Applications of fluorescent sensor based on 1H-pyrazolo[3,4-b]quinoline in analytical chemistry

Article abstract of DOI:10.1007/s10895-013-1251-5

Fluorescent dye 2-[(2-Hydroxyethyl)-(1,3-diphenyl-1H-pyrazolo[3,4-b] quinolin-6-ylmethyl)-amino]ethanol (LL1) was examined for its efficiency in the detection of small inorganic cations (lithium, sodium, barium, calcium, magnesium, cadmium, lead and zinc)

Full text of DOI:10.1007/s10895-013-1251-5

J Fluoresc
DOI 10.1007/s10895-013-1251-5
ORIGINAL PAPER
Applications of Fluorescent Sensor Based on 1H-pyrazolo
[3,4-b]quinoline in Analytical Chemistry
Marek Mac & Tomasz Uchacz & Andrzej Danel &
Hanna Musiolik
Received: 27 March 2013 /Accepted: 17 June 2013
#
The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Fluorescent dye 2-[(2-Hydroxyethyl)-(1,3-diphenyl-
1H-pyrazolo[3,4-b]quinolin-6-ylmethyl)-amino]ethanol (LL1)
was examined for its efficiency in the detection of small inor-
ganic cations (lithium, sodium, barium, calcium, magnesium,
cadmium, lead and zinc). The dye was synthesized in the
laboratory and investigated by means of both, steady-state and
time-resolved fluorescence techniques. This compound acts as a
fluorescent sensor suitable for detection of small inorganic cat-
ions (lithium, sodium, barium, calcium, magnesium, cadmium,
lead and zinc) in strongly polar solvent (acetonitrile). An elec-
tron transfer from the electro-donative part (receptor) of the
molecule to the acceptor part (fluorophore) is thought to be the
main mechanism that underlies functionality of the compound
as a sensor. This process can be retarded upon complexation of
the receptor moiety by inorganic cations. Relatively high sen-
sitivity but poor selectivity of the aminoalcohol thatcontains
indicator towards the two-valued cations was observed.
However, upon addition of some amounts of water the selec-
tivity of this sensor has been enhanced (especially towards
lead cation). The preliminary results in analytical application
of the sensor are discussed.
Introduction
Measurements of the cation concentration are of consider-
able interest and the subject of various investigations
conducted by chemists, biologists, clinical biochemists and
environmentalists. Some of the cations play a crucial role in
biological processes (Na+, K+, Ca2+, Mg2+, Zn2+). In medi-
cine some diagnostics are based on the detection of certain
cations and monitoring their concentration in blood and
urine. In psychiatry the control of the lithium concentration
is important for patients who are under the treatment for
manic depression. Some cations are toxic (Pb2+, Cd2+,
Hg2+) and early detection of their presence in organism is
of uttermost importance.
Among other sensors the fluorescent molecular sensors
(known also as indicators) are of considerable interest for the
detection of small amounts of cations such as H+, Li+, Na+,
K+, Ca2+, Mg2+, Zn2+, Pb2+, Al3+, Cd2+, etc. These are the
molecular compounds that fluorescence emission can be
strongly affected by the presence of inorganic cations.
Their high detection sensitivity permits the investigation of
non-fluorescent cations with the concentrations down to
10−6 M [1, 2]. Nevertheless, searches for new fluorescence
materials capable of extended sensitivity are still underway.
The principles underlying functionality of fluorescence
sensors are presented in several publications including
monographs and original papers [3, 4]. Beneath, only the
basic information necessary to understand the performance
of the sensors is briefly reviewed.
.
Keywords Fluorescent molecular probes Photoinduced
.
.
