A new fluorescent probe 2,7-dichlorofluorescein hydrazide for mercury quantification in aqueous medium has been described. It is based on the spirolactam ring opening of colorless and nonfluorescent 2,7-dichlorofluorescein hydrazide induced by Hg2+ ions through the hydrolytic cleavage of amide bond to produce green-colored highly fluorescent dichlorofluorescein in alkaline medium. The significant color change of this reagent in the presence of mercury ions can be used as a sensitive naked-eye detector. The working range, limit of detection, and relative standard deviations were found to be 0.2–20 ngmL−1, 0.042 ngmL−1, and 0.69% respectively. The proposed method is free from most of the common interfering ions present in the environmental samples. The developed method has been successfully applied to determine trace level mercury from water, soil, and industrial effluents.
Global mercury emissions have increased substantially in recent years due to increased human activity mainly through the emissions from coal burning power plants, gold mining operations, and industrial processes [
The metal ions can cleave and promote the hydrolytic cleavage of specific bonds like ester or amide bonds which are present in molecules like xanthenes and its derivatives accompanied by the change of spectroscopic properties of these molecules [
Fluorescence measurements were recorded using Ocean Optics Spectrofluorimeter (model USB 4000). Absorbance measurements were made using a Shimadzu Scanning Spectrophotometer (model UV-3101PC) with 1 cm quartz cuvettes. All pH measurements were carried out using Control Dynamics digital pH meter (model APX 175). All the infrared measurements were performed using Shimadzu Spectrometer (model FTIR-8400S). Mass spectral data was obtained using Thermo Finnigan Deca QXP Mass Spectrometer. Elemental analysis was carried out using Elementar (model Vario super user).
2,7-dichlorofluorescein hydrazide was prepared according to the literature reported (Scheme
Synthesis of 2,7-dichlorofluorescein hydrazide.
Aliquots of solutions containing mercury (concentration range of 0.2–20 ngmL−1) were taken into a series of 10 mL volumetric flasks. Then 2 mL of 10 mM 2,7-dichlorofluorescein hydrazide solution was added followed by 1 mL of buffer solution of pH 12. These solutions were allowed for 2 min and diluted up to the mark with distilled water. The green-colored species were excited at 502 nm, and the emitted fluorescence intensities were recorded at 520 nm.
Industrial effluents from the chrome plating and textile industry were collected in polyethylene containers. The solutions were filtered, and 50 mL of filtered solution was transferred into a beaker, 10 mL each of con. sulphuric acid and 30% H2O2 were added, and the solution was then heated on water bath until the foaming ceases in order to oxidize any elemental/ionic mercury (I) into mercuric (II) species. Then the solutions were cooled, and known aliquots were used for the analysis.
The water samples were collected using polyethylene containers from polluted lake (where painted clay idols were immersed after festival procession), unpolluted lake, and tap water supplied for drinking purpose. The water samples were filtered through Whatman filter paper to remove any suspended particulate matter. Then known aliquots of samples were used for mercury analysis.
The soil samples were collected from agricultural field and soil sludge samples from the pond bed where the painted clay idols were immersed. Both the samples were collected from the site and stored in polyethylene bags. The soil samples were air dried, and the known weight (100 g) of the sample was placed in 250 mL beaker and extracted four times with 10 mL portions of concentrated hydrochloric acid each time. The combined extract was boiled for about 30 min. Then the solution was cooled and diluted to 50 mL with distilled water. Then known aliquots of diluted samples were used for Hg2+ determination.
Xanthene-based derivatives are well-known chemosensors for the quantification of metal ions due to their specific quantitative reaction with metal ions through the spirolactam ring opening process [
Schematic representation of mechanistic pathway of 2,7-dichlorofluorescein hydrazide and its reaction with mercury ions.
Excitation and emission spectrum of ((a), (a′)) 2,7dichlorofluorescein hydrazide (10 mM) in the absence of mercury and ((b), (b′)) 2,7-dichlorofluorescein hydrazide (10 mM) in the presence of Hg2+ ions (100 ngmL−1).
