Selective fluorescence sensing of Cu(II) and Zn(II) using a simple Schiff base ligand: Naked eye detection and elucidation of photoinduced electron transfer (PET) mechanism

Article abstract of DOI:10.1016/j.saa.2015.02.013

A simple Schiff base compound 2-((cyclohexylmethylimino)-methyl)-naphthalen-1-ol (2CMIMN1O) has been synthesized and characterized by ¹H NMR, ¹³C NMR and FT-IR spectroscopic techniques. A significantly low emission yield of the compound has been rationalized in anticipation with photo-induced electron transfer (PET) from the imine receptor moiety to the naphthalene fluorophore unit. Consequently, an evaluation of the transition metal ion-induced modification of the fluorophore-receptor communication reveals the promising prospect of the title compound to function as a chemosensor for Cu²⁺ and Zn²⁺ ions selectively, through remarkable fluorescence enhancement as well as visual changes. While perturbation of the PET process has been argued to be the plausible mechanism behind the fluorescence enhancement, the selectivity for these two metal ions has been interpreted on the grounds of an appreciably strong binding interaction. Particularly notable aspects regarding the chemosensory activity of the compound is its ability to detect the aforesaid transition metal ions down to the level of micromolar concentration (detection limit being 2.74 and 2.27 ppm respectively), along with a simple and efficient synthetic procedure.

Full text of DOI:10.1016/j.saa.2015.02.013

Accepted Manuscript
Selective fluorescence sensing of Cu(II) and Zn(II) using a simple Schiff base
ligand: Naked eye detection and elucidation of photoinduced electron transfer
(PET) mechanism
Aniruddha Ganguly, Soumen Ghosh, Samiran Kar, Nikhil Guchhait
PII:
S1386-1425(15)00165-1
http://dx.doi.org/10.1016/j.saa.2015.02.013
SAA 13303
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 September 2014
9 December 2014
4 February 2015
Please cite this article as: A. Ganguly, S. Ghosh, S. Kar, N. Guchhait, Selective fluorescence sensing of Cu(II) and
Zn(II) using a simple Schiff base ligand: Naked eye detection and elucidation of photoinduced electron transfer
(PET) mechanism, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://
dx.doi.org/10.1016/j.saa.2015.02.013
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Selective fluorescence sensing of Cu(II) and Zn(II) using a simple Schiff base ligand:
Naked eye detection and elucidation of photoinduced electron transfer (PET)
mechanism
Aniruddha Ganguly, Soumen Ghosh, Samiran Kar‡, Nikhil Guchhait*
Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata-700009, India
*Corresponding author. Tel.: +913323508386; fax: +913323519755.
E-mail address: nguchhait@yahoo.com (N. Guchhait).
‡Present address: Research and Development section, Navin Fluorine International
Limited, Dewas Area, Madhya Pradesh, India

Abstract
A simple Schiff base compound 2-((cyclohexylmethylimino)-methyl)-naphthalen-1-ol
(2CMIMN1O) has been synthesized and characterized by 1H NMR, 13C NMR and FT-IR
spectroscopic techniques. A significantly low emission yield of the compound has been
rationalized in anticipation with photo-induced electron transfer (PET) from the imine
receptor moiety to the naphthalene fluorophore unit. Consequently, an evaluation for the
transition metal ion-induced modification of the fluorophore-receptor communication
reveals the promising prospect of the title compound to function as a chemosensor for
Cu2+ and Zn2+ ions selectively, through remarkable fluorescence enhancement as well as
visual changes. While perturbation of the PET process has been argued to be the
plausible mechanism behind the fluorescence enhancement, the selectivity for these two
metal ions has been interpreted on the grounds of an appreciably strong binding
interaction. Particularly notable aspects regarding the chemosensory activity of the
compound is its ability to detect the aforesaid transition metal ions down to the level of
micromolar concentration (detection limit being 2.74 and 2.27 ppm respectively), along
with a simple and efficient synthetic procedure.
Keywords: Schiff base, PET, Fluorophore-receptor communication, Chemosensor,
Transition metal ions.
1

