Colorimetry and phase transition characteristics in sensing ﬂ uoride anion based on hydrazide organogelators †

* a The ﬂ uoride anion sensing properties of BNB-t4 and BNBC-t8 consisting of hydrazide and azobenzene moieties both in solution and gel state, and the involved binding mechanism have been systematically investigated in this work. The remarkable changes in the absorption of receptor BNB-t4 with a terminal hydroxyl group demonstrate a colorimetric chemosensor with a higher sensitivity in sensing ﬂ uoride anions than that of BNBC-t8 with a terminal methoxy group. The detection limit of BNB-t4 for the analysis of F (cid:1) can reach as low as 4.27 (cid:3) 10 (cid:1) 8 M, while this value is 2.02 (cid:3) 10 (cid:1) 6 M for BNBC-t8. The results indicate that the F (cid:1) ion interacts with the amidic – NH and hydroxyl proton of BNB-t4 via hydrogen-bonding to give the stable 1 : 2 complex at the ﬁ rst equilibrium state, and further addition of F (cid:1) can induce deprotonation by forming HF 2 (cid:1) to establish a second equilibrium state. Meanwhile, the gel – sol transition of BNB-t4 has been successfully applied in sensing ﬂ uoride anions and thus makes BNB-t4 a naked-eye sensor. The color change of BNB-t4 induced by binding ﬂ uoride anions can be safely switched o ﬀ with the addition of HSO 4 (cid:1) , demonstrating an OFF – ON – OFF colorimetric sensor with a good reversibility.


Introduction
Anions play essential roles in many environmental, biological and industrial systems, and anion sensing has become one of the most attractive elds of supramolecular chemistry. [1][2][3][4] Fluoride is a small inorganic anion oen added to toothpaste due to its benecial role in dental health, and has also been used in the treatment of osteoporosis. 5,6 The right amount of uoride can prevent dental cavities, and in excess can lead to skeletal and dental uorosis in humans. 7 Hence, regulation and detection of uoride are crucial in various environmental and health applications. In past decades, the eld of anion sensors based on different signaling mechanisms, such as intramolecular charge transfer, [8][9][10] twisted intramolecular charge transfer, 11 photoinduced electron transfer, [12][13][14][15][16] metal-ligand charge transfer, 17-20 uorescence resonance energy transfer, 21-25 excited state proton transfer, [26][27][28][29][30][31] and excimer/exciplex formation, 32,33 has been well established and studied. However, most of the reported probes suffer from low sensitivity, slow response, or complicated synthetic procedures. Therefore, it is still a challenge to develop a highly selective and rapid detection method.
To date, many synthetic chemical receptors incorporating neutral or cationic N-H/F À hydrogen bonding donor groups (e.g., pyrrole, indole, ammonium, guanidinium, urea, thiourea, and hydrazide) have been reported. 20,[34][35][36][37][38] In contrast, receptors based on O-H/F À hydrogen bond interactions have been less exploited, [39][40][41][42] even though O-H shows higher acidity than N-H and the O-H/anion hydrogen bonding is almost as crucial as the ubiquitous N-H/anion interaction. 43 Stimuli-responsive organogel, structurally controlled by the assemblies of low-molecular-weight gelators (LMWGs) through noncovalent interactions is one of the most attractive examples due to its unique properties. 44 The gelation behavior involves the molecule self-assembly into three dimensional structures and is usually inuenced by the external conditions, and thus can be tuned by physical and chemical stimuli, such as temperature, 45 UV/visible light, 46 ions, 47-56 ultrasound, 57 and so on. Therefore, the self-assembly behavior, sol-gel transition, and colorimetric/uorescent change of LMWGs in gel or solution can be employed as molecular sensors in monitoring external stimuli. To create simple, convenient, and economical sensors requires successful fabrication of small molecular anion sensors into colorimetric test kits. 44 So far, a few reports on anion-tuning organogels have been reported for detecting uoride ion. [47][48][49] For instance, Lee and co-workers found that translucent colorless gel can be changed to liquid and showed a strong greenish uorescence with the presence of uoride anion. 47 Wu and co-workers developed a specic colorimetric and uorimetric sensor for detecting uoride anion. 48 Jiang and co-workers demonstrated that organogels based on salicylidene Schiff base showed a highly selective dual-responsive behavior to Zn 2+ and F À , respectively. 49 In the meantime, the detection limit for uoride anion in sol phase has been improved to micromole level, which is low enough to be applied in environmental or biological elds. 2 Although previous works presented a wide variety of anion sensors based on electrostatic interactions, hydrogen bond donor groups, Lewis acid groups and hydrophobic interactions, it is still a challenge to design and synthesize anion organogelators with specic selectivity and high sensitivity for sensing certain anions, because of their lower charge to radius ratio and highly solvated nature. 44 Especially, the gelators showing both reversible and highly sensitive colorimetric changes and sol-gel transition by anion stimuli are still limited to date.
