DMSO is used, it will compete with receptor molecule and affect
hydrogen bonding. Hence, visible color change and optical
signal did not occurr. With the addition of 4 Eq of anions
(1.5 ꢁ 10ꢀ3 M in ACN) to receptor 3 (2.5 ꢁ 10ꢀ5 M in ACN), the
yellow color of the receptor was changed into orange for Fꢀ,
AcOꢀ, H2POꢀ4 , and OHꢀ ions (Figure 2c). However, no color
change was observed for Clꢀ and Brꢀ ions. The more number
of anion detection by receptor 3 is due to the presence of the
chromogenic signaling unit (ꢀNO2) in it. Again, to check the
sensing ability of receptor 3 in aqueous medium, the anions
were prepared in DMSO/H2O (v/v) (9:1, 7.5:2.5, 5:5) with different
ratios. For receptor 3 and Fꢀ/AcOꢀ, the orange color of the
anion-receptor 3 complex turned into yellow (Supplementary
Figure 1b), resulting in the regeneration of the receptor molecule.
Finally, the regeneration of receptors 2 and 3 were occurred at
DMSO/H2O (7.5:2.5), which is due to the effect of water molecule
in hydrogen bonding of anion complex of receptors 2 and 3.
a. Color Changes observed upon the addition of 4 eq. of various anions (1.5 x 10-3 M) to
S1 (2.5 x 10-5 M) (from left to the right : Free S1, F-, Cl-, Br-, AcO-, H2PO4 , OH- )
-
b.Color Changes observed upon the addition of 4 eq. of various anions (1.5 x 10-3 M) to
1H NMR titration
S2 (2.5 x 10-5 M) (from left to the right : Free S2, F-, Cl-, Br-, AcO-, H2PO4 , OH- )
-
1H NMR technique is a powerful technique used to study the
molecular interaction between receptor 2 and anions. For the
further understanding of the detection nature of anions by
1
receptor 2, H NMR titration was carried out using the solution
of TBA salt of Fꢀ and AcOꢀ ions to receptor 2 in DMSO-d6
(Supplementary Figures 2 and 3). Commonly, two main effects
will takes place while adding anions to receptor molecules, that
is, downfield shifting of protons present with heteroatoms (OH,
NH, and SH) due to hydrogen bonding and deprotonation with
excess of anions. In the absence of Fꢀ or AcOꢀ ion, receptor 2
showed singlet at 12.69 ppm for OH proton and at 9.02 ppm
CH = N proton. Also, double doublet peak at 7.9–8.0 ppm was
observed due to the phenyl ring proton of azo benzene. In
the presence of 1 Eq Fꢀ and AcOꢀ ions, the –OH signal at
12.69 ppm disappeared, suggesting that the OH proton was
deprotonated by incoming anions. However, there was no
shift in imine and aromatic protons observed with 1 Eq of
Fꢀ and AcOꢀ ions. In addition, the signal corresponding to imine
c. Color Changes observed upon the addition of 4 eq. of various anions (1.5 x 10-3 M) to
S3 (2.5 x 10-5 M) (from left to the right : Free S3, F-, Cl-, Br-, AcO-, H2PO4 , OH- )
-
Figure 2. (a) Color changes observed on the addition of 4 Eq of various
anions (1.5 ꢁ 10ꢀ3 M) to S1 (2.5 ꢁ 10ꢀ5 M) (from left to the right: free S1,
Fꢀ, Clꢀ, Brꢀ, AcOꢀ, H2PO4ꢀ, and OHꢀ). (b) Color changes observed on the
addition of 4 Eq of various anions (1.5 ꢁ 10ꢀ3 M) to S2 (2.5 ꢁ 10ꢀ5 M)
(from left to the right: free S2, Fꢀ, Clꢀ, Brꢀ, AcOꢀ, H2POꢀ4 , and OHꢀ). (c)
Color changes observed on the addition of 4 Eq of various anions
(1.5 ꢁ 10ꢀ3 M) to S3 (2.5 ꢁ 10ꢀ5 M) (from left to the right: free S3, Fꢀ,
Clꢀ, Brꢀ, AcOꢀ, H2POꢀ4 , and OHꢀ).
