Speciation of Inorganic- and Methyl-Mercury in Biological Matrixes by

Article pubs.acs.org/ac

Speciation of Inorganic- and Methyl-Mercury in Biological Matrixes by Electrochemical Vapor Generation from an L‑Cysteine Modified Graphite Electrode with Atomic Fluorescence Spectrometry Detection Wang-Bing Zhang,* Xin-An Yang, Yong-Ping Dong, and Jing-Jing Xue Department of Applied Chemistry, Anhui University of Technology, Maanshan, Anhui 243002, P. R. China S Supporting Information *

ABSTRACT: A novel nonchromatographic speciation technique for ultratrace inorganic mercury (Hg2+) and methylmercury (CH3Hg+) in biological materials is developed and validated by electrolytic vapor generation (EVG) coupled with atomic fluorescence spectrometry (AFS). The studies show that CH3Hg+ and Hg2+ can be converted to Hg vapor efficiently on an Lcysteine modified graphite cathode, which has never been reported before. We observe that only Hg2+ can be converted efficiently to Hg vapor at low current mode (0.2 A). While at high current mode (2.2 A), both CH3Hg+ and Hg2+ can be reduced efficiently. As a result, we successfully establish an exact and sensitive method based on the current control to detect mercury speciation for the first time. The factors of electrolytic conditions have been optimized, and the potential mechanism is discussed. Under the optimal conditions, the detection limits (3s) of Hg2+ and CH3Hg+ in aqueous solutions are 0.098 and 0.073 μg L−1, respectively. The relative standard deviations for 6 replicate determinations of 2 μg L−1 Hg are determined as 3.2% and 4.7% for Hg2+ and CH3Hg+. The accuracy of the method is verified through the analysis of certified reference materials (CRM, NRCDORM-2), and the proposed method has been applied satisfactorily to the determination of mercury speciation in several seafood samples by calibration curve mode.

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Generally, the combination of chromatographic separation techniques such as gas chromatography (GC),4−8 highperformance liquid chromatography (HPLC), 9−18 ionic chromatography (IC),19 and capillary electrophoresis (CE)20 with a specific and sensitive detector including atomic absorption spectroscopy (AAS),9,20 atomic fluorescence spectrometry (AFS),6,10−19 inductively coupled plasma mass spectrometry (ICPMS),4,7,8 and microwave-induced plasma AES (MIP-AES)5 is a practical approach for mercury speciation analysis. An obvious advantage of chromatographic methods is the ability to distinguish between different mercury species in samples. However, a common disadvantage of these methods is that some complex and tedious pretreatment procedures are often involved in the separation of organic mercury.21 Hence,

ercury (Hg) is considered as a highly dangerous element because of its accumulative and persistent character in the environment and biota. The toxicity, bioavailability, and mobility not only depend on its concentration but also significantly depend on its chemical form.1 In general, organic mercury compounds, especially methylmercury (CH3Hg+), are more toxic than inorganic mercury (Hg2+). The main entry route of Hg into human body is through the food chain, especially via fish and shellfish which bioaccumulate this compound. Actually, all of the mercury released in the ecosystem undergoes biogeochemical transformation processes and can be converted into CH3Hg+ by microorganisms and microalgae in aquatic environments2 that result in CH3Hg+ constituting up to 90% of the total mercury in fish.3 For this reason, the total mercury and organomercury, especially CH3Hg+ levels in food, should be monitored and the detection methods should be routine and robust. © 2012 American Chemical Society

Received: July 12, 2012 Accepted: October 4, 2012 Published: October 4, 2012 9199

dx.doi.org/10.1021/ac3018923 | Anal. Chem. 2012, 84, 9199−9207

Analytical Chemistry

Article

precision, fast analysis, high generation efficiency, and ease of automation can also be achieved. In the last twenty years, more than fifty papers have been published related to electrochemical generation of volatile hydrides for sample introduction in atomic spectrometry. Among these works, EVG is mostly applied in analyzing inorganic ions as well as oxidation state speciation of elements such as As, Sb, and Se.37 Despite several determinations of Hg by the EVG technique44,45,53−55 being reported, no successful determination of Hg species has been achieved. Recently, Gan’s group investigated the EVG behavior of Hg and Sn on a polymer modified graphited electrode.55,56 They found that the modified cathode can raise the vapor generation efficiency of analyte; as well as, the stability of signal, the memory effect, and the sensitivity of the method have also been improved. Sulfhydryls are very specific and sensitive reagents for reaction with oganic mercurial compounds because of the strong Hg−S affinity in a wide pH range either for low molecular weight compounds like L-cysteine or for macromolecules like proteins.57−59 In this paper, we are trying to realize the conversion of CH3Hg+ to Hg vapor efficiently on an L-cysteine (L-cys) modified graphite cathode, investigate the different EVG behavior between Hg2+ and CH3Hg+ on the Lcys modified graphite cathode, and then establish an exact and sensitive method to detect mercury speciation. To the best of our knowledge, no study about the application of EVG for the determination of mercury speciation has been reported.