electron transfer Time resolved fluorescence
Pyrazolo[3,4-b]quinoline
:
:
M. Mac T. Uchacz (*) H. Musiolik
Faculty of Chemistry, Jagiellonian University,
Ingardena 3, 30-060 Krakow, Poland
e-mail: tomaszuchacz55@gmail.com
A general principle of operation of many PET fluorescent
indicators is based on significant enhancement of fluores-
cence emission upon addition of cations into fluorescing
medium. Molecule-cation complexes of various stoichiometry
:
T. Uchacz A. Danel
Department of Chemistry, University of Agriculture,
Balicka 122, 31-149 Kraków, Poland

J Fluoresc
can be formed in this process. Their high stability in both, the
ground and excited states is desired to permit fluorescence
emission followed the electronic excitation of the complex
(see Scheme 1). To understand fluorescence emission en-
hancement a detailed mechanism of the process has to be
understood. For the purpose of current investigation we recall
the mechanism determined for the molecular sensors based on
pyrazoloquinoline skeleton. The mechanism underlying func-
tionality of molecular sensors that are constructed on the basis
of pyrazoloquinoline skeleton was discussed in the previous
papers [5, 6]. It involves an electron transfer process, i.e. after
excitation, the pyrazoloquinoline derivative accepts one elec-
tron from the lone pair located at the nitrogen atom of the
donor receptor moiety, being simultaneously a cation receptor
subunit. It was found that previously constructed fluorescent
cation indicators are more sensitive to the presence of bivalent
cations such as Mg2+, Ca2+, Zn2+ than to monovalent cations
such as Li+ and Na+. Additionally, the binding constants
estimated from the concentration profiles of the fluorescence
changes are related to the charge density of the cations, so that
larger was the cation charge density, greater was the complex-
formation equilibrium constant.
We chose 1,3-diphenyl-1H-pyrazolo[3,4-b]quinoline as a
fluorophore because of its good emissive properties. The fluo-
rescence quantum yields and lifetimes are relatively independent
on solvent polarity and reach values up to 88 % and 25 ns,
respectively [7]. This fact results in effective photoinduced
electron transfer quenching in pyrazoloquinoline compounds
with electronically decoupled amino-recognition group and in
consequence easily detectable ON-OFF green fluorescence.
Another reason for selecting the pyrazoloquinoline is a relatively
simple synthesis. 1,3-diphenyl-1H-pyrazolo[3,4-b]quinoline is
received in reaction between commercially available substrates.
In view of the above, it is not surprising that pyrazoloquinolines
are extensively exploited for a long time as a luminescent
materials for fabrication of organic light emitting diodes
(OLED) [8], as dopants in PVK matrices [9] and polymers [10].
The paper investigates photophysical properties of the che-
lating sensor 2-[(2-Hydroxyethyl)-(1,3-diphenyl-1H-pyrazolo
[3,4-b]quinolin-6-ylmethyl)-amino]ethanol (LL1) and the
complexes formed between the sensor and several cations
such as Li+, Na+, Ba2+, Ca2+, Mg2+, Cd2+, Pb2+, Zn2+, Ag+
in acetonitrile and acetonitrile-water mixtures as well. To
elucidate the potential of LL1 for sensitive and selective
detection of cations we perform simple fluorimetric titrations.
Additionally, we examine also a possible application of the
dye (LL1) as an indicator in fluorimetric determination of Zn
and Pb bivalent cations by chloride anion titration in
acetonitrile.
Experimental
Synthesis of 2-[(2-Hydroxyethyl)-(1,3-Diphenyl-1H-Pyrazolo
[3,4-b]Quinolin-6-Ylmethyl)-Amino]Ethanol (LL1)
The synthesis reaction steps of LL1 are depicted in Scheme 2.
6-methyl-1,3-diphenyl-1H-pyrazolo[3,4-b]quinoline 1 was
obtained from the respective pyrazole and aniline precursors
[11]. The resulting compound 2 was brominated with NBS
(N-bromosuccinimide) in the presence of catalytic amount of
AIBN (2,2′-azoisobutyronitrile). The nucleophilic substitu-
tion of the bromomethyl group with diethanolamine in ace-
tonitrile gave the compound LL1. A more detailed descrip-
tion of the synthesis of this sensor was given in previous
publication [12].
Apparatus and Measuring Procedures
The solvents: dibutyl ether (DBE), butyl chloride (BuCl),
tetrahydrofuran (THF), acetone (ACE), acetonitrile (ACN),
and dimethylformamide (DMF) were of spectroscopic grade
and were used as received (all from Aldrich). All the solvents
used in this study did not show any traces of fluorescence.