Introduction
Fluorescent molecules, which are capable of performing light induced logic operations,
are of significant interest for their potential use as signaling systems competent of
responding in the presence of foreign analytes [1-3]. In general, such chemosensors are
simple conjunctions of a signaling unit, a recognition unit, and a linker connecting the
formers. The signaling unit is typically a fluorophore, and the recognition part in close is
usually a Lewis base site (that can coordinate to an ion, such as amines/imines) and the
linking unit called spacer plays the key role of establishing electronic communication
between the fluorophore and the receptor. In the absence of the guest ion, the emission
intensity of the fluorophore is generally very low (photoinduced electron transfer (PET)
is the most commonly encountered mechanism). However, in the presence of the guest
ion, the lone pair of the donor amine group is engaged, which leads to considerable
enhancement in the emission intensity of the fluorophore by turning off (or hampering)
the PET process and thereby constitutes the basis for actuating mechanism of functioning
as a sensor [4-8]. Further, a structural design based on fluorescence signaling guarantees
some additional edges such as specificity, sensitivity, real time monitoring with fast
response time and cost-effectiveness [1,9,10].
On the other hand, sensing of transition metal ions, especially Cu(II) and Zn(II) has long
been realized as an important goal in chemistry and biology with a view to their
important biological and environmental roles [1,11,12]. The close proximity of the values
of ionic radius and charge density as well as similar coordinating ability of both the
aforesaid ions, often leads to simultaneous sensing of both [13-15]. Now, till date, a great
number of methods have been developed for the detection of the aforementioned ions,
2

few of them being completely instrument-assisted techniques such as surface plasmon
resonance (SPR) or surface-enhanced Raman scattering (SERS) etc. However, the
requirement of sophisticated, expensive equipment, and highly trained personnel of the
aforementioned techniques restricts their application in facile and practical detection of
the transition metal ions thereby calling for relatively simpler chemical methods [16].
Keeping these in mind, the present work has been framed which describes the synthesis
and photophysical characterization of a simple Schiff base-derived new model compound
viz., 2-((cyclohexylmethylimino)-methyl)-naphthalen-1-ol (2CMIMN1O, Scheme 1).
Transition metal ion-induced modification of its photophysics has subsequently been
argued to be a promising possibility for the development of a new fluorescence
chemosensor for Cu(II) and Zn(II). The mechanism of the sensory action has been
rationalized in connection with PET mechanism. At the same time, the structural
simplicity as well as the ease of its synthetic procedure is noteworthy.
Experimental
Synthesis of 2CMIMN1O and materials
The detailed synthetic procedure and purification of the Schiff base 2CMIMN1O
is described in the supplementary material.
Spectroscopic grade acetonitrile (ACN) was purchased from Spectrochem, India
and used after proper purification according to standard procedures. The solvent was
found free from impurities and appeared transparent in the spectral region of interest. The
purity was also verified by recording the emission spectra in the studied spectral region.
The metal salts used in the present investigation are as follows: Zn(ClO4)2(H2O)6,
CrCl3(H2O)6, Cu(ClO4)2(H2O)6, Fe(ClO4)2(H2O)6, Ni(ClO4)2(H2O)6, Co(ClO4)2(H2O)6.
3

The metal salts were procured locally and used as received. Perchlorate salts have been
preferred because of the low coordinating ability of the anionic counterpart.
All other solvents and reagents such as dioxane (DOX), methanol (MeOH), hexane
(HEX), n-butanol (BuOH), tetrahydrofuran (THF) were of spectroscopic grade
(Spectrochem, India) and were used after proper distillation. Trifluoroacetic acid (TFA)
from E-Merck was used as received. Triple distilled water was used for preparing
aqueous solutions. d6-DMSO for NMR experiments was used as received from Sigma-
Aldrich, USA.
Instrumental details and methods
The absorption and emission spectra were recorded on Hitachi UV–Vis U-3501
spectrophotometer and Perkin-Elmer LS55 fluorimeter, respectively. Unless otherwise
mentioned, in all the measurements the sample concentration was maintained at 2× 10–6
M in order to avoid aggregation and reabsorption effects. All absorption and emission
spectral measurements were performed with proper background corrections and with
freshly prepared solutions only.
Fluorescence lifetimes were measured by the method of Time Correlated Single-
Photon Counting (TCSPC) using a HORIBA Jobin Yvon Fluorocube-01-NL fluorescence
lifetime spectrometer. The sample was excited using a laser diode at 375 nm and the
signals were collected at the magic angle of 54.7° to eliminate any considerable
contribution from fluorescence anisotropy decay [15]. The typical time resolution of our
experimental setup is ~ 100 ps. The decays were deconvoluted using DAS-6 decay
analysis software. The acceptability of the fits was judged by χ2 criteria and visual
4

inspection of the residuals of the fitted function to the data. Mean (average) fluorescence
lifetimes were calculated using the following equation [15]:
2
(1)
∑
α iτ i
τ av
=
∑ α iτ i
in which α is the pre-exponential factor corresponding to the ith decay time constant, τ .
i
i
Fluorescence quantum yield (Фf) was determined using recrystallized β-naphthol
(Фf = 0.23 in MCH) as the reference using the following equation [15]:
AS
ηS2
ηR2
(Abs)R
(2)
Φ S = Φ R
×
AR (Abs)S
×
where “A” terms denote the integrated area under the fluorescence curve, ‘‘Abs’’ denote
absorbance, η the refractive index of the medium and Ф, the fluorescence quantum yield.
Subscripts ‘‘S’’ and ‘‘R’’ stand for denoting respective parameters for the studied sample
and reference, respectively.
The cyclic voltammetric measurements were performed with a Sycopel model
AEW21820F/L instrument. The experiments were carried out in a standard three-
component cell equipped with a glassy carbon working electrode, a Pt wire as an
auxiliary electrode and Ag/AgCl reference electrode. The cyclic voltammograms were
acquired in a N2-bubbled DMSO solution containing ~ 0.2 M tetraethylammonium
perchlorate (TEAP) as supporting electrolyte. The scan speed was maintained at 50 mV/s.
All NMR spectra were recorded with TMS as internal standard on Bruker, AV
300 Supercon Digital NMR system using d6-DMSO as solvent as mentioned earlier.
Results and discussions
Absorption study
5

