A nanomolar detection of mercury(II) ion by a chemodosimetric rhodamine-based sensor in an aqueous medium: Potential applications in real water samples and as paper strips

Article abstract of DOI:10.1016/j.saa.2018.11.005

A new promising rhodamine based colorimetric and fluorometric chemosensor, RDV has been designed and synthesized for specific detection of Hg²⁺ ion. It acts as highly selective “turn-on” fluorescent chemosensor for Hg²⁺ ion without i

Full text of DOI:10.1016/j.saa.2018.11.005

Accepted Manuscript
A nanomolar detection of mercury(II) ion by a chemodosimetric
rhodamine-based sensor in an aqueous medium: Potential
applications in real water samples and as paper strips
Sagar K. Patil, Dipanwita Das
PII:
S1386-1425(18)30996-X
https://doi.org/10.1016/j.saa.2018.11.005
SAA 16569
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 19 September 2018
Revised date:
Accepted
date:
26 October 2018
3
November 2018
Please cite this article as: Sagar K. Patil, Dipanwita Das , A nanomolar detection of
mercury(II) ion by a chemodosimetric rhodamine-based sensor in an aqueous medium:
Potential applications in real water samples and as paper strips. Saa (2018), https://doi.org/
1
0.1016/j.saa.2018.11.005
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ACCEPTED MANUSCRIPT
A nanomolar detection of mercury(II) ion by a chemdosimetric rhodamine-
based sensor in an aqueous medium: potential applications in real water
samples and as paper strips
Sagar K. Patil and Dipanwita Das*
Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai 400 019,
India
Fax: (91)022-3361-1020
Email: dr.das@ictmumbai.edu.in
1

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Abstract
A new promising rhodamine based colorimetric and fluorometric chemosensor, RDV has
2
+
been designed and synthesized for specific detection of Hg ion. It acts as highly selective
2
+
“
turn-on” fluorescent chemosensor for Hg ion without interference from other competitive
2
+
metal ions in aqueous acetonitrile medium. The drastic color change with addition of Hg ,
2
+
2+
from colorless to pink, indicates RDV can acts as “naked-eye” indicator for Hg . The Hg
promoted selective hydrolysis of appended vinyl ether group in RDV followed by Hg2+
chelated complex formation with concomitant opening of spirolactam ring is the plausible
1
13
sensing mechanism. The detailed absorption, fluorescence, H NMR, C NMR and mass
spectrometry confirms the proposed sensing mechanism. The limit of detection (LOD) of
Hg2+ by RDV is 136 nM indicating the high sensitivity towards Hg . The RDV shows
consistent spectroscopic response in biological pH range 4-10. In addition to explore practical
2+
2
+
applicability of RDV, its paper strips have been made and used to detect Hg in pure water
solution up to 10 ppm level. Furthermore, the potential application of RDV for the sensing of
2
+
Hg in real water samples (tap water and drinking waters from different sources) were also
monitored and demonstrated.
Keywords:
Chemodosimeter
Oxymercuration
Colorimetric
Fluorescent
Paper strip application
2

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1
. Introduction
Mercury, highly toxic heavy metal, concerns great attention for scientific community due to
its widespread contamination in environment and biological systems. Both elemental and its
oxidised form Hg2+ can be converted to organic mercury form by microorganism in the
environment, makes it easy to accumulate in biological system through food chain [1-3].
Easy skin permeability of organic mercury can damage central nervous system and endocrine
system [4]. Inorganic mercury give serious effects on heart system, kidney and stomach.
Contamination of methylmercury to fish and grain reports epidemics in Japan and Iraq
respectively [5]. Prof. Karen Wetterhahn, an organometallic chemist, causes death due to skin
contact with highly poisonous methylmercury compound [6]. Beside these natural causes,
2
+
unnatural sources are also responsible for Hg poisoning including careless industrial waste
management, from coal-fired power plant [7], fossil fuel combustion [8] and gold mining [9].
Therefore, due to these adverse effects, scientific community concern more attention to
develop Hg2+ probe for environmental sensing, onsite application and most importantly
works in aqueous solution.
2
+
Conventionally, Hg quantified by atomic absorption spectroscopy (AAS) [10,11], emission
spectroscopy, inductively coupled plasma mass spectroscopy (ICP-MS) [12], electrochemical
measurement [13,14], gas chromatography etc. requires highly trained manpower,
sophisticated instruments and are time consuming processes with multistep analysis. In this
regard, optical chemosensor (optical and fluorescent) perceived great attention due to its high
selectivity, high sensitivity and on-site analysis with fewer resources. In literature, large
numbers of sulfur containing optical chemosensors are designed and reported, due to soft
Lewis basic nature of sulfur which can easily bind with soft Lewis acidic mercury ion [15-
1
8]. Prof. Koide et. al. reports mercury sensing based on oxymercuration reaction i.e.
hydrolysis of aryl vinyl ether, provides another approach to design mercury sensor [19].
3

