Article pubs.acs.org/JPCB
The Prospect of Salophen in Fluorescence Lifetime Sensing of Al3+ Tuhin Khan,* Shefali Vaidya, Darshan S. Mhatre, and Anindya Datta* Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai-400076, India S Supporting Information *
ABSTRACT: We have assessed the potential of salophen, a tetradentate Schiff base, in fluorescence sensing of Al3+ ions. While performing this investigation, we have noticed conflicting literature reports on the fluorescence spectral maximum and quantum yield of salophen. So, the compound has been purified by repeated crystallization. Fluorescence studies have been performed on samples in which the absorption and excitation spectra are completely superimposable. The purified compound exhibits a feeble fluorescence at 545 nm, associated with an ultrafast fluorescence decay. This is rationalized by excited state proton transfer and torsional motions within the molecule, which provide efficient nonradiative channels of deactivation of its excited state. The fluorescence quantum yield increases upon complexation of salophen with Zn2+ as well as Al3+. The increase is significantly more upon complexation with Al3+. However, fluorescence maxima are similar for the two complexes. This indicates that fluorescence intensity may not be a good parameter for Al3+ sensing by salophen, in the presence of a large excess of Zn2+. This problem can be circumvented if fluorescence lifetime is used as the sensing parameter, as the lifetime of the Al3+ complex is in the nanosecond time regime while that of the Zn2+ complex is in tens of picoseconds. The significant difference in the fluorescence quantum yield and lifetime between the two complexes is explained as follows: the Al3+ complex is monomeric, but the Zn2+ complex is dimeric. Quantum chemical calculations indicate a higher density of states near the locally excited state for the dimeric complex. This may lead to more efficient nonradiative pathways.
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aluminum could be a neurotoxin in Alzheimer’s disease, Parkinson disease, etc.13 It is known to inhibit plant growth in acidic soil, which makes up almost 40% of cultivable land.14 Al3+ is highly reactive due to its small size and high charge. Consequently, it can act as a competitive binder to essential metal ions with similar characteristics, e.g., Ca2+, Mg2+, and Fe3+.15,16 Unlike many other metal ions, detection of Al3+ is not straightforward. Because of its high hydration energy, it readily takes up water and reacts with it to form insoluble Al(OH)3. It lacks well-defined spectroscopic characteristics like charge transfer and does not form coordination complexes with most ligands. In recent times, these difficulties have been surmounted to design several fluorescence sensors of Al3+.17−20 Lee et al. have demonstrated the potential of salophen as a turn−on fluorescence sensor of Al3+ ion.21 More recently, Barboza et al. have reported a decrease in fluorescence quantum yield and lifetime of salophen with Zn2+.22 These two papers report contradictory values of fluorescence maximum (λems) and quantum yield (ϕf) of free salophen. While Barboza et al. have reported a strong emission with λems = 450 nm, Lee et al. and Kotova et al.23 have reported a feeble emission with λems = ca. 525 nm. This conflict between the two literature reports needs to be resolved. Moreover, Zn2+ is known to form emissive
he photophysics of inorganic metal complexes has been an active field of research for several decades and has given rise to various applications in artificial photosynthesis, photodynamic therapy, biological labeling etc. Fluorescence sensing of metal ions is one such application that has received considerable attention. This application builds upon the modulation of fluorescence intensity or lifetime of ligands upon complexation with metal ions.1 Selectivity and specificity are major issues that need to be considered for design of such ligands for metal ion sensing. Schiff bases constitute one of the most widely studied classes of ligands in this context, due to their ability to coordinate with a wide range of ions in various oxidation states.2 Their synthesis and chemical modification can be achieved with relative ease, providing a means for manipulation of the HOMO−LUMO gap.3,4 Salen (N,N′bis(salicylidene)ethylenediamine) and salophen (N,N′-bis(salicylidene)-o-phenyldiamine) are typical representatives of this class of ligands. Metal complexes of salen/salophen find application in catalysis, supramolecular chemistry,5 medicine, DNA intercalation,6 organic light emitting diode (OLED),7 electronics,8 sensing,9 and nonlinear optics (NLO).10 In the present study, our focus is on the complex of Salophen with Al3+ and Zn2+ ions. Aluminum is the most abundant metal in the earth’s crust (8.3%) and third most abundant element after oxygen and silicon. It has no known physiological role, but an average human consumes 3−10 mg of the metal every day, due to its high abundance.11,12 It has been proposed that © 2016 American Chemical Society
Received: June 10, 2016 Revised: August 6, 2016 Published: September 7, 2016 10319
DOI: 10.1021/acs.jpcb.6b05854 J. Phys. Chem. B 2016, 120, 10319−10326
Article
The Journal of Physical Chemistry B complexes with many ligands.24,25 If the fluorescence of salophen is indeed affected by Zn2+, then this could pose a serious hindrance to sensing of Al3+ by this Schiff base in solutions where Zn2+ is also present. Notably, zinc is the second most abundant d-block element in living organisms.26,27 The human body typically contains 2−3 g of zinc.28 With this background, we have synthesized salophen, purified it by repeated crystallization, and performed steady state and timeresolved fluorescence studies on the free ligand as well as the complexes with Zn2+ and Al3+. Before discussing our results, we present a brief overview of the photophysics of Schiff base compounds, which needs to be referred to during the rationalization of time-resolved spectroscopic data. The enol form of the Schiff base is predominant in solid and solution phase. The locally excited state formed upon π−π* transition of this form undergoes an ultrafast twist (torsional motion) and proton transfer in the time frame of ∼100 fs. The twisted enol form returns to the ground state via nonradiative pathway through a conical intersection. The cis-keto form formed as a result of excited state proton transfer emits at ∼550 nm with a lifetime of ∼10 ps. The cis-keto form further undergoes cis−trans isomerization to form a long-lived (∼ ms) trans photoproduct.29−31 Upon complexation, the twisting about the C−C bond is expected to be hindered significantly. This is likely to bring about an enhancement in fluorescence intensity and lifetime. The absence of proton transfer can also be expected to affect the photophysics of salophen in its metal ion complex. We have sought to understand the interplay of these processes in the complexed ligand.
Scheme 1. Reaction Scheme of Synthesis of Salophen and Its Metal Complex
methanol and diethyl ether over 2−3 days. The fine yellow single crystal thus formed was used for XRD and 1H NMR study. Characterization of Synthesized Compounds. 1H NMR studies of SalH2 was carried out in CDCl3 whereas SalZn and SalAl+ were carried out in DMSO-d6 in Bruker 400 MHz NMR spectrometer (Figure S1). An ESI−MS study was carried out with a Bruker maXis Impact mass spectrometer. A Rigaku Saturn CCD diffractometer, with a graphite monochromator (λ = 0.71073°) and Rigaku Crystal Clear-SM Expert 2.1 software, was used to collect the X-ray data. The structure was solved by direct methods and was refined by least-squares methods on F2 with the SHELXTL package. All non-hydrogen atoms were refined anisotropically. Steady State and Time-Resolved Fluorescence Spectroscopy. UV−visible absorption and emission studies were performed on a JASCO V-530 spectrometer and a Varian Cary Eclipse spectrofluorimeter, respectively. For the fluorescence study, band-pass of 5 nm was used for both excitation and emission. Fluorescence quantum yield (ϕf) was measured using Coumarin 30 as reference (ϕf = 0.307 in methanol) (additional details in the Supporting Infomration).34 The nanosecond lifetime was measured using time correlated single photon counting (TCSPC) spectrometer from IBH Horiba Jobin Yvon (FluoroCube). The sample was excited with a 375 nm diode laser (Horiba NanoLED) with a repetition rate of 1 MHz. The decay was collected at magic angle (54.7°) polarization with respect to vertically polarized excitation light. The full width at half-maximum (FWHM) of the instrument response function (IRF) was approximately 300 ps. The decays were fitted to single exponential function by iterative reconvolution using IBH DAS 6.2 software. The femtosecond optical gating (FOG) setup has been described elsewhere.35 A detailed description has been provided in the Supporting Infomration. SalH2 was excited at 385 nm while SalZn, SalAl+, and Sal2− were excited at 400 nm. A magic angle polarization was maintained to eliminate rotational anisotropy. The minimum possible laser power and integration time were used in order to avoid photodegrdation of the