The absorption spectra of 2CMIMN1O as obtained in various solvents are
displayed in Figure 1a, and the consequent spectroscopic parameters are summarized in
Table 1. The compound demonstrates three absorption bands viz. at ~ 290 nm, ~ 365 nm
and ~ 430 nm wavelength regions in non-polar solvents, whereas in polar solvents the ~
365 nm band is somewhat less prominent. To assign the observed spectral bands, we
consider the spectral characteristics of the parent compound 1-hydroxy-2-naphthaldehyde
(HN12) [17]. HN12 exhibits two distinct absorption bands at ~ 300 nm and ~ 370 nm
which are assigned to its open and intramolecularly hydrogen bonded closed form,
respectively. In a direct correspondence to the aforementioned spectral features of the
parent molecule HN12 [17], the ~ 365 nm absorption band for 2CMIMN1O has been
ascribed to the intramolecularly hydrogen bonded lowest energy conformer, i.e.
conformer I in Scheme 1, while the higher energy band at ~ 290 nm appears to reflect the
signature of the open conformer devoid of H-bonding (conformer II in Scheme 1). The
band at ~ 365 nm, as mentioned earlier is seen to be obscured on moving from non-polar
to polar solvents which is probably the outcome of an increasing degree of solute-solvent
interaction in polar media [15]. The lowest energy absorption band (at ~ 430 nm, vide
Figure 1a) has been attributed to the zwitterionic species of 2CMIMN1O (Scheme 1) via
a comparison with the spectra of structurally analogous compounds [18,19]. This
observation seems satisfactory on the ground that the zwitterionic species is capable of
delocalization of its formal charges resulting in a substantial stabilization. Additionally, it
is noted that the absorbance corresponding to the zwitterionic species is much greater as
compared to that for the closed form in polar solvents, while the reverse pattern is noted
in non-polar solvent (cf. Figure 1a). Such an observation is very likely since the charge
6

separated zwitterionic species is expected to be more stabilized and hence is expected to
be more populated in polar solvents than in non-polar solvents. Also, it is fascinating to
note that the absorption bands of 2CMIMN1O corresponding to the open (λabs ~ 290 nm)
and closed (λabs ~ 365 nm) conformers are relatively blue shifted compared to those of the
parent compound HN12 (λabs ~ 300 nm for the open form and λabs ~ 370 nm for the
closed form [17]). This observation is reasonable since the intramolecular hydrogen
bonding in 2CMIMN1O involves comparatively weaker acceptor nitrogen (whereas in
HN12 oxygen atom is the acceptor) and hence stabilization by hydrogen bonding is less
in case of 2CMIMN1O than the parent molecule HN12.
Emission study
The emission spectra of 2CMIMN1O following photoexcitation at both 365 nm
and 430 nm have been recorded in various solvents and the representative profiles
obtained with λex = 430 nm are illustrated in Figure 1b, while the corresponding
spectroscopic parameters are collected in Table 1. In all the solvents assayed other than
n-hexane, excitation at either of the two wavelengths produces identical spectral profile
with the emission maxima at ~ 480 nm, which is thus readily ascribed to the emission
from the locally excited state of the zwitterionic species (Scheme 1) suggesting that the
hydrogen bonded closed form of 2CMIMN1O undergoes a transformation to the
zwitterionic form on the excited state potential energy surface prior to emission. Within
the range of solvents assayed the emission maxima position is found to exhibit no
significant dependence (other than a minute blue shift) on solvent polarity (Figure 1b and
Table 1). The excitation spectra (Figure S2 in the supplementary information) obtained in
7