the development of nonchromatographic speciation technique, based on the different behaviors of chemical species before measurement by atomic spectrometry, is still attractive because of the simple, fast, and inexpensive methodologies. Typically, nonchromatographic analytical procedures include two steps; first, the direct detection of Hg2+ by a weak reductant reduction, while the CH3Hg+ can not be reduced by this reductant; second, detect total mercury with online or offline oxidation systems to convert CH3Hg+ to Hg2+. So far, the methods available for the oxidation of Hg compounds use strong acids22 and oxidants23 and high temperatures.24 However, these methods for mercury oxidation are a potential source of sample contamination, have potential analyte losses by volatilization and, additionally, have the potential generation of hazardous laboratory wastes using concentrated and corrosive regents.25 Currently, some oxidation processes, such as ultrasonic irradiation,26 UV radiation,27 and microwave digestion,28 are adopted to realize this conversion process. Chemical vapor generation (CVG) coupled with AFS or AAS represents the most powerful analytical tools for ultratrace determination of Hg in recent years. Sodium or potassium salts of tetrahydroborat(III) have usually been used as reducing agents for the reduction of Hg2+ to Hg vapor because it is simpler, faster, and efficient. However, these reducing agents are expensive and unstable and have low tolerances for interference of transition metals.29 Therefore, much research concentrated on looking for the innovation of a novel vapor generation technique to resolve these problems. For instance, Guo and Sturgeon found that the generation of reducing radicals from low molecular weight organic acids upon irradiation by UV sources can be used to reduce some heavy metal ions to their vapor, and then, they established a novel photochemical vapor generation (PCVG) technique in their excellent works.30,31 Zheng et al.32 noted that both CH3Hg+ and Hg2+ could be converted to Hg vapor under UV light irradiation, and only Hg2+ was converted to Hg vapor under natural light irradiation. Vieira et al.33 developed a new method for selective determination of CH3Hg+ using acetic acid. Briefly, tissues were digested in tetramethylammonium hydroxide with/ without acetic acid, respectively; CH3Hg+ or total mercury was selectively determined by exposure of the solutions to UV irradiation. Bendicho’s group developed a novel ultrasonic vapor generation (USVG) system for Hg.34,35 They believed that the free radical (H·) generated by the discomposure of formic acid under ultrasonic irradiation can reduce the Hg2+ to Hg. Thereafter, Ribeiro et al.36 found that the reduction of CH3Hg+ to Hg vapor by USVG occurs only at relatively high ultrasonic field density (>10 W cm−3), and for this reason, speciation of mercury is possibly analyzed by altering the ultrasonic field density. Noticeably, electrochemical vapor generation (EVG), as an important sample introduction technique, has also drawn more and more attention due to its merits. The basic principle of EVG relies on the reaction between H· derived from the cathode of an electrolytic cell and the analyte.37 Since this method requires only one simple electrolytic cell and a power supply, it is more efficient and greener than PCVG.38 Moreover, through the utilization of continuous flow/flow injection electrolytic cell, at least ten elements, including As,39−42 Bi,36 Cd,38,43 Hg,44,45 Ge,46 Pb,47 Se,48,49 Sb,39,50 Sn,51 and Te52 have been successfully reported using EVG coupled with different detection techniques, which has more application than USVG. Additionally, high sample throughput, good

