Microchemical Journal 103 (2012) 1–14

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Microchemical Journal

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Review article

Determination and speciation of mercury in environmental and biological samples byanalytical atomic spectrometry

Ying Gao a,b, Zeming Shi b, Zhou Long a, Peng Wu a, Chengbin Zheng a, Xiandeng Hou a,⁎a Analytical & Testing Center, and College of Chemistry, Sichuan University, Chengdu, Sichuan 610064, Chinab College of Nuclear Technology and Automation Engineering, Chengdu University of Technology, Chengdu, Sichuan 610059, China

⁎ Corresponding author. Tel.: +86 28 8547 0818; faxE-mail addresses: houxd@scu.edu.cn, houxiandeng@

0026-265X/$ – see front matter © 2012 Elsevier B.V. Aldoi:10.1016/j.microc.2012.02.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 January 2012Accepted 6 February 2012Available online 9 February 2012

Keywords:ReviewMercuryAtomic absorption spectrometryAtomic emission spectrometryAtomic fluorescence spectrometryAtomic mass spectrometrySample preparationCold vapor generation

Mercury and its compounds are ubiquitous in the environment. Much concern has been attracted to the de-termination of mercury and its species due to their high toxicity and biomagnification. The state of the arts ofmercury determination and speciation analysis and its applications in environmental and biological sciencessince 2008 are reviewed with 133 references. The methodological innovations in sample preparation, precon-centration, instrumentation and speciation analysis are summarized, and the future perspectives are brieflydiscussed and speculated.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. Total mercury determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3.1. Chemical vapor generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2. Instrumental developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.3. Pre−concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.3.1. Liquid–liquid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.3.2. Solid phase (micro-)extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4. Speciation analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.1. Chromatographic method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.1.1. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.1.2. High performance liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.1.3. Capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

4.2. Non-chromatographic methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115. Conclusions and future trends. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. Introduction

Mercury is regarded as one of the most toxic elements impacting onhuman and ecosystem health, which is released into the environment

: +86 28 8541 5695.yahoo.com.cn (X.D. Hou).

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from both anthropogenic and natural sources. As the result of popula-tion growth and urbanization, more and more human activities havebeen contributing to significantly elevated mercury emission. Morethan 2500 tons of mercury is emitted annually from global anthropo-genic sources [1]. Currently, mercury pollution becomes a worldwideenvironmental problem. The toxicity, bioavailability and mobility relynot only on their total concentration but also significantly on their

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chemical forms [2]. Generally, organic mercury compounds, especiallyin the form of MeHg+, are more toxic than inorganic mercury and ele-mental mercury, due to their high biomagnification factor (up to 106)in the food chain and their high liposolubility. However, all of the mer-cury released in the ecosystem undergoes biogeochemical transforma-tion processes and can be converted into MeHg+ by microorganismsandmicroalgae in aquatic environments [3]. Therefore, onlymonitoringthe totalmercury concentrations in the environment is not enough, andspeciation analysis provides more useful information to assess the tox-icity and health risks of mercury and further understand biogeochemi-cal cycling of mercury compounds. Consequently, tremendous effortshave been expended to develop reliablemethodologies formercury de-termination and speciation analysis. In fact, reviews on the determina-tion and speciation analysis of mercury species in natural waters havebeen published [4,5]. These two reviews paid particular attention tothe necessity for clean sampling procedures, proper sample storage,and the performance of different separation techniques including liquidchromatography (LC), gas chromatography (GC) and capillary electro-phoresis (CE).Measurement techniques for mercury species in ambientair was also discussed [6]. Solid phase microextraction (SPME) basedmercury speciation analysis and chemical vapor generation inductivelycoupled plasma mass spectrometry (CVG-ICP-MS) related mercury de-termination techniques were reviewed in 2008 and 2007, respectively[7,8]. Therefore, this review focuses on the determination and specia-tion analysis of mercury species since 2008, covering the major devel-opments in mercury determination and speciation analysis inenvironmental and biological samples by atomic spectrometry.

2. Sample preparation

Sample preparation is a key step for the accurate determination ofmercury in different sample matrice. For total mercury determina-tion, sample digestion prior to analysis is usually needed for the de-composition of organic compounds in sample matrix [9]. Oxygenflask combustion [10], microwave-induced combustion [11], as wellas sample preparation with tetramethylammonium hydroxide(TMAH) solubilization [12,13] are all found their analytical applica-tions for mercury determination in environmental and biologicalsamples. Microwave-assisted acid digestion, offering rapid sampledecomposition and high analyte recovery, is now still widely appliedto mercury analysis [14–17]. However, the development of on-lineand automated sample preparation methods to reduce sample analyt-ical time and simplify sample pretreatment process also draws muchattention. Han et al. reported an automated pressurized digestiontechnique based on electromagnetic induction acid-aided heatingfor on-line decomposition of solid samples [18]. High digestion effi-ciency was obtained due to the 0.36 MPa pressure and 135 °C reactiontemperature originated from the back pressure of a flow restrictor,which was installed downstream. They successfully applied this sys-tem to the determination of mercury in edible seaweeds. Leopold etal. utilized the extraordinary oxidation power of bromine monochlor-ide to develop a fully automated flow injection system for the trans-formation of dissolved mercury species to Hg2+ as well as theoxidation of dissolved organic carbon [19]. The comparison of the de-termination values by this method with those by standard EPA meth-od was performed to validate the accuracy of the technique. Inaddition, slurry sampling without complicated sample preparationprocess is another good alternative for conventional digestion ofmany type of samples. Aqua regia [20] and hydrochloric acid [21] so-lutions were used as the solid dispersion reagent for the determina-tion of mercury in serum blood and cereals by mercury cold vaporgeneration atomic fluorescence spectrometry (CV-AFS) and mercurycold vapor generation inductively coupled plasma mass spectrometry(CV-ICP-MS), respectively. Formic acid was also applied for the deter-mination of mercury in geological samples based on ultrasonic slurrysampling followed by photochemical vapor generation atomic

absorption spectrometric (photo-CVG-AAS) detection [22]. It isnoted that formic acid is used not only as a sample stability reagentbut also a photo-assisted reductant for formation of elementalmercury.

For mercury speciation analysis, microwave/sonication assistedacid leaching or alkaline dissolution methods are more favorableand have experienced sustaining increase in recent years because oftheir simple devices and high extraction efficiencies [23]. de Souzaet al. [24,25] and Batista et al. [26] used the mixture of mercaptoetha-nol, L-cysteine and hydrochloric acid for fast sonication assistant ex-traction of mercury species from hair, blood, and seafood within15 min. Rodrigues et al. [25] established a fast sample preparationprocedure for mercury species determination in blood samples byusing microwave assisted extraction with TMAH. Only 10 min wasneeded for sample pretreatment, resulting in time-saving and highsample throughput compared with previous extraction procedure.Gao et al. [27] compared different microwave/sonication assisted ex-traction methods for the analysis of mercury species in coal samples,and found that microwave assisted extraction at the power of 90 Wfor 5 min using TMAH as the extractant provided the most efficientextraction for methylmercury and ethylmercury. Simultaneous appli-cation of an ultrasonic bath and an ultrasonic probe for sample prep-aration can accelerate the extraction rate and improve the extractionefficiency of mercury species as well, especially for organic Hg. Basedon this fact, Zhang et al. proposed a dual frequency ultrasonic extrac-tion device for mercury species determination [28]. The extraction ef-ficiency of organic Hg was enhanced by at least 40%, compared withsingle ultrasonic probe at 20 kHz or single ultrasonic bath at 40 kHzunder the same conditions.