various solvents are also found to exhibit three bands at ~ 300 nm, ~ 380 nm and at ~ 430
nm, i.e. the excitation spectra corresponds well with the absorption spectra.
In case of n-hexane, however, photoexcitation at 365 nm produces a broad
emission band having the maximum at ~ 400 nm along with a broad shoulder ~ 445 nm
(To properly assign the emission bands, we deconvoluted the experimental spectrum into
individual Gaussian curves following the celebrated Marquardt-Levenberg algorithm,
vide inset of Figure 1b). The low intensity emission shoulder is anticipated to be the
signature of the zwitterionic form, since its population is expected to be quite low in n-
hexane (in ideal case, a zero population of the zwitterionic form is expected in a non-
polar solvent like n-hexane, but traces of water present in the solvent could result in some
discernible population of the zwitterionic form, as is predicted here). The emission band
~400 nm therefore must corroborate to the H-bonded closed form of the Schiff base.
The calculated values of quantum yields of the title compound in different
solvents are summarized in Table 1 and are found to be considerably lower as compared
to those of its parent molecule HN12 [17], which suggests that the compound is an
inefficient fluorophore as compared to its constituent compound.
Effect of protic acid on the spectral properties of 2CMIMN1O
The effect of addition of trifluoroacetic acid (TFA) on the absorption profile of
the compound (in acetonitrile medium) is displayed in Figure 2a, where an appreciable
blue shift from ~430 nm to ~ 405 nm is observed, which seems attributable to the
formation of a protonated species. However, on the emission profile, addition of acid
results in a remarkable enhancement of the intensity of the emission band with no
noteworthy shift of the emission maxima (Figure 2b) and the corresponding excitation
8

spectra is found not to corroborate well with the corresponding absorption spectra
suggesting the occurrence of some excited state affair. The underlying interpretation has
been argued in detail in the forthcoming sections.
Transition metal ion-induced modulation of photophysics of 2CMIMN1O
Steady-state spectral properties
In course of monitoring the effect of transition metal ions on the steady-state spectral
properties of 2CMIMN1O, the absorption spectra are found to show almost similar
observation of nominal or no decrement of absorbance in presence of the transition metal
ions (vide Figure S3 in the supplementary information), only Cu2+ being the exception
(Figure 3a). Gradual addition of Cu2+ ion to an actonitrile solution of the Schiff base
(2CMIMN1O) is found to accompany a decrease in absorbance at ~ 430 nm with a
concomitant appearance of a new band at ~ 570 nm which is expected to be the signature
of a ligand to metal charge transfer (LMCT). This observation leads to an immediate
formulation of a ratiometric sensing scheme for Cu2+ based on its response on the
absorption profile of 2CMIMN1O in terms of plotting the ratio of absorbance at 570 nm
and 430 nm (A570/A430) as a function of Cu2+ concentration, as depicted in Figure 3b.
Such ratiometric sensing via simple absorption measurement gives an additional thrust to
this study, as such a scheme is claimed to be more fruitful as an actuating tool due to its
independence from a number of common experimental and instrumental artifacts e.g.,
fluctuations in probe concentration and excitation source intensity, light scattering and so
forth, and hence the method does not call for frequent calibration during the estimation of
the analyte [20,21].
9

On the other hand, only Zn2+ and Cu2+ ions are found to impart noteworthy
modifications to the emission characteristics of 2CMIMN1O, while others fail to convey
any (vide Figure 3d). Since Zn2+ was found to impart no noteworthy modification in the
absorption profile of 2CMIMN1O other than a nominal decrement in absorbance (vide
Figure 3c), this significant dissimilarity of absorption spectral response of the sensor to
the presence of the two transition metal ions Cu2+ and Zn2+ can be used as an actuating
index to distinguish between these two ions spectroscopically. Furthermore, it also calls
for two different sensing mechanisms operating for the aforementioned ions. The
noteworthy emission intensity enhancement of the studied compound in presence of
increasing concentration of Cu2+ and Zn2+ is presented in Figure 4 (a and b respectively).
The corresponding excitation spectra (vide Figure S4 in the supplementary portion)
exhibit a notable disagreement with the corresponding absorption spectra in presence of
the two metal ions indicating that the transition metal ion-induced modification in the
emission characteristics of 2CMIMN1O corresponds to an excited state event.
Figure 4c depicts the visual change imparted by the ions on the compound. The
compound 2CMIMN1O shows a prominent colour change from yellow to purple in
presence of Cu2+ ion, which can be exploited to confirm the presence of Cu2+ ions in a
solution via visual inspection only, and in presence of both the ions (especially for Zn2+),
the compound exhibits a strong bluish green fluorescence under UV illumination.
Now, the modification of the emission profile of the title compound with both
Zn2+ and Cu2+ subsequently leads to the concept of utilizing such modulations for
exploiting the sensory action of the compound. The chemosensory emission spectral
response of 2CMIMN1O to the series of first row transition metal ions is constructed on
10