Fluorescence spectra of 2,7-dichlorofluorescien hydrazide in presence of variable mercury (II) concentrations (
Naked eye sensor (A), reagent only (B), reagent + Hg2+ (0.5 ppm) (C), reagent + Hg2+ (1.0 ppm) (D), and reagent + other interfering ions (500 ppm).
In order to prove that the reaction product obtained between the probe molecule and the metal ion is 2,7-dichorofluorescein, the reaction was carried out in bulk quantities. The product formed was isolated by extracting using ethyl acetate and purified by recrystallization. The recrystallized compound was characterized by FTIR study and elemental analysis.
The obtained compound was subjected to FTIR study by making the pellet after mixing with IR grade KBr in 1 : 100 ratio. The IR spectrum of the isolated product (2,7-dichlorofluorescein) did not show any significant peaks for N–H (primary amine) stretching at 3500 cm−1 (doublet) as well as N–C=O (amide) stretching at 1690 cm−1. However, characteristic significant peaks for 2,7-dichlorofluorescein have been observed. It has showed a strong stretching frequency at 3500 cm−1 due to OH moiety of COOH group. These studies have revealed that the compound formed is dichlorofluorescein (Figures
FTIR spectra of (a) 2,7-dichlorofluorescien hydrazide, (b) 2,7-dichlorofluorescien.
The CHN analysis of the synthesized 2,7-dichlorofluorescein and its authentic counterpart was carried out. The elemental analysis data has been found to be in good agreement with each other. The elemental analysis data for 2,7-dichlorofluorescein [C20Cl2O5H10] both synthesized compound as well as authentic samples are given as below. Synthesized: C-59.894, H-2.463; Authentic: C-59.884, H-2.493.
The spectral properties of the 2,7-dichlorofluorescein hydrazide usually depend on the pH as well as its concentration in solution phase because higher concentration of the dye may lead to molecular aggregation which in turn decreases the absorption. The presence of Hg2+ ion induces the ring opening of the probe molecule under selective pH condition. This pH selectivity can be used to prevent interference from the other metal ions by controlling the solution pH. The reaction variables have been carried out to get the maximum sample absorbance with the minimum blank value.
The reaction between 2,7-dichlorofluorescein hydrazide and mercury proceeds only in alkaline condition; hence, the effect of medium pH was studied in the range of 7–12. The fluorescence intensity values of 2,7-dichlorofluorescein gradually increased with the increase in pH, and the maximum fluorescence emission was observed between pH 9 and 12, and hence an optimum pH value was maintained at 12 by the addition of 1 mL of buffer (pH = 12) in all further studies. Therefore, initial studies were carried out using 10 ngmL−1 of mercury (II) in the presence of 0.5 mL of 10 mM 2,7-dichlorofluorescein hydrazide in 10 mL volumetric flask. The solutions were allowed for two minutes for the reaction completion, and the fluorescence values were measured at 520 nm.
The optimum concentration of the 2,7-dichlorofluorescein hydrazide required to react with 10 ngmL−1 mercury ion was examined by varying its concentration between 5 and 30 mM. The sample gave maximum fluorescence intensity beyond 20 mM concentration which was achieved by the addition of 2 mL of 10 mM 2,7-dicholorofluorescein in 10 mL volume.
In order to apply the proposed method to quantify mercury (II) ion at trace level from natural and environmental samples, the interference effect of common cations and anions that normally exist in natural water samples was studied. The interfering species were added in their respective salt forms, and its impact on the signaling behavior of 2,7-dichlorofluorescien hydrazide with Hg2+ ion was studied. The spectral interference of several metal ions with 2,7-dichlorofluorescien hydrazide is insignificant when compared to mercury (II) including Cu2+ at pH 12 (Figure
Interference study.
