Preparation of 4,4′-bis-(carboxyl phenylazo)-dibenzo-18-crown-6 dye and its application on ratiometric colorimetric recognition to Hg2+

Article abstract of DOI:10.1016/j.saa.2011.03.054

A multifunctional dye, 4,4′-bis-(carboxyl phenylazo)-dibenzo-18- crown-6 dye (BCADC) was designed and prepared via diazotization and coupling reaction of dibenzo-18-crown-6 with p-amino benzoic acid. The dye, combining crown ether ring, azo and carboxyl g

Full text of DOI:10.1016/j.saa.2011.03.054

Spectrochimica Acta Part A 79 (2011) 661–665
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Preparation of 4,4ꢀ-bis-(carboxyl phenylazo)-dibenzo-18-crown-6 dye and its
application on ratiometric colorimetric recognition to Hg2+
Zhengquan Yan∗, Lei Hu, Li Nie, Hong Lv
Department of Material and Chemical Industry, West Anhui University, Lu’an 237012, China
a r t i c l e i n f o
a b s t r a c t
A multifunctional dye, 4,4ꢀ-bis-(carboxyl phenylazo)-dibenzo-18-crown-6 dye (BCADC) was designed
and prepared via diazotization and coupling reaction of dibenzo-18-crown-6 with p-amino benzoic
acid. The dye, combining crown ether ring, azo and carboxyl group, exhibits well-defined Hg2+-selective
ratiometric colorimetric behavior, with the maximum absorbance peak changing from 354 nm to
408 nm exclusively. Under the optimum conditions, the recognition to Hg2+ has a linear range of
2.5–58 × 10−7 mol L−1 with a 0.9978 correlation coefficient. The method was applied to analyse 3 envi-
ronmental water samples with a detection limit of 2.9 × 10−8 mol L−1 and a relative standard deviation
(R.S.D.) lower than 3.7% (n = 5). The action mechanism between BCADC and metal ions was discussed by
means of Job’s plots and theoretical calculations.
Article history:
Received 11 February 2011
Received in revised form 21 March 2011
Accepted 24 March 2011
Keywords:
4,4ꢀ-Bis-(carboxyl
phenylazo)-dibenzo-18-crown-6
Azo dye
Ratiometric
© 2011 Elsevier B.V. All rights reserved.
Colorimetric sensors
Recognition
Hg2+
1. Introduction
ing process, and even only to utilize naked eyes instead of the
complex instrument [2,12,13]. However, the sensitivity of common
Hg2+ is one of the environmentally most toxic and dangerous
heavy metal ions because of the high affinity for thiol groups in pro-
teins and enzymes. Especially, Hg2+ can accumulate in the human
body, which leads to the dysfunction of cells and consequently
causes a wide variety of diseases in the brain, kidney, and central
nervous system even in a low concentration [1,2]. However, despite
the toxicity, mercury and mercuric salts are still widely used in
industrial process and a high percentage of mercury contamination
has happened here and there in many products of daily life such
as paints, electronic equipment, and batteries [3,4]. Accordingly,
the accurate and facile detection of Hg2+ is of great importance.
Classic detection methods include atomic absorption spectrometry
[5], X-ray fluorescence spectrometry [6] and electro-chromic tech-
niques [7], most of them require the complicated instrumentation
and involve some cumbersome laboratory procedures. Although
several papers describe fluorescent chemosensors which offer the
advantages of rapid, sensitive and nondestructive detection meth-
ods [8–10], these sensor systems for Hg2+ have typically suffered
from unfavorable fluorescence quenching due to spin orbit inter-
actions [11].
colorimetric sensors is low and the detections are conducted in
nonaqueous solutions. So in view of the selective signal of Hg2+ in
aqueous solution, the present work concerns a multifunctional dye
that exhibits high selectivity and sensitivity to Hg2+ achieved by
using a crown-ether ring as the anchoring group with Hg2+ based
on the oxygen affinity nature as well as the size of the mercuric
ion [14], –N N– group as the connecting group to reduce other
coexisting ion interferences [15], and –COOH group to increase the
solubility in aqueous solution.