Here, as an attempt to obtain a smart uoride anionresponsive gelator with potential anion sensing applications, we had designed and synthesized a gelator bearing phenol O-H and hydrazide for hydrogen bond donor subgroups, N-(3,4,5tributoxyphenyl)-N 0 -4-[(4-hydroxyphenyl)azophenyl] benzohydrazide (BNB-t4), and a control molecule, N-(3,4,5-octanoxyphenyl)-N 0 -4-[(4-methoxyphenyl)azophenyl] benzohydrazide (BNBC-t8) (Scheme 1). 46,58 The characteristics and binding mechanism of BNB-t4 with uoride anion have been systematically investigated in this work. The results indicate that BNB-t4 gels exhibit both colorimetric and gel-sol transition sensing properties upon addition of uoride anion. The remarkable change in the absorption of receptor BNB-t4 with terminal hydroxyl groups demonstrates a colorimetric chemosensor with higher sensitivity in sensing uoride anion than that of BNBC-t8 with terminal methoxy groups. In addition, the color change of BNB-t4 induced by binding uoride anion can also be safely switched off with the addition of HSO 4 À , demonstrating an excellent OFF-ON-OFF colorimetric sensor with a robust reversibility.

Results and discussion
During anion recognition, the interaction between receptor and target anion can lead to changes in molecular electronic ground state, and thus in the absorption properties. To this end, UV-vis spectrometry was performed to evaluate the recognition ability of BNB-t4 toward F À , Cl À , Br À , I À , CH 3 COO À (AcO À ), HSO 4 À and H 2 PO 4 À in CHCl 3 and DMSO, respectively (using their tetrabutylammonium salts as the sources). As shown in Fig. 1a, the selective binding of BNB-t4 in CHCl 3 with anions displayed intense variations in absorption spectra upon addition of the same amount of F À , AcO À and H 2 PO 4 À at room temperature, while no obvious colorimetric changes were observed in the presence of Cl À , Br À , I À and HSO 4 À . With the addition of 30 equiv. F À ion, the characteristic absorption maximum at 356 nm of BNB-t4 is dramatically decreased and a new broad band around 460 nm appears, indicating the formation of new complexes. The color of solution is also changed from light yellow to orange, which could be easily observed by the naked eyes. The selective binding of BNB-t4 in DMSO with anions shows the similar phenomena, in addition to having a deeper color change (from light yellow to crimson upon addition of 30 equiv. of F À , AcO À , H 2 PO 4 À and OH À ) (Fig. S1 †). Furthermore, the pH dependence of BNB-t4 in HEPES buffer solution system was examined using UV-vis spectroscopy. The results indicate that BNB-t4 binding process with F À can take place at the pH value ranging from 11 to 14 ( Fig. S2 †). However, when pH value of buffer solutions was 11, the characteristic absorption Scheme 1 Molecular structures of BNB-t4 and BNBC-t8. Fig. 1 UV-vis spectra of (a) BNB-t4 (1 Â 10 À4 mol L À1 ) and (b) BNBC-t8 (1 Â 10 À4 mol L À1 ) in CHCl 3 upon addition of 30 equiv. of various anions (F À , Cl À , Br À , I À , AcO À , HSO 4 À and H 2 PO 4 maximum at 485 nm of BNB-t4 in HEPES buffer solution system was almost unchanged with the addition of OH À ion (Fig. S3 †). So, changing the pH would be an optional and feasible way to overcome the shortcomings of the chemosensor in sensing uoride anion from OH À . Specically, to clarify the effect of the hydroxyl group in the BNB-t4 unit on responses to anions, we replaced the hydroxyl group with a methoxy group to form BNBC-t8. As expected, the selective binding of BNBC-t8 with anions only displayed an obvious variation in absorption spectra upon addition of the same amount of F À and H 2 PO 4 À at room temperature (Fig. 1b). With the addition of 30 equiv. F À ion, compared to that of BNB-t4, less spectral change for BNBC-t8 was observed, indicating a lower sensitivity to F À . The color of the BNBC-t8 in CHCl 3 with anions was changed from light yellow to yellow, less color change than that of BNB-t4 and consistent with the absorption spectral observations. The results indicate that the introduction of hydroxy facilitates the binding-induced changes in optical signals, which is in good agreement with previous reports. 5,49,50 Similar phenomena were also observed for H 2 PO 4 À responsive process (Fig. 1b).