the basis of variation of relative fluorescence intensity of the compound (i.e. I/I0 where I0
and I are fluorescence intensities in the absence and presence of a defined amount of the
transition metal ion, respectively) as a function of concentration of the metal ions and is
illustrated in Figure 4d. The figure clearly advocates for the selective chemosensory
response of the title Schiff base to Cu2+ and Zn2+ ions only, to different extent though. It
is also exciting to note that the proposed sensor is able to sense Cu2+ and Zn2+ selectively
up to a sufficiently low concentration, in our case in the micromolar order, the
corresponding detection limit being 2.74 and 2.27 ppm respectively for Cu2+ and Zn2+
ions as calculated using the reported procedure [20,21].
Mechanism of sensory action
The mechanism of sensory action of the studied compound, as well as the effect
of protic acid has been attempted to be rationalized on the basis of PET mechanism,
which is a commonly exploited strategy for metal sensing fluorosensors showing
intensity enhancement in the presence of protic acid and metal ion(s) [1,8,9,10]. In order
to substantiate the hypothesis, the following series of experiments and arguments have
been undertaken. Firstly, we focus on establishing the efficiency of the studied molecular
architecture to function as a suitable PET system. A search for the extent of electron
deficiency of the fluorophore unit in 2CMIMN1O has been employed as the actuating
index for this purpose [1,8,9,10]. A direct evidence of the electron deficient nature of the
fluorophore unit comes from a scrutiny of the quantum yield values which are
considerably less than that of the parent compound HN12 [17] as stated earlier.
Furthermore, cyclic voltammetric measurements have been performed to assess the
electron deficiency of the fluorophore unit in 2CMIMN1O. The Ered(fluoro) appears at –
11

0.652 V (irreversible) while the oxidation potential is observed at Eox(fluoro) = 0.681 V
(irreversible). Now, with the measured reduction potential coupled with the spectral data,
a quantitative estimate of the thermodynamic driving force for PET process in
2CMIMN1O can be achieved in terms of the free energy change (∆G) of the process
[8,9,10]. The ∆G value has been calculated using the equation:
∆G = 23.06[Eox (recep) − Ered ( fluoro)
]
− E0,0
(3)
where, Eox(recep) and Ered(fluoro) represent the oxidation potential of the receptor moiety
and the reduction potential of the fluorophore, respectively whereas E0,0 represents the
energy of the fluorescent state [8,9,10]. The value of E0,0 used in the calculation has been
estimated from location of the first peak position in the fluorescence spectrum (λem ~ 480
nm). Using Eox(recep) = 0.49 V [8,9,10] (for triethylamine, literature SCE value corrected
for Ag/AgCl electrode by substracting 0.27 V), ∆G value is estimated to be – 25.83
kcal/mol, which dictates the thermodynamic feasibility of the PET process in the studied
molecular system.
As stated in our previous publication [17], the parent fluorophore HN12 is found to
undergo usual quenching as induced by the transition metal ions Zn2+ and Cu2+. The
extent of quenching has been quantitatively estimated using the Stern–Volmer equation
[15]:
(4)
I0
I
= 1 + K SV [Q]
in which KSV is the Stern–Volmer quenching constant and [Q] is the molar concentration
of the quencher [15]. Cu2+ ion (with one unpaired electron) is found to be a more efficient
quencher for HN12 than Zn2+ ion (KSV = 7.75 × 104 M−1 for Cu2+ and 1.13 × 103 M−1 for
Zn2+ [17]). Thus interaction between the fluorophore (HN12 unit of 2CMIMN1O) and
12

transition metal ions (Cu2+ and Zn2+) should result in fluorescence quenching. Therefore
to counter the above observations we are left with the possibility that the interaction
between the receptor (imine moiety of 2CMIMN1O) and the metal ions leads to
fluorescence enhancement. Hence it is presumed that it is the net result of these two
opposing interactions that plays the governing role behind the resultant consequence
(quenching or enhancement) of interaction of 2CMIMN1O with the transition metal ions
(Cu2+ and Zn2+). Since the fluorescence yield of the title compound is quite low in the
absence of the guest, the presence of a communication (PET) between the flurophore and
the receptor is expected which results in the decrement of fluorescence yield. Thus, it is
not unlikely that the interaction with the guest ion (H+ or transition metal ions) will lead
to modulation of the fluorophore–receptor communication in a manner that will facilitate
fluorescence enhancement by impairing the aforesaid communication [1,8,9,10].
Time-resolved fluorescence study
Fluorescence lifetime measurements serve as a sensitive indicator of the local
environment of a fluorophore, and it is responsive to excited state affairs [8,10,15]. Thus
in order to attain a deeper insight into the photophysics of 2CMIMN1O and its
modulation in the presence of protic acid as well as transition metal ions, fluorescence
lifetimes has been monitored under differential circumstances. Figure 5a represents the
time resolved fluorescence decay profile of 2CMIMN1O under different experimental
conditions and the relevant data are compiled in Table 2. The compound demonstrates a
complicated tri-exponential decay pattern in all the solvents assayed which reflects the
presence of a short-lived major component (~ 40 ps, with the corresponding amplitude
being ~ 96% in ACN), along with two comparatively long lived components. Such a
13