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Reaction is specific towards the mercury due to π electrophilicity of mercury ion and reaction
can proceed by methyl mercury species. So far, very few reports are available on this type of
mercury sensors [20-22].
Rhodamine has excellent photophysical properties, like long absorption and emission
wavelength, high molar extinction coefficient value and high quantum yield [23,24] which
makes it a competent optical probe for d-block metals [25]. Opening of non-fluorescent
spirolactam ring to highly fluorescent ring opened amide scaffold is a promising tool for the
recognition mechanism [26]. Several rhodamine based receptors were reported in literature
for sensing of palladium [27-30], copper [31-34], zinc [34], cadmium [35-37], chromium [38-
4
0] and also for mercury. For mercury, however, in most of the cases, the mechanism is
governed by chelation [40], i.e. metal complex formation with rhodamine receptor where
specificity and the detection limit are still questionable. Hitherto, no rhodamine-based
receptor was reported based on chemdosimetric oxymercuration approach towards specific
sensing of Hg2+ ion at nM detection level. In the last decade, some chemosensors are
2
+
designed and synthesised for Hg based on different functionalities [41-54], however, most
of these suffer certain limitations like high detection limits [41-43,46,47,50,52], poor
solubility in aqueous media [41,42,45-47,49], interference by other metal ions [51,53], unable
2
+
to detect Hg in wide pH range [41,49,54] and not suitable for practical applications [44, 46-
9, 52, 53] (Table S1). Therefore, the chemosensor for Hg2+ which conquer the above-
4
mentioned limitations are of high demand. Herein, we design a turn-on probe explicit for
2
+
Hg among all other metal ions under physiological conditions. A rhodamine appended vinyl
ether probe for selective Hg2+ recognition, (E)-3',6'-bis(ethylamino)-2',7'-dimethyl-2-(2-
((2(vinyloxy)benzylidene)amino)ethyl)spiro[isoindoline-1,9'-xanthen]-3-one (RDV) has been
synthesized which gives “turn ON” emission response explicitly for Hg2+ ion over other
metal ions. The details sensing mechanism has been investigated by UV-Vis absorption
4

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1
spectroscopy, emission spectroscopy, H NMR, mass spectroscopy techniques. Additionally,
2
+
RDV is explored by making its test paper strip to detect Hg in pure water samples up to 10
ppm level for practical application. Moreover, potentiality of RDV was also tested in real
2
+
water samples for the monitoring of Hg ion.
2
. Experimental section
2
.1 Materials and instrumentations
Solvents and reagents of analytical grade were procured from commercial sources and were
used as received. Spectroscopic grade CH CN and deionised water were used for absorption
3
1
13
and emission study. H NMR spectra and C NMR spectra were recorded on 400 MHz and
1
00 MHz respectively in CDCl
3
and CD CN solvents. Chemical shifts are reported in parts
3
per million (δ) relative to tetramethylsilane as an internal standard. Coupling constant (J)
values are reported in hertz (Hz). HR-MS of the product was done commercially on 6550
iFunnel Q-TOF of Agilent Technologies. The equimolar solution of RDV and Hg2+ in
2
+
CH
3
CN was prepared to measure the mass of RDV+Hg complex. Elemental analysis (C, H
and N) data were obtained from Vario Micro cube instrument.
2
.1.1. Absorption and Emission Study
Shimadzu UV-1800 Spectrophotometer (with 1.0 cm pathlength quartz cuvette) was used to
record UV-vis absorption. Emission studies were performed on Varian Cary Eclipse
Fluorimeter. 0.1 M stock solution of RDV prepared in spectroscopic grade CH
3
CN solvent
-5
was used to make another solution of 2 × 10 M in CH
3
CN + H
2
O (3:7 v/v) solvent to
perform spectroscopic measurements. For spectrophotometric titration, 0.1 M solutions of
metal salts were prepared in deionised water. Each reading was measured at the interval of 30
2
+
min after Hg addition.
5