To further reduce the sample preparation time, Torres et al.developed a total and inorganic mercury determination method bypreparation of biological samples as slurries in water instead of ininorganic acid and subsequent quantification with CV-AAS [29]. Nostanding time is required prior to the determination of Hg2+, result-ing in reduction of sample preparation time and minimization of thechance of sample contamination. For total mercury measurement, arapid oxidation of organic mercury with KMnO4 was needed. Themethod was applied to analysis of different reference materials,with analytical results in good agreement with the certified values.

The increasing interest in metallomic research needs more gentledigestion to preserve complete molecular information. Enzymatic hy-drolysis, which can split analytes frommacromolecules by adding en-zymes into sample solutions without altering their chemical forms,seems to be a good choice. Lemes et al. developed a method for thedetermination of MeHg-cysteine complexes, MeHg-glutathione com-plexes as well as MeHgX and inorganic HgX in a dogfish muscle sam-ple by HPLC-ICPMS after enzymatic hydrolysis with trypsin [30]. Theanalytical results offered the first evidence for the presence and dom-inance of MeHgCys in fish muscle. Later, Chung et al. used pancreatinand hydrochloric acid digestion for the determination of MeHg+ andEtHg+ in foods with GC-ICP MS [31]. However, the long sample prep-aration time (5–24 h) and low analyte recoveries are the major draw-backs of enzymolysis for mercury determination. The use ofultrasound can accelerate the enzymatic digestion process and short-en the extraction time to a few minutes [32]. However, there are stillno reports concerning its analytical application for mercury specia-tion analysis.

3. Total mercury determination

3.1. Chemical vapor generation

Chemical vapor generation (CVG) remains the most popular andsuccessful sample introduction approach for trace Hg determination,which can easily separate analytes from the troublesome sample ma-trix and improve the sample introduction efficiency compared to the

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traditional pneumatic nebulization. Among CVG techniques, coldvapor generation (CV) using tetrahydroborate (THB) or SnCl2 as a re-ductant is presently considered to be the most widespread one for thedetermination of trace/ultratrace mercury in varied matrix includingnatural water, biological samples, geological samples, and so on. Butinterferences from transitionmetals in THB system often decrease de-tection sensitivity and reproducibility, leading to incorrect analyticalresults. Moreover, contamination from THB reagent and the instabili-ty of its solutions limit the determinable concentration range of mer-cury. Additionally, the toxicity and low vapor generation efficiencyapparently restrict the wide application of SnCl2 system [33]. As aconsequence, other CVG strategies have been developed for mercurydetermination in analytical atomic spectrometry, including electro-chemical vapor generation, photo-CVG, ultrasound-promoted CVG,and so on.

Electrochemical vapor generation is a cost/labor-effective tech-nique for Hg cold vapor generation which eliminates the used of ex-pensive reducing agent and its concomitant contamination, leadingto low blank and excellent limit of detection. The cathode materialand electrolyte are the most concerned issues that significantly con-tribute to the vapor generation efficiency of mercury. A polyanilinemodified graphite electrode was used by Jiang et al. as cathode mate-rial for Hg electrochemical vapor generation followed by AFS deter-mination [34]. The use of polyaniline modified electrode provideshigher sensitivity, better stability and lower memory effect comparedwith a conventional graphite electrode. The developed technique wassuccessfully applied to the determination of mercury in human hairs.Zhang et al. reported an electrolytic CVG system for mercury vaporgeneration on Pt/Ti cathode in the presence of formic acid catholyte[35]. They found that formic acid catholyte favored the mercuryvapor generation and elimination of interferences from the transitionmetal ions. The influences of the experimental conditions on the CVGwere investigated; the detection limit of 1.4 ng L−1 was achievedunder optimal conditions. The authors also investigated the effectsof different buffer solutions and traditional inorganic acid on mercuryelectrolytic CVG efficiency [36]. Phosphate buffer solution (PBS)showed good performance in increasing mercury CVG efficiency andreducing the impact of cathode erosion. However, the desire of care-ful selection of cathode material and the request of frequent condi-tioning of the cathode surface are still a challenge, which needed tobe settled down to further improve the reproducibility and reducethe memory effect for mercury determination.

Photo-CVG, utilizing free radicals produced by photo-redox reac-tions as reductants, appears as a promising greener technique forsample introduction. For example, Hg and its species can be efficient-ly converted into Hg0 by hydrogen and carboxyl radicals arising fromphotodecomposition of low molecular weight organic compounds (e.g., alcohol, formic acid, acetic acid or formaldehyde). By direct coldvapor generation of mercury from alcohol medium, Li et al. [37] de-veloped a sample matrix-assisted method for the ultrasensitive deter-mination of mercury in wine and liquor samples. Mercury wasconverted into Hg0 with the assistance of sample matrix under UV ir-radiation, resulting in reagent-free green analytical methods. Later,Liu [38] successfully applied this technique for the determination ofmercury in wine vinegar samples with satisfactory results. In fact,early studies in environmental sciences and physical chemistry haveshown that Hg2+ can be reduced to atomic mercury vapor in distilledwater and natural water through several complicated photochemicalprocedures [39]. Based on those research results, Xia et al. [40] furtherexplored the analytical application of the matrix assisted photo-CVGtechnique to quantify Hg2+ in natural water samples. Bendicho etal. [41] utilized the synergic effect of photo-CVG and ultrasoundassisted gas–liquid separation for improved determination of mercu-ry in biotissues. In this processes, formic acid not only served as aphoto-assisted reagent for Hg reduction but also as a solubilizer forthe biological tissues. Ribeiro et al. [42] examined the feasibility of

using CVG for sample induction into furnace atomization plasmaemission spectrometry for mercury determination. Both conventionalCVG and photo-CVG systems were investigated. They found that the70 W low power He plasma could tolerate to water vapor introducedfrom gas–liquid separator, thus the dehydration process was unnec-essary. The detection limits were 250 pg mL−1 and 240 pg mL−1 formercury determination after photo-CVG and SnCl2 reduction, respec-tively. However, the use of THB-acid system produced too much hy-drogen, extinguishing the He plasma.

