Exclusive detection of sub-nanomolar levels of palladium(II) in water: An excellent probe for multiple applications

Article abstract of DOI:10.1002/asia.201402635

A new colorimetric probe has been developed for the detection and estimation of Pd^II at sub-nanomolar concentrations. The probe consisted of rhodamine (signaling unit), which was linked with a bis-picolyl moiety (binding site) through a phenyl

Full text of DOI:10.1002/asia.201402635

FULL PAPER
DOI: 10.1002/asia.201402635
Exclusive Detection of Sub-Nanomolar Levels of Palladium(II) in Water: An
Excellent Probe for Multiple Applications
[
a]
[a]
[a]
[a, b]
Namita Kumari, Nilanjan Dey, Krishan Kumar, and Santanu Bhattacharya*
Abstract: A new colorimetric probe
tion of a prominent pink color. The ex-
cellent selectivity of the probe towards
Pd over Pd or Rh ensured its poten-
tial utility for the detection of residual
palladium contamination in pharma-
ceutical drugs and in Pd-catalyzed reac-
tions. The probe showed a “turn-on”
(bright yellow) fluorescence upon the
addition of Pd , which made it suitable
for the detection of Pd contaminants in
mammalian cells.
has been developed for the detection
II
II
0
II
and estimation of Pd at sub-nanomo-
II
lar concentrations. The probe consisted
of rhodamine (signaling unit), which
was linked with a bis-picolyl moiety
(
binding site) through a phenyl ring.
Keywords: fluorescence
dium · rhodamine · sensors · water
·
palla-
II
Pd induced opening of the spirolac-
tam ring of the probe with the genera-
Introduction
maceutical ingredients has been restricted to less than
[7]
1
0 ppm.
Various analytical methods, such as atomic absorption
Palladium is one of the most important platinum-group ele-
ments (PGEs) because of its unique chemical and physical
properties. It is widely used in organic synthesis in cata-
lysts for many cross-coupling reactions, such as the Buch-
wald–Hartwig, Heck, Sonogashira, and Suzuki–Miyaura re-
spectroscopy, X-ray fluorescence spectroscopy, and plasma
emission spectroscopy, have long been used to detect vari-
ous ions and analytes, even at sub-micromolar concentra-
tions. However, these methods require extensive sample
preparation, expensive instrumentation, and are often not
cost-effective and may not be widely accessible. To over-
come these difficulties, over the past few years, there has
been increasing focus on the design and synthesis of various
small-molecule-based probes that can detect Pd by using
simple UV/Vis and fluorescence spectrophotometry. In
2007, Koide and co-workers reported the first fluorescein-
based Pd probe for the detection of palladium below its per-
[1]
[
2]
actions. One of its best-known applications is in vehicle-ex-
haust systems, in which it helps to decrease the emission of
deadly gaseous pollutants, such as carbon monoxide, hydro-
[3]
carbons, and nitrogen oxides. However, its extensive use
has resulted in a rapid increase in the concentration of palla-
dium in the environment, which can lead to serious health
[4]
hazards. Moreover, owing to its greater solubility gradient
II
compared to other PGE ions, Pd has a higher biological
[8a]
uptake and a higher accumulation propensity in the food
chain. It binds with thiol-containing amino acids (cystine,
homocysteine, methionine, glutathione, etc.) or proteins
casein, silk fibroin, etc.) because of its high thiophilic
nature. Thus, a restriction has been imposed on the maxi-
mitted level in water. However, to achieve saturation in
the spectroscopic response, 1 h incubation time with em-
ployment of high pH was required (borate buffer at pH 10).
Subsequently, these authors also developed a library of
other probes for the detection of palladium in various sam-
ples within significantly lower detection times (about
[
5]
(
[
5]
mum dietary intake of palladium of <15 mg per person per
day. Even the amount of residual palladium in active phar-
[
6]
[8]
15 min) at pH 7.
Following this report, considerable recent effort has been
[9]
made towards the naked eye detection of Pd. The diverse
sensing pathways employed by these probes often include
II
Pd complexation or Pd-catalyzed reactions that modulate
+
+
[
a] Dr. N. Kumari, N. Dey, K. Kumar, Prof. Dr. S. Bhattacharya
Department of Organic Chemistry
Indian Institute of Science
[10]
their photophysical properties.
mainly been exploited for the detection of palladium. The
first involves the interaction of Pd with p-electron-rich
functionalities, such as propargyl, allyl, and nitrile groups, of
which the hydrolysis of propargyl ethers could be catalyzed
by both Pd and Pd species, thereby rendering similar opti-
cal responses. The second approach is the oxidative insertion
of Pd and the third is the coordination of Pd through mul-
tiple coordination sites. In this instance, rhodamine-based
sensors are advantageous as signaling moieties, because they
Three procedures have
II
Bangalore 560012 1 (India)
Fax : (+91)080-2360-0529
E-mail: sb@orgchem.iisc.ernet.in
[
b] Prof. Dr. S. Bhattacharya
0
II
Jawaharlal Nehru Centre of Advanced Scientific Research
Bangalore 560012 (India)
0
II
+
[
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201402635.
