Selective colorimetric sensors for cyanide and acetate ion in partially aqueous medium

ESI Fig.1. UV-Vis spectra of L1 and L2. ESI Fig.2. H-NMR spectra of L1. ESI Fig.3. C-NMR spectra of L1. ESI Fig.4. H-NMR spectra of L2. ESI Fig.5. C-NMR spectra of L2. ESI Fig.6. IR spectra of L1. ESI Fig.7. IR spectra of L2. ESI Fig.8. A plot of (A Amin/Amax Amin) vs log A to calculate LOD of L1 with (a) CN ion (b) AcO ion. A plot of (A Amin/Amax Amin) vs log A to calculate LOD of L2 with (c) CN ion (d) AcO ion. ESI Fig.9. Job’s plot of L1 with CNand AcOion. ESI Fig.10. Job’s plot of L2 with CNand AcOion.


Introduction
In supramolecular chemistry, the development of chromogenic sensor is of great interest in anion sensing due to their chemical and biological applications [1].The most hazardous cyanide ion widely used in the various chemical industries (140,000 tons of cyanide per year) causes the severe pollution of water supplies [2][3].Cyanide is extremely toxic, increases environmental pollution due to its industrial use mainly production of textiles, papers and plastics.Importantly, cyanide rigorously suppresses the transport of oxygen as it binds with cytochrome c oxidase and effects electron transfer from cytochrome c oxidase to oxygen in mitochondria [4].Acetate ion is more important in living organisms as acetyl coenzyme [5].
There are many conventional detection methods for quantitative determination of cyanide and acetate ion mainly based on electrochemical and voltammetric techniques [6][7][8][9][10].Due to their cost and sophisticated instruments, detection of cyanide and acetate ion by absorption or fluorescence or both studies are easier and growing very fast [11][12][13][14][15].So the development of colorimetric anion sensor is more attractive due to no requirement of expensive equipment as color changes can be easily detected by the naked-eye.Generally, the chromogenic anion sensor consisted many pathways as: displacement of a metal complex (Fig. 1), anion receptor and chromophore, these sensors having chromogenic centre that is covalently bonded to the receptor unit.The receptor unit or binding sites are based on hydrogen bond donor group, generally -OH and -NH group of phenols, sulphonamide, urea, thiourea [16][17][18][19].Compared to the above traditional chemosensor method, an alternative chemodosimeter approach based on an irreversible specific chemical reaction has emerged as an active research area of significant importance.However, considerable efforts have been devoted to the development of a chemodosimeter for anions.Many examples are available about colorimetric sensors for acetate and cyanide ion in the literatures [20][21][22][23][24][25][26][27][28].
In this paper, we described two phenol based azo dyes L1 and L2 which selectively sense biologically important CN -and AcO -ion in partially aqueous medium without any interference of different anions.Two azo dye based ligands were synthesised having phenolic group for better sensing ability towards anions, resulting in the enhancement of push-pull approach of intramolecular charge transfer (ICT), which reproduced red-shifted absorption.
Azo dye ligand L2 with -NO2 units (electron-withdrawing substituent) performed high selective binding ability towards cyanide ion over other studied ion.

Reagents and Instruments
Analytical grade anion salts are purchased from Merck.Phenol, 2-aminothiazole and 2amino-5-nitrophenol were purchased from Sigma-Aldrich.CHNS analysis was recorded on an Elementar model Vario EL-III.IR spectra were obtained using a Perkin Elmer FT-IR 1000 spectrophotometer as films between KBr. 1 H NMR spectra were recorded on Bruker AVANCE 500 MHz spectrometer and Zeol 400 MHz spectrometer.UV-vis spectra were recorded on Shimadzu, UV-3600 double beam spectrophotometer using 10 mm path length of silica cell.DFT computational studies were obtained with Gaussian 09 W programme in gas phase using a B3LYP function with 6-31G(d,p) basis set for L1, L2, L1+CN -and L2+CN -.
The mixture was stirred for half an hour at 0º C (Solution 1).For the coupling reaction, the mixture of 0.9 gm of phenol and 10 ml of 0.1 M NaOH were taken in a separate flask and maintain the temperature at 0º C (Solution 2).Solution 2 was added in solution 1 while stirring at 0º C. The formed precipitate washed with distilled water and recrystallised using ethanol and collect the orange powdered product (Scheme 1

2-((4-hydroxyphenyl)diazenyl)-5-nitrophenol (L2)
Took, 2 gm of 2-amino-5-nitrophenol and dissolved in 24 ml of H2SO4 and then add 0.6 gm of sodium nitrite (NaNO2) in 10 ml water in dropwise manner on continuous stirring.The mixture was stirred for half hour at 0º C (Solution 1).For the coupling reaction, the mixture of 1.8 gm of phenol and 10 ml of 0.2 M NaOH were taken in a separate flask and mentain the temperature of this solution at 0º C (Solution 2).Solution 2 was added slowly on stirring at 0º C to the solution 1.The occurred precipitate washed with distilled water and recrystallised by ethanol and collect yellow-orange powder (Scheme 1