As a matter of fact, the generation of free radicals and electronsalso occurs in different discharge processes. Many innovated mercu-ry vapor generation techniques have been developed on the basis ofdifferent discharge techniques, as shown in Fig. 1. Wu et al. [43]presented a laboratory-built mini-dielectric barrier discharge(DBD) for high yield production of Hg0. Mercury solution was intro-duced into the low temperature argon plasma via the mini-DBD, inwhich Hg2+ was transformed into Hg0 without or with formicacid. The vapor generation efficiency was significantly enhanced inthe presence of formic acid and superior to conventional SnCl2–HCl system. The technique is featured low power consumption,cost-effectiveness, green analytical method, and ease of operation.Furthermore, a particularly simple design of electrolyte cathodeatmospheric-pressure glow discharge (ELCAD) named solution-cathode glow discharge (SCGD) has been developed for mercuryCVG [44]. The discharge is ignited and sustained between the tipof tungsten rod and the outlet of a capillary tube using for samplesolution delivery. When sample solutions were fed into the dis-charge, dissolved mercury species in solution were directly trans-formed into volatile Hg vapor without the need of a chemicalreducing agent and subsequently transported into a commercialICP-AES for the detection by an argon stream after gas–liquid sepa-ration. The use of low molecular weight compounds facilitated mer-cury reduction, with a vapor generation efficiency enhanced by afactor of 2–3, compared to that from 0.1 M HNO3 electrolyte.Under optimal conditions, the detection limit was found to be0.7 μg L−1 in the continuous sample introduction mode. Lately, anew design of electrolyte cathode glow discharge technique was de-veloped by Shekar et al. [45] by using a V-groove to the liquid glass-capillary to eliminate plasma fluctuations arising from the variationin the gap between solid anode and liquid cathode, thus leading to astable plasma even at low flow rates (0.96 mL min−1). Subsequent-ly, they explored the application of this new design for the determi-nation of mercury in hepatitis-B vaccine with AES detection [46].This new technique obviated the need of sample digestion and theuse of chemical reagents for organic mercuric compounds decompo-sition and mercury CVG. Consequentially, the risk of analytes con-tamination as well as analysis time was greatly reduced. Themethod was successfully applied to the analysis of mercury in dif-ferent biological certified reference materials.

dos Santos et al. [15] reported a newmethodology for mercury de-termination based on reduction of mercury at high pH solution. It isworthwhile to note that a reducing agent, like TBH and SnCl2, is notnecessary here for mercury vapor generation. The scheme was ap-plied to the analysis of biological samples with pleasant results. How-ever, the mechanism of this CVG procedure is not clear yet.

3.2. Instrumental developments

The innovation of analytical instruments either in components orthe whole devices is another important aspect in the advancementof atomic spectrometry. Ramezani et al. [47] reported a methodusing a low density polyethylene window instead of quartz windowfor the mercury determination by CV-AAS. The reliability was evalu-ated by the determination of mercury in different certified referencematerials and real samples. Obviously, the low density polyethylenefilm is cheaper than quartz.

Fig. 1. Schematic diagrams of the discharge induced vapor generation system: A. DBD reactor [43]; B. closed SCGD cells [44]; and C. new ELCAD-AES system [45].B, reprinted with permission from [44]. Copyright (2008) American Chemical Society. and C, reprinted with permission from [45]. Copyright (2009) American Chemical Society.

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The development of miniaturized analysis systems, which over-come the problems associated with high cost and large size of manycurrent equipments, becomes an important trend in analytical chem-istry. To achieve miniaturized analytical systems, the successful min-iaturization of main components of instruments is critical.Microplasmas based analytical techniques have attracted much atten-tion due to their reduced gas and power consumption, small size, andrelatively low manufacturing cost. A low-power, low-temperaturemicroplasma source based on DBD, was developed as an emissionsource for optical emission spectrometry (OES) by Zhu et al. [48].The microplasma is produced within a glass cell between two elec-trodes and can be operated without the removal of residual watervapor. Therefore, conventional sample introduction can be used. TheDBD induced microplasma has relatively modest gas temperatureand low continuum and structured background emission from 200to 260 nm. The DBD source has been applied to the determinationof Hg vapor derived from SnCl2 system after separating from the sam-ple solution by a commercial ICP concentric pneumatic nebulizer, andoffers detection limits from 14 (He-DBD) to 43 pg mL−1(Ar-DBD).Later, they reconstructed the cold DBD device and employed it as avaporization technique for mercury determination [49]. The plasmawas generated concentrically in-between two quartz tubes. One ofthe quartz tube embedded with a copper electrode was serviced asa sampling tool, allowing sample coating onto the outer surface ofquartz tube. Then, the quartz tube was directly introduced into theDBD chamber; the sample solution coated on the quartz tube wasquickly vaporized and atomized when igniting the plasma. Underthe optimized conditions, the limits of detection were 0.02 ng mL−1

for Hg2+, MeHg+ or EtHg+. Furthermore, the low consumption ofsample volume (~6 μL) and power (b5 W) makes the proposed tech-nique more attractive.

At the same time, Wang et al. presented a miniaturized OES sys-tem for mercury determination based on DBD induced microplasmatechniques [50]. In this portable system, a small Ar-DBD wasemployed as the radiation source and a portable charge-coupled de-vice (CCD) spectrometer as the detector. The plasma is ignited andmaintained by a neon power supply with a 35 kHz high-frequencydischarge. A stable and homogeneous microplasma is observed at1.20–1.80 kV. Sample introduction is achieved with mercury cold

vapor generation by a microsequential injection unit. The emissionline of mercury at 253.65 nm clearly isolated from the backgroundwas adopted for quantification. Under optimized experimental condi-tions, a detection limit of 0.2 ng mL−1 was achieved, which is compa-rable with other OES system for mercury determination. Recently,Puanngam et al. [51] further developed the minimized system andapplied it for automated determination of atmospheric Hg0 aftergold coated tungsten filaments entrapping. As described by Wang etal., the preconcentration process was beneficial for eliminating thelarge background fluctuations in the DBD emission arising from ambi-ent humidity and further improving analytical sensitivity [50]. Afterpreconcentration for 2 min, the estimated detection limit was0.12 ng L−1 for Hg0 determination and the linear range was extendedto 6.6 ng L−1.

The need of downscaling chemical assays also promotes the devel-opment of microfluidic systems, which offers simplified and automat-ed analytical procedures, compacted apparatus as well as enhancedrepeatability of sample operation as a result of its permanent rigidstructure. Aristidis et al. [52] proposed a lab-on-a-valve (LOV) plat-form which incorporated a sorbent microcolumn and membranelessgas–liquid separation for mercury CV-AFS assays. The proposed sys-tems facilitated on-line sample processing and trace mercury deter-mination on a LOV manifold. The preconcentration of mercury wascarried out onto the surfaces of reversed-phase co-polymericOasis™ HLB beads. The absorbed analytes was eluted by a mixtureof HCl and HNO3, subsequently mixed downstreamwith SnCl2 to gen-erate Hg cold vapor in an integrated reaction chamber/gas liquid sep-arator. The produced Hg cold vapor was transported to a peripheralAFS for detection. The detection limit of 0.04 μg L−1 and repeatabilityof 3.8% at a concentration level of 10.0 μg L−1 were obtained, respec-tively. With the rapid development of miniaturized analysis systems,their analytical applications, ranging from environmental analysis tofood safety, can be possible.

3.3. Pre−concentration

Because of the extremely low concentration levels of mercury inmany type of samples and its potentially complicated matrix, the pre-concentration and separation of the analytes prior to measurement

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are frequently demanded even with a highly sensitive and sophisti-cate detection system. Twomain approaches for the preconcentrationof trace elements from water have dominated the literature duringthis review period: liquid–liquid extraction (LLE), and solid phase ex-traction (SPE). The trial of potential chelating agent for LLE or chem-ical modifier for SPE adsorbents, the development of green/reagent-free methods as well as the miniaturization of existing techniquesseem to be the main trend for mercury preconcentration.