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Santanu Bhattacharya et al.
typically exhibit a “turn-on” response. Moreover, their facile
synthesis and highly sensitive photophysical properties con-
[10]
tribute to their frequent application.
However, most of
the reported rhodamine-based sensors pose serious draw-
backs, including prolonged incubation time, responses only
in organic or organic/water mixtures, potential interference
II
with other PGE ions, and no discrimination between Pd
and Pd .
0
Considering these practical challenges in the detection of
palladium, we have designed a new probe for the efficient
II
II
sensing of Pd . This probe shows selective detection of Pd
in water, as well as at physiological pH 7.4, with a “turn-on”
signal in both UV/Vis and fluorescence spectroscopy. More-
over, this probe can be used multiple times for the detection
II
of Pd ions. The real-time application of the probe has been
II
examined by determining residual Pd in Pd-catalyzed reac-
tions, pharmaceutical products, real-life water samples, and
also in glassware that was used for Pd-catalyzed reactions
following washing. This probe also showed excellent selec-
II
0
tivity for Pd over Pd , a property rarely manifested by
other probes. Suitable portable test strips have been pre-
pared by using filter paper to check the fast-track detection
of Pd ions. Furthermore, the probe was also found to be effi-
II
cient in detecting Pd contaminants in mammalian cells.
Figure 1. a) Changes in the UV/Vis spectra of compound 1 (10 mm) in
+
2+
water (pH 7.4) upon the addition of various cations (1 equiv; Ag , Cd
,
2+
2+
2+
2+
2+
2+
2+
2+
3+
3+
3+
2+
Co , Cu , Hg , Ni , Pb , Mg , Mn , Zn , Al , Cr , Fe , Pt , and
Results and Discussion
Synthesis
2
+
Pd ). Inset shows the color change of compound 1 upon the addition of
Pd . b) Normalized absorbance of compound 1 (10 mm) at 533 nm upon
2
+
the addition of various cations.
Following our interest in designing small-molecule-based
[
11]
probes,
herein we report the synthesis of a new rhoda-
the solution resulted in a visible color change from colorless
to bright pink (Figure 1, inset). This color change was fur-
ther monitored by UV/Vis absorption spectroscopy, as
shown in Figure 1. The addition of Pd to an aqueous solu-
tion of the probe (10 mm) resulted in an approximate 55-fold
mine derivative (1), as shown in Scheme 1 (for details, see
the Experimental Section). The structure of the probe was
characterized by H and C NMR spectroscopy and by
HRMS (see the Supporting Information).
1
13
II
increase in the absorbance at 533 nm. The selectivity of the
II
probe towards Pd was also investigated. The absorbance
Absorption and Emission Spectroscopic Studies
spectra were recorded upon the addition of various other
cations to the probe solution (Figure 1b). The observed
changes in absorbance upon the addition of other cations
A solution of compound 1 in water was both colorless and
non-fluorescent, thus indicating the presence of a closed spi-
rolactam ring structure of the probe. The addition of Pd to
II
II
were negligible compared to that with Pd . Further interfer-
ence was studied in the pres-
ence of excess amounts of other
cations. The probe showed
a similar increase in absorbance
II
owing to Pd , even in the pres-
ence of excess amounts of other
cations (see the Supporting In-
formation, Figure S1). Thus,
this probe showed excellent se-
II
lectivity towards Pd ions.
UV/Vis titration of com-
pound 1 was performed with
II
the gradual addition of Pd (see
the Supporting Information,
2 4 3
Scheme 1. Synthesis of compound 1: a) K HPO /MeCN , reflux, 24 h; b) POCl /DMF, 908C, 2 h; c) NH
2ꢀ
MeOH, reflux, 2 h; d) MeOH, AcOH (cat.), RT, 4 h.
NH2/
Figure S2). Saturation in ab-
sorbance was observed upon
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Santanu Bhattacharya et al.
II
the addition of only 1 equivalent of Pd (see the Supporting
Information, Figure S2b). The stoichiometry of the interac-
tion was determined from a Job plot analysis, which showed
II
a 1:1 interaction between the probe and the Pd ions, as ex-
pected (see the Supporting Information, Figure S3a). The
respective binding constant was calculated by using the
Benesi–Hildebrand equation for 1:1 stoichiometry. Probe
II
1
showed a good binding affinity toward Pd ions, with
a binding constant of logK=4.73
ing Information, Figure S3b).