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EXPERIMENTAL PROCEDURES Instrumentation. Atomic fluorescence measurements of Hg were carried out with an AFS-230 double-channel nondispersive atomic fluorescence spectrometry (Beijing Kechuanghaiguang Instrument Co., Beijing, China) equipped with a model IFIS-C intermittent flow reactor (Xi’an Mairui Electronic Science and Technology Co., Xi’an, China). A mercury high performance hollow cathode lamp (General Research Institute for Nonferrous Metals, Beijing, China) was used as the radiation source. A JYL-350-type food making machine (Joyoung small household electrical appliances Co., Ltd. Shandong, China) and a 800-type centrifuge (Shanghai Medical Instruments Group Corp., Ltd. Surgical Instruments Factory, Shanghai, China) were also used for sample preparation. A homemade disk shaped thin-layer electrolysis cell similar to that described in our previous work38 was applied as generator for Hg vapor generation. Its photograph is shown in Figure S1 of the Supporting Information. Power supply for the electrolytic cell was DH1719A-3 constant current and constant voltage unit (Beijing Dahua Wireless instrument Co, Beijing, China) working in the constant current mode. IFIS-C peristaltic pumps were used to transport the samples. All X-ray photoemission spectra (XPS) measurements were recorded in an ESCALAB 250 spectrometer (Thermo-VG Scientific), which incorporated an Al Kα (hv-1486.6e eV) X-ray source and operated in the CAE mode. The pass energy was set to 20.0 eV, and the energy step size was 50 meV. Before recording the spectra, the samples were stored in a vacuum of better than 5 × 10−10 mbar in order to degas. During the measurement, the vacuum in the spectrometer was better than 1 × 10−10 mbar. All IR spectrum measurements were recorded in a Nicolet 6700 Fourier Transform Infrared Spectrometer (Thermo Scientific). The IR spectrum of L-cys, the L-cys 9200



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modified graphite electrode, and the used graphite electrode was obtained by making KBr tablet. Reagents. All chemicals are of analytical-reagent-grade. High purity deionized water is used throughout (Hangzhou Wahaha Group Co., Ltd. Hangzhou, China). The Hg2+ standard solution is prepared by serial dilution of a 1000 mg L−1 mercury stock solution (Dr. Ehrenstorfer GmbH-Bgm.Schlosser-Str. bA-86199 Augsburg, Germany). The CH3Hg+ stock solution (50 mg L−1) is prepared by dissolving methyl mercury chloride (Dr. Ehrenstorfer GmbH-Bgm.-Schlosser-Str. bA-86199 Augsburg, Germany) in methanol solution (Sinopharm Chemical Reagent Co., Ltd.). An L-cys solution is prepared by dissolving solid L-cys (Sinopharm Chemical Reagent Co., Ltd.) in 0.01 M HCl solution. High-purity HCOOH, HCl, H2SO4, and HNO3 are used throughout. Argon of 99.99% purity (Nanjing special gases factory Co., Ltd., Nanjing, China) is used as the carrier gas. Procedures. Immobilization of L-cys on Graphite Electrodes. The bare graphite electrode (GE) is commercially available from the local market. It was polished to the size of about 50 mm × 10 mm × 1 mm by abrasive paper and fine alumina slurries on a polishing cloth. Then, the electrode is rinsed with water, sonicated for 10 min, rinsed with methanol, and allowed to dry. Prior to immobilization, the GE is initially oxidized by a +1.8 V, 6 min potential step in 0.1 mol L−1 H2SO4 to create carboxylic acid surface groups. The electrodes are then removed from the H2SO4 solution and immersed in 1.5 g of L-cys in a 100 mL solution for 8 h at room temperature to ensure peptide bond formation between the surface groups of the GE and the amine terminus of L-cys. After immobilization, the electrode is rinsed with deionized water. The immobilization procedures are shown in Figure S2 of the Supporting Information. Sample Preparation. Three samples (including mussel, razor clam, and baby clam) are purchased in local supermarkets (Maanshan, China) and transported to the laboratory in an ice compartment. All samples, previously homogenized, are overdried at 40 °C for 48 h, then stored in closed polyethylene boxes, and kept in a refrigerator at −18 °C until use. A homemade device with double-frequency ultrasonic is used for sample preparation, and the treatment process of samples is described in our previous work.57 Briefly, approximately 1 g of sample is weighed into the glass vessel, and 10 mL of 6 mol L−1 HCl extraction solvent is added to the vessel, sealed with the PVC screw-cap, and then double-frequency sonicated for 1 min by the above device. The extracted suspension is then centrifuged at 4000 rpm for 3 min. Five mL of supernatant is then quantitatively transferred into a 25 mL colorimeter tube. The extraction process above is repeated one more time. The two parts of the supernatant are combined, made up to the certain volume with 2.0 mol L−1 HCOOH, and then stored at 4 °C until analysis. Analytical Procedures. The analytical procedures of EVGAFS of Hg are schematically shown in Figure 1. First, deionized water is rapidly transported into the cell to check the sealing performance. Second, the anodic electrolyte (0.5 mol L−1 H2SO4) is continuously introduced into the anode chamber of the cell by the peristaltic pump, at a flow rate of 5.5 mL min−1. The anodic electrolyte is reclaimed and recycled. The catholyte (1.0 mol L−1 HCOOH) or sample solution is transported at the same flow rate by the other peristaltic pump to a T-tube and then carried into the cathode chamber of the cell by Ar gas flow. After the chamber is filled, the electricity