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2
+
Job plot was carried out by preparing a series of solutions of RDV and Hg ion in such a
2
+
way that sum of concentration of receptor and Hg is constant. The graph was plotted as
2
+
3
2+
2+
corrected absorbance (OD300nm × XHg × 10 ) versus molar fraction (XHg ) of Hg [55].
Limit of detection (LOD) has been calculated based on emission studies at 530 nm
wavelength using following equation [56],
Limit of Detection (LOD) = (3 × SD)/slope
Where, SD is standard deviation calculated by taking 15 consecutive scans of blank receptor
and the slope is obtained by linear fit graph of emission intensity of receptor versus
2
+
concentration of Hg added.
2
.1.2. Preparation of paper strip
Paper strip was prepared by immersing filter paper in 0.01 M solution of RDV in CH
2
Cl and
2
2
+
followed by air drying. These strips were kept in solutions of Hg of different concentration
prepared in pure water such as 0, 10, 20, 100 and 200 ppm. The appearance of color changed
from white to pink.
2
2
2
.2. Syntheses
.2.1. 2-(2-bromoethoxy)benzaldehyde (1)
-hydroxybenzaldehyde (5 mmol, 0.610 g) and 1,2-dibromoethane (25 mmol, 4.700 g) were
dissolved in dry CH
and refluxed for 18 hours under inert atmosphere. The reaction mixture was cooled to room
temperature and filtered, washed with CH CN and evaporated to dry. The desired product
3
CN (50 mL). To this, anhydrous K
2
CO
3
(7.5 mmol, 1.035 g) was added
3
was purified by column chromatograph on silica gel using mixture of petroleum ether and
ethyl acetate (95:5) as an eluent to get white solid as a pure product. Yield 0.798 g, (70 %);
o
1
Color: White. m. p. 52-54 C H NMR (400 MHz, CDCl
3
) δ in ppm 10.57 (s, 1H), 7.88 (d, J
6

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=
7.5 Hz, 1H), 7.58 (t, J = 7.0 Hz, 1H), 7.10 (t, J = 7.1 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 4.45
(t, J = 5.4 Hz, 2H), 3.74 (t, J = 5.4 Hz, 2H).
2
.2.2. 2-vinyloxybenzaldehyde (2)
To a stirred solution of compound 1 (5 mmol, 1.140 g) in DMSO (10 mL), potassium tert-
butoxide (6 mmol, 0.673 g) was added and stirred at room temperature for 1 hour. The
reaction mixture was diluted with ethyl acetate (50 mL) and washed with cold H
2
O (30 mL ×
3
) and brine (30 mL × 3). The organic layer was dried over Na SO and concentrated by
2
4
vacuum evaporation. The desired product was purified by column chromatograph on silica
gel column using petroleum ether and ethyl acetate mixture (95:5) to get liquid compound 2.
1
Yield 0.290 g, (39%); Color: Yellow liquid. H NMR (500 MHz, CDCl
3
) δ in ppm 10.44 (s,
1
H), 7.87 (dd, J = 7.7, 1.8 Hz, 1H), 7.56 (ddd, J = 8.4, 7.4, 1.8 Hz, 1H), 7.17 (dt, J = 8.3, 0.7
Hz, 1H), 7.08 – 7.05 (m, 1H), 6.70 (dd, J = 13.7, 6.1 Hz, 1H), 4.85 (dd, J = 13.7, 2.1 Hz, 1H),
4
.59 (dd, J = 6.0, 2.0 Hz, 1H).
2
.2.3. N-(rhodamine-6G)lactam-ethylenediamine (3)
o
Rhodamine 6G (2.09 mmol, 1.00 g) was dissolved in ethanol at 80 C and to it 1,2-
ethylenediamine (10.43 mmol, 0.70 mL) diluted in 5 mL ethanol was added dropwise. The
reaction mixture was refluxed for 24 hours till the complete disappearance of fluorescence in
the solution. The reaction mixture was allowed to cool in deep fridge to form white colored
precipitation. The solid was filtered, dried in vacuo and recrystallized in CH
3
CN to afford
pure product. Yield 0.743 g, (78%); Color: White. m. p. 220-222 C H NMR (400 MHz,
CDCl ) δ in ppm 7.92 (d, J = 5.2 Hz, 2H), 7.49 – 7.40 (m, 4H), 7.06 (d, J = 5.3 Hz, 2H), 6.34
s, 4H), 6.22 (s, 4H), 3.51 (s, 4H), 3.24 – 3.16 (m, 9H), 2.35 (t, J = 6.4 Hz, 4H), 1.90 (s,
o
1
3
(
1
2H), 1.32 (t, J = 6.9 Hz, 12H).
7