A
H
U
G
R
N
U
G
The spectroscopic properties of rhodamine-based sensors
are known to be influenced by the pH value of the
[
12]
medium.
sensing process. Pd was added to solutions of compound
at different pH values and the corresponding absorption
spectra were recorded. Compound 1 showed the efficient
Thus, we investigated the effect of pH on the
II
1
2
+
detection of Pd above pH 4 (see the Supporting Informa-
tion, Figure S4) and changes were even observed in the ab-
sorption spectra upon the addition of Pd up to pH 9. Thus,
II
II
the probe could effectively detect Pd over a broad pH
range (pH 4.5–9).
We further investigated the emission properties of the
probe upon the addition of various cations. The solution of
compound 1 on its own was non-fluorescent. However, the
II
addition of Pd ions to the solution resulted in the instanta-
Figure 2. a) Changes in the fluorescence spectra of compound 1 (1 mm,
neous generation of a bright-yellow emission (Figure 2,
inset). The emission spectra showed an approximate 10-fold
l
ex =520 nm) in water (pH 7.4) upon the addition of various cations
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
(
1 equiv; Ag , Cd , Co , Cu , Hg , Ni , Pb , Mg , Mn , Zn ,
II
3+
3+
3+
2+
2+
increase in emission intensity upon the addition of Pd
Al , Cr , Fe , Pt , and Pd ). Inset shows the change in fluorescence
2+
of compound 1 upon the addition of Pd under the irradiation of a UV
lamp (365 nm). b) Corresponding normalized emission intensity ratio of
compound 1 (1 mm) at 555 nm upon the addition of various cations.
(
Figure 2). None of the other cations induced any change in
the emission intensity of the probe under identical condi-
tions.
The fluorescence titration experiments were performed
II
with the gradual addition of Pd ions to the solution of
yield of 0.05, whereas the metal complex showed a quantum
yield of 0.24.
Furthermore, the reversibility of formation of the 1/Pd
probe 1 (see the Supporting Information, Figure S5). Satura-
tion was observed upon the addition of only 1 equivalent of
metal ions. The minimum detectable concentration of Pd
by the probe was 0.59 nm (see the Supporting Information,
II
II
complex was investigated by the addition of ethylenediami-
netetraacetic acid (EDTA). The addition of EDTA resulted
in the complete recovery of the molecular fluorescence (see
the Supporting Information, Figure S10). This result indicat-
ed that the probe may be used multiple times for the detec-
2+
Figures S6–S8), much lower than the permitted level of Pd
[8]
in drinking water.
The mechanism of fluorescence enhancement upon bind-
ing with Pd was investigated by performing time-dependent
emission decay measurements (TCSPC). The fluorescence
decay of compound 1 was fitted to a tri-exponential function
with time constants of 0.89 (97.5%), 1.64 (2.4%), and
.27 ns (0.1%). However, upon binding with Pd , probe
II
II
tion of Pd ions. Notably, most of the previously reported
II
Pd sensors suffered from poor discrimination ability be-
II
0
tween Pd and Pd . Therefore, it was important to examine
0
the effect of Pd on the optical response of the probe. The
II
0
0
addition of Pd induced a very small change in both the ab-
1
only showed a single-exponential decay profile, with
sorption and emission spectra, which were negligible com-
pared with the changes on the addition of Pd (see the Sup-
II
a time constant of 0.78 ns (see the Supporting Information,
Figure S9). The shorter average decay time in the metal
complex (0.78 ns) compared to in the free molecule
porting Information, Figure S11). This result confirmed the
II
0
excellent selectivity of the probe towards Pd ions over Pd .
Furthermore, the effect of various counteranions was also
(
0.92 ns) was correlated with opening of the spirolactam
II
ring. Tight binding of the probe to Pd (higher binding con-
stant) converted the “free-hanging” rhodamine moiety into
part of the extended electronic conjugation. In turn, this
change resulted in an increase in the number of non-radia-
tive decay channels and a shortening of the average life-
investigated by using solutions of PdCl , Pd ACHTUNGTERNNUG( OAc) , and [Pd-
2 2
AHCTUNGTRNNEG(U PPh ) Cl ]. The individual addition of each salt solution to
3 2 2
the probe solution resulted in similar increases in the ab-
sorption and emission intensities of compound 1 (see the
Supporting Information, Figure S11). This result indicated
[13]
II
time.
The quantum yields were calculated for both the
that the binding process of Pd with the probe occurred in-
probe and the metal complex; probe 1 showed a quantum
dependently of the counteranion.
