A Novel Quinoxaline-Rhodamine Conjugate for a Simple and Efficient Detection of Hydrogen Sulphate Ion

This work presents the development of a quinoxaline and rhodamine conjugate system that acts as a colorimetric chemosensor for hydrogen sulfate (HSO− 4 ) ions in methanol media. This sensor has been characterized both theoretically and experimentally. The detection limits for HSO− 4 are small as 0.71 μM and 3.8 μM for the absorption and emission experiments, respectively. The effectiveness of the probe in recognizing HSO− 4 both in gel and solid phase is evaluated as well. Thus, this works presents a simple strategy to detect the environmental HSO− 4 pollutant event at tiny concentrations.


Introduction
The exponential increase of environmental pollution has forced researchers to rethink the modes of identification of the hazards through quick yet accurate methods [1,2]. In this framework, the emergence of fluorescent/colorimetric chemosensors for cations/anions/ neutral molecules has recently driven groundbreaking advances in the field by providing a plethora of efficient sensing systems with sometimes exceptional selectivity and sensitivity [3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Among the various anions that need to be detected, hydrogen sulfate (HSO − 4 ) ions are of particular interest because: (i) they play major roles in biological systems; (ii) they impart toxicity as pollutant; and (iii) they are essential as sulphate binding proteins in the physiological transport system [17][18][19][20][21]. In addition, HSO − 4 ions have numerous applications in industrial sectors such as agricultural fertilizers, industrial raw materials, nuclear fuel wastes, etc. [22,23]. It is also notable that HSO − 4 dissociates at high pH to generate toxic sulfate (SO 2− 4 ), which can cause skin and eye irritations and even respiratory paralysis or other severe disorders [24][25][26][27][28][29]. The large standard Gibbs energy of hydration (−1080 kJ·mol −1 ) and small pK a value of 1.99 (in aqueous medium) of the HSO − 4 anion also imply major constraints for its recognition and separation from an aqueous media [30][31][32]. Accurate and easy detection of HSO − 4 is consequently essential to monitor and control several hazardous effects.
Recent research efforts to target HSO − 4 have been mostly focused on chemosensors based on various fluorophores and chromophores, e.g., oxindole, boron-dipyrromethene (BODIPY), diarylethene, azomethine, indolium, naphthalimide, benzothiazolein, as well as different media, e.g., acetonitrile:water, ethanol and tetrahydrofuran (see Table S1). However, only a few probes have been reported in the literature for the detection of HSO − 4 [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. New strategies are therefore required to fill that gap in the currently available methods of detection. Among all possible alternatives, colorimetric detection is one of the most preferable alternatives as it allows an easy and rapid detection by the available methods of detection. Among all possible alternatives, colorimetric detection is one of the most preferable alternatives as it allows an easy and rapid detection by the naked eye without any expensive instruments. Accordingly, we have chosen an effective colorimetric signaling unit, rhodamine derivative, for the anion. Rhodamine moiety has already proved its potential in the field of chemosensors due to the signature photophysical outcomes of the fragmented spirolactum ring [52]. Herein, we propose a novel synthetic route by integrating quinoxaline and rhodamine moieties to generate the probe molecule, QRH, for selective detection of the anion HSO − 4 in methanol. We have also been able to reproduce the sensing phenomenon in gel droplet and paper strip formats.

Experimental Section
Synthesis of the novel ligand (hereafter labeled as QRH) used indeno [1,2−b]quinoxalin−11−one (compound 1) and Rhodamine B hydrazide (compound 2) as starting materials (see Scheme 1).The resulting product was completely characterized by spectroscopic and X-ray crystallography techniques. The nature of the main excited states was also assessed by means of theoretical calculations within the framework of the density functional theory (DFT). See Appendix A for further experimental and computational details.

Synthesis and Characterization
Simple condensation of compound 1 and compound 2 yielded QRH as illustrated in Scheme 1. The resulting product is fully characterized by FTIR, 1 H, 13 C NMR, ESI−MS spectra (Figures S1-S3, S5) and single crystal XRD method before its application. The FTIR data confirmed the generation of Schiff base by the appearance of the characteristic peak of C=N at 1609 cm −1 . From 1 H and 13 C NMR data the aromatic proton signals appeared at δH 8.06-6.1. The ESI−MS spectra shows 671.32 amu as the same as the calculated value for [C43H39N6O2] + species. Characterization was continued with single crystal X-ray diffraction experiment to determine the structure of the probe. The red colored single crystal was obtained by layering technique using the solvents chloroform and ether. As illustrated in Figure 1, QRH crystallizes in triclinic space group P-1 with a chloroform molecule as solvent of crystallization.