For fluorescence quantum yield and fluorescence lifetime
measurements the solutions of the dye were degassed using
multiple freeze-pump-thaw cycles. The sample concentra-
tion of the dyes (LL1 and fluorophore) for spectroscopic
Scheme 1 Principle underlying
metal ion sensing by fluorescent
PET (photoinduced electron
transfer) indicators
cation
recognition
group
fluorophore

J Fluoresc
Scheme 2 a 180–190 °C; b
NBS/AIBN/CCl4, c diethanol-
amine/K2CO3
OHC
Cl
Ph
Ph
N
Me
Me
a
+
N
N
NH2
N
N
Ph
Ph
1
b
HO
Ph
Ph
c
N
Br
N
N
N
N
N
N
Ph
Ph
OH
LL1
2
Ph
N
N
Ph
N
fluorophore
measurements was ca. 4×10−5 M (this corresponds to absor-
bance of ca. 0.2 at the excitation wavelength used for the fluo-
rescence investigations). Lithium, sodium, barium, magnesium,
calcium (tetrahydrate) lead (x-hydrate), cadmium, zinc
(hexahydrate) perchlorate, and zinc chloride, tetrabuty-
lammonium chloride (TBACl) (Aldrich) were used as received.
In an independent measurement it was found that a small addi-
tion of deionised water could not influence significantly the
fluorescence of the dye alone in acetonitrile solution. The fluo-
rescence titration experiments were not proceeded by the
degassing procedure.
correction for spectral sensitivity) were measured by means of
Hitachi F7000 fluorometer. For time-resolved fluorescence
measurements (time-correlated single photon counting tech-
nique) a picosecond diode laser (λ=400 nm, 70 ps pulse
duration) (IBH-UK) was used as the excitation source. For
steady-state fluorescence measurements a 365 or 380 nm line
was used. The fluorescence quantum yield measurements
were carried out with quinine sulphate in water (Φfl=0.51)
[13] as an actinometer.
Joe-Jones Method
The fluorescence measurements of the titration between LL1-
Pb2+ complex and tetrabutylammonium chloride (TBACl) were
carried out on ultracentrifuged samples. The others fluorimetric
titrations were performed in situ.
The absorption spectra were recorded using a Shimadzu
UV-2101 PC spectrometer and the emission spectra (with the
The equilibrium constants (K1) for complexation of the li-
gand by alkali cations are calculated by fitting Eq. 1 to the
relative fluorescence intensities in the presence of different
concentration (cM) of the employed alkaline metal perchlo-
rates [14],
ꢀ
ꢁ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Φð∞Þ− Φð0Þ
2cL
2
ΦðcM Þ ¼ Φð0Þ þ
cL þ cM þ 1=K1− ðcL þ cM þ 1=K1Þ −4cLcM
ð1Þ
where Φ(0), Φ(∞) and Φ(cM) is the fluorescence intensity of
the dye alone, with a large concentration of the salt and with
the cM concentration of the salt, respectively, cL indicates the
analytical concentration of the ligand.

J Fluoresc
For 2:1 complexation the binding constant K2 is defined
Job’s Method
by Eq. 2 [1]:
Â
Ã
Stoichiometry of the fluorescent complexes can be
established by measuring the fluorescence of the mixtures
of fluorophore (sensor) (L) and metal cations (M) provided
that the sum of the concentration of the sensor and the cation
is constant, i.e. cL+cM=C. This method is called Job’s
method or method of continuous variations [1].
MLþ2
K2 ¼
ð2Þ
2
½Lꢀ ½Mþꢀ
The Eq. 1 converts into more complex one for 2:1 com-
plexation scheme [3, 4]:
2x
Let us assume the following equilibrium:
ΦðcM Þ ¼ Φ0 þ ð Φ∞− Φ0Þ
ð3Þ
cL
lL þ mMnþ⇔LlMm
nþ
where x denotes the concentration of the complex ML+. This
2
quantity can be obtained numerically by solving the third
degree polynomial:
We measure the fluorescence of the mixtures at different
values of the parameter x, defined as
ax3 þ bx2 þ cx þ d ¼ 0
with the following parameters:
cM
x ¼
ð4Þ
cM þ cL
The position of the maximum of fluorescence on x is
related to l/m.
If we assume the 1:1 stoichiometry of a complex the
dependence of the integrated fluorescence on x has a follow-
ing form:
a
b
c
¼
¼
¼
− 4 K2
4 K2 ðcL þ cM Þ
À
Á
ð3bÞ
2
− K2cL þ 4K2cLcM þ 1
2
d ¼ K2 cL cM
0
. 1
#
ꢂ
.