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Santanu Bhattacharya et al.
the significance of the bis-picolyl unit in the binding with
the Pd ions. Thus, we concluded that the probe interacted
Mechanism of Detection
II
The recovery of the molecular signal upon the addition of
EDTA supported the reversible ring-opening mechanism of
the spirolactam, rather than an ion-catalyzed hydrolysis
with the palladium atom through its picolyl unit, which in
[10n]
turn results into opening of the spirolactam ring.
2+
To rationalize the mode of interaction with Pd , density
functional theory (DFT)-based computational studies were
performed at the B3LYP level of theory with the 6-31G*
basis set for C, H, and N atoms and the LANL2DZ basis set
for Pd atoms. The optimized structures showed close prox-
imity of the aniline nitrogen (dPdꢀN1 =2.11 ꢁ) and picolyl ni-
trogen atoms (dPdꢀN2 =2.039 ꢁ, dPdꢀN3 =2.04 ꢁ) to the Pd
center (Figure 4). The N1ꢀPdꢀN2 bond angle was about
2+
mechanism. Furthermore, the MS (ESI) spectrum of 1+Pd
complex showed prominent peak at m/z=854.2
L(Pd)Cl] with appropriate isotopic distributions, thereby
a
+
[
ensuring 1:1 complexation of the probe to the metal ion (see
the Supporting Information, Figure S12). The IR spectra of
2+
both the probe and the 1+Pd complex were recorded in
water. No significant changes were observed in the frequen-
ꢀ
1
cy of the carbonyl stretch (1650 cm ; see the Supporting In-
formation, Figure S13), thus indicating the non-involvement
of the carbonyl group in the complexation of the probe with
palladium ions.
1
Furthermore, a H NMR spectrum of the probe was also
II
recorded in the presence of Pd in D O/[D ]DMSO. Signifi-
2
6
cant downfield shifts were observed for the signals of pro-
tons H , H , and H , which clearly suggested the involve-
a
d
e
ment of the picolyl nitrogen atoms in the coordination with
II
Pd (Figure 3). Furthermore, the disappearance of proton
H confirmed the involvement of the picolyl moiety in the
f
II
interaction of the probe with Pd .
The involvement of the picolyl nitrogen atoms was further
probed by synthesizing a control molecule that contained
a phenyl ring instead of a bis-picolyl unit (see the Support-
ing Information, Figure S14). This molecule did not show
II
any change upon the addition of Pd ions, which indicated
2
+
Figure 4. DFT-optimized structures of probe 1 and the 1/Pd complex, as
calculated by using the B3LYP/6-31G* basis set for probe 1 and the
2
+
B3LYP/LANL2DZ basis set for the 1/Pd complex.
9
68, thus indicating the formation of a square-planar com-
2+
plex with Pd . The effect of electronic push in opening of
the spirolactam ring was ascertained from variation in the
2+
N4ꢀC1 distance in the isolated probe and its metal (Pd )
complex. Shortening of the N4ꢀC1 bond length (1.47 ꢁ to
1
.39 ꢁ) upon complexation with palladium suggested signifi-
cant enhancement in the extent of p bonding.
The presence of an orthogonal spirolactam ring in free
probe 1 prevented electronic “mixing” of the signaling (xan-
thene) and recognition units (bis-picolyl). In the free probe,
the HOMO was concentrated on the electron-rich xanthene
moiety, whereas the LUMO was mainly situated on the bis-
2
+
picolyl fragment. The addition of Pd induced opening of
the spirolactam ring and increased the effective conjugation.
Furthermore, the HOMO–LUMO band-gap was significant-
ly lower in the metal complex (1.67 eV) than in the free
probe (4.14 eV, Figure 5). This lowering of the band-gap was
also supported by the experimentally observed bathochro-
mic shift in the UV/Vis spectra of probe 1 upon the addition
1
Figure 3. H NMR spectra of compound 1 (10 mm in [D
6
]DMSO) after
2
+
the addition of Pd a) 0, b) 0.25, c) 0.5, and d) 1 equiv. Inset shows the
molecular structure of probe 1 and partial H NMR spectra of probe
before and after the addition of Pd (1 equiv).
1
II
1
II
of Pd .
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Santanu Bhattacharya et al.
indicated that probe 1 could effectively estimate the concen-
II
tration of Pd ions in real-life water samples.
Then, we investigated whether the probe could detect re-
sidual palladium present in glassware in which a reaction
II
had been carried out using a Pd reagent after normal labo-
ratory washing. A visual color change would help to identify
2+
the presence of trace amounts of Pd in reusable glassware.