On the basis of the ideas above, in this work, a multifunctional
chemosensor 4,4ꢀ-bis-(carboxyl phenylazo)-dibenzo-18-crown-6
dye (BCADC) was selected and prepared (Scheme 1). The compound
effectively signals Hg2+ exhibiting well-defined, visible, Hg2+
-
selective ratiometric chromogenic behavior with the maximum
absorbance at 354 nm decreasing and that at 408 nm increasing
accordingly. The selectivity to Hg2+ was not significantly affected
in the presence of common physiologically and environmentally
important alkali, alkaline earth, rare earth and transition metal
ions, such as K+, Mg2+, Ca2+, Ba2+, Na+, Ni2+, Cd2+, Ag+, Cu2+
,
Pb2+, Al3+, Co3+, Fe3+, Eu3+ and Dy3+. The detection limit was
2.9 × 10−8 mol L−1, which is comparable to the toxicity level of
Hg2+ in drinking water (3.0 × 10−8 mol L−1) defined by Word Health
Organization (WHO) [16]. The action mechanism of BCADC and
metal ions was discussed by means of Job’s plots and theoretical cal-
culations. The proposed method was applied in the analysis of Hg2+
in environmental aqueous samples and the results were satisfying.
Recently, colorimetric sensors have drawn more and more
attention to decrease the experimental cost, to simplify the sens-
∗
Corresponding author. Tel.: +86 564 3305801.
E-mail address: yzhq2008@yahoo.com.cn (Z. Yan).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.03.054

662
Z. Yan et al. / Spectrochimica Acta Part A 79 (2011) 661–665
Scheme 1. Synthetic route and serial number of aromatic ring of the target compound.
2. Experimental
2.1. Reagents
Dibenzo-18-crown-6, p-amino benzoic acid and all the other
chemicals were of AR grade and were used as received from
Sinopharm Chemical Reagent Co. Ltd. Water used throughout was
doubly deionized.
A 1.0 × 10−6 mol L−1 Hg2+ standard solution for testing was pre-
pared in doubly deionized water at room temperature and diluted
to appropriate concentration daily. 5.0 × 10−4 mol L−1 (BCADC)
stock solution was prepared in DMSO at room temperature and
stored at room temperature. NaAc–HAc or phosphate buffers were
prepared by the mixture of 0.01 mol L−1 of HAc solution and
0.01 mol L−1 of NaAc solution or 0.01 mol L−1 Na2HPO4 solution and
0.01 mol L−1 KH2PO4 solution to the desired pH.
Fig. 1. UV–vis absorption spectra of BCADC in the absence (- - -) and presence (––)
of Hg2+ (CHg = 5.8 × 10−6 mol L−1, pH 5.0; CBCADC = 5.0 × 10−5 mol L−1).
2+
flask. The mixture was stirred thoroughly and finally diluted to
10 mL with doubly deionized water. After incubating for 15 min,
the absorbance spectra were measured from 250 nm to 500 nm and
the band-slit was set as 2.0 nm. The ratio (A408/354) of the absorb-
ing intensity at 408 nm to that at 354 nm was used for quantitative
analysis.
2.2. Apparatus
IR spectra were recorded on a Perkin Elmer Model 882 infrared
spectrometer, mixed with KBr and pressed into pellets, scanning
from 4000 to 500 cm−1 1H NMR were recorded using a Bruker
.
AMX-400 spectrometer operating at 400 MHz, with tetramethyl-
silane(TMS) as a reference and DMSO-d6 as solvent. Elemental
analysis was conducted using an Elemental Vario EL-III apparatus.
UV–vis spectra were recorded on a Shimadzu UV-265 spectrometer
using a 1-cm square quartz cell. All pH measurements were made
with a PHS-25 pH meter.