1.4
S3
0.8
S2
F-
-
1.2
F
Cl-
-
OH
Br-
-
4
0.6
1.0
H PO
AcO-
2
-
AcO
OH-
-
0.8
Cl
H2PO4-
-
(Br )
0.4
0.2
0.0
0.6
0.4
0.2
0.0
300
400
500
300
400
500
600
Wavelength(nm)
Wavelength(nm)
a
b
Figure 3. Absorption spectra of (a) receptor 2 in DMSO and (b) receptor 3 in ACN recorded (2.5 ꢁ 10ꢀ5 M) after the addition of 4 Eq of all anions
(1.5 ꢁ 10ꢀ3 M).
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S. SUGANYA AND S. VELMATHI
(CH = N) proton at 9.00 ppm had a upfield shift to 8.82 ppm with
the addition of 4 Eq of Fꢀ and AcOꢀ ions. Similarly, aromatic
rings proton at 7.9–8.0 ppm also got chemical shift in upfield to
7.87 with the addition of 4 Eq of Fꢀ or AcOꢀions. 1H NMR
titration concluded that the disappearance of OH signal and
chemical shifting of other protons present in receptor 2 system
by Fꢀ and AcOꢀ ions is mainly because of the detection property
of anion by receptor 2 via the deprotonation of OH group of
receptor 2, which leads to the formation of phenolate ion. Hence,
the electrodensity of aromatic system will be high and act as a p
conjugation with aromatic ring.
at 520 nm was observed for Fꢀ and AcOꢀ ions (Figure 3a). The
formation of new band at 520 nm is due to the breakage of
intramolecular hydrogen bonding and the formation of new com-
plex between anion and receptor molecule. On the incremental
4 eq
70
70
0 eq
60
50
60
40
50
30
20
40
10
UV-vis titration
0
30
0
1
2
3
4
5
no of eq
20
10
0
The anion detection was further confirmed by optical spectros-
copy. The spectroscopic titrations were carried out in DMSO
medium for receptors 1 and 2 and ACN for receptor 3. In the
absence of anion, both receptors 1 and 2 showed band at
375 nm, which is due to intramolecular hydrogen bonding
present within the receptor molecule. With the addition of
4.0 Eq of Fꢀ, Clꢀ, Brꢀ, AcOꢀ, OHꢀ, and H2PO–4 (1.5 ꢁ 10ꢀ3 M
in DMSO) into the solution of receptors 1 and 2 (2.5 ꢁ 10ꢀ5 M
in DMSO), receptor 1 showed a new band at 520 nm only with
Fꢀ ion; and for receptor 2, a new and strong absorption peak
440 460 480 500 520 540 560 580 600
Wavelength(nm)
Figure 6. Emission spectra of receptor
1 recorded in DMSO
(2.5 ꢁ 10ꢀ5 M) after the addition of 0–4 Eq of Fꢀ (1.5 ꢁ 10ꢀ3 M) with the
excitation wavelength of 420 nm.
0.16
0.20
0.14
1.0
0.12
0.10
1.0
0.15
0.08
0.8
0.10
0.06
0.04
0.8
0.05
0.02
0.6
0.00
0.6
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
no of eq
4 eq. F-
no of eq
4 eq. AcO-
0.4
0.2
0.0
0.4
0.2
0 eq. AcO-
0 eq. F-
0.0
300
400
500
600
300
400
500
600
Wavelength(nm)
Wavelength(nm)
a
b
Figure 4. Absorption spectra of receptor 2 recorded in DMSO (2.5 ꢁ 10ꢀ5 M) after the addition of 0–4 Eq of (a) Fꢀ and (b) AcOꢀ (1.5 ꢁ 10ꢀ3 M).