Figure 1. Schematics design of manifold and instrumental setup used for EVG-AFS determination of Hg. GLS, gas liquid separator; AFS, atomic fluorescence spectrometry.

power is set at a constant current of 0.2 A for Hg2+ detection. When CH3Hg+ is determined, the power is set at a constant current of 2.2 A. The product in cathode chamber is directly carried into the GLS for the gas−liquid separating. Lastly, the gas phase is delivered into the atomizer and determined by AFS. At the same time, oxygen produced in the anode chamber is driven out along with the anodic electrolyte and then diffused to the surroundings at the outlet of the transferring tube. The reaction can be described as follows: +4e

Hg 2 + + 2H+ ⎯⎯⎯→ Hg + H 2

(1)

−4e

2H 2O ⎯⎯⎯→ O2 + 4H+

(2)

The method of additions was used for quantitative analysis. The optimized operating conditions used for the EVG-AFS system are summarized in Table S1, Supporting Information.

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RESULTS AND DISCUSSION Characteristics of L-cys Modified Graphite Cathode. The chemical state of elements in the L-cys modified graphite cathode is further investigated by XPS. The wide scan XPS spectrum (Figure 2A) of L-cys modified graphite cathode shows photoelectron lines at a binding energy of about 163, 285, 401, and 531 eV attributed to S 2p, C 1s, N 1s, and O1s, respectively. In the spectrum of S 2p (Figure 2B), the peaks of S 2p3/2 and S 2p1/2 are located at 163 and 164 eV when the electrode is exposured to cysteine. The solution containing 2 ng mL−1 Hg 2+ is injected into the cell continually, and the cathode has been working for 3 h. We can find from Figure 2C that S content decreases from 7.98% to 0.4% and C content increases from 54.97% to 92.66%, indicating that the modified L-cysteine has been lost. In order to study the adsorption behaviors of CH3Hg+ and Hg2+ on the modified cathode during electrolysis, higher characteristic peaks at Hg 4f are achieved. Before the XPS study, the 10 μg L−1 Hg2+ and CH3Hg+ standard solutions are introduced into the EVG cell and determined for 5 cycles, respectively, according to the analytical procedures. Figure 2D displays the peak of Hg 4f7/2 located at 101.7 eV, showing that the Hg is adsorbed on the surface of cathode during electrolysis. 9201



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Figure 2. XPS spectra: (A) wide scan, (B) S 2p spectra, (C) wide scan after cathode working for 3 h, and (D) Hg 4f spectra.

ence of transition metals by carefully selecting the cathode material. Pt and carbon in different forms have been most commonly used for Hg vapor generation because of its electrochemical inactivity and better EVG efficiency than other cathodes (Figure 4). We first compare the performance

Figure 3a−c shows the IR spectrum of cathode before and after modification. In comparison with pure L-cys (Figure 3b),

Figure 3. IR spectra: GE after anodic activation (a); pure L-cys (b); Lcys/GE (c); L-cys/GE after working for 1 h (d), and (e) L-cys/GE after working for 3 h.

Figure 4. Effects of the different cathodes on the fluorescence intensity of 2 μg L−1 Hg2+ and CH3Hg+. Catholyte flow rate of 5.5 mL min−1; electrolytic current of 2.2 A; 300 mL min−1 Ar. Instrumental conditions are as mentioned in Table S1, Supporting Information.