complex decay pattern can be explained by a through space electron transfer mechanism
involving the overlap of the lone pair orbital of the imine nitrogen and the π-orbitals of
the fluorophore ring. The long-lived components arise from species in which the donor
and acceptor groups are well separated in space, while the predominant short-lived
component represents the quenched fluorophore (via a photoinduced charge transfer
mechanism), having the donor imine moiety in close proximity of the acceptor [8,10,15].
Interestingly, with increasing solvent polarity the amplitude corresponding to the PET
quenched fluorophore drastically increases which is not unlikely owing to the effect of
polar solvents to ensure a smoother electron transfer process and is also confirmed by the
decrement in the corresponding lifetime value from non-polar to polar media.
The overall decay behaviour of 2CMIMN1O in the presence of the aforesaid
transition metal ions and protic acid undergoes noteworthy modifications (vide Figure 5b
and Table 2). In presence of both Zn2+ ion and protic acid (H+ ion), the decay
characteristic of the compound is amply modified and it is observed that with the addition
of the mentioned ions, the amplitude corresponding to the short-lived PET-quenched
component is found to fade away gradually and at a certain concentration of the ions (~
30 µM for both Zn2+ H+ ions), the decay shows a single-exponential characteristic with a
lifetime of ~ 1.7 ns which closely resembles to one of the lifetime components obtained
for the bare compound. This situation corresponds to the complete recovery of
fluorescence and the observed lifetime component thus corresponds to the fluorophore in
some sort of a locked conformation due to binding with the ion(s). As obvious, further
addition of the ions, leads to expected quenching of the fluorescence lifetime. However,
in case of Cu2+ ion, even up to the level of saturation of interaction, the basic complicacy
14

of the decay profile persists, though an overall increase of the average fluorescence
lifetime is distinctly detectable (from 2.94 ns to 7.02 ns in the presence of 30 µM Cu2+,
vide Table 2) and the fact that Cu2+ ion can also efficiently perturb the PET mechanism is
evident from the noticeable fall of the amplitude associated with the short-lived decay
component along with its increasing magnitude. The overall scenario thus could be linked
to the greater binding efficiency of the Schiff base with Zn2+ than Cu2+ which is further
substantiated from the observation reported in Figure 4d showing that in the presence of
the same concentration (30.0 µM) of the metal ions (Cu2+ or Zn2+) fluorescence
enhancement occurs to a significantly greater extent for Zn2+.
A more critical analysis of the data has been undertaken with a view to mark out the
contributions from radiative (kr) and nonradiative (knr) decay constants according to the
following equations [15]:
〈τ f 〉
kr =
(5)
(6)
Φ f
−1
kr + knr = 〈τ f 〉
A careful examination of the data (vide Table 3) reveals that interaction of 2CMIMN1O
with the ions results in only a nominal depletion of non-radiative decay rates, while there
is a striking enhancement of the radiative decay rates, which appears to play the essential
role behind the observed fluorescence enhancement. Though one might be argue that a
binding interaction of 2CMIMN1O with the transition metal ion(s) would impart rigidity
to the entire molecular framework thereby reducing the rotation/vibrational degrees of
freedom and hence would contribute towards the observed fluorescence enhancement,
15

this explanation does not seem to be adequate for explaining the high magnitude of the
observed fluorescence enhancement.
Strength of 2CMIMN1O – metal ion interaction and the probable functional groups
involved in the interaction
Herein, we endeavor to estimate the strength of 2CMIMN1O–metal ion
interaction assuming a simple complexation reaction between the two moieties involved.
An assessment of the emission intensity data using the modified Benesi-Hildebrand
equation has been exploited for this purpose. A thorough discussion on Benesi-
Hildebrand equation is avoided since it is routine and abundantly available in the
literature [9]. We thus start with the equation [9]:
(7)
1
1
1
=
+
n +
(
I − I0 ) (I∞ − I0 ) (I ∞ − I0
)
K
[M
]
in which I0, I and I∞ are the emission intensities, respectively, in the absence of, at an
intermediate and infinite concentration (indicating saturation of interaction) of the metal
ion (Mn+). A linear regression for the plot of [I-I0]-1 vs. [Mn+]-1 for both Cu2+ and Zn2+ ions
(Figure 5c) dictates a 1:1 association between 2CMIMN1O and each of the two metal
ions. Assuming a binding interaction between the two partners, the strength of binding
has been quantitatively evaluated in terms of the corresponding binding constant K,
obtained via the intercept to slope ratio of the Benesi-Hildebrand plot. The values
obtained are (4.93
0.4) × 104 M-1 for Zn2+ and (1.88
0.4) × 104 M-1 for Cu2+.
Accordingly the free energy change for the process of complexation reaction is calculated
= – 26.87 kJ mol–1 for Zn2+ and – 24.38 kJ/mol–1 for Cu2+, i.e. a
to be
∆G = −RT ln K
negative free energy change which dictates the thermodynamic feasibility for the
occurrence of the binding interaction. Also a relatively stronger binding of the title
16