1.0
0.8
0.6
0.4
0.2
1.5
1.0
0.5
0.0
S3
10:0
9:1
7.5:2.5
5:5
S2
10:0
9:1
7.5:2.5
5:5
300
400
500
600
700
300
400
500
600
Wavelength(nm)
Wavelength(nm)
a
b
Figure 5. Absorption spectra of (a) receptor 2 in DMSO and (b) receptor 3 in ACN recorded (2.5 ꢁ 10ꢀ5 M) before and after the addition of 4 Eq
of Fꢀ (10: 0, 9:1, 7.5:2.5, 5:5 DMSO/ACN/H2O 1.5 ꢁ 10ꢀ3 M).
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J. Mol. Recognit. 2013; 26: 259–267

COLORIMETRIC CHEMO SENSORS
addition of 0–4 Eq of Fꢀ ion to receptor 1, an increase in the inten-
sity of the band at 520 nm (Supplementary Figure 4); and on the
incremental addition of Fꢀ/AcOꢀ to receptor 2, there is an increase
in the intensity of band at 520 nm and a decrease in the intensity of
the band at 375 nm with the isosbestic point at 422nm is observed
(Figures 4a and 4b).
The effect of water content in the anion complex of receptor 2
is also performed to monitor the sensitivity and stability of
the receptor toward Fꢀ/AcOꢀ ions in aqueous medium. To
find the sensing action of receptor 2 in aqueous medium, H2O
content was adjusted from DMSO to 9:1 DMSO/H2O mixture.
Then the yellow color of the receptor changed into orange for
Fꢀ and AcOꢀ ions. Accordingly, UV-vis titration showed the
newly formed band at 520 nm only for Fꢀ and AcOꢀ ions
(Supplementary Figure 5). With the incremental addition of
0–4 Eq of Fꢀ/AcOꢀ ion (9:1 DMSO/H2O), there is an increase
in the intensity of the newly formed band at 520 nm, and
the band at 375 nm is decreased (Supplementary Figures 6a
and 6b). Further, the water content was adjusted from DMSO
to 9:1, 7.5:2.5, and 5:5 (DMSO/H2O) mixtures, and the intensity
of the band at 375 nm is increased and at 520 nm is decreased
and attains the receptor peak. This highly indicates that the
water molecule will destroy the guest–host complex and re-
generate the free receptor 2 species with the intramolecular
hydrogen bonding as shown in (Figure 5a).
Table 1. Binding constant values for receptors 1, 2, and 3
with corresponding anions by the Benesi–Hildebrand plot
Anions
Binding
Receptors
(DMSO/H2O v/v)
constants(Ka)
S1
S2
S2
S2
S2
S3
S3
S3
S3
Fꢀ(10:0)
Fꢀ(10:0)
Fꢀ(9:1)
AcOꢀ(10:0)
AcOꢀ(9:1)
Fꢀ
9.568 ꢁ 103
1.709 ꢁ 104
1.238 ꢁ 104
1.49 ꢁ 104
8.37 ꢁ 103
2.484 ꢁ 104
6.585 ꢁ 104
4.47 ꢁ 103
7.94 ꢁ 103
The similar titration was repeated for receptor 3 in ACN
medium with same concentration. Receptor 3 shows bands at
370 nm in the absence of anion, which is corresponding to the
intramolecular hydrogen bonding present within the molecule.
AcOꢀ
OHꢀ
H2POꢀ4
S3
800
S2
F-
Cl-
Br-
AcO-
H2PO4
OH-
F-
200
Cl-
700
600
500
400
300
200
100
0
Br-
160
AcO-
-
H2PO4
OH-
120
80
40
440
480
520
560
600
440
480
520
560
600
Wavelength(nm)
Wavelength(nm)
a
b
Figure 7. Emission spectra of (a) receptor 2 in DMSO and (b) receptor 3 in ACN recorded (2.5 ꢁ 10ꢀ5 M) after addition of 4 Eq of all anions
(1.5 ꢁ 10ꢀ3 M).