the S−H band appears at 2550 cm−1, similarly. The bands at 1588 cm−1 (CO) and 1386 cm−1 (C−O) in the IR spectrum of pure L-cys shift positively to 1716 and 1476 cm−1, respectively. Compared to GE, the −NH band appears in the range of 3030 to 2900 cm−1 in Figure 3c. All the evidence indicates that the L-cys modified on the cathode successfully. Figure 3d,e shows the IR spectrum of L-cys/GE after electrolysis. The S−H band disappears in Figure 3d during the electrolysis process, suggesting the rupture of the S−H bond in L-cys/GE and the formation of the Hg−S bond at the same time. Figure 3e shows that the modified L-cysteine has been lost after a long time use. Effect of Cathode on the EVG’s Performance of Hg2+ and CH3Hg+. The EVG technique has been shown to be a suitable alternative because it not only obviates the need for chemical reducing reagents but also tolerates higher interfer-

of several metals and the L-cys modified graphite electrode (Lcys/GE) as cathodes with both Hg2+ and CH3Hg+ in order to find a suitable one for speciation analysis of Hg. All electrodes are initially tested as 1 cm × 5 cm foils with a Pt anode; 1.0 mol L−1 HCOOH and 0.5 mol L−1 H2SO4 are used as the catholyte and analyte, respectively, and a constant current of 2.2 A is used. The response of different cathodes toward 2 μg L−1 Hg2+ and CH3Hg+ are shown in Figure 4 with the Hg2+ response of a graphite cathode taken as unity. Among all the materials, Pt produced the most intensive response for Hg2+, and the results are similar to our previous work.44 Hg2+ signal on L-cys/GE cathode is statistically equivalent with that on the graphite. 9202



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Figure 5. Effects of electrolytic current (A) and catholyte concentration (B) on the fluorescence intensities of 2 μg L−1 Hg2+ and CH3Hg+. Solid line and dotted line are expressed as applied currents of 2.2 and 0.2 A, respectively. 1.0 M HCOOH is used as catholyte (A); Catholyte flow rate of 5.5 mL min−1; 300 mL min−1Ar.

Hg2+ signals on Pb, Au, and Ag are more negative than that on graphite. Among these cathodes explored in present work, the efficiency of CH3Hg+ on L-cys/GE modified cathode is the highest. Generation from CH3Hg+ implies that the use of L-cys/ GE cathode to achieve efficiency is similar to that obtained from Hg2+. Noticeably, the CH3Hg+ can almost not be converted to Hg vapor on any unmodified cathodes. According to the principle of hard and soft acids and bases, Hg (a soft acid) prefers to bind with sulfur (a soft base) through covalent attachment.60 Angeli et al. studied the behavior of organic mercury with thiol systeine in CVG-AFS and found that the C−S−Hg bond is more stable to the reduction than the C−Hg bond.61 Wang et al. have proposed an online HPLC−CVG-AFS method for separation and determination of Hg2+, CH3Hg+, and ethylmercury, which uses cysteine in the mobile phase of chromatography, without an oxidation system. They speculated that cysteine plays an important role in the digestion of organic mercury and could weaken the C−Hg bond by forming complexes with organic mercury, subsequently broken by BH4− and HCl.58 Parks et al.62 has proved quantitatively that coordination of R-Hg 2+ by two cysteine thiolates is necessary and sufficient to activate the Hg−C bond. They have brought up the mechanism that the coordination of R-Hg2+ induces redistribution of electron density into Hg2+ and the leaving group carbon. Jiang’s group63 proposed a mechanism for thiol-assisted PCVG of CH3Hg+, in which, the formation of CH3Hg-thiol complexes resulted in increasing electro density of Hg and then reduced CH3Hg+ to Hg vapor more easily. At the same time, the whole process of EVG could be described as an electrodeposition step and an electrochemical reduction step. According to their views and combined with our experimental data, we deduce that the Hg atom in CH3Hg+ prefers to bond with S atom in L-cys forming CH3Hg−thiol complexes by S−Hg−C bond on the cathode surface, first resulting to weaken the Hg−C bond. Then, the reductive radicals (like H·) generated by electrolysis attack the Hg−C bond in complexes and transform it into Hg vapor directly (Figure S2, Supporting Information). Certainly, the role of L-cys in the conversion from CH3Hg+ to Hg vapor by electrolysis is unclear, and it should be deeply studied. We also research the effects of graphite with/without anodic activation on the signal intensity and lifetime of modified electrode and results are shown in Figure S3 of the Supporting