compound with Zn2+ ion is manifested through a higher binding constant value as
compared with Cu2+ ion.
To unveil the functional groups of 2CMIMN1O that are involved in the
interaction with the transition metal ions, the 1H NMR spectroscopic technique has been
exploited. It should be emphasized here that since the sensing mechanism in this case is
related to an excited state phenomenon, one must not expect substantial changes in the
NMR spectra which proliferates only ground state interactions. Now, as can be seen from
1
Figure 6, The H NMR spectra of 2CMIMN1O is found to exhibit some distinct
modifications of chemical shift upon interaction with Zn2+ ion connecting to the
possibility of ground state complexation. In the presence of Zn2+ ion, the peak
corresponding to the -OH proton (δ 13.18) is found to disappear indicating deprotonation
at that site which is quite likely in case of such complex formation involving the oxygen
atom. Furthermore, such complexation of the title compound with Zn2+ ion is expected to
reduce the electron density of the coordination sites and thus induce a down-field shift of
the nearby proton signals [22]. In our case, prominent down-field shift of the –CH=N–
proton (δ 8.24→8.43, vide Figure 6) is observed accounting for the electron deficiency of
the corresponding site thereby suggesting the involvement of the imine nitrogen in
complexation. Likewise, down-field shifts for H3 and H4 protons (δ 6.67→6.99 and δ
7.04→7.28) are also noteworthy. Interestingly, in absence of Zn2+ ion, both the H5 and H8
protons behave as magnetically equivalent and thus appear as a triplet near δ 7.55, while
in the presence, the concerned protons become non-equivalent and appear respectively at
δ 7.74 and δ 7.59 as triplets. The above observation can be rationalized invoking the fact
that the H8 proton is in a closer proximity to the coordination site than the H5 proton
17

which results in a more prominent down-field shift of the former due to a reduced
electron density at the coordination sites as mentioned earlier. As expected, the protons in
the remote part of the molecular framework from the site of interaction do not experience
notable change in chemical shift.
Conclusion
A Schiff base-derived new model compound, 2CMIMN1O has been synthesized
and its photophysics has been characterized by steady-state absorption, emission and
time-resolved emission spectroscopic techniques. The fluorescence yield of the
compound is found to be considerably low compared to its constituent compound, HN12,
which has been rationalized in connection with the operation of PET mechanism from the
imine moiety to the fluorophore unit in the multi-component system of interest. The
specific structural architecture of the title compound in fluorophore-spacer-receptor
(FSR) format paves way for a perusal of the perturbation of its fluorescence properties
with the addition of transition metal ions. The compound reveals significant fluorescence
enhancement in the presence of Cu2+ and Zn2+ ions selectively. Hence, this compound has
subsequently been argued to be a promising candidate for the development of Cu2+ and
Zn2+ ion selective fluorescence chemosensor. The prominent difference on the absorption
profile (as well as the naked eye colour change) of 2CMIMN1O in the presence of Cu2+
and Zn2+ ions also paves way for demarcating the response of the sensor compound to the
two transition metal ions. Additional edges of 2CMIMN1O to act as a fluorescence
chemosensor are its relatively simple structure and facile synthetic route. Also such
commendable selectivity in generating sensing responses indicates the possibility of
viable expansion of the technique to further consequences in sensory research.
18

Acknowledgements
AG and SG gratefully acknowledge Senior Research Fellowships respectively from CSIR
and UGC, New Delhi, Govt. of India.
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21

Figure captions:
Figure 1: (a) Absorption spectra and (b) emission spectra (λex = 430 nm) of 2CMIMN1O
in different solvents as specified in the figure legend. The inset corresponds to the
emission spectra of the compound in n-hexane (λex = 365 nm). The sample concentration
was maintained at 2×10–6 M.
Figure 2: Effect of increasing concentration of acid (TFA) on the (a) absorption profile
and (b) emission profile of 2CMIMN1O in ACN solvent. (sample concentration was
maintained at 2×10–6 M).
Figure 3: (a) Absorption spectra of 2CMIMN1O in the presence of increasing
concentration of Cu2+ ion. Direction of arrow indicates increasing concentration of Cu2+
(0.0, 6.67, 16.12 and 30.01 µM). (b) The ratiometric sensing scheme for Cu2+ based on its
response on absorption profile in terms of plotting the ratio of absorbance at 570 and 430
nm (A570/A430) as a function of Cu2+ concentration. (c) Absorption spectra of
2CMIMN1O in the presence of 30.0 µM concentration of Zn2+ ion. (d) Emission spectra
(λex = 420 nm) of 2CMIMN1O in ACN solvent and in the presence of 30.0 µM of
different transition metal ions as indicated in the figure legends (sample concentration
was kept at 2×10–6 M) .
Figure 4: Emission spectra of 2CMIMN1O in ACN solvent in the presence of increasing
concentration of transition metal ions, (a) Cu2+ and (b) Zn2+ (λex = 365 nm). Curves (i) →
(xi) correspond to [Mn+] = 0, 1.54, 3.26, 6.67, 12.24, 15.88, 18.09, 21.01, 23.68, 26.86
and 30.03 µM and curves (i) → (xiii) correspond to [Mn+] = 0, 0.76, 1.54, 3.26, 6.67,
12.24, 15.88, 18.09, 21.01, 23.68, 26.86, 30.03 and 33.35 µM. (c) The photograph of
(left) 2CMIMN1O alone in ACN solvent and in the presence of Cu2+ ion and (right) the
22