ꢃ
"
1
2
ꢄ
ꢅ
2
Φ∞ Φ0−1 Φ0
B
B
@
C
C
A
1
1
ΦðxÞ− Φ0ð1−xÞ ¼
1 þ
−
1 þ
K1C
−4xð1−xÞ
ð4aÞ
2
K1C
donating macrocyclic recognition unit on the energetics of the
optical transitions within the fluorophore. For instance, the
maxima of the absorption and fluorescence bands of LL1 in
acetonitrile were found at 397 nm and 481 nm, whilst those of
the fluorophore are centred at 395 nm 476 nm (see Fig. 1). It can
The meaning of the parameters which appeared in Eq. 4a
has been explained in the previous section, except of Φ0(1−x)
defined as the fluorescence intensity measured in the absence
of cation at every concentration of the sensor.
The values of the binding constants K1 and K2 were obtained
from the fluorescence intensities using a nonlinear least-square
fitting routine coded in FORTRAN.
1.2
2.5
1.0
0.8
2.0
0.6
Results and Discussion
0.4
1.5
UV–vis Absorption and Fluorescence Emission
0.2
Spectroscopy Studies
0.0
1.0
450
500
550
600
650
λ [nm]
The absorption spectrum of LL1 in acetonitrile consists of two
separate bands located between 250 and 325 nm and above
350 nm, respectively (Fig. 1). The first one is a structured band
with a maximum at 260 nm (ε=57400 dm3mol−1cm−1) while
the second one is a Gaussian-like band having maximum at
397 nm (molar absorption coefficient ε=7400 dm3mol−1cm−1).
A simple comparison of the absorption spectra of LL1 and the
parent molecule 1,3-diphenyl-1H-pyrazolo[3,4-b]quinoline
(fluorophore of LL1) reveals a negligible effect of the electron-
0.5
0.0
250
300
350
400
450
λ /nm
Fig. 1 Room absorption and fluorescence (inset) spectra of 1,3-diphenyl-
1H-pyrazolo[3,4-b]quinoline (solid), LL1 (dash), LL1+Mg(ClO4)2 (dash
dot dot) in acetonitrile. The concentration of Mg(ClO4)2 is equal to 4.104 M

J Fluoresc
Table 1 The maximum of the
first absorption (λabs) band and
fluorescence (λfl) band, fluores-
cence quantum yields (Φfl) and
lifetimes (τfl) for LL1 fluorescent
sensor as a function of dielectric
permittivity of the solvent (εs).a
denotes the magnitude of the
long-life component of the bi-
exponential fluorescence decay
function
Solvent
λ
abs [nm]
λfl [nm]
Φfl
τfl [ns]
εs
3.08
DBE
BuCl
THF
ACE
403
402
401
398
456
464
472
478
0.61
0.59
0.34
0.051
19.09
17.96
19.89
7.37
7.52
0.63
20.29(a)
21.01
ACN
DMF
397
400
481
482
0.040
0.049
0.76
22.01(a)
36.63
38.25
0.52
19.25(a)
also be inferred that the absorption spectra of LL1 are rather
insensitive to the solvent polarity.
the results described in this paper are related to the acetoni-
trile solutions.
Polarity of the solvents strongly influences the fluorescence
lifetimes and quantum yields of LL1, likewise the previously
investigated other dyes containing pyrazoloquinoline skeleton
[3, 4]. Such dependences of the above mentioned fluorescence
quantities on the dielectric permittivity parameter (εs) are
tabulated in Table 1.
The reduced quantum yield of LL1 in acetonitrile
(Φfl=0.04) as compared to the fluorophore part (Φfl=0.75 -
determined experimentally) and simultaneous decrease of Φfl
and τfl with increasing solvent polarity suggest the existence
of quenching via an electron-transfer process from the electron-
ically decoupled nitrogen atom, located at diethanolamine, to
the fluorophore.
The fluorescence decays are monoexponential in nonpo-
lar and medium polar solvents. However, in highly polar
solvents the decays are clearly biexponential which may
indicate the existence of two different ground-state con-
formers, related to the endo/exo isomerism [15]. The differ-
ences in position and direction of the lone pair at the nitrogen
atom and its degree of pyramidalization can be responsible
for various electron transfer quenching activities.
A small bathochromic shift of the fluorescence maximum
of LL1, observed during the solvatochromic studies, points
to a partial charge-transfer character of the transition within
the pyrazoloquinoline fragment.
Upon addition of cations to the acetonitrile solution of
LL1, the absorption spectrum does not change significantly
(Fig. 1). The structured band in the UV region (250–325 nm)
turns now into a relatively broad band.