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2
.2.4. Synthesis of RDV
To a stirred solution of N-(rhodamine-6G)lactam-ethylenediamine (3) (1.0 mmol, 0.456 g) in
0 mL MeOH, 2-vinyloxybenzaldehyde (2) (1.0 mmol, 0.148 g) in MeOH was added
3
dropwise at room temperature and stirred for 24 h. The resulted white precipitate was filtered,
washed with cold MeOH to isolate pure RDV and dried under vacuum. The crystal suitable
for X-ray diffraction analysis was obtained by slow evaporation of saturated acetonitrile
o
1
solution of RDV. Yield: 0.322 g, (55 %); Color: White. m. p. 190-192 C H NMR (500
MHz, CDCl ) δ in ppm 8.38 (s, 1H), 7.95 – 7.92 (m, 1H), 7.82 (dd, J = 7.8, 1.7 Hz, 1H), 7.44
m, J = 3.5, 1.7 Hz, 2H), 7.33 (ddd, J = 8.2, 7.4, 1.8 Hz, 1H), 7.08 – 7.02 (m, 2H), 6.94 (dd, J
3
(
=
8.2, 0.8 Hz, 1H), 6.60 (dd, J = 13.7, 6.1 Hz, 1H), 6.35 (d, J = 2.5 Hz, 2H), 6.24 (d, J = 0.6
Hz, 2H), 4.69 (dd, J = 13.7, 1.9 Hz, 1H), 4.44 (dd, J = 6.1, 1.9 Hz, 1H), 3.51 – 3.36 (m, 6H),
1
3
3
.20 (m, J = 12.7, 6.5 Hz, 4H), 1.87 (s, 6H), 1.32 (td, J = 7.1, 2.8 Hz, 6H). C NMR (101
MHz, CD CN) δ in ppm 170.35, 167.53, 156.45, 155.55, 153.87, 151.68, 148.71, 147.89,
32.43, 131.85, 131.21, 128.22, 128.13, 127.27, 126.12, 123.48, 122.32, 118.45, 116.94,
3
1
1
05.72, 95.88, 94.81, 64.65, 59.00, 40.81, 37.92, 15.97, 13.69. HRMS Calcd for C37
H
38
N
4
3
O :
+
[
M+H] m/z 587.30, found 587.30. Anal. found: C 75.50, H 6.33, N 9.65. Anal. Calcd for
C
37
H
38
N
4
3
O : C 75.74, H 6.53, N 9.55.
3
.
Results and discussion
3
.1. Synthesis
The starting materials 2-(2-bromoethoxy)benzaldehyde (1), 2-vinyloxybenzaldehyde (2) and
N-(rhodamine-6G)lactam-ethylenediamine (3) were synthesized by following the previous
1
reports with slight modification [57,58,26] and well characterized by H NMR (Figs. S1-S3)
(see Experimental Section).
8