To quantitatively examine the binding properties of BNB-t4 and BNBC-t8 to uoride anion, we performed the titrated absorption spectral analysis at a low concentration (1 Â 10 À5 M) ( Fig. 2 and 3). As shown in Fig. 2, the solution of BNB-t4 in chloroform is light yellow with a dominant absorption maximum at 356 nm (p-p* transition) and an absorption tail around 450 nm. With increasing amount of F À added, the absorbance at 356 nm gradually decreased and red-shied to 372 nm along with a clear isosbestic point at 382 nm, when 4 equiv. of uoride anions was added. This process corresponds to the formation of a BNB-t4-F À complex by hydrogen bonding during the rst course of titration (0-4 equiv.). With the continuous addition of uoride anion to 12 equiv., the absorption of p-p* transition was shied to 404 nm and a new isosbestic point was shied to 355 nm in the second course. Meanwhile, a new broad absorption band at 460 nm emerged during the whole titration course, demonstrating the formation of another complex and HF 2 À by deprotonation of -NH subgroup. 48,59 The deprotonation of BNB-t4 upon addition of F À ion was eventually conrmed by 1 H NMR titration as given below (see Fig. 5). Concurrently, the colorimetric changes are striking and visible, from almost colorless to yellow with the addition of uoride anion. Plotting the A 0 /(A 0 À A) quantity for absorbance at 356 nm against the reciprocal of [F À ] 2 gives a good linear response (Fig. 2b), and shows a 1 : 2 ratio for the complex composition of BNB-t4 and the F À ion in chloroform. 59 Similarly, this 1 : 2 ratio of complex composition for BNB-t4 with F À ion was also obtained in DMSO (see Fig. S4 †). Furthermore, the stoichiometric ratio between BNB-t4 and the F À ion was determined by Job's plot and indicated a 1 : 2  binding mode (Fig. 4a), which is consistent with the Benesi-Hildebrand plot observations. Likewise, two isosbestic points at 302 nm and 391 nm were observed in the absorption titration experiments of BNBC-t8 (Fig. 3a). Plotting the A 0 /(A À A 0 ) quantity of absorbance at 450 nm against the reciprocal of [F À ] (Fig. 3b), by contrast, gives a 1 : 1 ratio for the complex composition of BNBC-t8 and the F À ion. Moreover, the absorbance value approached the maximum when the molar fraction of BNBC-t8 was 0.5 as shown in Job's plots (Fig. 4b), indicating the formation of a 1 : 1 complex between BNBC-t8 and F À . Taking together, these results suggest that both phenol O-H and hydrazide subgroups of BNB-t4 can recognize the added F À through hydrogen bonding interaction. Furthermore, the sensitivity and selectivity of BNB-t4 towards anions can also be characterized by the association constant K s of complex formation between a proton donor and an anion. 