Naked Eye Experiments:
The recognition and chromogenic sensing ability of azo dye L1 and L2 (20 µM) were checked with the sodium salt of a series of anions F -, Cl -, Br -, CN -, H2PO4 -, HPO4 2-, SO4 2-, SO3 2-, AcO - , N3 -and SCN -(200 µM) in DMSO/H2O-HEPES (v/v; 1:1, pH = 7.3±0.2) solution.By examine the chromogenic properties of azo dyes L1 and L2, it was found that both ligands showed instant color changes from yellow to violet with more basic anion, CN -/AcO -and other anions showed no such color changes (Fig. 2).Basicity of anions is known to be in order of CN -> AcO -> N3 -> F -> H2PO4 -> Cl -> Br -> I - [30] and the ability to form hydrogen bond in order of F -> AcO -> H2PO4 -> N3 -> CN -so that both anions with more basic and least H-bonding character might deprotonate the phenolic hydrogen rather than forming H-bonding.Further the sensitivity of azo dyes with CN -and AcO -ion was analysed by absorption spectra.

UV-Vis Analysis:
Upon addition of different anion like F − , Cl − , Br − , AcO − , HPO4 2-, H2PO4 − , CN -and N3 -(200 µM), changes in UV-vis absorption spectra of L1 and L2 (20 µM) with CN -/AcO -ions were found.Azo dye L1 exhibited two absorption peak at 253 (π-π* transition), 393 nm (n-π* transition) and L2 exhibited three absorption peak at 213 (σ-σ* transition), 262 (π-π* transition) and 374 nm (n-π* transition).L1 showed new absorption band with CN -and AcO - ion at 486 and 483 nm respectively hence L2 also showed same behaviour with CN -and AcO - ion and found a new absorption band at 435 and 437 nm respectively.With this absorption studies, it was confirmed that both azo dye ligands selectively detect cyanide and acetate ion.This new absorption band may occur because of the abstraction of proton with cyanide and acetate ion.The titrations of L1 (20 µM) were performed in DMSO/H2O-HEPES (v/v; 1:1, pH = 7.3±0.2) solution with CN -and AcO -ion.For the titration, a series of spectra were taken and started by adding the little amount of the anion solution with the help of microsyringe in the quartz cuvettes containing the solution of azo dye (Fig. 3a-3b).The series of UV-vis spectra were taken after each addition and absorbance values were recorded.L1 exhibits changes in absorption spectra upon addition of CN -ion, the band at 393 nm were quenched with consequently increases in the absorption band at 486 nm due to interaction of CN -ion with ligand.Titration experiments for L2 with CN -and AcO -ion was performed in the same way (Fig. 4a-4b).The absorption spectra of L2 shown the changes upon addition of CN -ion, the band at 374 nm were quenched with consequently increases in absorbance at 435 nm due to interaction of CN -ion with ligand.Three isosbestic point were found and showed that free azo dye and adduct (dye + anion) was in equilibrium (L1+CN -; 417, 301, 258; L1+AcO -; 433, 298, 267; L2+CN -; 400, 300, 270; L2+AcO -; 399, 301, 273).The binding constant of CN -and AcO -ion with L1 and L2 was calculated using BH-plot (Inset; Fig. 3 and   4) and found to be 1.6 × 10 3 , 8.0 × 10 2 , 8.4 × 10 3 , and 1.7 × 10 2 respectively.The most important parameter of chemosensor, limit of detection, for cyanide and acetate ion by using L1 and L2 were also determined from absorption titrations based on reported procedure [31][32].The obtained results from absorption titrations were normalised between maximum and minimum absorbance intensities.A plot of (A -Amin/Amax -Amin) vs log A -(A= CN -, AcO -) in DMSO-H2O (1:1, v/v) gave a linear curve and the point at which linear line crossed to x-axis was familiar to detection limit (ESI Fig. 8).Table 1 lists the detection limit of CN -and AcO - ions presented by L1 and L2.The detection limit in DMSO-H2O (1:1, v/v) was determined to be 87 nM (L1 + CN -), 83 nM (L1+ AcO -), 81 nM (L2 + CN -), 89 nM (L2+ AcO -) respectively.

Intereference study on sensor performance:
As we use the both azo dyes for the detection of cyanide and acetate ion, we also tested the absorption response of L1 and L2 towards various anions (N3 -, HPO4 2-, H2PO4 -, SCN -, AsO2 -, Cl -, Br -, SO4 2-, SO3 2-, NO2 -, NO3 -and F -) and we found that there was no significant changes in the absorption spectra in the presence of various anions under the same conditions.L1 and various other anions (Fig. 5).We have done competitive experiments [33] in the presence of detecting anion (100 µM) and other anions (100 µM) with azo dyes (20 µM) and we found that the absorption spectra of azo dyes with detecting anions remained unaffected with the addition of other anions.This demonstrated that both azo dyes could be used to quantitative detection of cyanide and acetate ion concentration with great selectivity.In the figure 5-a and 5-b, the blue bar indicates the absorption intensity of CN -with azo dye L1 and L2 and the red bar indicates the absorption intensity of azo dyes with CN -ion in the presence of interfering ions.