Interferent | Concentration in |
---|---|
(ngmL−1) | |
F−, I−, Cl−, CH3COO−, |
3000 |
|
2500 |
|
2000 |
Fe2+, Fe3+, Ni2+, Zn2+, K+, Li+, Ca2+, Na+, Hg+, Pb2+ | 2000 |
Cd2+, Mg2+, Co2+, Ni2+, Al3+, Cr6+, Mn2+ | |
Cu2+ | 500 |
Fluorescence spectra of (a) Probe alone, (b) Probe + Fe2+, Fe3+, Pb2+, Cd2+, Ni2+, Hg+, Mn2+, Zn2+ (500 ng), (c) Probe + 500 ng Cu2+, (d) Probe + 20 ng Hg2+.
The proposed method has been validated by determining trace level mercury concentrations from a variety of natural samples like water, soil and industrial effluents. The water and soil, sludge samples were collected from the lake polluted due to the dumping of the clay idols after festival procession. These clay idols slowly dissolve in water releasing pigments containing toxic metal ions like mercury, lead, arsenic, and so forth into water as well as soil bed of the lake. The mercury content in these water and soil sludge samples of the lake was analyzed by the procedure described elsewhere. Recovery studies were also carried out by spiking the samples with known concentrations of mercury to these samples, and the results have been compared with that of standard method [
Determination of mercury from different sample matrices.
Sample | Hg2+ originally found (ngmL−1) |
Total Hg found (ngmL−1) |
Recovery (%) | ||||||
---|---|---|---|---|---|---|---|---|---|
Proposed method | Standard method |
|
Hg2+ added (ngmL−1) | Proposed method | Standard method |
|
Proposed method | Standard method | |
(1) Chrome plating industry effluent | 18 ± 1.6 | 18 ± 1.6 | 1.3 | 1.0 | 19 ± 1.1 | 18.5 ± 1.1 | 1.00 | 100 | 97.3 |
(2) Textile industry effluent | 10 ± 1.2 | 10 ± 2.4 | 2.0 | 1.0 | 10.9 ± 1.9 | 12 ± 1.6 | 1.18 | 99.0 | 109 |
(3) Lake water | ND | ND | — | 10.0 | 9.9 ± 1.9 | 9.9 ± 1.9 | 1.00 | 99.5 | 99.5 |
(4) Tap water | ND | ND | — | 15.0 | 14.9 ± 1.7 | 14.9 ± 1.6 | 1.06 | 99.6 | 99.6 |
(5) Polluted lake water | 10.1 ± 1.5 | 10.2 ± 1.9 | 1.26 | 1.00 | 10.9 ± 1.6 | 11 ± 1.2 | 1.30 | 99.0 | 100 |
(6) Soil sludge* | 0.5 ± 0.01 | 0.5 ± 0.014 | 1.4 | 1.00 | 1.5 ± 0.05 | 1.5 ± 0.05 | 1.00 | 100 | 100 |
(7) Agricultural soil* | ND | ND | — | 10.0 | 10.0 ± 1.2 | 10 ± 1.9 | 1.58 | 100 | 100 |
ND: not detected (
* ngg−1.
A simple and sensitive protocol has been developed for mercury quantification using 2,7-dichlorofluorescein hydrazide as a fluorescent probe based on the spirolactam ring opening of the molecule by the metal ion in alkaline medium at nanogram level. The proposed method can be used as a naked-eye sensor in industrial atmospheres as a prewarning signal whenever the high levels of mercury have been discharged in industrial effluents. The molecule is highly selective to divalent mercury, and most of the common metal ions present in water do not interfere significantly including copper which can be prevented by pH control. The developed method has been successfully applied to determine trace level mercury from industrial effluents as well as water samples. The results obtained by the proposed method have been compared with standard method, and the results are in good agreement. The precision of the method was evaluated by calculating the
The authors acknowledge the financial support and award of the fellowship to K. Sureshkumar by the University Grants Commission (UGC), New Delhi, India.