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Scheme 1 Synthesis of chemodosimeter RDV.
The desired RDV was synthesized by the condensation reaction between 2 with 3 in MeOH
at room temperature with stirring for 24 h which precipitated white colored compound,
1
13
designated as RDV. It was well characterized by H NMR, C NMR, HRMS analysis (Figs.
13
S4-S6) and by elemental analysis (see Experimental Section). C NMR shows characteristic
3
signal at 64.65 ppm for sp C-8 carbon indicates spirolactam form of RDV in solution (Fig.
S5) [59]. The crystal structure of RDV was determined by Single Crystal X-ray Diffraction
Study which confirms the spirolactam form of RDV (Fig. 1). Crystal structure also shows
that both the planes of aromatic rings in rhodamine moiety are perpendicular to each other.
The pendant vinyl ether group was clearly observable in crystal structure with the bond
distance of C18-C19 1.175 (11) Å. The crystallographic data for RDV were summarised in
Table 1.
9

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Fig. 1 ORTEP representation of chemosensor RDV. Ellipsoids are drawn at 40 % probability.
Table 1. Crystallographic data for RDV
RDV
Formula
Fw
C
37
H
38
N
4
O
3
586.71
T(K)
293(2) K
Triclinic
P-1
Cryst. Syst.
Space group
a /Å
9.6613(12)
b /Å
c /Å
α (º)
β (º)
γ (º)
V /Å
12.4438(17)
13.7295(19)
89.608(8)
74.410(7)
84.868(7)
1583.3(4)
1.231
3
-3
D
Z
c
(mg m )
2
-1
μ (mm )
0.079
F (000)
624
θ range (deg)
Data/restraints/params
GOF on F
2.941 to 24.999
5579/2/409
1.139
2
R indices (all data)
Largest diff. peak/ hole/e Å−3
CCDC deposition number
R1 = 0.1236, wR2 = 0.1939
0.319/-0.304
1840839
1
0

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3
.2. Absorption and Emission Study
From the UV-Vis absorption and fluorescence study, it was revealed that the RDV exits as a
colorless, non-fluorescent spirolactam form. It exhibits a strong intense absorption band at
3
01 nm in CH
significant band beyond 380 nm in CH
RDV with addition of different metal solutions like Na , K , Ca , Mg , Fe , Co , Ni ,
3
CN:H
2
O (3:7) solvent system (Fig. 2). Spirolactam ring does not show any
3
CN solvent [60]. The changes in absorption spectra of
+
+
2+
2+
2+
2+
2+
+
2+
2+
2+
2+
2+
Ag , Zn , Cd , Pb , Cu and Hg were examined in CH
3
CN:H
2
O (3:7) solvent system.
The UV-Vis absorption study reveals no significant changes when aqueous solution of Na+,
+
2+
2+
2+
2+
2+
+
2+
2+
2+
2+
K , Ca , Mg , Fe , Co , Ni , Ag , Zn , Cd , Pb , Cu were added separately (10
Equivalents) to the probe, RDV in CH CN:H O (3:7) (Fig. 2) while a new band at 530 nm
appears with addition of Hg in aqueous solution of RDV. The concomitant appearance of
30 nm band with color transformation to pink is certainly a characteristic of ring opened
amide form [61].
3
2
2
+
5
-5
Fig. 2 Changes in UV-vis spectra of RDV (2 × 10 M) in CH
3
CN:H O (3:7) with the
2
presence of different metal ions.
1
1

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2+
Fig. 3 represents systematic titration of RDV with addition of aqueous Hg ion. A new band
2
+
at 530 nm arises with incremental addition of Hg ion and gets saturated at 20 equivalents of
2
+
2+
Hg . The solution becomes colorless to pink with addition of Hg ions while with the rest of
metal ions no such color change was observed (Fig. 4a) which indeed indicates the “naked
2
+
eye” sensing ability of RDV towards Hg . The changes in spectroscopic titration and pink
-5
Fig. 3 Changes in UV-vis spectra of RDV (2 × 10 M) in CH
3
CN:H O (3:7) with the
2
2
+
addition of Hg ions.
2
+
color formation attributed to the interaction of Hg ions with RDV, causes conformational
change in rhodamine moiety from spirolactam ring form to ring opened amide form. The
2
+
selectivity of RDV with Hg ions also investigated with other interfering metal ions. The
2
+
absorbance intensity at 530 nm for RDV does not abrogate significantly for Hg ion due to
+
+
2+
2+
2+
2+
2+
+
2+
2+
2+
presence of other metal ions like Na , K , Ca , Mg , Fe , Co , Ni , Ag , Zn , Cd , Pb
2
+
and Cu (Fig. 4b). This indicates the receptor RDV works as highly selective colorimetric
system for Hg2+ ion to analyse sample matrix with interfering ions. The binding
stoichiometry was investigated by Job plot method, suggesting 1:1 binding between RDV
2
+
and Hg ion (Fig. S7).
1
2