However, the addition of cations changes the fluorescence
properties considerably (Fig. 2.). The locations of the band
maximum in the fluorescence spectra of solutions containing
and not containing inorganic cations are only slightly differ-
ent (shifted bathochromically in the presence of the cations
by ca. 10 nm, see inset in Fig. 1) but upon addition of the
cations the intensity of the fluorescence rises dramatically
(Fig. 2). For example, in the presence of 10 equivalent of
Mg2+, the mixture shows an intense green fluorescence and a
10-fold enhancement in the fluorescence intensity at 490 nm.
Similar behaviour has been observed in the experiments
when other cations were added. The examples of the titration
curves are presented in Fig. 3.
It can be noticed that the bivalent cations have stronger
influence on the fluorescence than the monovalent ones. The
values of the binding constants that were calculated from the
salt concentration dependencies of the integrated fluorescence
8000
0.01 M
6000
Mg2+
Stability of the Fluorescent Complex (LL1Men+)
in Acetonitrile
4000
LL1+Mg2+ LL1
0.0 M
2000
0
Because of the large reduction of the fluorescence lifetime
and quantum yields reduction, there is an opportunity to
apply the molecule LL1 as a PET fluorescent probe in highly
polar solvents (dielectric permittivity larger than 7).
Acetonitrile was chosen as a solvent for all measurements
conducted in this investigation owing to its high dielectric
constant, lack of hydrogen bond formation and good solu-
bility of the inorganic salts used in the experiments. Thus, all
450
500
550
/nm
600
650
700
λ
Fig. 2 Fluorescnce spectra of LL1 in acetonitrile in the absence and
presence of magnesium perchlorate (concentration of the salt spans the
region of 0–0.01 M)

J Fluoresc
cations such as Mg2+, Zn2+, Cd2+ and Pb2+. Moreover, the
equilibrium constants is found in good agreement with charge
density of the cation by taking into account the complexes
with alkaline and transition metals separately. For practical
applications, the detection limit for this sensor evaluated by
fluorescence titration was estimated at 4×10−7 M, and is 100
times lower than that of the ligand concentration.
a
b
70
60
50
40
30
20
10
Mg(ClO )
4
2
Ba(ClO )
4
2
AgClO
4
0.0
5.0x10-3
1.0x10-2
1.5x10-2
2.0x10-2
salt concentration/M
Stability of the Fluorescent Complex (LL1Men+)
in Acetonitrile-Water Mixtures
400
350
300
250
200
150
100
50
The selectivity of the LL1 can be improved by performing
fluorimetric measurements in acetonitrile-water mixtures. By
adding small amounts of water to the acetonitrile solution of
the complex LL1 with magnesium cation can quench fluores-
cence of the complex almost completely (Fig. 4). The fluores-
cence of the magnesium complex was immediately quenched
in the mixture containing 0.1 molar fraction of water (B),
while the complexes with other bivalent cations are almost
unaffected and the fluorescence intensity stays the same as in
pure acetonitrile (A). However, a systematic increase of water
concentration quenches fluorescence of other complexes ex-
cept of the Pb2+-LL1 complex (C). This is probably the result
of different ability of the cations to hydrate.
3
-1
K
=1.6x10 /M
LL1+Ba
0
0.0
0.2
0.4
0.6
0.8
1.0
[Ba2+]/[Ba2++LL1]
Fig. 3 a Dependence of the integrated fluorescence intensity of LL1 as
a function of magnesium, barium and silver perchlorate concentration
in acetonitrile. The solid line represent the values calculated from Eq. 3
with the association constant K2=7.9×108 M−2. b Job’s plot of Ba2+
versus ([LL1]+[Ba2+] =80 μM) at 490 nm emission wavelength and
presentation of the fitted function (4a) to experimental data
Hence, a simple addition of water to the mixture of the
cations can change the selectivity of the dye making it more
prone to form complex only with Pb2+ cation.
intensities, are collected in Table 2. The stoichiometry of the
complexes have been established by the analysis of the Job’s
plots obtained from the fluorimetric titration of the mixtures of
the acetonitrile solutions of the dye (LL1) and that of perchlo-
rates (Fig. 3b). It has been found that in the case of Mg or Zn
cation the 2:1 complexes are formed in the ground state. For the
other cations the LL1 forms complexes with 1:1 stoichiometry.