Thus, PdCl (2 mg) was stirred in THF for 1 h in four differ-
2
ent round-bottomed flasks. Then, each flask was washed in
various ways (see the Experimental Section). After washing,
the probe solution was poured into each of the washed
flasks and the optical spectra were recorded (see the Sup-
porting Information, Figure S18). The experiments showed
that washing with a laboratory detergent (labolin) or water
II
was not sufficient to completely remove the residual Pd
Figure 5. Energy diagrams of the HOMO and LUMO of a) probe 1 and
from the glassware. However, washing with acetone was
able to remove most of the residual palladium ions, owing
to the higher solubility of Pd in acetone.
2
+
b) the formation of the 1/Pd complex, as calculated by using the
B3LYP/6-31G* basis set for probe 1 and the B3LYP/LANL2DZ basis set
II
2
+
for the 1/Pd complex.
Palladium-catalyzed cross-coupling reactions are widely
[15]
used in the pharmaceutical industry for drug discovery.
Therefore, these drugs are often contaminated with trace
amounts of palladium. Herein, the application of the probe
was further explored in the determination of the level of
contamination of palladium ions in the end products. For
this investigation, aspirin was chosen as model drug and
a known amount of Pd was added externally (for details,
see the Experimental Section). When the probe solution
was added to this mixture, a visible color change was ob-
served. The extent of spectroscopic change was similar to
that with the positive control sample (i.e., with the metal
ion alone), which demonstrated the robustness of this probe,
even in heterogeneous media (see the Supporting Informa-
tion, Figure S19).
Various Applications of the Probe
The unique properties of palladium among the PGEs lead
to its versatile application in diverse industrial processes,
ranging from catalytic converters in the automobile industry
to cross-coupling reactions in pharmaceutical drug design.
Thus, monitoring of the palladium level in different environ-
mental samples, industrial waste, or pharmaceutical residues
is essential to decrease its further accumulation in the bio-
logical food chain. To this end, efficient colorimetric sensors
may be more useful than other sophisticated techniques,
owing to their cost-effectiveness and ease of handling.
2+
The higher solubility gradient of Pd compared to other
closely associated PGEs (such as Rh and Pt) is the reason
Owing to the lethal effects of palladium, various govern-
ments have imposed restrictions on the allowable level of
residual Pd in the end products of Pd-mediated CꢀC cross-
2
+
for the increased contamination of Pd in drinking water
near industrial areas. Therefore, the estimation of Pd levels
ꢀ
1
(
must be <100 ngg ) in different natural water sources is
coupling reactions (5–100 ppm). Therefore, it is important to
monitor the amount of palladium present in the end reac-
tion mixture. To address this problem, we chose the well-
crucial to ensuring quality control. For this purpose, we col-
lected water from two commonly used water sources, a city
water supply tap and a nearby pool. Then, a known amount
known Suzuki coupling reaction, because it used Pd ACHTUNGTRENNUNG( OAc)
2
II
of Pd ions (higher than the permitted level) was added ex-
as a catalyst. Emission spectra were recorded for the reac-
tion mixture, as well as after standard work-up (for details,
ternally to each of these samples. The change in absorbance
upon the addition of the probe was recorded immediately in
both cases. Linear fits were obtained when changes in the
absorbance at 535 nm were plotted against the concentra-
II
see the Experimental Section). The Pd concentrations
before and after work-up were determined by using a cali-
bration curve (as obtained from the titration studies). After
II
II
tion of added Pd ions (0.75–3.75 mm), with a correlation co-
work-up, the residual Pd content was significantly lower
efficient of >0.99, which confirmed that this sensor could be
used for the detection of Pd , even in unknown samples
than the initial value, although the result was not quantita-
tive because some of the Pd had been reduced into Pd
II
2+
0
(
see the Supporting Information, Figure S15). Then, we
during the reaction.
II
compared the estimated concentration of Pd from the
changes in the absorption spectra against the actual amount
added. A linear response in both cases suggests that the
probe may be able to estimate Pd concentration without
any interference (see the Supporting Information, Fig-
ures S16 and S17). The minimum detectable concentrations
In addition to these results, to obtain an “in-situ” estima-
tion of heavy-metal-ion contamination, we developed porta-
ble paper strips. These filter-paper strips were initially
soaked in a solution of the probe and dried in air. After
that, they were dipped in solutions with different concentra-
II
2
+
tions of Pd ions (0.5 and 1 equiv) and a distinct color
change with bright-yellow emission was immediately ob-
served (Figure 6).
II
of Pd by the probe were approximately 60 nm (tap water)
and 63 nm (pool water). All of these observations clearly
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Santanu Bhattacharya et al.
opening of the spirolactam ring. The exact mode of metal-
1
ion coordination was further investigated by H NMR spec-
troscopy, mass spectrometry, and theoretical investigations.