3. Results and discussion
3.1. The UV–vis absorption spectrum of BCADC
The UV–vis absorption spectra of BCADC in the absence and
presence of Hg2+ in HAc-NaAc (pH 5.0) solutions were shown in
Fig. 1.
2.3. Preparations of BCADC
It can be seen from Fig. 1 that BCADC has two absorbance peaks
between 250 nm and 500 nm. The first absorption peak at 288 nm
is attributed to the ␲–␲ electron transition in non-conjugated p-
amino benzoic acid moiety. The second absorption peak at 354 nm
results in the ␲–␲ electron transition in the huge ␲-conjugated
As shown in Scheme 1, p-amino benzoic acid (0.686 g, 5 mmol)
was dissolved in an ice–water solution of 15% sodium nitrite
(0.38 g, 5.5 mmol). After cooling to 0 ◦C, the solution was added
to concentrated hydrochloric acid (1.2 mL) and stirred for 30 min.
The excess nitrous acid was destroyed with about 5 mg urea.
The mixture was then added drop wise to 10 mL buffered
aqueous solution (KH2PO4/Na2HPO4, pH 6) containing dibenzo-
18-crown-6 (1.56 g, 5 mmol) and stirred for another 2 h at 0–5 ◦C.
The resultant precipitate was filtered and purified by column
chromatography on silica gel (eluent: petroleum methanol/ethyl
acetate = 1:9) to provide light yellow crystal BCADC in the yield of
68.5%.
system, N,N-di-phenyl azo, with εmax about 7.5 × 104 L mol−1 cm−1
.
In the presence of Hg2+, the absorbance at 288 nm keeps unchanged
nearly, however, the absorbance at 354 nm obviously red shifts to
Mp, 202–203 ◦C; IR (KBr), ꢀ (cm−1): 2500–3500 (–COOH), 1697
(C O), 1604 (N N), 1258 (C–O–C). 1H NMR (DMSO-d6, 400 Hz) ı
(ppm): 12.5 (s, 2H, COOH), 7.8 (d, J = 8.5 Hz, 4H, H5), 7.5 (d, J = 8.4 Hz,
4H, H4), 7.0–6.8 (m, J = 5.6 Hz, 6H, H1,2,3), 4.1 (t, J = 2.7 Hz, 8H, CH2),
3.9 (t, J = 2.5 Hz, 8H, CH2). Anal. Calcd for C34H32N4O10: C, 62.19; H,
4.91; N, 8.53. Found: C, 62.73; H, 5.04; N, 9.01.
2.4. Procedures of detection
For Hg2+ detection, 1.0 mL HAc–NaAc (pH 5.0), 1.0 mL 5.0 ×
10−4 mol L−1 of BCADC and 1.0 mL of the appropriate concentra-
tion of Hg2+ solution were transferred into a 10 mL volumetric
Fig. 2. Effect of pH on the absorbance of BCADC at 408 nm (pH 5.0;
CBCADC = 5.0 × 10−5 mol L−1, CHg = 5.8 × 10−6 mol L−1).
2+

Z. Yan et al. / Spectrochimica Acta Part A 79 (2011) 661–665
663
Fig. 3. Effect of different iron ions on the UV–vis absorbance spectrum of BCADC
Fig. 6. Job’s plot of BCADC–Hg2+ system. [BCADC] + [Hg2+] = 6.0 × 10−5 mol L−1, in
(from up to down: blank, K+, Mg2+, Ca2+, Ba2+, Na+, Ni2+, Cd2+, Ag+, Cu2+, Pb2+, Al3+
,
aqueous 10% DMSO solution at pH 5.0.
Co3+, Fe3+, Eu3+ and Dy3+), pH 5.0; CBCADC = 5.0 × 10−5 mol L−1.
3.3. Effect of coexisting foreign substances
408 nm, a visible spectrum, which allows a naked-eye detection of
Hg2+ in aqueous solution.