photograph of 2CMIMN1O alone in ACN solvent and in the presence of Zn2+ ion under
UV illumination (sample concentration was maintained at 2×10–6 M). (d) Intentiometric
chemosensory emission spectral response of 2CMIMN1O in the presence of different
transition metal ions (concentration of each of transition metal cation = 30.0 µM).
Figure 5: Typical time-resolved fluorescence decay profile of 2CMIMN1O (a) in various
solvents as indicated in the figure legend (sample concentration 2× 10–6 M) and (b) in the
presence of 30.0 µM of Cu2+, Zn2+ and H+ ions in ACN solvent (sample concentration
2×10–6 M). (c) Benesi-Hildebrand plot ([I-I0]-1 vs. [Mn+]-1) for complexation between
2CMIMN1O and the transition metal ions ((i) Cu2+ and (ii) Zn2+) as derived from
emission spectral data.
1
Figure 6: H NMR spectra of 2CMIMN1O in the (i) absence and (ii) presence of Zn2+
ion.
23

Table 1: Spectroscopic parameters for 2CMIMN1O in various solvents as obtained
from absorption and emission measurements
Solvent
Absorption λ
Fluorescence λ Quantum yield
fc
(nm)
(nm)a
(Φf)b
λ1
λ2
λ3
HEX
THF
294
292
289
291
288
292
365
360
361
357
367
358
435
432
434
430
430
432
400/447
479
0.144
0.081
0.113
0.063
0.081
0.094
32.69
77.54
68.91
82.37
77.54
34.13
DOX
ACN
482
470
MeOH
BuOH
469
482
a: λex = 365 nm for non-polar solvents and λex = 430 nm for polar solvents.
b: Quantum yields are × 10-2 order.
c: f is the factor by which the fluorescence yield of 2CMIMN1O is lower than its parent
compound, HN12.
24

Table 2: Time-resolved fluorescence decay parameters of 2BIMN1O in different
environments
b
b
b
Environment
τ1
τ2
τ3
α1
α2
α3
χ2
〈τf〉
(ns)
(ns)
(ps)
(ns)
HEX
DOX
THF
1.82
1.54
1.44
1.86
1.39
1.78
1.76
1.61
6.44
7.11
7.74
8.56
6.69
11.38
–
609
569
683
63
0.377
0.262
0.196
0.172
0.035
0.267
1.000
1.000
0.054
0.045
0.051
0.016
0.007
0.069
–
0.569
0.694
0.754
0.814
0.958
0.664
–
2.67 1.06
2.79 1.02
3.16 1.05
3.49 1.01
2.94 1.09
7.02 1.08
1.76 1.01
1.61 1.03
MeOH
ACN
aCu2+
aZn2+
aH+
37
204
–
–
–
–
–
a: in the presence of 30.0 µM TFA or metal ion in ACN.
b: error in measurement 3%.
25

Table 3: Fluorescence quantum yield (Φf) and kinetic fluorometric and nonradiative
parameters for 2BIMN1O in the presence and absence of transition metal ions in
ACN solvent
Environment
τav (ns)
kr (s-1)
knr (s-1)
Φf
ACN
Zn2+
Cu2+
0.063 × 10-2
0.355
2.94
1.76
7.02
2.14 × 105
2.01 × 108
1.79 × 107
3.39 × 108
3.72 × 108
1.25 × 108
0.126
26

100
80
60
40
20
0
0.4
0.3
0.2
0.1
0.0
HEX
HEX
THF
120
90
60
30
0
(b)
(a)
Fitted peaks
ACN
MeOH
BuOH
THF
MeOH
ACN
375
450
525
600
DOX
Wavelength (nm)
400
450
500
550
600
650
280
320
360
400
440
480
Wavelength (nm)
Wavelength (nm)
Figure 1
27

0.8
0.6
0.4
0.2
0.0
350
300
250
200
150
100
50
i
(b)
(a)
xiii
v
i
0
280
320
360
400
440
480
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
Figure 2
28