Information. This study documents that the modified cathode after anodic activation has better stability and longer lifetime. How to Analyze the Hg Species? We can find from Figure 5A that the response from both Hg2+ and CH3Hg+ increases remarkably as the current increases from 0.05 to 1.0 A. While at applied currents lower than 2.0 A, Hg2+ has perceptibly greater response than CH3Hg+, and they become equal when currents are higher than 2.2 A. Importantly, at a current of 0.2 A, the response to CH3Hg+ is essentially zero, while that to Hg2+ still remains significant. This dramatic difference in the behavior at applied currents of 0.2 and 2.2 A provides an opportunity to make measurements of Hg2+ and CH3Hg+, without any prior chemical redox conversions. Compared with the method of replacing the cathode, this method based on the current control to detect mercury speciation is simple, fast, and operable. We observe the effect of type of the catholyte on the Hg vapor generation efficiency. Traditionally, the catholytes have four types such as inorganic acid, buffer solution, alkaline, and organic acid.41 The signal intensity of Hg2+ in HCOOH media by EVG is higher than that in inorganic acidic medium like H2SO4 (Figure S4, Supporting Information). Here, we chose a 0.5 mol L−1 H2SO4 as the anodic electrolyte and HCOOH as the catholyte. The concentration of HCOOH is optimized. Figure 5B shows that the signal intensity for 2 μg L−1 CH3Hg+ solutions increases proportionally with the HCOOH concentration increase to 1.0 mol L−1 and decreases thereafter. The same curve is obtained for Hg2+ detection. With the applied current of 0.2 A, the signal intensity of Hg2+ increases remarkably with the HCOOH concentration up to 1.0 mol L−1 and increases slightly in the range of 1.0−2.0 mol L−1. At the same time, the response of CH3Hg+ is almost zero. Hence, with a 1.0 mol L−1 HCOOH catholyte and applied current at 2.2 A, both Hg2+ and CH3Hg+ respond equally, and these are then chosen as the total Hg detection conditions. At the applied current of 0.2 A within the same catholyte, only the Hg2+ response is arrived at and these are then chosen as the Hg2+ detection conditions. L-cys Concentration and Memory Effect. On the basis of that mentioned above, the conversion from CH3Hg+ to Hg vapor by EVG is dependent on the Hg binding with sulfur through covalent attachment. The mass of L-cys modified on the graphite surface is important. Due to the fact that the concentration of L-cys in the solution used for preparation of 9203



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Figure 6. Effects of L-cys concentration in the solution for preparation of the electrode on the determination of 2 μg L−1 Hg2+ and CH3Hg+ (A) and the memory effect of modified cathode on the signal intensities of 2 μg L−1 Hg2+ and CH3Hg+ (B). Electrolysis current 2.2 A; 1.0 M HCOOH is used as catholyte; catholyte flow rate of 5.5 mL min−1; 300 mL min−1Ar. Instrumental conditions are as mentioned in Table S1, Supporting Information.

the electrode is the first factor of the mass of L-cys modified on the graphite surface, the effect of L-cys concentration on the response of Hg2+ and CH3Hg+ is studied in the range of 0−20 g L−1. We find from Figure 6A that the response of Hg2+ changes slightly with L-cys concentration. The signal intensity of CH3Hg+ increases obviously with L-cys concentration from 0 to 12 g L−1 and then levels off. Therefore, 15g L−1 L-cys is chosen for further experiments. Moreover, reports in the literature claim that mercury determinations suffer from serious memory effects, in contrast to the determination of many other elements. Thus, we discuss the potential memory effect of the EVG system, and results are shown in Figure 6B. First, 2 μg L−1 Hg2+ or CH3Hg+ standard solutions are introduced continually into the EVG cell and electrolyzed for 3 times determination in the optimum conditions. Second, the electrolysis procedure is ceased, and the sample solution is vented totally. Third, blank catholyte (1.0 mol L−1 HCOOH) is pumped into the cell and then electrolyzed for 3 times determination. Lastly, 4 μg L−1 Hg2+ or CH3Hg+ standard solution replace blank solution and detected 3 times. The results in Figure 6B show that the fluorescence signal intensities of blank catholyte are less than 20% and 22% of that of 2 μg L−1 Hg2+ and CH3Hg+ standard solution, respectively, and less than 14% and 15% of that of 4 μg L−1 Hg2+ and CH3Hg+ standard solution, respectively. Compared with our pervious works, the memory effect of Hg2+ on the modified cathode is more than that on the unmodified cathode. It is probably the covalent attachment between Hg and sulfur resulting in the adsorption of Hg2+ and CH3Hg+ on L-cys modified graphite electrode. Since the adsorption of Hg forming Hg vapor by the further electrolysis, this memory effect has disappeared. Effect of Carrier Gas Flow Rate and Sample Flow Rate. Argon is usually used as carrying gas to deliver the generated gaseous mercury vapor to the detector and is introduced traditionally through two locations: sample outlet and sample inlet. The introduction place of carrier gas is not taken into consideration because it does not influence the signal intensity of analytes. However, we find that either Hg2+ or CH3Hg+ has almost zero response when the carrier gas is introduced from the sample outlet (not going through the cathode compartment of the cell) but strong signals of both are detected when the carrier gas is introduced from the sample inlet (entering the cathode cell). We deduce there are two reasons. One, the lifetime of Hg vapor is short44 and the vapor cannot transfer to