To demonstrate the selectivity of BCADC for Hg2+, we have inves-
tigated the colorimetric response of BCADC to other physiologically
and environmentally important alkali, alkaline earth, rare earth and
transition metal ions, such as K+, Mg2+, Ca2+, Ba2+, Na+, Ni2+, Cd2+
,
3.2. Effect of pH value
Ag+, Cu2+, Pb2+, Al3+, Co3+, Fe3+, Eu3+ and Dy3+ in aqueous solutions
containing 5.0 × 10−5 mol L−1 BCADC at pH 5.0.
As pH of the system has a significant influence on the interaction
between BCADC and Hg2+ and the detection sensitivity, the effect
of pH on the detection was investigated in the range of pH 4.0–9.0.
As shown in Fig. 2, it can be easily seen that pH of the solution plays
an important role in the interaction between BCADC and Hg2+. The
absorbance at 408 nm vary gradually with the decrease of pH and
reaches the maximum when pH is ca 5.0. The reason may be that at
pH < 5.0, oxygen atoms in crown ring are easily protonated, which
reduces their coordination with Hg2+. When pH is more than 5.0,
the solubility of Hg2+ in aqueous solution decreases. Both above
decrease the interaction between crown ether rings and Hg2+, so
the absorbance at 408 nm decreases.
Fig. 3 shows the relative changes in the absorbance intensity
of BCADC with the addition of 1 equiv of different metal ions. The
additions of these metal ions except Hg2+ all show negligible red-
shifts in the absorption of BCADC and the change in the absorbance
intensity at 408 nm is less than 5% relative to Hg2+ at the same
conditions. It means that the selectivity of BCADC to Hg2+ is well.
We also find that some high valence metal ions, especially
rare earth elements, i.e., Eu3+ and Dy3+ can make the absorbance
of BCADC at 354 nm quench. The reason may be that some high
valence metal ions, i.e., Eu3+ and Dy3+ can coordinate with –N
bond to make the terminal phenyl rings distorted. While in the
N
Fig. 4. The schematic diagram of the complex between BCADC and rare earth elements and Hg2+
.
Fig. 5. (a) The absorbance spectra of BCADC in different Hg2+ concentrations: 0, 25, 40, 60, 80, 100, 140, 170, 200, 250, 300, 350, 400, 480, 580 × 10−7 mol L−1. (b) The linear
relationship between the A408/354 of the system at 408 nm. pH 5.0; CBCADC = 5.0 × 10−5 mol L−1
.

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Z. Yan et al. / Spectrochimica Acta Part A 79 (2011) 661–665
Table 1
Determination results for environmental water samples (n = 5).a
Samplesb
CHg in sampleb (10−9 mol L−1
)
Spiked (10−9 mol L−1
)
Found (10−9 mol L−1
)
Recovery (%)
R.S.D. (%) (n = 5)
2+
1(the Pi River)
2 (underground water)
3 (tap water)
425.1
948.4
0.00
500.0
500.0
500.0
881.6
1487.5
493.0
95.3
102.7
98.6
2.0
0.9
3.1
a
pH 5.0; CBCADC = 5.0 × 10−5 mol L−1
.
b
The environmental water Hg2+ concentration determined using BCADC with the proposed method. The real values are the table values × 10−2 mol L−1 for the detected
water samples were concentrated 100 times.
Fig. 7. The optimized structures of the complexes between BCADC and rare earth elements and Hg2+
.
presence of Hg2+, Hg2+ can coordinate with oxygen atoms in crown
ether ring, not with nitrogen atoms in –N N bonds, based on the
oxygen affinity nature as well as the size of the mercuric ion [14]
(Fig. 4).
mined by using Job’s plot. For the Job plot analyses, a series of
solutions with varying mole fraction of Hg2+ ions were prepared
by maintaining the total BCADC and Hg2+ concentration con-
stant (6.0 × 10−5 mol L−1). The absorbance intensity at 408 nm was
measured for each solution. A 1:1 stoichiometry, not 1:2 for the
complex between BCADC and Hg2+, drawn from Job’s plots [17] in
Fig. 6, confirms that Hg2+ coordinates with oxygen atoms in crown
ether rings, not with nitrogen atoms in –N N bonds, as proposed
above.