II
The excellent selectivity of the probe towards Pd over
II
0
other closely associated metal ions, such as Pt , Pd , and
II
Rh , ensured its high potential for versatile real-life applica-
2+
tions. The probe could be used as an indicator for Pd ions
in real water samples. Further applications of the sensor
have been demonstrated for the detection of residual palla-
dium contaminants in pharmaceutical drugs or in certain
palladium-catalyzed cross-coupling reactions. The “turn-on”
response of the probe was also employed for examining
Figure 6. Photographs of test strips of probe 1 (1 mm) for the detection of
2
+
Pd in water: a) in daylight and b) under a UV lamp (365 nm) upon
2
+
soaking in 0, 0.5, and 1 mm solutions of Pd (from left to right).
II
Pd -ion contaminants in cellular samples, thus also indicat-
ing its potential for in vivo applications.
2+
The application of probe 1 for imaging intracellular Pd
ions was also investigated by using confocal microscopy. The
experiments were performed on a cancer cell line (HeLa
2+
cells, Figure 7). First, the cells were incubated with Pd
Experimental Section
Materials
All of the solvents and reagents were purified and dried by using usual
methods. All of the starting materials were obtained from well-known
commercial suppliers and used as received. A stock solution of com-
pound (1) was made in DMSO and the final concentration of DMSO for
all the studies was about 0.1%.
Typical Sensing Procedure
Sensing of the different metal ions in water was performed by adding
a solution of probe 1 in DMSO (5 mL/1 mL) to pure water to a final
ꢀ6
volume of 1 mL ([1]=10ꢂ10 m for the UV/Vis experiments, [1]=1ꢂ
ꢀ
6
1
0
m for the fluorescence experiments), followed by the addition of a so-
+
2+
2+
2+
2+
2+
lution of the metal ions in water (Ag , Cd , Co , Cu , Hg , Ni
,
,
Figure 7. Confocal microscopy images of HeLa cells with the probe.
2
+
2+
2+
2+
3+
3+
3+
Pb , Mg , Mn , Zn , Al , Cr , and Fe ). The solutions of PdCl
Pd(PPh Cl ], Pd(OAc) , and PtCl
ture procedures. The effect of pH value on the sensing process was
studied by using different buffer solutions with various pH values
HCO Na/HCl buffer for pH 2–4.5, CH CO Na/HCl buffer for pH 5–6.5,
Tris/HCl for pH 7–9, Na ·10H O/NaOH for pH 10–12). All of the
2
Panel 1: cells incubated with probe 1 alone; panel 2: cells incubated with
[
A
H
U
G
R
N
U
G
3
)
2
2
A
H
U
G
E
N
N
2
2
were prepared according to litera-
2
+
2+
Pd alone; panel 3: cells incubated with both the probe and Pd . Confo-
cal microscopy images from the A) yellow and B) blue channels (staining
with DAPI) and C) an overlay of images (A) and (B).
[
16]
(
2
3
2
2
B
4
O
7
2
studies at pH 7.4 were performed in tris buffer (10 mm).
(
1 mm) for 2 h. Then, they were treated with a solution of
Instrumentation
compound 1 (10 mm) and incubated for a further 4 h. Con-
trol experiments were performed with the metal ion alone
and with the probe alone, which were incubated with cells
for the same period of time as mentioned above. After proc-
essing (see the Supporting Information), the samples were
examined under a confocal microscope. The images showed
no fluorescence for the cells that were incubated with the
probe alone. However, bright-yellow fluorescence was ob-
served for the cells that were incubated in the presence of
1
13
H and C NMR spectra were recorded on a 400 MHz/100 MHz Bruker
Advance DRX 400 spectrometer. HRMS analysis was performed on a Q-
TOF YA263 high-resolution spectrometer. IR spectra were recorded on
a PerkinElmer FTIR spectrometer BX. UV/Vis absorption spectra were
recorded on a Shimadzu UV-2100 spectrophotometer. Fluorescence spec-
tra were recorded on a Cary-Eclipse spectrofluorophotometer. The slit-
widths for the fluorescence experiments were kept at 5 nm (excitation)
and 5 nm (emission) and the excitation wavelength was set at 520 nm for
all of the fluorescence experiments.
2+
Sample Preparation for Various Applications
Pd ions. Therefore, this probe may also be useful for the
2+
II
detection of cellular Pd contaminants.
1) Estimation of residual Pd in laboratory glassware: A solution of
PdCl (2 mg) in THF (10 mL) was added to four different round-bot-
2
tomed flasks and the solution was stirred at RT for 1 h. Then, the solu-
tion was removed from each flask and the palladium content were deter-
mined: 1) in a flask that hadn’t been washed and in flasks that were
Conclusion
2
) washed with water; 3) brushed with detergent (labolin), followed by
In summary, we have developed a new rhodamine-based
probe that contained a bis-picolyl binding unit for the effec-
tive sub-nanomolar detection of Pd in water at physiologi-
washing with water; and 4) brushed with detergent, followed by washing
with water and acetone. Then, water (5 mL) was added into all of the
flasks. After that, probe 1 was added to each flask ([1]=5.0 mm for the
UV/Vis studies, [1]=1 mm for the fluorescence studies) and their corre-
II
cal pH value. The instantaneous “turn-on” response of the
probe—both in terms of color and fluorescence emission—
sponding spectra were immediately recorded.