Synthesis and Characterization
Simple condensation of compound 1 and compound 2 yielded QRH as illustrated in Scheme 1. The resulting product is fully characterized by FTIR, 1 H, 13 C NMR, ESI−MS spectra (Figures S1-S3, S5) and single crystal XRD method before its application. The FTIR data confirmed the generation of Schiff base by the appearance of the characteristic peak of C=N at 1609 cm −1 . From 1 H and 13 C NMR data the aromatic proton signals appeared at δ H 8.06-6.1. The ESI−MS spectra shows 671.32 amu as the same as the calculated value for [C 43 H 39 N 6 O 2 ] + species. Characterization was continued with single crystal X-ray diffraction experiment to determine the structure of the probe. The red colored single crystal was obtained by layering technique using the solvents chloroform and ether. As illustrated in Figure 1, QRH crystallizes in triclinic space group P-1 with a chloroform molecule as solvent of crystallization.

Crystal Structure Description
The crystal structure depicts the spirolactum ring and three rings in the xanthene moiety are non-planar due to the presence of sp 3 spiro carbon. The five-member lactum ring and the six-member xanthene rings are nearly perpendicular (87.20 • ) [53]. Here the condensed planar quinoxaline conjugate makes an angle of 59.82 • with the lactum ring. Due to the presence of a large number of π-rings, the molecule undergoes intermolecular π···π stacking interaction between two naphthalene-phenyldiammine moieties in reverse. The two stacking interactions take place between phenazine···phenazine ring (distance 3.899(3) Å, slippage 1.592 Å) and phenyl···cyclopentane ring (distance 3.756(3) Å, slippage 1.272 Å) along b-axis. The C-H···π between the molecule forms pseudodimeric structure along c-axis ( Figure S14).

Crystal Structure Description
The crystal structure depicts the spirolactum ring and three rings in the xanthene moiety are non-planar due to the presence of sp 3 spiro carbon. The five-member lactum ring and the six-member xanthene rings are nearly perpendicular (87.20°) [53]. Here the condensed planar quinoxaline conjugate makes an angle of 59.82° with the lactum ring. Due to the presence of a large number of π-rings, the molecule undergoes intermolecular π•••π stacking interaction between two naphthalene-phenyldiammine moieties in reverse. The two stacking interactions take place between phenazine•••phenazine ring (distance 3.899(3) Å, slippage 1.592 Å) and phenyl•••cyclopentane ring (distance 3.756(3) Å, slippage 1.272 Å) along b-axis. The C-H•••π between the molecule forms pseudodimeric structure along c-axis ( Figure S14).