3.3.1. Liquid–liquid extractionCloud point extraction (CPE) utilizing “could point” phenomenon

of surfactant for preconcentration is a “green chemistry” approachas it is solventless extraction. Yuan et al. [53] presented an approachfor the determination of trace mercury by AFS in environment sam-ples after complexation of Hg2+ with dithizone and micelle-mediated extraction of the resulted complex with Triton X-114. Thelimit of detection obtained under the optimal conditions is 5 ng L−1.Depoi et al. [54] and Shah et al. [55] enriched Hg2+ from honey andbroiler chicken tissues using O,O-diethyldithiophosphate (DDTP) asa complexing agent and Triton-X-114 as the extractant for CV-ICP-OES and CV-AAS measurement. The limits of detection (LOD) were2.2 ng g−1 and 0.4 ng g−1 for honey samples and broiler chicken, re-spectively. Ionic liquid extraction (ILE) is also regarded as an alterna-tive to traditional LLE. The inherent characteristics of ionic liquid,such as water stability, negligible vapor pressure, and changeable vis-cosity and density, make ILE to be an attractive technique in separa-tion processes. Without the need of generating cloud point byheating or adding large amount of electrolytes, the ILE process is sim-pler compared to CPE. Martinis et al. [56] used 1-butyl-3-methylimi-dazolium hexafluorophosphate ([C4mim][PF6]) to quantitativelyextract mercury after forming its complex with 2-(5-bromo-2-pyri-dylazo)-5-diethylaminophenol (Hg-5-Br-PADAP). Subsequently, theanalyte was back-extracted with 9.0 mol L−1 HCl from the ionic liq-uid phase into an aqueous media. A detection limit of 2.3 ng L−1

was obtained by CA-AAS determination under optimal conditions.Miniaturized preconcentration methods, involving liquid-phase

microextraction (LPME) and solid-phase microextraction (SPME),have achieved expansive development due to their high preconcen-tration factors and minimized organic reagents consumption. LPMC,such as singles drop microextraction (SDME), hollow fiber liquidphase microextraction (HFMC) and dispersive liquid–liquid microex-traction seem (DLLME), is particularly suitable for ETAAS and ETV-ICP-MS for its tiny volume of the enrichment phase. Lopez-Garcia etal. [57] presented an approach for the preconcentration of trace mer-cury with solidification of floating undecanoic acid droplet and deter-mination by ETAAS. The weak acid, with a melt point temperature of28–31 °C, acted both as the complexing and extracting agent in thisexperiment. Under the optimal conditions, a detection limit of70 ng L−1 with an enrichment factor of 430 was obtained. Bagheriet al. [58] combined SDME with ETAAS for the determination oftrace mercury in water samples. A microdrop of m-xylene containingdithizone was applied as the complexing and extraction agent. Opti-mized experimental parameters led to a high enrichment factor of970 and a detection limit of 0.01 μg L−1. Despite reducing the con-sumption of volatile organic compounds, the use of conventional vol-atile solvents in LPME is inevitable in many cases. Therefore, thedevelopment of more greener and more efficient extraction schemesis still demanded.

3.3.2. Solid phase (micro-)extractionSolid phase (micro-) extraction (SPE/SPME) is the most popular

sample preconcentration method for its simplicity, high enrichmentfactor and low consumption of organic solvents. Publications onSPE/SPME for mercury determination in environmental and biologicalsamples were summarized in Table 1. The typical sorbents are de-rived from chemical or physical immobilization of suitable organic

agents to different solid surfaces on solid supports. As shown inTable 1, undoubtedly, sulfur containing molecules as ligands are pre-ferred for mercury preconcentration because of their high affinity formercury species leading to high preconcentration factors and high se-lectivity for Hg.

Also, based on the high affinity of mercury on the surface of gold, areagent-free method was reported by Leopold et al. for the determi-nation of dissolved mercury in natural waters by atomic fluorescencespectrometry [59,60]. The nano gold-coated silica adsorbent directlyadsorbed mercury species from acidified aqueous solution due tothe catalytic activity surface of nano-structure gold [59]. Subsequent-ly, liberation of trapped mercury was carried out by thermal desorp-tion. As a result, no reagents were needed for species conversion,preconcentration, sample storage and desorption, benefiting to re-duced contamination and lowered blank. Consequently, a very im-pressive LOD for Hg was achieved at 180 pg L−1, by using a samplevolume of 7 mL (Fig. 2).

On the other hand, the exploration of new materials, especiallynanometer sized materials, as the support phase is another active re-search area in SPE/SPME for mercury determination. The use of nano-particles leads to higher extraction capacity/efficiency and rapiddynamics of extraction originated from the higher surface area to vol-ume ratio and short diffusion route. Zhang et al. developed a methodfor the preconcentration of mercury on the surface of nano silicamodified with 2,6-Pyridine dicarboxylic acid [61]. After careful inves-tigation of analytical parameters affecting analyte recoveries, such asacidity, shaking time, eluent condition, sample volume, sample flowrate, and influence of potentially interfering ions, a maximum uptakecapacity for mercury was found to be 92 mg g−1 at pH 3. Moreover,the use of magnetic nano-materials as solid support phase has pro-moted an increasing application of SPE in elements preconcentration.Target analytes adsorbed on the superparamagnetic particles can bequickly separated from the matrix in a magnetic field, which facilitiesthe SPE processes and largely reduces analysis time. Zhai et al. utilized1,5-diphenylcarbazide doped magnetic Fe3O4 nanoparticles as extrac-tant for the preconcentration of mercury from aqueous solution [62].Under optimal conditions, the maximum adsorption capacity was220 μmol g−1, with a detection limit (3σ) of 0.16 μg L−1 for CV-AASdetermination. Sodium dodecyle sulfate-coated magnetite nanoparti-cles were also proposed for trace mercury determination in environ-mental samples [63]. The detection limit of 0.04 ng mL−1 wasachieved for ICP-OES determination after preconcentration.

Due to biocompatible ability and easy control of magnetic nano-particles, the application of magnetic SPME in microfluidic analysishas been extended. Chen et al. developed a chip-based magneticSPME method for Cd, Pb and Hg determination in cultured cells by in-tegration of micro sample introduction and magnetic SPME on amicrofluidic chip, coupled to electrothermal vaporization (ETV)-ICP-MS detection [64]. Under an external magnetic field, γ-mercaptopropyltrimethoxysilane (g-MPTS) modified silica-coatedmagnetic nanoparticles self-assembled in microchannels to form asolid phase packed column. The chip-based technique allowed for alimit of detection down to 0.86 ng L−1 and an enrichment factor of42 for mercury determination.