59 As mentioned above, the hydroxyl and hydrazide subgroups in BNB-t4 are the dominant sites for binding uoride anion, the calculated association constant K s is as large as 5.54 Â 10 9 M À2 , suggesting efficient and sensitive recognition for F À . In comparison, the association constant of BNBC-t8 for F À was determined to be the value of 3.20 Â 10 3 M À1 . Thus, the much stronger binding ability of BNB-t4 to F À compared to that of BNBC-t8 can be attributed to O-H/F intermolecular hydrogen bonding. As an important index of chemosensors, the detection limit of receptor BNB-t4 in solution for sensing F À can also be obtained from the plot of absorption as a function of F À concentration (Fig. S5 †), 41,59 which was found to be 4.27 Â 10 À8 M in chloroform. In comparison, the detection limit of BNBC-t8 for F À was determined with the value of 2.02 Â 10 À6 M (Fig. S6 †), two order smaller than that of BNB-t4. As a consequence, the BNB-t4 with terminal hydroxyl group is more sensitive to uoride anion than the BNBC-t8 with terminal methoxy group, which can be conrmed by a more visible color change. This high sensitivity for uoride anion makes the BNB-t4 receptor a competitive candidate for environmental detection. In contrast to uoride detection, the obtained binding ratio is 1 : 1 for BNB-t4 with both AcO À and H 2 PO 4 À anions in chloroform, and the corresponding complex constants are 7.5 Â 10 4 (M À1 ) and 2.0 Â 10 3 (M À1 ), respectively ( Fig. S7 and S8 †). Therefore, these results indicate that BNB-t4 with terminal hydroxyl groups is more sensitive to uoride than to other anions.
To investigate the interaction active sites of BNB-t4 that bind with uoride anion, the 1 H NMR titration experiments were carried out in DMSO-d 6 . However, aer the addition of 10 equiv. uoride anion, the existence of a weak peak at 10.50 ppm of BNB-t4 indicates that not all of the phenol O-H and hydrazide N-H experienced a deprotonation process. In the NMR spectra of BNBC-t8 with different amount of F À anion, a weak peak at 10.41 ppm was also observed upon the addition of 6 equiv. F À , suggesting only one of hydrazide N-H underwent deprotonation (Fig. S9 †). Meanwhile, a new weak triplet signal appeared at d 16.43 ppm, 16.13 ppm and 15.83 ppm (Fig. 5g), clearly demonstrates the formation of HF 2 À . Moreover, the spectral shis of aromatic phenyl rings (H d , H 3 and H u ) linked to the hydroxyl and hydrazide group are shown in Fig. S10. † The continuous increase of F À concentration resulted in the protons H d , H 3 and H u of the phenyl rings to be out of the same chemical environment, and thus the up-eld shis of resonance signals for the H d , H 3 and H u were observed in the NMR spectra. Combining all the previous observations and obtained results, the sensing behavior of BNB-t4 for uoride anions can be schematically described in Scheme S1. † Due to the existence of hydrogen bonding subgroups -OH and -NH for uoride anion in BNB-t4, the addition of F À induces the rebalancing among hydrogen bonding, van der Waals and p-p stacking in  solution. Moderate amount of F À ion establishes a hydrogenbonding interaction with BNB-t4 to generate a stable 1 : 2 complex, while excess amount of uoride anion causes one of the NH group to deprotonate, and the other NH group together with the oxygen atom nearby deprotonated nitrogen atom forms an intramolecular hydrogen bonding. Concomitantly, the recognition of BNB-t4 to uoride anion leads to colorimetric changes visible to naked eyes in solution. In contrast with the situation of BNBC-t8, the hydrogen bonding subgroup -NH dominates the recognition of uoride anion, demonstrating the 1 : 1 ratio for the complex composition of BNBC-t8 and F À . Therefore, it can be concluded that the terminal hydroxyl group of BNB-t4 effectively improves the binding ability and the detection limit in sensing uoride anion, resulting in a more obvious color change that can be sensed by naked eyes. The good performance in binding/sensing uoride anion over other anions including acetate and phosphate makes BNB-t4 a highly sensitive and selective chemosensor.