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MATERIALS AND METHODS Synthesis of Salophen and Its Complexes. All chemicals were used without further purification unless mentioned otherwise. Solvents were of spectroscopic grade. In order to synthesize salophen (SalH2), an ethanolic solution of 2 mL of salicyaldehyde (SD Fine Chemicals, Mumbai, India) was added dropwise with constant stirring at room temperature to 25 mL of an ethanolic solution of 1 g of 1,2-phenylenediamine (SigmaAldrich, USA).32 The mole ratio in the final mixture was 2:1 (Scheme 1). A bright yellow precipitate of SalH2 was formed in about 5 min. The reaction mixture was further stirred for 3−4 h. The precipitate was filtered under suction and repeatedly washed with cold ethanol and diethyl ether. The solid dry mass was further air-dried for 1−2 days. Finally, it was purified by repeated crystallization from a hot ethanolic solution. The fine crystals formed thereafter were washed with ditheyl ether and air-dried. These crystals were used for all further studies. Zn(salophen) (SalZn) was synthesized by the following procedure: To an ethanolic solution of 100 mg of crystallized salophen was added 70 mg of Zn(OAC)2·2H2O (Merck, USA) in ethanol dropwise, to maintain a mole ratio of 1:1.32 The yellow precipitate thus formed was further stirred for 3−4 h, followed by filtration and washing with cold ethanol and diethyl ether. For the synthesis of Al(salophen) (SalAl+), to a methanolic solution of 100 mg of crystallized salophen was added 120 mg of Al(NO3)3·9H2O (Merck, USA) in methanol dropwise, in order to maintain a mole ratio of 1:1.33 The resultant yellow solution of the complex was stirred for 3−4 h followed by filtration and drying under reduced pressure. The solid mass was further washed with dicholoromethane and then decanted and dried under reduced pressure. The crude complex was then crystallized through a slow diffusion method with 10320
DOI: 10.1021/acs.jpcb.6b05854 J. Phys. Chem. B 2016, 120, 10319−10326
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The Journal of Physical Chemistry B samples. No concentration dependence was observed in the kinetic profiles. The decay traces were fitted to a linear sum of three exponentials with iterative reconvolution of a Gaussian function of FWHM = 275 fs, using Igor Pro 6.3 platform. For the study of mixture of zinc and aluminum ion to a total concentration of 50 μM salophen in acetonitrile mixture of Zn2+ and Al3+ added with final concentration of Zn2+ and Al3+ being 20 μM and 10 μM respectively. A control experiment was performed with individual salts at the same concentration. For both steady state and time-resolved measurement an excitation wavelength of 375 nm is used and 500 nm as emission wavelength is used for lifetime measurement. Aluminum salt was dried under desiccator for 3−4 days. Quantum Chemical Calculations. Computational calculation was performed using Gaussian 09 package whereas GaussView05 was used for drawing and visualization purpose.36 Density functional theory (DFT) calculations were carried out at the B3LYP level of theory with 6-31G(d) as the basis. The excited state energy was calculated with time-dependent DFT. Solvent effect (methanol) was taken care of by using the polarization continuum model. During geometry optimization, a frequency calculation was also carried out to make sure that the structure is at minima.
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Figure 1. Absorption (blue solid line), emission (red solid line), and excitation (red circles) spectra of (A) as synthesized/as received SalH2, (B) SalH2 crystallized once, (C) SalH2 crystallized thrice, and (D) filtrate of SalH2 crystallization process in acetonitrile. λex and λem are the excitation and emission wavelength for the corresponding emission and excitation spectra. The excitation and emission spectra are peak normalized. The absorption spectra are normalized at the lowest energy peak.