800
4 eq. F-
800
700
4 eq. AcO-
700
600
800
700
600
500
400
300
200
100
0
800
700
600
500
400
300
200
100
0
600
500
400
300
200
100
0
0 eq. F-
500
400
300
200
100
0
0 eq. AcO-
0
1
2
3
4
no of eq.
0
1
2
3
4
no of eq.
440
480
520
560
600
440
480
520
560
600
Wavelength(nm)
Wavelength(nm)
a
b
Figure 8. Emission spectra of receptor 2 recorded in DMSO (2.5 ꢁ 10ꢀ5 M) after the addition of 0–4 Eq of (a) Fꢀ and (b) AcOꢀ (1.5 ꢁ 10ꢀ3 M) with the
excitation wavelength of 420 nm.
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S. SUGANYA AND S. VELMATHI
While adding 4 Eq of anions to receptor 3, a new band at 420 nm
for Fꢀ, AcOꢀ, OHꢀ, and H2PO–4 was noticed (Figure 3b). However,
in this case, during the incremental addition of 0–4 Eq anions to re-
ceptor 3, the intensity of both band at 370 nm and the band at
420nm is increased. The main reason for the enhancement in
the intensity of the band at 370 nm is due to the formation of
six-membered transition state complexes between receptor 3
and anions (Supplementary Figures 7a–7d).
given in the Table 1. Job’s plot studies (Supplementary Figure 9)
was performed to find out the stoichiometry of the complex formed
between receptors 2 and 3 and anions, and it was found that the
complex formed was in 1:1 stoichiometry.
Emission spectral studies
Emission spectroscopy will give more information about the anion
sensing properties of the receptors. Emission titration was carried
When the same titration was performed in the 9:1 ACN/H2O
medium, a selective recognition of Fꢀ and AcOꢀ was ob-
served (with the addition of 4 Eq) from light yellow to intense
yellow, and optical changes are also noticed with the
addition of 4 Eq of anions to receptor 3. The intensity of
both bands at 370 and 420 nm is increased (Supplementary
Figure 8). As mentioned earlier, when the water content
was adjusted from ACN to 9:1, 7.5:2.5, and 5:5 (ACN/H2O)
mixtures, the intensity of the band at 370 and 420 nm is
decreased and attains the receptor peak. This suggests that
the water molecule will destroy the guest–host complex
and regenerate the free receptor 3 species with the intramo-
lecular hydrogen bonding (Figure 5b).
Table 2. Quantum yield and quenching constant values for
receptors 1, 2, and 3 with corresponding anions
Receptors
Quantum yield Quenching constants (Ksv)
S1
0.009
0.078
0.016
0.65
0.61
0.20
0.05
0.04
0.123
0.18
—
—
—
—
—
S1 + Fꢀ
S2
S2 + Fꢀ
S2 + AcOꢀ
S3
—
The binding ability of sensors 1–3 toward various anions
was studied using UV-vis titration experiments by nonlinear
least squares fitting curve at 535, 520, and 420 nm for recep-
tors 1, 2, and 3, respectively. The association constants
(Ka) were calculated using the Benesi–Hildebrand plots, which are
S3 + Fꢀ
S3 + AcOꢀ
S3 + H2POꢀ4
S3 + OHꢀ
3.029 ꢁ 104
3.978 ꢁ 104
9.776 ꢁ 103
1.841 ꢁ 103
0 eq F-
0 eq AcO-
4 eq AcO-
180
160
140
120
100
80
180
160
200
200
160
120
80
140
4 eq F-
120
100
160
80
60
60
40
120
40
0
1
2
3
4
0
1
2
3
4
no of eq.
no of eq.
80
40
0
40
0
440
480
520
560
600
440
480
520
560
600
Wavelength(nm)
Wavelength(nm)
a
b
180
0 eq H2PO4-
160
180
0 eq OH-
200
160
120
80
160
200
160
120
80
4 eq H2PO4-
140
4 eq OH-
140
0
1
2
3
4
120
100
80
no of eq.