To test the ability of receptor BNB-t4 as a colorimetric for uoride anion against other anions, we carried out competition experiments in the presence of F À mixed with various anions. As shown in Fig. 6, the uoride anion induced absorption spectral response demonstrates little change with the addition of the other coexistent anions except for HSO 4 À . The experimental results indicate that the receptor BNB-t4 has a good selectivity for F À ion in the presence of other anions, making it very useful in practical applications. With the addition of HSO 4 À , there is no obvious change observed in the absorption spectrum of BNB-t4 without the presence of uoride anion, indicating a very weak recognition of BNB-t4 to HSO 4 À (Fig. 6). However, it is very interesting to notice that the absorption spectral change of BNB-t4 induced by F À ion can be efficiently recovered to the initial state with the addition of 10 equiv. HSO 4 À . In other word, the bonded uoride anion with BNB-t4 can be totally released by the addition of HSO 4 À ion. Correspondingly, the color change of BNB-t4 induced by binding F À ion can also be safely switched off with the addition of HSO 4 À (the inset of Fig. 6), demonstrating a typical OFF-ON-OFF colorimetric sensor with a great recycling feature. Furthermore, the absorption spectral change of BNB-t4 induced by binding F À ion can also be recovered with the addition of H + , such as HClO 4 and methanol (MeOH) (Fig. S11 †). The gelation properties of BNB-t4 were discussed in our previous work, and the results indicate that BNB-t4 is capable of forming stable gels in moderately polar solvents such as dichloromethane, chloroform, and aromatic solvents. 46 The introduction of anions is expected to break the balance among the involved driving forces and induces the phase transition to sense the specic anion in turn. The effect of anions on BNB-t4 gelator was revealed by the gelation experiments in the presence of uoride anion. When 5 equiv. of F À was added into the chloroform organogel of BNB-t4 at 25 C, a thin column of winecolored solution immediately appeared at the upper part and then the gel underwent a gradual decomposition of the gelatinous state in 25 min, yielding a wine-colored solution (Fig. 7). This observation clearly indicates that the force balancing in BNB-t4 gel is destroyed by the introduction of F À and thus causes the dramatic phase transition from gel to sol with the concomitant color change visible to the naked eyes. The changed color of solution can be readily recovered by adding proton reagents such as MeOH. Similar phenomena were also observed upon the addition of solid TBA salts of AcO À or H 2 PO 4 À (Fig. S12 †), but the corresponding phase transition took a much longer time. This different response rate can be thus used to discriminate uoride anion from acetate and phosphate anions. However, the color transition and gel decomposition was not observed with the addition of Cl À , Br À , I À and HSO 4 À at the identical condition ( Fig. S13 †), indicating the selectivity of BNB-t4 gelator for the recognition of anions. In addition, these results also demonstrate the anions such as F À , AcO À and H 2 PO 4 À , rather than TBA cations, are responsible for the transformation from organogel to solution.
The uoride anion induced gel-sol transition can be further conrmed by scanning electron microscopy (SEM) study. Without the presence of F À ion, the morphology of BNB-t4 xerogel from chloroform (Fig. 8a) shows entangled and dense bers. In contrast, these bers are morphed into bent and swelled sausage structure to expel the gelated solvent (Fig. 8b) with the addition of uoride anion. Based on the fact that uoride anion has strong attacking ability to -NH and -OH Fig. 6 Competitive selectivity of BNB-t4 (1 Â 10 À5 mol L À1 ) towards F À (10 equiv.) in the presence of other anions (10 equiv.) in CHCl 3 . Inset: the image of BNB-t4 (1 Â 10 À5 mol L À1 ) in CHCl 3 (left), upon addition of 10 equiv. of F À (middle) and BNB-t4 treated with F À (10 equiv.), then added HSO 4 À (10 equiv.) (right). subgroups in BNB-t4, this morphology transformation suggests that F À can be inserted into the molecular assembly by hydrogen binding with the receptor BNB-t4, similar to the binding mode in solution. As a result, the host-guest binding destroys the balance of intermolecular interactions and initiates the driving force competition with the introduction of F À . It is however worthwhile to mention that this phase transition could also be partly attributed to the deprotonation of -NH and -OH subgroups, especially in the case of excess F À . As a consequence of the deprotonation, a new established balance among the driving forces consisting of hydrogen bonding, van der Waals and p-p stacking results in the formation of nal solution phase.