One important conclusion emerges from the data collected
in Table 2: the compound LL1 in acetonitrile exhibits a great
sensitivity but poor selectivity to the presence of bivalent
Application of LL1 to the Fluorimetric Titration of Cations
The fluorimetric titration of LL1-Pb2+ complex by tetra-
butylammonium chloride (TBACl) was performed in aceto-
nitrile. Addition of TBACl to the solution of Pb(ClO4)2 causes
a precipitation of PbCl2 with a residue that is hardly soluble in
Table 2 Binding constants i.e.
Cation(X)
Radius (Å) [16]
Charge density of cation [17]
K1(LL1M+n)/M−1 or K2(LL12M+n)/M−2
equilibrium constants for the re-
action between different inor-
ganic cations and the compound
LL1 in ACN determined from
fluorimetric titrations.(a)
Li+
Na+
Ca2+
0.59
1.02
1.0
1.47
1.03
2.02
K1=12.7
K1=2.8
K1=1.2×104
K1=1.6×104(a)
K1=1.7×103
K1=1.6×103(a)
K2=2.7×109
K2=1.4×1010
K1=3.3×105
K1=4.2×105(a)
K1=1.5×105
K1=2.1×105(a)
K1=84.1
Obtained from the Job’s method
Ba2+
1.33
1.49
Mg2+
Zn2+
Cd2+
0.66
0.74
0.97
3.03
2.7
2.06
Pb2+
Ag+
1.2
1.67
0.78
1.29

J Fluoresc
8000
7000
6000
5000
4000
3000
2000
1000
0
and further
A
B
PbCl2 þ 2Cl−⇔PbCl42−
C
Pb2+
Due to the progressive reaction, the concentration of the
fluorescent complex LL1-Pb2+ decreases which results in the
reduction of fluorescence intensity. Hence, the concentration
of the lead cations may be calculated from the equivalence
point, that can be determined by analysis of the first deriva-
tive of titration curve (Fig. 5 - inset)
Another example of the fluorimetric titration is the reac-
tion of zinc perchlorate (Zn(ClO4)2) with TBACl in acetoni-
trile. In this system a precipitation of the zinc chloride, that is
soluble in acetonitrile, is not observed but the product reacts
with an excess of chloride anions to form an anion complex
(ZnCl4)2- [19]. Again, this anion does not form a fluorescent
complex with the LL1 dye. The fluorimetric titration profile
is displayed in Fig. 6.
Zn2+
Cd2+
Mg2+
no
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Molar fraction of water
Fig. 4 Dependence of the integrated fluorescence of the complex
LL1Me2+ on concentration of water in ACN-H2O mixtures
acetonitrile. Further addition of Cl- anions dissolves the pre-
cipitate due to formation of the anion complex Pb(Cl4)2- [18].
The titration process can be monitored by fluorescence tech-
niques in the presence of nonfluorescent LL1 dye (Fig. 5).
Both PbCl2 and Pb(Cl4)2- cannot form a fluorescent complex
with LL1.
As shown in Fig. 5, up to the equivalence point the chloride
anions consume mostly the lead cations that are non-bonded
to the sensor (being in an excess), thus the fluorescence
intensity remains nearly constant. In the vicinity of the equiv-
alence point, the fluorescent complex reacts with the excess of
the chloride anions as follows:
In this case, the titration curve illustrating the reaction
between Zn(LL1)2 and chlorides is more complex. Up to
2+
the equivalence point chloride anions reacts only with free
zinc cations non-bonded to the LL1 dye (being in excess).