Application of headspace-SPE/SPME (HS SPE/SPME) to trace mer-cury preconcentration is another typical approach for mercury deter-mination. Noble metals including gold, palladium and silver areobviously favorable solid sorbents for trapping mercury vapor. Mou-savi et al. [65] employed silver wool solid sorbent for retaining mer-cury vapor prior to AAS determination, providing a detection limitof 3 ng L−1 for mercury. Madden et al. suggested trapping mercuryvapor generated from photo-CVG onto a palladium coated graphitefurnace and AAS for determination [66]. Under optimized conditions,a detection limit of 0.12 μg L−1 was achieved, which was 9 times bet-ter than that of previous method without preconcentration. Romeroet al. used different Pd-based substrates for the microextraction of

Table 1Solid phase preconcentration for mercury in environmental and biological samples.

Extractant Support material Eluent Detectionmethod

LOD Sample Volume Real samples Ref.

5,5′-dithiobis(2-nitrobenzoicacid)

Octadecyl silicamembrane disks

1 mol L−1 HCl AAS 2 ng L−1 – Water samples [68]

1-acylthiosemicarbazide Activated carbon 2% CS(NH2)2 and2.0 mol L−1 HCl

ICP-OES 0.12 μg L−1 300 mL Natural waters [69]

Gold Silica Heating AFS 180 pg L−1 7 mL Natural waters andwastewaters

[59]

2-mercaptobenzimidazole Agar powder 3 mol L−1 HCl AAS 0.02 μg L−1 250 mL Drinking waters, wastewatersand fish samples

[70]

N-(2-chlorobenzoyl)-N′-Phenylthiourea

Sulfur powder 3 mol L−1 HCl AAS 0.012 μg L−1 250 mL Water samples,otolithes, platicephalusand oyster

[71]

2-(2-oxoethyl)hydrazinecarbothioamide

Silica gel 0.5 mol L−1 HCl and1% CS(NH2)2

ICP-OES 0.1 μg L−1 100 mL River water and tap water [72]

Mercaptopropyltrimethoxysilane Silica coated magneticnanoparticles

ETV-ICP-MS 0.86 ng L−1 0.5 mL Cells [64]

Ion-exchange resin AG 1× 4 microcolumn 0.05% L-cysteine and0.5 M HNO3

MC-ICP-MS – – Freshwaters [73]

Sodium dodecyle sulfate Magnetitenanoparticles

1-propanol ICP-OES 0.04 μg L−1 1000 mL Tap water, well water andmineral waters

[63]

1,5-diphenylcarbazide Magneticnanoparticles

0.5 mol L−1 HNO3 CV-AAS 0.16 μg L−1 200 mL Natural water andplant samples

[62]

2,6-Pyridine dicarboxylic acid Nanometer-sized silica 0.1 mol L−1 HCl and3% thiourea

ICP-OES 0.09 μg L−1 50 mL Environmental andbiological samples

[61]

Nano-structured gold Silica heating AFS 80 pg L−1 7.0 mL Natural waters [74]Sulfhydryl cotton fiber 0.1 mol L−1 HCl and

0.1 mol L−1 NaClCV-AAS 0.025 μg L−1 6.6 mL Edible seaweeds [75]

– Active carbon HNO3 CV-AAS 10 ng L−1 25 mL Tap water, hair samples [76]– Silver wool Heating AAS 3 ng L−1 50 mL Water and wastewater [65]Palladium Graphite furnace Heating AAS 0.12 μg L−1 50 mL – [66]– Palladium wire Heating AAS 90 ng L−1 5 mL Lobster hepatopancreas and

mussel tissue[67]

6 Y. Gao et al. / Microchemical Journal 103 (2012) 1–14

mercury vapor prior to releasing into a modified absorption cell forAAS detection [67]. It was found that Pd wire offered the best perfor-mance in respect to sensitivity and fiber lifetime, while Pd-coatedSiO2 fibers were more flexible for SPME.

4. Speciation analysis

4.1. Chromatographic method

The combination of chromatographic separation techniques withan element specific detector is a practical approach for mercury spe-ciation analysis [77]. ICP-MS and CV-AFS remain the most powerfuldetectors owning to their superior sensitivity. The separation power

Fig. 2. (A) Flow injection manifold for the on-line determination of total mercury by SPMEcoated silica. (1) silicone plugs; (2) quartz glass tube (i.d. 8 mm, wall thickness 0.5 mm); (wall thickness 0.5 mm).Reprinted with permission from [59]. Copyright (2008) American Chemical Society

and compatibility with an atomic spectrometer are the most impor-tant criteria for selection a separation technique for the hyphenation.Most separation methods discussed here can be directly connected toan atomic spectrometric instrument by using conventional or com-mercially available interfaces. Table 2 shows selected applicationsfor mercury speciation in environmental and biological samples.

4.1.1. Gas chromatographyThe coupling of GC with atomic spectrometers makes this tech-

nique a popular tool for mercury speciation [78,79]. The high separa-tion power of GC and the excellent detectability of modern atomicspectrometers are beneficial for mercury speciation analysis. As GCis usually utilized for volatile and semi-volatile compounds analysis,

onto catalytic active nano-gold collectors. (B) Photograph of the collector with gold-3) quartz wool wads; (4) gold-coated silica; and (5) quartz glass capillary (i.d. 1 mm,

Table 2Chromatographic techniques for mercury speciation analysis.

Analyte Matrix Derivatization Columns Preconcentration Detector Limit of detection Ref.

Gas chromatographyHg2+, MeHg+ Tuna fish,

human hairNaBPr4 MXT (Silcosteel) – MC-ICP-MS (IDA) – [88]

MeHg+, EtHg+ Fish, soil,sediment, water

NaBPh4 Fused-silica Purge-and-trap AFS, ICP-MS AFS:MeHg+: 0.03 ng L−1,EtHg+: 0.03 ng L−1

ICPMS:MeHg+: 0.02 ng L−1

EtHg+: 0.01 ng L−1

[87]

Hg2+, MeHg+, EtHg+ Fish, meat,soil, tree bark

Magnesiumbromide

DB-17 – ICP-MS Hg2+: 0.2 pg g−1,MeHg+: 0.09 pg g−1,EtHg+: 0.1 pg g−1

[83]

MeHg+, EtHg+ Fish, egg NaBPr4 Fused-silica HS-SPME Py-AFS MeHg+: 0.04 ng g−1,EtHg+: 0.13 ng g−1

[80]

Hg2+, MeHg+ – NaBEt4 Packed 15% OV3on Chromosorb

Purge-and-trap MC-ICP-MS (IDA) – [85]

MeHg+ Human blood NaBEt4 Rtx-1701 – MS, ICP-MS ICPMS: 300 ng L−1

MS: 500 ng L−1[89]

Hg2+, MeHg+ Freshwater NaBEt4 Packed 15% OV3on Chromosorb

Purge-and-trap ICP-MS (IDA) Hg2+: 0.074 ng L−1

MeHg+: 0.005 ng L−1[84]

MeHg+ Seafood NaBPh4 Phenomenex ZB1 – AES 6.1 ng g−1 [82]MeHg+,total Hg

Human hair NaBEt4 packed 10% OV3on Chromosorb

Headspacesampler trap

AFS, AAS MeHg+: 0.04 ng g−1 (AFS)total Hg: 1.5 ng g−1 (AAS)

[78]