0
1
2
3
4
no of eq.
40
40
440
480
520
560
600
440
480
520
560
600
Wavelength(nm)
Wavelength(nm)
c
d
Figure 9. Emission spectra of receptor 3 recorded in ACN (2.5 ꢁ 10ꢀ5 M) after the addition of 4 Eq of (a) Fꢀ, (b) AcOꢀ, (c) H2POꢀ4 , and (d) OHꢀ
(1.5 ꢁ 10ꢀ3 M) with the excitation wavelength of 400 nm.
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J. Mol. Recognit. 2013; 26: 259–267

COLORIMETRIC CHEMO SENSORS
1.0
0.8
0.6
0.4
0.2
0.0
S2
S2+F-
S2+F-+TP
S2+TP
300
400
500
600
Wavelength(nm)
a
b
Figure 10. (a) UV-vis spectrum and (b) proof of F- ion detection in TP of receptor 2 (2.5 ꢁ 10ꢀ5 M in DMSO) (A), 2+ TP (20 mg/ml in DMSO) (B), 2 + F- ion
(1.5 ꢁ 10ꢀ3 M in DMSO) (C) and 2+ F- ion+TP (D).
by OHꢀ ion (Equation (1)), and the formation of anion
complexes of receptor 3 is taking place at equilibrium. The
H
R
C
reason behind this enhancement and quenching in the
fluorescence intensity may be due to the formation of anion
complexes of receptor molecules 1–3 through intermolecular
hydrogen bonding, which leads to nonradiative decay Internal
Charge Transfer (ICT) from the excited state.
N
H
N
N
O
R=NO2
AcO- in ACN
R=Cl
AcO- in DMSO
R
H
R
C
N
N
N
N
H
N
N
-
-
½LHꢃ þ OH ⇌½L ꢃ þ H2O !
(1)
O
O
H
H
O
O
O
H3C
O
The quantum yield of receptors 1–3 and their anion complexes
were calculated using the emission titration method with rhoda-
mine B as a standard. Quenching constants (Ksv) of receptor 3
and anion complexes of receptor 3 were also calculated using
the Stern–Volmer equation (Eq 2) (Supplementary Figure 10), and
their values are given in Table 2. The quenching rate constants
were found higher for the Fꢀ and AcOꢀ ions than for the H2POꢀ4
and OHꢀ ion complexes of receptor 3.
CH3
H2O
H
R
C
H2O
N
H
N
N
O
+ Aqs. AcO-
-
Figure 11. The proposed mechanism for the complex formed between
receptors 2/3 and acetate anion.
F0=F ¼ 1 þ Ksv½Qꢃ !
(2)
With this knowledge, we can design the biologically active
fluorescence sensor containing OH binding site for the
detection of biologically important anions. On the basis of
the previously mentioned results, the mechanism behind the
anion sensing property of receptors 2 and 3 in organic and
semi-aqueous medium will be proposed as shown in Figure 10.
On the basis of the binding constant values, the order of
binding affinity of receptors toward anions is S3 > S2> > S1
(Figure 11). The main reason for this order is the presence of Cl
and NO2 substituents in S2 and S3. Because of their electron
withdrawing nature, it will enhance the acidity of the hydroxyl pro-
ton present in receptors 2 and 3 and form an effective hydrogen
bond with the anions. By increasing the electron-withdrawing
properties, we can be able to tune strong anion complexes
with higher binding constants. The binding order of anions for
receptors is as follows (in either DMSO/ACN or DMSO/ACN/water):
AcOꢀ > Fꢀ > OHꢀ, H2PO4ꢀ > Clꢀ, Brꢀ, and Iꢀ.