Naked Eye Sensing
The developed sensor, QRH, was examined in the presence of several metal ions (M n+ = Na + , K + , Ca 2+ , Cr 3+ , Cd 2+ , Hg 2+ , Pb 2+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ and Al 3+ ) available in their chloride and perchlorate salts ( Figure S10) and anions (terabutylam- in methanol at room temperature. The sensor shows a distinct amount of response in bare eyes for HSO − 4 ion from yellow to pink in methanol and shows a pink fluorescence under UV light irrespective of other anions in the respective solution phase (Figure 2). Based on these preliminary results, the response of the probe towards HSO − 4 was investigated in detail by means of the absorption and emission spectral outputs in methanol at room temperature.  − 4 We observed that the absorption spectral output of QRH (10 -6 M) shows peaks at 357 nm and 275 nm, and upon addition of HSO  Figures S6 and S7). These spectral outputs are mainly governed by the opening of spirolactum ring in presence of HSO − 4 , resulting in ring conjugation that is responsible for generation of absorption band at 558 nm and emission band at 609 nm ( Figures S6 and S7) [54]. That ring opening phenomenon and the resultant conjugation increases the electron density throughout the rhodamine moiety and this can be monitored by NMR titration in  Figures S6 and S7). These spectral outputs are mainly governed by the opening of spirolactum ring in presence of HSO − 4 , resulting in ring conjugation that is responsible for generation of absorption band at 558 nm and emission band at 609 nm ( Figures S6 and S7) [54]. That ring opening phenomenon and the resultant conjugation increases the electron density throughout the rhodamine moiety and this can be monitored by NMR titration in CDCl 3 where the related aromatic proton peaks of the probe show a somewhat downfield shift ( Figure S13).  (Figures S11 and S12). Sahoo et al. reported a rhodamine 6G hydrazide sensor for HSO − 4 but the detection limit was 16.70 µM in absorbance which is higher than our probe (see Table S1). Therefore, due to the presence of the quinoxaline moiety, the sensitivity towards HSO − 4 has improved. The fluorescence quantum yields were calculated to be 0.004 and 0.32 in the absence and presence of HSO − 4 ions (6 equiv.) by using rhodamine B as the standard (Φ, 0.64 in methanol), respectively.          Figure 5 summarizes the numerical outcomes of the performed theoretical calculations. The left panel shows the computed absorption profile for the neutral-closed spirolactum ring form of QRH (black line). As one can see, the theoretical profile matches the experimental measures, with an intense peak at ca. 300 nm and a clean spectrum beyond 500 nm. On the contrary, if calculations are carried out with the protonated-open spirolactum ring, a new band appears at that region, which is consistent with our experimental observations. Such dissimilarity can be analyzed through the nature of the first excited state. According to the time dependent (TD) DFT calculations, the parent QRH presents the first S 0 →S 1 excitation at 526 nm. However, such allowed transit is associated with a very low absorbance, which is characterized by the oscillator strength value (f = 0.0043). However, when QRH adopts the protonated-open spirolactum ring form, the first excitation is largely activated (f = 0.2333), and eventually produces the observed change in the absorption spectrum. That first excitation corresponds to the HOMO→LUMO transition. The frontiers orbitals are displayed on the right panel of Figure 5. A close inspection reveals that the S 0 →S 1 gap can be interpreted as a π-π* transition with a charge transfer contribution; the HOMO is mainly located in the bottom aromatic entity while LUMO spreads in both system aromatic systems. The computed absorption spectra consequently confirm that the new band arises from the opening of the spirolactum ring due to the activation of the charge transfer phenomena; overall the structure of them is the QRH ligand.

Theoretical Study and the Elucidation of a Proposed Model
As the preceding section describes, the experimental results have already pointed towards the existence of open spirolactum ring to form the probe, QRH, due to the presence of absorbance and fluorescence peaks at 558 and 609 nm, respectively. The precursor compound 1 and compound 2 also checked in the presence of HSO − 4 and neither showed any characteristic peaks in both absorbance and emission as QRH at experimental concentration. Rhodamine undergoes lactum opening in the presence of acidic protons. Although hydrogen sulfate is an anion, it still is pretty acidic (pK a~1 -2). Thus, the ring-opening of rhodamine is sensitive to the acidity of the proton, not the anion per se, which is why there is no emission turn-on in the presence of other anions. From the theoretical study, we also found that the protonated-open spirolactum ring form of QRH exists where the HSO − 4 can pair up as a counter anion but not in direct interaction with the probe (Scheme 2). This proposed model can be further reinforced by the ESI−MS spectrum where we got the peak for the protonated-open spirolactum form of the probe, [C 43 H 39 N 6 O 2 ] + along with HSO − 4 , Na + and solvent methanol molecule at 823.96 amu ( Figure S4). Therefore, we may presume that the generation of protonated-open spirolactumring form of QRH in association with HSO − 4 counter anion produces a stable ensemble and consequently results in the visual color change (Scheme 2). the experimental measures, with an intense peak at ca. 300 nm and a clean spectrum beyond 500 nm. On the contrary, if calculations are carried out with the protonated-open spirolactum ring, a new band appears at that region, which is consistent with our experimental observations. Such dissimilarity can be analyzed through the nature of the first excited state. According to the time dependent (TD) DFT calculations, the parent QRH presents the first S0S1 excitation at 526 nm. However, such allowed transit is associated with a very low absorbance, which is characterized by the oscillator strength value (f = 0.0043). However, when QRH adopts the protonated-open spirolactum ring form, the first excitation is largely activated (f = 0.2333), and eventually produces the observed change in the absorption spectrum. That first excitation corresponds to the HOMO→LUMO transition. The frontiers orbitals are displayed on the right panel of Figure 5. A close inspection reveals that the S0S1 gap can be interpreted as a π-π* transition with a charge transfer contribution; the HOMO is mainly located in the bottom aromatic entity while LUMO spreads in both system aromatic systems. The computed absorption spectra consequently confirm that the new band arises from the opening of the spirolactum ring due to the activation of the charge transfer phenomena; overall the structure of them is the QRH ligand.  As the preceding section describes, the experimental results have already pointed towards the existence of open spirolactum ring to form the probe, QRH, due to the presence of absorbance and fluorescence peaks at 558 and 609 nm, respectively. The precursor compound 1 and compound 2 also checked in the presence of HSO − 4 and neither showed any characteristic peaks in both absorbance and emission as QRH at experimental concentration. Rhodamine undergoes lactum opening in the presence of acidic protons. Although hydrogen sulfate is an anion, it still is pretty acidic (pKa ~1-2). Thus, the ring-opening of rhodamine is sensitive to the acidity of the proton, not the anion per se, which is why there is no emission turn-on in the presence of other anions. From the theoretical study, we also found that the protonated-open spirolactum ring form of QRH exists where the HSO