MeHg+, EtHg+ Dogfish muscle,dogfish liver,synthetic water

NaBEt4 TRB-5DB-5MSMXT-1

– Pyro-AFSMSICP-MS

Pyro-AFS: MeHg+: 1.0 pg,EtHg+: 1.8 pgMS:MeHg+: 0.7 pg,EtHg+: 1.2 pgICP-MS:MeHg+: 0.05 pg,EtHg+: 0.06 pg

[90]

MeHg+, EtHg+ Foods NaBPh4

NaBPr4DB-5MS – ICP-MS MeHg2+: 0.3 ng g−1

EtHg2+: 0.3 ng g−1[31]

Hg2+, MeHg+, EtHg+ Whole blood NaBPr4NaBEt4

Capillary(Crossbond 100%dimethyl polysiloxane)

– ICP-MS NaBPr4:Hg2+: 50 fg,MeHg+: 50 fg,EtHg+: 20 fgNaBEt4Hg2+: 40 fg,MeHg: 40 fg+

[79]

Hg2+, MeHg+, C3H7Hg+ Biological SRMs NaBEt4 Packed column,capillarycolumn

Purge-and-trap AFS, ICP-MS Packed:AFS:MeHg+:0.042 pg,C3H7Hg+: 0.27 pg; ICPMS:MeHg+: 0.030 pg,C3H7Hg+: 0.25 pg.capillary:AFS:MeHg+: 0.25 pg,C3H7Hg+: 0.53 pg; ICPMS:MeHg+: 0.06 pg,C3H7Hg+: 0.08 pg

[86]

Hg2+, MeHg+ Dolphin liver NaBPr4 – – ICP-MS (IDA) – [81]

High performance liquid chromatographyHg2+, MeHg+ Seawater – Alltima HP C-18 (RP) SPME ICP-MS Hg2+: 0.07 ng L−1,

MeHg+: 0.02 ng L−1[95]

MeHg+, EtHg+ Sediment, coal – Shim-pack CLC-ODS (RP) – CV-AFS – [27]Hg2+, MeHg+, EtHg+ Flour – Perkin-Elmer C8 (RP) – CV-ICP-MS – [116]Hg2+, MeHg+,EtHg+, PhHg+

Seafood – Shim-pack CLC-ODS (RP) – Photo CVG-AFS Hg2+: 85 ng L−1

MeHg+: 33 ng L−1 EtHg+:29 ng L−1

PhHg+: 38 ng L−1

[105]

Hg Brain cytosol – TSK-GEL G3000SWxlColumn (SEC)

– ICP-MS 0.2 ng [91]

Hg2+, MeHg+, PhHg+ Water, humanhair, fish

– Discovery C18 (RP) CPE ICP-MS Hg2+: 6 ng L−1 MeHg+:13 ng L−1 PhHg+: 8 ng L−1

[99]

Hg2+, MeHg+ Urine – MSpak SP-80 4B (IE) – ICP-MS Hg2+: 100 ng L−1

MeHg+: 30 ng L−1[104]

Hg2+, MeHg+,EtHg+, PhHg+

Drinking water,sediment

– Nucleosil 100–5 C8 (RP) SPE ICP-MS Water:Hg2+: 4.6 ng L−1,MeHg+: 5.2 ng L−1

sediment:MeHg+: 500 ng L−1

EtHg+: 800 ng L−1

PhHg+: 1000 ng L−1

[101]

(continued on next page)

7Y. Gao et al. / Microchemical Journal 103 (2012) 1–14

Table 2 (continued)

Analyte Matrix Derivatization Columns Preconcentration Detector Limit of detection Ref.

HgX, MeHgX,MeHgCys, MeHgGlu(X=H2O, OH−,or Cl−)

Fish muscle – Luna C18(2) (RP) – ICP-MS HgX: 450 ng L−1 MeHgX:280 ng L−1,MeHgCys: 220 ng L−1,MeHgGlu: 230 ng L−1

[30]

Hg2+

MeHg+

EtHg+

Liquid cosmetic – XBbridge TM C18 Ionic liquid-basedDLLME

ICP-MS Hg2+: 1.3 ng L−1

MeHg+: 7.2 ng L−1

EtHg+: 5.4 ng L−1

[102]

Hg2+, MeHg+ Fish tissue – Gemini C18 (RP) – ICP-MS Hg2+: 800 ng L−1,MeHg+: 700 ng L−1

[117]

Hg2+, MeHg+, EtHg+ Fish tissue,human hair

– Eclipse XDB C18 (RP) – CV-AFS Hg2+: 400 ng L−1

MeHg+: 200 ng L−1

EtHg+:400 ng L−1

[109]

Hg2+, MeHg+, EtHg+ Hair – C18 (RP) – ICP-MS Hg2+: 15 ng g−1,MeHg+: 10 ng g−1 EtHg+:38 ng g−1

[24]

Hg2+, MeHg+ Fish – PRP X-200 (IE) – photo-CVG-AFS Hg2+: 100 ng g−1,MeHg+: 80 ng g−1

[103]

Hg2+, MeHg+ Human urineand serum

– PhenomenexLuna C18 (RP)

– ICP-MS Hg2+: 0.3 ng, MeHg+: 1 ng [118]

Hg2+, MeHg+ Blood – C18 (RP) – ICP-MS Hg2+: 100 ng L−1,MeHg+: 250 ng L−1

[25]

MeHg+, EtHg+ Urine – Brownlee AnalyticalC-18 (RP)

SPME ICP-MS MeHg+: 60 ng L−1

EtHg+: 60 ng L−1[97]

Hg2+, MeHg+, EtHg+ Fish andlobster tissue

– Shim-packCLC-ODS (RP)

– CV-AFS Hg2+: 100 ng L−1

MeHg+: 50 ng L−1

EtHg+: 70 ng L−1

[110]

Hg2+, MeHg+, EtHg+ Water – Zorbax XDB-C18 (RP) SPE ICP-MS Hg2+: 3 ng L−1 MeHg+:3 ng L−1 EtHg+: 3 ng L−1

[100]

Hg2+, MeHg+, EtHg+ Seafood – C8 (RP) – ICP-MS Hg2+: 0.25 ng g−1,MeHg+: 0.1 ng g−1,EtHg+: 0.20 ng g−1

[26]

Mercury bindingprotein

Rat tissue – Symmetry ShieldRP18 (RP)

– ICP-MS(IDA)

b200000 ng L−1 [92]

Hg2+, MeHg+, EtHg+ Seafood – ZORBAX SB-C18 (RP) – SCGD-AFS Hg2+: 670 ng L−1

MeHg+: 550 ng L−1 EtHg+:1190 ng L−1

[106]

Hg2+, MeHg+ Environmentalwater

– XBridge™ C18 (RP) DLLME ICP-MS Hg2+: 7.6 ng L−1, MeHg+:1.4 ng L−1

[98]

Hg2+, MeHg+ Dolphin liver – Superdex 200 (SEC) – ICP-MS (IDA) – [81]

Capillary electrophoresisHg2+, MeHg+ Goldfish – Fused-silica – AAS Hg2+: 27000 ng L−1 MeHg+:

35000 ng L−1[113]

Hg2+, MeHg+ River water – Fused-silica – ICP-MS Hg2+: 12000 ng L−1

MeHg+: 9700 ng L−1[114]