out similar to UV-vis spectroscopy under same condition with
excitation wavelength around 420 nm for receptors 1 and 2
and 400 nm for receptor 3. On the addition of 4 Eq of anions
(1.5 ꢁ 10ꢀ3 M) to receptors 1 and 2 (2.5 ꢁ 10ꢀ5 M), only Fꢀ ion
shows emission maxima for receptor 1 at 480 nm (Figure 6);
Fꢀ and AcOꢀ ions show emission maxima for receptor 2 at
480 nm (Figure 7a). With the incremental addition of 0–4 Eq of
Fꢀ ion to receptor 1, enhancement in the fluorescence
intensity was observed. Similarly, enhancement in the fluores-
cence intensity was noticed during the incremental addition of
0–4 Eq of Fꢀ and AcOꢀ ions to receptor 2 (Figures 8a–8b).
However, receptor 3 showed fluorescence quenching instead
of enhancement toward Fꢀ, AcOꢀ, OHꢀ, and H2POꢀ4 ions at
480 nm (Figure 7b). This quenching mechanism of receptor 3
in the presence of anions is due to the presence of ꢀNO2
functionality as fluorescence quencher. When the titration of
receptor 3 was carried out with the incremental addition
of 0–4 Eq of Fꢀ, AcOꢀ, OHꢀ, and H2POꢀ4 ions, the intensity of
the band at 480nm is decreased (Figures 9a–9d). Simulta-
neously, by the addition of OHꢀ ion to receptor 3, a new band
was created at 540 nm with fluorescence enhancement as well
as the band at 480 nm with fluorescence quenching. This
result confirms the deprotonation of highly acidic OH group
Qualitative detection of Fꢀ ion in toothpaste
To extend the molecular recognition properties of receptor 2 in
real sample (Kumar et al., 2010), we have carried out the qualita-
tive detection of Fꢀ ion experiment in commercially available
toothpaste. The result was analyzed by UV-vis spectroscopic
J. Mol. Recognit. 2013; 26: 259–267
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S. SUGANYA AND S. VELMATHI
a
b
Figure 12. The absorbance at 520 nm for receptor (a) 2 and at 420 nm (b) 3 after the addition of all anions.
method. The samples used for UV-vis titration are toothpaste in
DMSO (20mg/ml), 4Eq Fꢀ (1.5 ꢁ 10ꢀ3 M in DMSO), and receptor 2
(2.5 ꢁ 10ꢀ5 M in DMSO). The absorption measurements clearly ]
show that the Fꢀ ion complex of receptor 2 has less absorbance
than that of the Fꢀ ion complex of receptor 2 with toothpaste
solution. Also, the formation of a new band at 520 nm in UV-vis
spectrum only with the addition of toothpaste solution to
receptor 2 indicates that the Fꢀ ion present in the toothpaste
was detected by receptor 2 (Figure 12). To device a sensor
system for the Fꢀ ion detection under aqueous medium is
aimed for future research.
Receptors 2 and 3 showed distinct color changes for Fꢀ and
AcOꢀ in 9:1 DMSO/H2O mixture. The strong affinity of receptors
2 and 3 toward Fꢀ and AcOꢀ ions in competitive solvent mixture
indicates the stability of the complex formed between receptor
molecules and anions. In comparison, receptor 3 shows high
affinity toward Fꢀ and AcOꢀ ions with 1:1 stoichiometric ratio,
which is proved by the higher binding constant values. This is
mainly because receptor 3 contains highly acidic OH group
through NO2 substituent in it. Finally, receptor 2 was applied
for the qualitative detection of Fꢀ ion in commercially available
toothpaste sample.
CONCLUSION
Acknowledgement
S.V. thanks Defence Research and Development Organization
(DRDO) (grant no. EPIR/ER/1006004/M/01/1333) for a major
sponsored project and for financial assistance.
In conclusion, we have developed a series of simple and novel
colorimetric probes 1–3, which are applied for the detection of
various anions in organic and organic–aqueous mixture medium.
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COLORIMETRIC CHEMO SENSORS
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