Thus the fluorescence intensity remains nearly constant. At
the equivalence point it is very likely that the following
reaction occurs:
ZnðLL1Þ22þ þ 2Cl−⇔ZnLL1Cl2 þ LL1
and the fluorescence intensity is reduced up to c.a. 50 % of
the initial level. This assumption was proven in the indepen-
dent titration of LL1 by ZnCl2. It was found out that ZnCl2
can be effectively bound to a chelate (K1 is equal 2.28 x104
PbLL12þ þ 2Cl−⇔PbCl2 þ 2LL1
7x103
6000
5000
0,0
6x103
-5,0x104
5x103
4000
3000
2000
1000
0
0
-1,0x105
-1x105
-2x105
-3x105
-4x105
-5x105
4x103
-1,5x105
0,00
0,05
0,10
0,15
0,20 0,25
3x103
2x103
1x103
VTBACl /ml
-0,02 0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14
VTBACl/ml
-0.02 0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
VTBACl/ml
VTBACl/ml
Fig. 5 Dependence of the integrated fluorescence of the mixture of
LL1 (4×10−5M) and Pb(ClO4)2 (1×10−3M) on the volume of the added
TBACl (concentration 2×10−2M). Initial volume of the mixture of LL1
and Pb(ClO4)2 is 1 ml. Inset: the first derivative of the titration curve
with a minimum (equivalence point) c.a. 0.09 ml of TBACl
Fig. 6 Dependence of the integrated fluorescence of the mixture of
LL1 (4×10−5M) and Zn(ClO4)2 (1×10−3M) on the volume of the added
TBACl (concentration 2×10−2M). Initial volume of the mixture of LL1
and Zn(ClO4)2 is 1 ml. Inset: the first derivative of the titration curve
with a minimum (equivalence point) c.a. 0.11 ml of TBACl

J Fluoresc
Scheme 3 Reactions that occur
during the titration of
PET
Zn(LL1)22+ complex by TBACl
H
H
O
O
N
N
N
Zn 2+
N
N
N
N
N
O
O
H
H
PET
TBACl
+
PET
H
O
Cl
Cl
N
N
Zn
N
N
O
H
TBACl
+
PET
H
O
N
2-
+
N
N
Zn(Cl
)
4
N
O
H
M−1) and likewise in the first equivalence point (Fig. 6), the
intensity of the fluorescence (at the high ZnCl2 concentra-
tion) is almost twice lower than the maximal intensity of the
Zn(LL1)2 complex (both experiments were performed at
the same concentration of the dye).
Further addition of chloride anions leads to the formation
of an anion complex (ZnCl4)2− and weakly fluorescent LL1
dye. The process has been depicted in scheme 3.
Again, this hypothesis was confirmed by the reaction of
ZnCl2 with TBACl in the presence of LL1 dye (Fig. 7)
The present investigation show analytical capabilities of
applied fluorescence technique. It can be used for instance
for monitoring of a simple exchange reaction where product
could be traced by the fluorimetric method. Moreover, the
fluorimetric titration experiment shows also that the binding
of Zn2+ or Pb2+ to the investigated sensor is reversible in the
presence of Cl- anions, which is of particular importance if
the sensor will be widely employed in the detection of
specific analysis.
4,0x103
3,5x103
3,0x103
2,5x103
2,0x103
1,5x103
1,0x103
2+
5,0x102
0,0
0,0
0,2
0,4
0,6
0,8
1,0
1,2
VTBACl /ml
Fig. 7 Dependence of the integrated fluorescence of the mixture of
LL1 (4×10−5M) and ZnCl2 (8×10−5M) on the volume of the added
TBACl (concentration 5×10−4M). Initial volume of the mixture of LL1
and ZnCl2 is 2 ml

J Fluoresc
5. Mac M, Uchacz T, Wróbel T, Danel A, Kulig E (2010) New
fluorescent sensors based on 1H-pyrazolo[3,4-b]quinoline skele-
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7. Mac M, Uchacz T, Andrzejak M, Danel A, Szlachcic P (2007)
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Conclusions
The photophysical properties of 2-[(2-Hydroxyethyl)-(1,3-
diphenyl-1H-pyrazolo[3,4-b]quinolin-6-ylmethyl)-
amino]ethanol (LL1) relevant to that of fluorescent indicator
of inorganic cations were investigated. It was found that this
dye exhibits a large sensitivity with respect to bivalent cat-
ions such as Mg2+, Zn2+, Cd2+ and Pb2+ and its selectivity to
detection of bivalent cations can be easily improved by addi-
tion of a small amount of water.
The fluorimetric titration (precipitation of hardly soluble lead
chloride) using the LL1 dye was performed. The results of the
investigation show that this fluorimetric method can be promis-
ing for sensitive and selective analysis of cationic species. Its
potential importance for application in analytical chemistry and
in a range of environmental analyses shall be stressed as well.
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Acknowledgments Drs Andrzej Turek and Marek Tulej are acknowl-
edged for fruitful discussions and help in planning the experiments.
This work was partly financed by the basic funding of the University of
Agriculture (DS 3133/KCh/2012).
(2002) 1,3-Diphenyl-1H-pyrazolo[3,4b]quinoline:
a
versatile
fluorophore for the design of brightly emissive molecular sensors.
Org Lett 4:4647–4650
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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