8 Y. Gao et al. / Microchemical Journal 103 (2012) 1–14

the derivatization of mercury to volatile and thermally stable speciesprior to GC separation is always the most important step. Aqueousethylation using sodium tetraethylborate (NaBEt4) is a very well ac-cepted strategy for mercury speciation analysis. After ethylation, vol-atile mercury species can be preconcentrated by SPME or directlysubject to GC-related detection. However, in case of EtHg+ as one ofthe target analytes, both Hg2+ and EtHg+ can be transferred to thesame product, diethylmercury. Therefore, aqueous ethylation doesnot fit for the purpose of the detection of Hg2+ and EtHg+ simulta-neously. The alternatives are the use of sodium tetrapropylborate(NaBPr4) [80,81] and sodium tetraphenylboron (NaBPh4) [31,82]. Al-though providing precise and reliable results by aqueous propylationof mercury with NaBPr4, the lack of commercially available reagentand its unstable property are still problems. Recently, Yan et al. firstlyused butyl magnesium bromide as a derivatization reagent to avoidthe loss of species specific information of analytes and employed syn-thesized Pr3PbCl as an alternative internal standard to improve theanalytical precision and accuracy for the simultaneous determinationof different Pb and Hg species [83]. Moreover, an alternative thermo-diffusion interface (TDI) was designed and constructed for highly ef-fective transportation of derivatized analytes from capillary GC toICP-MS. The developed method has been successfully applied to si-multaneous speciation analysis of Pb and Hg in biological and

environmental samples, providing detection limits of these elementalspecies down to the pg/g levels.

Significant methodological improvement in GC-based speciationtechniques has been represented by the implementation of species-specific isotope dilution [84] and the combination of GC–ICP-MS/AFS with advanced preconcentration and matrix separation tech-niques based on head-space SPME or purge-and-trap technologies[80,85,86]. By combining the purge-and-trap preconcentration withaqueous phenylation, Mao et al. proposed a newmethod for determi-nation of trace levels of organomercury species [87]. Phenylationproducts of MeHg and EtHg were first separated by capillary GC andthen detected by AFS and ICP-MS. Quite similar LODs from 0.01 to0.03 ng L−1 were obtained by both techniques for MeHg and EtHg.Jackson et al. utilized species-specific isotope dilution, purge-and-trap and GC–ICP-MS for the determination of ultratrace MeHg+ infreshwater samples with a detection limit as low as 0.003 ng L−1 [84].

The study of stable isotopic fractionation of mercury can offer apowerful tool to track mercury transformations in biogeochemicalcycle. The combination of GC with multicollector-ICP-MS(MC-ICP-MS) opens a new door for the study of species-specific stable isotopegeochemistry of mercury. However, the short available time for iso-tope ratio measurements, the limited quantities of analytes, and theisotope ratio drift during analyte transient passage are the main

9Y. Gao et al. / Microchemical Journal 103 (2012) 1–14

limitations for the accurate determination of isotope ration by GC–MC-ICP-MS. Epov et al. developed a new method to obtain preciseand accurate species-specific Hg isotope values from consecutive GCtransient signals by coupling GC with a commercially available MC-ICP-MS [88]. To afford optimal counting statistics, the use of isother-mal temperature programs to extend the elution of the Hg species,the proper selection of the peak integration window as well as thepreconcentration of real samples are critical. The validation of GC–MC-ICP-MS measurements was performed by the analysis of differentstandards and real samples, and subsequent comparison with previ-ously published results and with conventional nebulization and CVGsample introduction techniques. The species-specific δ valuesachieved by the proposed method in secondary fractionated refer-ence standard and environmental matrix were in good agreementwith those obtained by different techniques. Dzurko et al. also usedGC–MC-ICP-MS for the determination of mercury specific isotoperatio after purge-and-trap preconcentration [85]. They evaluated theaccuracy and precision of different isotope ratio calculating methodsincluding peak area ratio, average peak area ratio and peak apexratio. It was found that average peak area method yielded the bestprecision and the closest isotope ratio in relation to values obtainedby continuous flow CV measurements. They also utilized a mass biascorrection and reference standard addition for getting better repro-ducibility and understanding of the isotope composition of inorganicmercury and its methylated reaction product.

Besides ICP-MS measurement, Hg derivatives are quantified byAES, MS as well as AFS after GC separation. Hippler et al. comparedtwo methods for the determination of methyl mercury in wholeblood samples based on two different mass spectrometric detectiontechniques (GC–MS and GC–ICP-MS) [89]. The results from thesetwo independent methods correlated well, indicating the high accu-racy of MeHg determination. Berzas Nevado et al. [90] evaluated ad-vantages and disadvantages of three hyphenated techniques formercury speciation analysis of different sample matrices using GC–MS, GC–ICP-MS and GC-pyro-AFS detection. All of them were validat-ed with respect to precision (RSD b5%), which were confirmed by theF-test. After all, these three systems are all sensitive enough for mer-cury speciation of environmental samples, with GC–MS and GC–ICP-MS offering isotope analysis capabilities by using species-specificisotope dilution analysis, while GC-pyro-AFS remains the most costeffective alternative.

4.1.2. High performance liquid chromatographyHPLC is a powerful separation technique offering several advan-

tages over GC separation. It can be directly applied to non-volatilecompounds of high and low molecular weight, providing a great ver-satility derived from different separation modes (reverse phase, anionexchange and size exclusion). It can be easily “on line” coupled to anelement specific detector for mercury detection for analysis of envi-ronmental and biological samples.

Reverse phase (RP) columns with a mobile phase containing anorganic modifier and a chelating or ion pair reagent are often usedfor HPLC separation of mercury species in environmental and mostbiological matrix. However, for the study of mercury toxicity, metab-olism and transformation in organisms, size exclusion chromato-graphic (SCE) hyphenated to ICP-MS has shown its superiority inanalysis of Hg binding biomolecules. Generally, to guarantee the spe-cies integrity during the whole analytical procedure is one of the keypoints associated with the determination of metal-binding biomole-cules. Pedrero et al. combined mercury isotopic tracers with GC–ICP-MS and SCE–ICP-MS to evaluate the sample treatment procedurefor Hg containing biomolecules in dolphin liver [81]. The results indi-cated that the Hg-biomolecules entities could be degraded understrong sonication conditions. In addition, specific Hg species affinitywith different bimolecular weight fractions was demonstrated byuse of isotopic tracers.

Wang and his co-workers proposed a method for the determina-tion of mercury-containing protein fractions in the brain cytosol ofthe maternal and infant rats by SEC-ICP-MS with the postcolumn iso-tope dilution [91]. The enriched spiking 198Hg was continuouslymerged with the eluent after column separation and then the isotopefractions were on-line detected by ICP-MS. The detection limit of Hgwas 0.2 ng. The analytical results showed that the existing differentmercury-containing protein fractions between maternal and infantrats in brain cytosol would be helpful to understand the differentialtoxicity between mothers and their offspring. Later, they made useof the proposed mercury-binding protein quantification method forthe study of the distribution patterns of mercury species in organictissues and the subcellular fractions of maternal and infant rats afterexposure to MeHg+ [92].