RESULTS AND DISCUSSION Salophen (SalH2) has an absorption maximum at 330 nm, with a molar absorption coefficient of 17000 M−1cm−1 and a shoulder at 370 nm. A very weak band is observed at 450 nm in polar solvents like acetonitrile and methanol, but not in nonpolar solvents like n-heptane and toluene (Figure S2). This weak band can be assigned to the polar cis-monoketo form.37,38 The crude reaction product, as well as the as received sample from Sigma−Aldrich, exhibits a strong 460 nm emission in acetonitrile, when excited at the absorption maximum. This is in agreement with some earlier reports.22,39−41 However, the excitation spectra of the crude as synthesized/as received salophen recorded with λem = 460 nm are very different from the corresponding absorption spectra (Figure 1A). So, it appears that the 460 nm emission arises from a highly fluorescent impurity. Upon repeated crystallization, the signature of the impurity becomes negligible. A feeble emission (ϕf = ca. 1.4 × 10−4 in acetonitrile) with λem = 545 nm (Figure 1B,C) is obtained, using λex = 370 nm. This is in agreement with the position of the emission maxima reported by Lee et al. in solution and Kotova et al. in solid state.21,23 For λex = 320 nm, the impurity emission is observed for once−crystallized salophen, albeit to a lesser extent (Figure 1B). For thrice−crystallized salophen, the excitation spectrum for λem = 560 nm is superimposable with the absorption spectrum (Figure 1C). The emission peak for 10 μM and 90 μM solutions of salophen are found to be identical (Figure S3). Hence, the possibility of aggregation of salophen in solution is ruled out since the solubility of salophen in acetonitrile is >30 mM. Interestingly, the filtrate obtained during crystallization exhibits a strong fluorescence at 460 nm. The absorption spectrum of the filtrate is superimposable with the excitation spectrum of the highly emissive impurity (Figure 1D). Thus, the filtrate is found to be enriched in the impurity. The fluorescence decay of the highly emissive impurity is single exponential, with a lifetime of 3.6 ns in acetonitrile (Figure S4), while the fluorescence of purified salophen decays within tens of picoseconds (vide inf ra). Excitation spectra of the crude product, monitored with λem = 450 nm (Figure 1), indicate that
the impurity does not absorb wavelengths longer than 350−360 nm. So, excitation wavelengths of 370−385 nm have been used for all our studies discussed below, in order to ensure selective excitation of salophen, even if the impurity is present in traces. Besides, the choice of this excitation wavelength allows us to avoid excitation of minute amounts of salicylaldehyde (λabs = 320 nm) that may be present in solutions of salophen. It may be noted here that the absorption spectrum as well as the spectral and temporal characteristics of fluorescence of the impurity suggests that it is likely to consist of one or more benzimidazole derivatives42,43 which are reported to form as major products44 of the reaction between the same reactants at different reaction condition. It is worth noting that N,N′bis(salicylidene)-p-phenylenediamine and salicylideneaniline which falls in the same family of Schiff base emits in the region of 550 nm.30,37 Steady state spectra undergo remarkable changes upon complexation with Zn2+ as well as with Al3+ (Figure 2A−C). The absorption spectra of the complexes are red-shifted to 390 nm (Figure 2, parts B and C), which is close to the absorption maximum of salophen anion, Sal2−, prepared by treating SalH2 with the non-nucleophilic base NaH in tetrahydofuran (THF) (Figure 2D).45 Notably, there is a strong band in the 420−450 nm region for the complexes, where the absorption of the cisketo form of SalH2 occurs. This band is present, albeit to a very small extent, in the absorption spectrum of SalH2 in methanol (Figure S2). The absorption band of the enol form is absent in the complexes, as there is no enolic proton. Consequently, there is no possibility of ESIPT unlike in the free ligand. It may be mentioned here that the absorption is predominantly due to intraligand π−π* transitions.46 The emission maximum of the 10321
DOI: 10.1021/acs.jpcb.6b05854 J. Phys. Chem. B 2016, 120, 10319−10326
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The Journal of Physical Chemistry B
is encouraging from the point of view of fluorescence sensing. However, a difficulty arises due to the almost identical position of the emission maxima of the two complexes. Large excess of Zn2+ can interfere with the Al3+ sensing ability of SalH2. Since zinc is a more abundant metal than aluminum in human body, this difficulty could be nontrivial during in vivo detection of Al3+. Time resolved fluorescence studies have been performed in order to understand how fluorescence lifetimes are affected upon complexation. The motivation for this study is 2-fold. If the lifetimes are significantly different in the two complexes, then they can be used as an effective parameter for selective sensing of one of the metal ions. Besides, these studies provide an insight into the effect of complexation on the excited state dynamics of fluorophore. The fluorescence of SalH2 decays within 60 ps while that for Sal2− decays within 40 ps. The ultrafast decay (Figure 3A) can be attributed to efficient
Figure 2. Absorption (blue), emission (red solid line), and excitation (red circles) spectra of (A) SalH2, (B) SalZn, (C) SalAl+ in acetonitrile and (D) Salophen anion (Sal2−) in THF. λex and λem are the excitation and emission wavelength for the corresponding emission and excitation spectra. The excitation and emission spectra are peak normalized. The absorption spectra are normalized at the lowest energy peak.