2+
2
) Estimation of Pd contamination in pharmaceutical drugs: Aspirin
II
upon the addition of Pd was found to be due to reversible
(500 mg) was dissolved in MeOH by stirring overnight. Then, the solu-
&
&
Chem. Asian J. 2014, 00, 0 – 0
6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

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Santanu Bhattacharya et al.
1
3
tion was filtered and, after filtration, the solvent was evaporated. The res-
idue was dissolved in water (50 mL) to prepare stock solution
(5 mm) was added. Then, probe 1 was
added and the changes were monitored by UV/Vis and fluorescence
spectroscopy (see the Experimental Section).
(s, 1H); C NMR (75 MHz, CDCl
3
): d=56.9, 111.9, 120.6, 122.4, 126.4,
a
132.0, 137.1, 149.8, 153.0, 157.1 ppm; IR (neat): n˜ =2735.3, 1670.2, 1598.5,
ꢀ
1
ꢀ1
(
10 mgmL ), to which PdCl
2
1523.4, 1396.9, 1227.7, 1169.2, 817.3, 750.0 cm ; MS
A
H
U
G
R
N
N
(ESI): m/z: 326
+
+
[
M+Na] ; HRMS: m/z calcd for C19
found: 326.1266.
Rhodamine hydrazone (2): Rhodamine-6G hydrazone (4) was prepared
17 3
H N ONa: 326.1269 [M+Na] ;
2
+
3
) Estimation of Pd in the reaction end products: To detect the amount
[
12, 20]
II
according to a literature procedure.
tomed flask, rhodamine-6G (5, 0.479 g, 1 mmol) was dissolved in EtOH
15 mL). To that, excess hydrazine monohydrate (85%, 1.5 mL) was
added dropwise with vigorous stirring at RT. Then, the mixture was
heated at reflux for 2 h and then cooled overnight. The resulting precipi-
tate was filtered and washed three times with EtOH/water (1:1, 10 mL).
After drying under vacuum, the reaction afforded rhodamine-6G hydra-
zone. Yield: 0.369 g, 80%; H NMR (400 MHz, CDCl
.1 Hz, 6H), 1.92 (s, 6H), 3.22 (q, J=6.9 Hz, 4H), 3.56 (br s, 4H), 6.26 (s,
H), 6.39 (s, 2H), 7.06 (t, J=3.3 Hz, 1H), 7.45 (t, J=4.6 Hz, 2H),
Briefly, in a 50 mL round-bot-
of residual Pd in a reaction mixture, a Suzuki coupling reaction was per-
[
17]
formed.
a non-nucleophilic carbonate base. Thus, a mixture of 4-bromoanisole
0.5 mmol), phenylboronic acid (0.75 mmol), Pd(OAc) (0.25 mol%),
CO (1.0 mmol), and water (1.0 mL) was stirred at 1008C under a N
atmosphere for 1 h. Then, the reaction mixture was added to brine
10 mL) and extracted with EtOAc (3ꢂ10 mL). The organic solvent was
2
Pd ACHTUNGTRENNUNG( OAc) was used in this reaction as a pre-catalyst with
(
(
K
A
H
U
G
R
N
U
G
2
2
3
2
(
1
3
): d=1.32 (t, J=
concentrated under vacuum to afford the desired product (18% yield).
An aliquot was taken directly from the reaction mixture and probe 1 was
added to it. Similarly, an aliquot was removed after work-up of the reac-
7
2
7
.96 ppm (t, J=3.2 Hz, 1H); IR (neat): n˜ =3370.6, 2921.5, 1621.4, 1516.6,
1270.3, 1203.6, 1017.8, 742.4 cm ; HRMS: m/z calcd for C H N O :
451.2110 [M+Na] ; found: 451.2106.
2+
0
ꢀ1
tion. During the reaction, most of the Pd was converted into Pd and,
upon quenching, the remaining active catalyst precipitated as palladium
2
6
28
4
2
+
[
18]
black. This palladium species became insoluble and, therefore, was un-
Compound 1: Rhodamine hydrazone (47 mg, 0.11 mmol) was mixed with
aldehyde 3 (33 mg, 0.11 mmol) in MeOH (4 mL). Glacial acetic acid (2–
detectable by this procedure in the presence of probe 1.