Materials
All the solvents for spectral measurements were of spectroscopic grade and used as received. Tetrabutylammonium salts were all >98% pure and dried in vacuum overnight before use. The solutions used in titrations were prepared from freshly opened solvent bottles.

Synthesis and characterization
The synthesis and characterization of receptors BNB-t4 and BNBC-t8 were described in our previous work. 46,58 The UV-vis absorption spectra were obtained on a Cary 5000 UV-vis-NIR spectrometer with quartz cuvettes of the appropriate path length (0.1-1 cm) at room temperature. 1 H NMR spectra were recorded with an Avance-400 400 MHz spectrometer using tetramethylsilane (TMS) as an internal standard. Field emission scanning electron microscopy (FE-SEM) images were taken with a JSM-6700F apparatus. Samples for FE-SEM measurement were prepared by wiping a small amount of gel or solution onto a silicon plate and followed by drying in a vacuum for 12 h at room temperature. Spectrophotometric titrations. Titrations were performed at room temperature in chloroform. In a typical experiment, the solution of the receptor (BNB-t4 and BNBC-t8) was titrated with a 200-fold concentrated solution of the tetrabutylammonium salt of the desired anion. The pH dependence experiment was performed in HEPES buffer solution system (DMSO : H 2 O ¼ 80 : 20 [v/v], containing 0.01 mol L À1 HEPES, pH ¼ 1-14).
Gelation test. Weighted gelator was mixed in a cap sealed test tube [3.5 cm (height) Â 0.5 cm (radius)] with an appropriate amount of solvent, and the mixture was heated until the solid dissolved. The sample vial was cooled to 4 C and then turned upside down. When a clear or slightly opaque gel formed, the solvent therein was immobilized at this stage.

Conclusions
The dually responsive properties of BNB-t4 bearing hydrazide and azobenzene groups in the recognition of uoride anion have been systematically studied in this work. The selective response to uoride anion over other anions indicates that the BNB-t4 is a good candidate in recognizing uoride anion. The interactions of uoride anion with amidic -NH and hydroxyl proton of BNB-t4 via hydrogen bonding and deprotonation cause the formation of new complexes and regulate their electronic states and molecular congurations. As a consequence, the remarkable changes in the absorption of receptor BNB-t4 with terminal hydroxyl group demonstrate a colorimetric chemosensor with better selectivity and higher sensitivity in sensing uoride anion than BNBC-t8 with terminal methoxy group. It can be concluded that the introduction of hydroxyl group greatly improves the recognition ability and detection limit of BNB-t4 for sensing uoride anion in solution. Meanwhile, the anion induced gel-sol transition of BNB-t4 selfassembly demonstrates a potential application in sensing uoride anion through the noticeable color change and phase transition. Meanwhile, the proposed sensing mechanism and binding ratio for BNB-t4 with uoride anion has been used to interpret the observations both in solution phase and in gel phase. Moreover, the color change of BNB-t4 induced by binding uoride anion can be safely switched off with the addition of HSO 4 À , indicating that the BNB-t4 is a typical OFF-ON-OFF colorimetric sensor with a great reversible feature. Therefore, the dual responsive receptor BNB-t4 with high selectivity and sensitivity for uoride anion could be a competitive chemosensor candidate for environmental applications.

Conflicts of interest
There are no conicts to declare.