the detector if the distance between the detector and introduction place is too long. Two, the adsorption of the Hg vapor on the cathode surface and the walls of the transportation lines during the transferring process lead to the loss of Hg. We evaluate Ar flow rate ranging from 300 to 800 mL min−1. The relationship between the flow rate of carrier and the signal is shown in Figure 7A. It is observed that, when the Ar flow rate

Figure 7. Effects of carrier gas flow rate (A) and sample flow rate (B) on the fluorescence intensities of 2 μg L−1 Hg2+ and CH3Hg+. Solid line and dotted line are expressed as applied currents of 2.2 and 0.2 A, respectively. Instrumental conditions are as mentioned in Table S1, Supporting Information.

increases constantly, the fluorescence signal of either Hg2+ or CH3Hg+ decreases remarkably. Excessive high Ar flow rate can dilute the concentration of Hg before Hg vapor is transported efficiently. Therefore, the Ar flow rate is selected as 300 mL min−1 which is the smallest value of our equipment. Since the sample flow rate affects the transport of the species toward the cathode surface primarily, the changes observed in sensitivity will be produced by variations in the efficiency of the analyte reduction process. The effects of sample flow rate from 1.4 to 8.5 mL min−1 on response of Hg2+ and CH3Hg+ are studied. From Figure 7B, we can find that the signal intensity of Hg2+ increases still with increasing the sample flow rate. CH3Hg+ increases remarkably with the increasing sample flow rate until 5.5 mL min−1, after which the signal decreases. Therefore, the sample flow rate of 5.5 mL min−1 is chosen. At 9204



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Table 1. Effect of Coexisting Ions on the Determination of 2μg L−1 CH3Hg+ and Hg2+ recovery (%)

recovery (%)

species

concentration (mg L−1)

Hg2+

CH3Hg+

species

concentration (mg L−1)

Hg2+

CH3Hg+

Ni2+

1 5 20 100 1 5 20 100 1 5 20 100

103 95 97 84 97 104 111 86 106 115 94 69

107 109 114 82 94 102 113 79 93 88 77 42

Pb2+

0.2 0.4 0.8 10 0.2 0.5 1 10 1 5 20 100

94 83 54 47 95 77 62 52 103 108 94 75

92 71 45 29 91 70 55 24 105 101 88 53

Fe2+

Fe3+

Cu2+

Co2+

Where CHg represents the concentration (μg L−1) of Hg. Equation 3 described the linear graphs for the determination of Hg2+ under an electric current of 0.2 A, and eq 4 described the linear graphs for the determination of total Hg (T-Hg) with electric current at 2.2 A. The calibration graphs are linear from 0.5 μg L−1 up to 10 μg L−1 for Hg. Repeatability, expressed as the relative standard deviation (RSD), is 4.7% (n = 6) for 2 μg L−1 CH3Hg+ standard and 3.2% (n = 6) for 2 μg L−1 Hg2+ standard. The limits of detection (LODs), using the definition 3s/m (s is the standard deviation and m is the slope of the calibration graph), are 0.073 and 0.098 μg L−1 for CH3Hg+ and Hg2+, respectively. The detailed data are plotted in Figure S6 of the Supporting Information. The influence of CH3Hg+ on the efficiency of generation of Hg2+ should be considered, especially when the CH3Hg+ content in the real samples is several fold higher than the Hg2+ content. The response of different mixed solution of Hg2+ and CH3Hg+ are shown in Figure S7 (Supporting Information) with the Hg2+ response taken as unity. It is found that the influence problem of CH3Hg+ on Hg2+ determination may be able to be solved by reducing the current. The signal intensities of Hg2+ and CH3Hg+ vs number of injections are evaluated, and the results are shown in the Figure S8, Supporting Information. This result documented that the new prepared cathode can be used for more than 20 cycles. Analysis of Real Samples. To evaluate the usefulness of the developed method for the determination of Hg2+ and CH3Hg+, a certified reference material (CRM, DORM-2 dogfish muscle, NRCC, Ottawa, Canada) and three seafood samples purchased from local markets are analyzed. Because of the significant interferences arising from the presence of some metal ions including Cu2+ and Pb2+, the method of additions is used to reduce this influence. The reliability of the developed method is tested with recovery experiments by adding Hg2+ and CH3Hg+ standard solutions into the sample solution before the sample preparation begins. The analytical results and recoveries are listed in Tables 2 and 3, and their recoveries are satisfactory. t tests are performed, and no significant difference is found between the certified values and obtained values in this work. The relative standard deviations for 5 replicate determinations of Hg2+ and total Hg in real samples are 6.6% and 8.1%, respectively. In this paper, we first research the EVG behavior between Hg2+ and CH3Hg+ on an L-cys modified graphite cathode. The results show a dramatic difference between Hg2+ and CH3Hg+ at applied currents of 0.2 and 2.2 A and then provided an