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(a)
(b)
Fig. 4 a) Changes in color of chemosensor RDV in CH
3
CN:H O (3:7) with the addition of 20
2
2
+
+
+
2+
2+
2+
equivalents of metal ions. i) Free RDV; ii) Hg ; iii) Na ; iv) K ; v) Ca ; vi) Mg ; vii) Fe ;
2
+
2+
+
2+
2+
2+
2+
viii) Co ; ix) Ni ; x) Ag ; xi) Zn ; xii) Cd ; xiii) Pb ; xiv) Cu . b) Absorption of RDV at
5
30 nm in CH CN: H O (3:7) with addition of 20 equivalents of various cations (pink bar)
3
2
2
+
and those after addition of Hg (maroon bar).
To explore RDV for practical application, the absorbance study of RDV at different pH was
performed in the absence and presence of Hg2+ ion (Fig. S8). The native receptor has no
2
+
absorbance at 530 nm in pH range 4-10 while with addition of Hg ion a new band generates
around 530 nm with the same intensity. This result indicates that RDV can be used to detect
2
+
Hg in relevant biological pH 4-10.
The selective interaction of RDV with Hg2+ ion over other metal ions has been also
investigated by emission spectroscopy. Fig. 5 indicates RDV is non-emissive in nature, while
with the addition of Hg2+ ion generates a new band centred at 560 nm (λex, 500 nm).
However, no significant emission enhancement was observed with the addition of other metal
2
+
ions to RDV solution. With the incremental addition of Hg to RDV enhances emission
1
3

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(a)
-5
Fig. 5 Changes in emission spectra of RDV (2 × 10 M) in CH
3
CN:H O (3:7) with the
2
presence of different metal ions.
intensity at 560 nm and get saturated at 20 equivalents of Hg2+ ion resulted in 140 times
enhancement in fluorescence (Fig. 6a). The non-emissive RDV in spirolactam ring form
2
+
interacts with Hg to form highly fluorescent ring opened amide form. The selectivity of
Hg2+ ion by RDV in presence of other interfering metal ions was also investigated by
fluorescence study. With the presence of excess of interfering metal ions, emission intensity
2
+
at 560 nm of RDV for Hg ion does not altered significantly (Fig. 6b) indicates RDV indeed
2
+
acts as selective fluorescent sensor for Hg . Further, fluorescent response of RDV towards
2
+
2+
Hg at variable pH performed in the absence and presence of Hg (Fig. S9), shows RDV
can acts as fluorescent sensor in biological pH range 4-10.
2
+
The limit of detection has been calculated by emission response of RDV towards Hg ions to
2
+
cover linear range from 7-12 equivalents of Hg and it was found as low as 136 nM (Fig.
S10).
1
4

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(
a)
(b)
-5
Fig. 6 a) Changes in emission spectra of RDV (2 × 10 M) in CH
3
CN:H
addition of Hg ions (λex, 500 nm); b) Emission response of RDV at 560 nm in CH
3:7) with addition 10 equivalents of various cations (blue bar) and those after addition of
2
O (3:7) with the
2
+
3
CN+H O
2
(
2
+
Hg (light red bar).
To determine reaction kinetics, time dependant absorbance study was performed on
2
+
chemodosimeter RDV with Hg . It has been observed that the absorbance at 530 nm for
-5
2+
RDV (2 × 10 M) in presence of 20 equivalents Hg has been increased with time and get
saturated after 30 minutes (Fig. S11). It has been confirmed that the reaction between RDV
1
5