zinc complex of salophen (SalZn) occurs at 505 nm (Figure 2B). Its fluorescence quantum yield is greater than that of SalH2 by an order of magnitude (ϕf = 1.1 × 10−3 in acetonitrile). The aluminum complex, SalAl+, exhibits an emission with maximum at 495 nm (Figure 2C) and ϕf = 7.4 × 10−2 in acetonitrile (Table 1). This 500 fold enhancement of emission with Al3+ is Table 1. Steady State Photophysical Parameters of Salophen, Its Complexes (in ACN), and Salophen Anion (in THF) sample
λabs (nm)
λems (nm)
ϕf
SalH2 SalZn SalAl+ Sal2−
330 390 385 380
545 505 495 490
∼1.4 × 10−4 1.1 × 10−3 7.4 × 10−2 −
Figure 3. Fluorescence decay profile of SalH2, SalZn, and SalAl+, at the respective emission maxima, recorded by (A) the FOG technique (normalized) and by (B) the TCSPC technique. Additionally, the fluorescence decay of Sal2− has been shown in part A and that of SalH2 crystal, superimposed with SalH2 in ACN, has been shown in part B.
in line with earlier reports of SalH2 being a turn on fluorescent sensor for Al3+.21 The position of the emission maximum is close to that of Sal2− in both the cases (480 nm, Figure 2D). The slight red shifts are likely to be due to change in energy levels complexation with the metal ions. The increase in ϕf due to complexation is explained as follows: The low quantum yield of Schiff bases has been assigned to the flexible structure of these molecules, which facilitate segmental motion like photoisomerization about the CN bond and rotation about the C−C single bond. Rigidification of the molecule, by complexation with metal ions, leads to the suppression of such segmental motion and hence cuts down the associated nonradiative channels of deactivation of the excited state, leading to an increase in fluorescence quantum yield and lifetime. The significantly greater degree of enhancement of fluorescence intensity upon complexation of salophen with Al3+
nonradiative deactivation of the excited state due to torsional motions about the bonds, as reported for other Schiff bases.30 The faster decay in Sal2− may be attributed to a more flexible structure than that of SalH2, due to the lack of the phenolic hydrogen atoms and consequent intramolecular hydrogen bonding. It may be noted that in earlier studies, the longest component of a very similar Schiff base salicyledeneaniline, has been attributed to the cis-keto form. The shortest component has been assigned to the enol form and the intermediate component has been assigned to the emission from vibrationally excited cis-keto form.30 10322
DOI: 10.1021/acs.jpcb.6b05854 J. Phys. Chem. B 2016, 120, 10319−10326
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The Journal of Physical Chemistry B
respectively). In this experiment, we find that in steady state emission spectra it is difficult to identify either of the two metalcomplexes (Figure S5). But in time-resolved measurement, a clear signature of the Al3+ of nanosecond lifetime is observed (Figure 4). The associated lifetime is 1.6 ns, which is the same
The fluorescence decay becomes slower upon complexation with Zn2+, indicating the suppression of nonradiative processes that occur prior to complexation. The decays are multiexponential in both the cases (Table 2). The longest lifetime Table 2. Kinetic Parameters of Salophen, Its Complexes, and anion FOG
a
sample
λex/λem (nm)
SalH2
385/550 ACN
SalZn
400/510 ACN
SalAl+
400/490 MeOH
Sal2−
400/490 THF
TCSPC τ (ps) 0.2 5.0 14.8 0.6 5.4 34.4 1.2 14.4 ∼ns 0.4 2.6 9.2
a
(0.26) (0.21) (0.53) (0.38) (0.15) (0.46) (0.13) (0.05) (0.82) (0.39) (0.33) (0.28)
λex/λem (nm)
τ (ns)
375/550 ACN