3
drops) was added and the mixture was stirred at RT for 4–5 h. After
completion of the reaction, the precipitate was filtered and washed with
MeOH until it was completely pure, as determined by TLC. Yield:
1
5
5 mg, 70%; H NMR (400 MHz, CDCl
3
): d=1.31(t, J=8 Hz, 6H), 1.86
(
(
s, 6H), 3.19 (q, J=4 Hz, 4H), 4.87 (s, 4H), 6.35 (d, J=10 Hz, 4H), 6.56
d, J=4 Hz, 2H), 7.02–7.04 (m, 1H), 7.24 (d, J=8 Hz, 4H), 7.34 (d, J=
9
1
[D
6
Hz, 2H), 7.44–7.46 (m, 2H), 7.67 (t, J=8 Hz, 2H), 7.99 (t, J=4 Hz,
Fluorescence Decay Experiments
1
3
H), 8.33 (s, 1H), 8.60 ppm (d, J=2 Hz, 2H); C NMR (100 MHz,
]DMSO): d=14.1, 16.9, 37.4, 65.5, 95.8, 104.9, 117.7, 122.0, 123.3,
126.9, 127.9, 129.4, 132.2, 147.3, 151.3, 152.0, 165.1 ppm; IR (neat): n˜ =
Fluorescence lifetime values were measured by using a time-correlated
single-photon-counting (TCSPC) fluorimeter (Horiba Jobin Yvon). The
system was excited with a 400 nm nano-LED with a pulse duration of
ꢀ
1
3814.6, 2917.5, 1696.5, 1518.6, 1203.0, 1093.8, 718.0 cm ; HRMS: m/z
calcd for C32
+
1
.2 ns (slit width: 2/2, lem =560 nm). The average fluorescence lifetimes
H
31
N
5
O
2
: 714.3531 [M+H] ; found: 714.3531.
(
t
av) for the exponential iterative fitting were calculated from the decay
times (t
i
) and the relative amplitudes (a
i
) by using Equation (1), where
a
1
–
3
are the relative amplitudes and t –
1 3
are the lifetime values.
2
2
2
t
av ¼ ða
1
t
1
þa
2
t
2
þa
3
t
3
Þ=ða
1
t
1
þa
2
t
2
þa
3
t
3
Þ
ð1Þ
Acknowledgements
1
H NMR Titration Studies
S.B. thanks DST (J.C. Bose Fellowship) for financial support. N.D. thanks
CSIR for an SRF and N.K. thanks IISc for a research associateship (RA).
1
H NMR titration experiments were performed for compound 1 (8 mm)
2+
6 2 3 2
in a [D ]DMSO/D O (9:1) mixture with Pd (in CD OD/D O, 1:3, with
NaCl). All of the spectra were recorded under identical conditions.
[
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[
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4
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3
J=7.65 Hz, 2H), 8.57 ppm (d, J=4.5 Hz, 2H); C NMR(75 MHz,
CDCl ): d=57.2, 112.4, 117.1, 120.7, 121.9, 129.2, 136.8, 148.1, 149.6,
58.8 ppm, IR (neat): n˜ =3062.4, 1590.9, 1506.1, 1434.7, 1351.8, 1047.1,
3
1
7
2
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50.1 cm ; HRMS: m/z calcd for C18
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and POCl (3.54 g, 23.0 mmol) and stirring at RT for 30 min, followed by
3
heating at 908C for 2 h. After completion of the reaction, the mixture
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H NMR (300 MHz, CDCl
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): d=4.83 (s, 4H), 6.80 (d, J=9.0 Hz, 2H),
7
.22–7.28 (m, 4H), 7.65–7.71 (m, 4H), 8.63 (d, J=3.9 Hz, 2H), 9.74 ppm
Chem. Asian J. 2014, 00, 0 – 0
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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: June 9, 2014
Revised: July 10, 2014
Published online: && &&, 0000
&
&
Chem. Asian J. 2014, 00, 0 – 0
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ÝÝ These are not the final page numbers!

FULL PAPER
Sensors
Namita Kumari, Nilanjan Dey,
Krishan Kumar,
Santanu Bhattacharya*
&&&& — &&&&
Exclusive Detection of Sub-Nanomolar
Levels of Palladium(II) in Water: An
Excellent Probe for Multiple Applica-
tions
II
On the level: A rhodamine-based
probe with a bis-picolyl binding unit
has been used for the effective sub-
towards Pd , both in terms of color
and fluorescence emission. The appli-
cation of this probe has been demon-
strated by detecting Pd in real water
II
II
nanomolar detection of Pd in water
at physiological pH value. The probe
showed instant “turn-on” response
samples, pharmaceutical drugs, and
cells lines.
Chem. Asian J. 2014, 00, 0 – 0
9
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