ACCEPTED MANUSCRIPT
2
+
-1
and Hg is first order and based on this, the observed rate constant was 0.1385 min (Fig.
S11).
2
+
The plausible mechanism for the selective detection of Hg is the hydrolysis of rhodamine
appended vinyl ether functionality due to Hg2+ (oxymercuration) [19,20] followed by
chelated complex formation with subsequent spirolactam ring opening. The mass peak m/z =
5
73.3063 (Fig. S12) in High Resolution Mass Spectra (HRMS) confirms the formation of
2
+
spirolactam ring opened chelated complex formation due to Hg . To confirm the proposed
1
13
mechanism the H NMR, C NMR and IR spectral analysis of RDV were performed with
2
+
absence and presence of Hg .
1
2+
The H NMR spectral titration was performed with the incremental addition of Hg to the
1
RDV in CD
3
CN solvent. The partial H NMR spectrum of RDV (Fig. 7) shows the signal at
4
0
.69 ppm and 4.44 ppm corresponds to –CH=CH
2
which gets disappeared with addition of
.5 equivalents of Hg2+ ions and concomitantly a new small intense signal at 9.94 ppm
2
+
appears for the phenolic O−H which became more intense with further addition of Hg ion
up to 1.5 equivalents. It clearly indicates hydrolysis of vinylic ether occurred due to presence
2
+
of Hg during course of reaction.
In 13C NMR titration (Fig. S13), the native receptor RDV shown signal at 65.01 ppm
3
corresponds to tertiary carbon (C8 in crystal structure, sp hybridized) of spirolactam ring
2
+
gets disappeared upon addition of Hg ions. It confirms that the spirolactam ring opens with
1
6

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1
Fig. 7 Partial H NMR titration spectra of RDV in CD CN in (1) absence; (2) 0.5 equivalent;
3
2
+
(
3) 1.0 equivalent and (4) 1.5 equivalent of Hg ions.
mercury interaction and the formation of coordinated complex with O atom of –CONH group
2
+
2+
of rhodamine moiety with Hg . The IR spectra of the RDV and RDV+Hg in KBr pallet
were performed to observed changes in C=O frequency. The characteristic frequency
-1
stretching frequency at 1684 cm due to C=O amide functionality was red shifted to 1658
-1
2+
cm in the presence of Hg (Fig. S14). The shifting towards lower energy signifies the
2
+
polarization of C=O group by coordination with Hg .
1
13
Based on the H NMR, C NMR, HRMS and IR spectral analysis the plausible mechanistic
2
+
2+
pathway for Hg recognition is the represented in scheme 2. The Hg promoted hydrolysis
2
+
of vinyl ether followed by formation of Hg chelated complex with spirolactam ring opening
give rise to pink colored highly fluorescent solution.
1
7

ACCEPTED MANUSCRIPT
Scheme 2
3
3
.3. Practical Applications
.3.1. Test Paper Strip
The drastic change in color by RDV probe in aqueous solution motivated us to prepared test
strip experiment in pure water. The test paper strips were prepared by immersing filter papers
in the CH
2
Cl solution of RDV (0.001 M) and air dried. When these paper strips were
2
2
+
immersed in the aqueous solutions of Hg with different concentration, the color changes
were observed represented in Fig. 8.
2
+
Fig. 8 Colorimetric detection of Hg at different concentrations from 0, 10, 20, 100 and 200
ppm in water.
1
8

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The color of the strips was altered from white to pink even at as low as 10 ppm concentration
2
+
2+
of Hg . It provides an on-sight application of paper strip detection for Hg ion by naked eye
at significant lower concentration without any additional equipment, making RDV is a
promising viable chemosensor for Hg2+ ion. So far, the reported rhodamine based
2
+
chemosensor for detection of Hg in different solvents may not be convenient for on-site
application.
2
+
3
.3.2. Hg detection in real water samples
2
+
The ability of RDV to detect Hg in real water samples was investigated using the spike-
and-recovery method. The three water samples have been collected from Lab tap water
located at ICT Chemistry Department and other two water samples are drinking water from
2
+
Hostel no. 1 and 5 at ICT Mumbai, India. Table 2 shows the detail results for spiked Hg and
2
+
recovered Hg .
Table 2. Determination of Hg from different water samples by using probe RDV
2
+
2
+
2+
Sample
Hg spiked (M)
Hg recovered (M)
Recovery (%)
92.71
.00 × 10-
.00 × 10
.10 × 10-
5
6.49 × 10
9.55 × 10
1.11 × 10
1.36 × 10
1.73 × 10
1.92 × 10
2.05 × 10
6.27 × 10
9.75 × 10
1.11 × 10
1.34 × 10
1.47 × 10
1.89 × 10
2.02 × 10
6.49 × 10
9.31 × 10
-5
7
9
1
-
5
4
4
4
4
4
5
5
4
4
4
4
4
5
5
4
4
4
4
-5
106.11
100.91
104.62
115.33
112.94
107.89
89.57
108.33
100.90
103.07
98
111.17
106.31
92.71
103.44
109.09
107.68
98
-4
-
-4
Tap Water
1.30 × 10
.50 × 10-
.70 × 10-
.90 × 10-
-4
1
1
1
7
9
1
-4
-4
-
-5
.00 × 10
.00 × 10-
-5
.10 × 10-
-4
-
-4
Drinking Water 1
Drinking Water 2
1.30 × 10
.50 × 10-
-4
1
1
1
7
9
1
-
-4
.70 × 10
.90 × 10-
.00 × 10-
-4
-5
-
-5
.00 × 10
.10 × 10-
-4
1.20 × 10
1.40 × 10
1.47 × 10
1.71 × 10
-
-4
1.30 × 10
-
-4
1
1
1
.50 × 10
.70 × 10-
.90 × 10-4
-4
100.58
103.15
-4
1.96 × 10
1
9