Theoretical calculations have been carried out to further under-
stand the nature of the bonding between BCADC and Hg2+. The
complexes shown in Fig. 4 were optimized using the B3LYP/6-
31G level of theory and method implemented in the Gaussian 03
suite of program [18]. That Hg2+ can selectively coordinate with
oxygen atoms in crown ether ring, not with nitrogen atoms in
–N N bonds, may be attributed to the oxygen affinity nature as
well as the size of the mercuric ion [14]. As shown in Fig. 7, once
the binding of Hg2+ with BCADC, the stability and conjugated sys-
tem of the complex both increase owing to the formation of six
rigid five-membered rings. So the absorbance of BCADC is red-shift.
On the other hand, the coordination of some high valence metal
ions such as Eu3+ and Dy3+ with –N N bond makes the terminal
phenyl rings distorted which results in the original conjugated sys-
tem destroyed and so the absorbance at 354 nm deduced and even
quenched.
3.4. Analytical parameters and samples detection
Fig. 5a shows the absorbance spectra of BCADC at dif-
ferent concentrations of Hg2+ between 2.5 × 10−7 mol L−1 and
5.8 × 10−6 mol L−1. From the spectra, the calibration graph, the
detection limit and precision for Hg2+ detection under the opti-
mum conditions can be obtained (Fig. 5b). A linear relationship
between BCADC and Hg2+ concentration is exhibited in the range
of 2.5–58 × 10−7 mol L−1 with a correlation coefficient of 0.9978.
The regression equation is A408/354 = −1.46 × 10−1 + 7.73 × 10−3 c
(10−7 mol L−1). Based on the definition of detection limit, three
times of average deviation of absorbing intensity ratio (A408/354
)
of the intensity at 408 nm to that at 354 nm in 20 blank samples
without Hg2+ used here, the limit of detection (LOD) for Hg2+ is up
to 2.9 × 10−8 mol L−1, which is comparable to the toxicity level of
Hg2+ in drinking water (3.0 × 10−8 mol L−1) defined by Word Health
Organization (WHO) [16].
To further evaluate the feasibility, the proposed method has
been applied to analyse 3 environmental water samples from the
Pi River, underground water and tap water in campus, respectively.
All the samples were obtained by filtering several times and con-
centrated by 100 times. The Hg2+ concentrations in the sample are
determined colorimetrically using a standard addition method by
measuring A408/354 of BCADC at 408 nm and 354 nm with the above
mentioned procedures.
Table 1 shows the results obtained in the three independent
samples above. For recovery studies, some known concentrations
of Hg2+ were added to the environmental water samples and the
total Hg2+ concentration determined following the method pro-
posed above. The recoveries of different known amounts of Hg2+
spiked were obtained from 95.3% to 102.7% with a satisfying ana-
lytical precision (R.S.D. ≤ 3.7%), which validated the reliability and
practicality of this method.
4. Conclusions
In conclusion, we have designed and demonstrated the use of
BCADC as a novel ratiometric colorimetric sensor for the detection
of Hg2+ in aqueous solutions. BCADC displays unusual selectivity for
Hg2+ due to the special oxygen affinity nature and size of mercury
ion. Under the optimum conditions, the method has a linear range
of 2.5–58 × 10−7 mol L−1 with a 0.9978 correlation coefficient. The
limit of detection was 2.9 × 10−8 mol L−1 meeting the toxicity level
of Hg2+ in drinking water (3.0 × 10−8 mol L−1) defined by Word
Health Organization (WHO) [16]. The presented method has been
applied successfully to the determination of Hg2+ in environmental
samples. The reaction mechanism between BCADC and metal ions
was discussed by means of Job’s plots and theoretical calculations.
Further studies are in progress to explore the application of BCADC
in detection of rare earth metals.
3.5. Mechanisms of the action
To investigate the nature of the bonding between BCADC and
Hg2+, the binding stoichiometry of BCADC with Hg2+ was deter-

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665
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