1 H-NMR titration:
To know the mechanism of interaction between azo dye and cyanide ion, 1 H NMR spectra of L1 and L2 were recorded in absence and presence of cyanide ion.L1 shows NMR peak at δ 10.796 ppm for -OH proton and azo dye L2 shows NMR peak at 10.884 ppm and 8.973 ppm for hydroxy protons and the rest peak of NMR assigned for aromatic region.After adding CN -ion solution to L1 and L2, shake the solution for a while and 1 H NMR at room temperature were taken which showed all the peaks of NMR were shifted to the upfield region in both azo dye due to the abstraction of phenolic proton or formation of phenolate ion which increased the electronegativity on π-e -cloud of aromatic region.The peak of -OH proton of (10.796 ppm) L1 and (10.884 and 8.973 ppm) L2 was found to be completely disappeared and aromatic proton signals of L1 and L2 slightly shifted to upfield region (Δδ = 0.1 ppm and 0.08 ppm respectively).When adding more amount of CN -ion solution, aromatic protons of L1 and L2 were dramatically shifted into a high field region (Δδ = 0.2 ppm and 0.17 ppm) due to the possible phenolate ion formation.The 1 H NMR studies, clearly illustrated that the both L1 and L2 were interacted cyanide ion via.deprotonation of -OH group (Fig. 6 and 7).From the careful analysis of 1 H NMR spectra, the colorimetric change could take place partly through proton abstraction.We reasoned that the basic cyanide anions (pKa<9.4)are expected to readily abstract the acidic proton of OH group (pKa<4).

Theoretical Calculations:
To understand the recognition behaviour of phenol based azo dyes L1 and L2 with cyanide ion, theoretical studies have been done.The optimizied geometry of L1, L2, L1+CN -and L2+CN -was obtained in gas phase on Gaussian 09 W computational program [34].HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) of L1, L2, L1+CN -and L2+CN -obtained by optimised geometry and found that the free ligands L1 and L2 have higher energy gap (ΔE = 2.362 and 2.236 eV respectively) between HOMO and LUMO then the adduct L1+CN -and L2+CN -(ΔE = 1.865 and 1.773 eV respectively, Table 2).These computational calculations were performed using Gaussian 09 with B3LYP/6-31G (d,p) basis set.The deprotonation of OH proton of L1 and L2 concludes the decrease in energy gap of L1+CN -and L2+CN -.The theoretical calculation leads to the success of experimental results.Fig. 8 shows the optimized structure and Fig. 9 shows HOMO-LUMO energy level diagram of L1, L2 and their cyanide adduct.

Measurement of Anions with Test strips:
The test strips were prepared by immersing the whatman filter paper in DMSO solution of azo dye L1 and L2.The paper strips were subsequently dried in air to figure out the "dipstick" method suitability for the detection of both anions [35].The coated test strips were further immersed in the aqueous solution of CN -and AcO -ion solution of different concentration and suddenly a fast color change was found from light yellow to violet red (Fig. 10).The development of such a "dip-stick" approach is extremely attractive for "in-thefield" measurements as it does not require any additional equipment.

Fast Response
To know the fast response time of chemosensor L1 and L2 towards cyanide ion, changes in absorption spectra was monitored with time.In this sensor, detection of CN -ion with L1 and L2 was found to be very fast.New absorption band of L1 and L2 with CN -ion was found at the higher intensity (plateau region) within 10 s and remains stable for 8-11 min.A curve between time v/s ratiometric absorption intensity reveals that reaction was completed in 10 s (Fig. 11).

Analytical Application
To know the applicability of the sensor L1 and L2, it was further used for the quantitative determination of cyanide ion in drinking water.The sample of drinking water was prepared by adding a known amount of cyanide ion (2 µM).The experiments were performed using absorption spectra of 2 ml volume of sensor sample after adding a known concentration of cyanide ion which found within the linear calibration range.The experiment was repeated three times and the average concentration of cyanide ion in drinking water was found to be 1.9 µM.Then, the recovery percentage performed well with relative standard deviation lower than 2%.

Conclusion
Highly selective chromogenic azo dyes L1 and L2 was designed that can detect both CN -and AcO -ion in 50% aqueous medium via proton abstraction.The stoichiometry of L1 and L2 with CN -/AcO -ion was confirmed in account of Job's plot.i.e. 1:1 and 1:2 respectively.The more basic cyanide and acetate ion abstract the more acidic proton of the molecule and allow the formation of phenolate ion.Thus the accumulation of the negative charge on the whole molecule exhibited a large significant red shift in CT band and major changes in color.LOD for CN -ion with L1 and L2 was found to be 87 and 81 nM and for AcO -ion with L1 and L2 was found to be 83 and 89 nM respectively.Therefore, the system used to detect the WHO suggested maximum allowed cyanide concentration in drinking water (1.9 mM).Tables Table 1.Stoichiometry, binding constant, detection limit values for obtained adduct of L 1 and L 2 with CN -and AcO -ions.

S.
No.