ACCEPTED MANUSCRIPT
The observed recovery factor ranges from 92.71-115.33 % indicates the potential
2
+
applicability of chemodosimeter RDV to detect Hg for real water sample.
4
.
Conclusions
In summary, we demonstrated a newly designed and synthesized colorimetric and
fluorometric chromophore RDV based on rhodamine 6G appended vinyl ether moiety for
2
+
selective recognition of Hg in aqueous acetonitrile system. RDV exhibited high sensitivity
2
+
and selectivity for Hg ion over series of other metal ions with its detection limit as low as
2
+
1
36 nM. RDV can detect Hg in physiological pH range 4-10. The spectroscopic analysis
2
+
2+
supports Hg promoted hydrolysis of vinyl ether followed by Hg complex formation in
spirolactam ring opened form. In addition, we prepared paper strip of RDV for detection of
Hg2+ as practical colorimetric sensor for on-site measurement upto 10 ppm level in pure
2
+
water. Potential applications have been shown for signalling Hg in real water samples by
RDV.
Acknowledgement
DD is gratefully acknowledged to Department of Science and Technology (DST), Govt. of
India for DST-INSPIRE faculty Award (IFA-13CH-94) and research grant. SKP is grateful to
University Grant Commission (UGC) for providing UGC-BSR research fellowship.
Sophisticated Analytical Instrumentation Facility (SAIF), IIT Madras is acknowledged for
providing SC-XRD data. Department of Pharmaceutical Sciences and Technology, ICT,
Mumbai is also thankful due to carrying out NMR titration experiments.
2
0

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7

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Table of Content
A nanomolar detection of mercury(II) ion by a chemdosimetric rhodamine-
based sensor in an aqueous medium: potential applications in real water
samples and as paper strips
Sagar K. Patil and Dipanwita Das*
Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai 400 019,
India
Fax: (91)022-3361-1020
Email: dr.das@ictmumbai.edu.in
A rhodamine appended vinyl ether probe (RDV) for selective Hg2+ recognition has been
2
+
synthesized which gives “turn ON” emission response for Hg ion in aqueous medium over
other metal ions in a chemodosimetric approach. The LOD is 136 nM. RDV has been
2
+
explored for paper strip application as well as Hg analysis from real water samples.
2
8

ACCEPTED MANUSCRIPT
A nanomolar detection of mercury(II) ion by a chemdosimetric rhodamine-
based sensor in an aqueous medium: potential applications in real water
samples and as paper strips
Sagar K. Patil and Dipanwita Das*
Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai 400 019,
India
Fax: (91)022-3361-1020
Email: dr.das@ictmumbai.edu.in
Highlights

A rhodamine appended vinyl ether probe (RDV) for selective Hg2+
recognition has been synthesized.
2
+

RDV gives “turn ON” emission response for Hg ion in aqueous
medium over other metal ions in a chemodosimetric approach.
The LOD value is as low as 136 nM.


RDV has been explored for paper strip application as well as Hg2+
analysis from real water samples.
2
9