Application
Encouraged by the colorimetric visual HSO − 4 detection ability of the probe, QRH, in methanol solution, we decided to assess the performance of QRH in mediums other than the solution phase. The sensing property of QRH was evaluated in solid and gel forms [56]. The preparation methods are discussed in Appendix A. As shown in Figure 6c, a significant color change occurs on test paper from yellow to pink. In Figure 6a

Application
Encouraged by the colorimetric visual HSO − 4 detection ability of the probe, QRH, in methanol solution, we decided to assess the performance of QRH in mediums other than the solution phase. The sensing property of QRH was evaluated in solid and gel forms [56]. The preparation methods are discussed in Appendix A. As shown in Figure 6c, a significant color change occurs on test paper from yellow to pink. In Figure 6a,b, both in daylight and under UV light, after addition of HSO − 4 , the probe undergoes a noticeable change implying suitability of the methods.
Encouraged by the colorimetric visual HSO − 4 detection ability of the probe, QRH, in methanol solution, we decided to assess the performance of QRH in mediums other than the solution phase. The sensing property of QRH was evaluated in solid and gel forms [56]. The preparation methods are discussed in Appendix A. As shown in Figure 6c, a significant color change occurs on test paper from yellow to pink. In Figure 6a,b, both in daylight and under UV light, after addition of HSO − 4 , the probe undergoes a noticeable change implying suitability of the methods.

Conclusions
In this work, a quinoxaline-rhodamine integrated colorimetric sensor for selective detection of HSO  4 ] ensemble. The detection limit of the probe is 7.1 × 10 −7 M in methanol medium. In addition to solution phase detection, the probe shows its versatility in both gel droplet and solid paper strip forms. All accumulated data confirm the wide applicability of the proposed probe for detecting hydrogen sulphate ion in real life samples at a reduced cost.

Conclusions
In this work, a quinoxaline-rhodamine integrated colorimetric sensor for selective detection of HSO − 4 is reported. We demonstrated the detailed characterization of the probe by FTIR, 1 H, 13 C NMR, ESI-MS and single crystal XRD method. The probe shows effective recognition towards HSO − 4 aside from the other anions. The experimental as well as theoretical results provided evidence in favor of the spirolactum ring opening in the presence of HSO − 4 anion, for example, by exhibiting the signature spectroscopic outputs for the open form. We have, therefore, proposed a model consisting of a stable [probe·HSO − 4 ] ensemble. The detection limit of the probe is 7.1 × 10 −7 M in methanol medium. In addition to solution phase detection, the probe shows its versatility in both gel droplet and solid paper strip forms. All accumulated data confirm the wide applicability of the proposed probe for detecting hydrogen sulphate ion in real life samples at a reduced cost.