the same time, the vapor generation efficiency of Hg2+ and CH3Hg+ on the modified cathode decreases from 92.4% to 52.3% and 95.5% to 50.7%, respectively, and the detailed data are plotted in Figure S5 of the Supporting Information. Here, the vapor generation efficiency is calculated by determination of analytes remaining in the waste liquid. Interference Studies. Transition metal (Cu, Co, Ni, and Fe) ions are usually considered as serious interference because these ions are easily reduced to their metallic states or colloidal forms during electrolysis, which scavenge or decompose the volatile analyte forms before the accomplishment of phase separation in EVG methods.44 To assess the effect of the presence of these transition metal ions and hydride-forming elements in solution on Hg determination by EVG-AFS, considering that the matrix of seafood samples is complex, the effects of several ions at three different concentrations on the determination of 2 μg L−1 Hg2+ and CH3Hg+ are studied, respectively. The effect is expressed as the recovery in the presence of interfering ions relative to the interference-free response. Variations of less than 10% of recovery may be considered insignificant. The results are shown in Table 1. It can be seen that Cu2+ and Pb2+ cause the most serious interference. The presence of 1.0 mg L−1 Cu2+ produced 38% and 45% signal suppression on Hg2+ and CH3Hg+ signals, while 0.8 mg L−1 Pb2+ can lead to over 45% signal suppressions on both CH3Hg+ and Hg2+ signals. The effects of hydride-forming elements on the determination of Hg species are also studied. The results are listed in Table S2 of the Supporting Information. 1.0 mg L−1 Bi3+, Cd2+, Ge4+, Se4+, Sn2+, and Zn2+ and 0.1 mg L−1 As3+ have no significant influence on the measurement of CH3Hg+ and Hg2+. Moreover, Na+, K+, Ca2+, Mg2+, Ba2+, Al3+, CO32−, and H2PO4− did not interfere with the determination. Analytical Figures of Merit. The analytical characteristics of electrochemical cold vapor generation are evaluated under optimal operating conditions. Total Hg is measured from different sets of Hg2+ and CH3Hg+ standards separately at an applied current of 2.2 A. Statistically indistinguishable calibrations for Hg2+ and CH3Hg+ are obseved, suggesting that either Hg2+ or CH3Hg+ standards can be used as calibrant for total Hg measurement. The linear graphs correspond to the equation: If = −8.22 + 345.43

C Hg , r = 0.9974

(3)

If = 38.09 + 675.68

C Hg , r = 0.9931

(4) 9205



Article

Table 2. Analytical Results for Hg2+ and CH3Hg+ Determination in the CRM, DORM-2a 2+

−1

Hg (μg g ) T-Hg (μg g−1)

certified value24

foundb

foundc

0.710 ± 0.412 4.64 ± 0.26

0.702 ± 0.248 4.59 ± 0.42

0.713 ± 0.219 4.66 ± 0.23

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a The value was mean ± standard deviation. bResults obtained by EVG-AFS. cResults obtained by CVG-AFS.51

Table 3. Analytical Results of the Determination of Hg2+ and CH3Hg+ in Biological Samples CH3Hg+a

Hg2+ sample

found (μg kg−1)

recovery (%)b

found (μg kg−1)

recovery (%)

mussel razor clam baby clam

Speciation of Inorganic- and Methyl-Mercury in Biological Matrixes by

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