Quantitative proteomics is expected to offer new functional in-sight into biological processes and the identification of diagnosticor prognostic disease markers [93]. More and more sophisticatedanalytical methods have been developed in order to obtain reliablequantitative results. The specific reactions between monofunctionalorganic mercury and sulfhydryl in proteins provides the opportuni-ty for the labeling of proteins with mercury species and absolutequantification of proteins by ICP-MS. Wang et al. presented thequantitative analysis of proteins via MeHg+ labeling and integratedapplication of molecular and elemental mass spectrometry [93]. Thesimple 1:1 stoichiometry between MeHg+ and sulfhydryl made theprotein quantification easily. According to the known number of –SH per protein, the absolute protein concentration can be obtainedvia Hg determination using SEC–ICP-MS. Bovine pancreatic ribonu-clease A, lysozyme and insulin were taken as model proteins foranalysis and their corresponding absolute detection limits (3 s)were 0.6, 1.2 and 0.4 pmol, respectively. In order to avoid directuse of extremely toxic MeHg+, they synthesized methylmercur-ithiosalicylate and 204Hg-enriched methylmercurithiosalicylate forlabeling peptides and proteins and subsequently for label-specificisotope dilution analysis using ICP-MS [94]. MeHg+ andMe204Hg+ were dynamically released from methylmercurithiosali-cylate and 204Hg-enriched methylmercurithiosalicylate in solutionand subsequently binding to proteins and peptides.

In many cases, direct coupling of HPLC to a detector is not sensi-tive enough to analyze real samples, especially for environmentalwater samples analysis, preconcentration methods such as CPE, SPE/SPME as well as DLLME before chromatographic separation are usual-ly necessary [95–102]. Post-column vapor generation is also oftenused to improve the sensitivity and decrease the matrix effects [96].Based on photo-CVG as an interface to on-line coupled ion chroma-tography with AFS/ICP-MS, methods were developed for rapid analy-sis of different Hg species in varied samples [103–105]. Afterseparation, both inorganic mercury and organic species are reducedby formic acid in mobile phase under UV radiation to produce Hg0

on-line, which is subsequently swept into AFS/ICP-MS for measure-ment by argon carrier gas. SCGD was also used as interface to on-line combine HPLC with AFS for mercury speciation [106]. With anSCGD induced vapor generation system, the decomposition and re-duction of organic mercury species could be completed in one step.

However, in traditional CV system, an extra step is required for theconversion of organomercury species to Hg2+ after separation to ob-tain higher sensitivity, because of mercury species-depended vaporgeneration efficiency. Chemical oxidation, UV irradiation, microwaveheating, or an external heat source is used to facilitate the decompo-sition of organomercury species [4,107]. Bramanti group [108] pro-posed a fully integrated UV/microwave (MW) photochemicalreactor for on-line decomposition and determination of p-Hydroxymercurybenzoate (PHMB) and its thiolic complexes (thiol-PHMB) by CV-AFS detection. PHMB and thiol-PHMB complexes arequantitatively converted into Hg2+ after chromatographic separationby the UV/MW reactor, with a yield between 91% and 98%. Hg2+ was

10 Y. Gao et al. / Microchemical Journal 103 (2012) 1–14

reduced to Hg0 with NaBH4 solution in a knitted reaction coil anddetected by AFS. The proposed method was applied to the determina-tion of PHMB-tagged thiols in human plasma, blood and wine. How-ever, the long reaction time of chemical oxidation and thecomplicated system design of UV/microwave assisted decompositionis still a problem. To simplify the speciation analysis procedure, Yin etal. developed a method based on HPLC-direct CVG-flame atomizationAFS for mercury speciation analysis without post-column digestion[109]. Mercury species were reduced to their hydrides by reactionwith KBH4 after chromatographic separation, further atomized inflame atomizer and determined by AFS. Alternatively, by adding L-cysteine into mobile phase, organic mercury compounds could also

Table 3Non-chromatographic techniques for mercury speciation analysis.

Analyte Preconcentration Separation

Hg2+

MeHg+SPE on a sheep woolpacked minicolumn

Reductions of Hg2+ with SnCl2on-line oxidation of MeHg+ witBr−/BrO3

− before reaction withHg2+

orgHgCV ionic-liquid assistedheadspace single dropmiroextraction (HS SDME)

Reductions of Hg2+ with SnCl2photo-oxidation of orgHg withlamp for 3 h before reduction w

Hg2+,total Hg

– Reduction of Hg2+ with SnCl2 adetermination of total Hg with

Hg2+

MeHg+– Determination of Hg2+ with Sn

and quantification of sum of Hgand MeHg+ with NaBH4

Hg2+

total Hg– Reduction of Hg2+ with SnCl2, o

oxidation of orgHg with KMnO4

then reduction with NaBH4.

Hg2+,MeHg+

– Direct reduction of Hg2+ in slursamples by NaBH4, and reductioMeHg+ after oxidation with KMfor 2 min.

Hg2+

total Hg– Conversion of organic Hg to Hg

on-line mixing with L-cys, andsubsequent reduction with NaB

Hg2+

MeHg+On-line preconcentration ina coiled reactor (CR)

Selective photo desorption of Hand MeHg+ from CR in differenconcentration of DDTC

Hg2+

MeHg+– Reduction of Hg2+ and MeHg+

low concentration of NaBH4, ansubsequent determination of Hgand MeHg+ with W-coil atomizroom temperature and 500 °C, r

Hg2+

MeHg+– Formation of volatile mercury s

with low concentration of NaBHsubsequent determination of HgMeHg+ without or with DBD at

Hg2+

MeHg+Preconcentration of Hg2+ andMeHg+ by cys-fiberpacked mini column

Determination of hg2+ and totacold atomization mode and flamatomization mode of AFS after rwith low concentration of nabh

MeHg+ Hollow fiber three phasemicroextraction (HF-LLLME)and hollowfiber two phase microextraction(HF-LPME)

Selective extraction of MeHg+ b

Hg2+

MeHg+Selective preconcentrationof Hg2+ onion imprinted microbeads

Determination of Hg2+ in nondsample and detection total merdigested sample.

Hg2+

Org HgPreconcentration of Hg2+ on a [1,5-bis(2pyridyl)-3sulphophenylmethylene] thiocarbonohydrazydelfunctionalized chelating resin

Selective retention of Hg2+ on tchelating resin and previous oxof org Hg before preconcentrati

Hg2+

MeHg+Retention of Hg2+ and MeHg+ on aStaphylococcus aureus loaded DowexOptipore V-493 micro column

Selective and sequential elutionand Hg2+ with 0.1 mol L−1 HCl2 mol L−1 HCl

Hg2+

Me Hg+Preconcentration of mercury vapor ona gold gauze inside a graphite tube

Selective extraction of MeHg+ bchloroform and back extracted1% L-cys

be on-line converted to Hg0 and introduced directly to AFS for detec-tion without any post column oxidation process or additional inter-face [110].

4.1.3. Capillary electrophoresisAmong all the separation methods, CE is a relatively new and de-

veloping technique, but it has already shown a great potential formercury speciation [111,112]. Rapid separation with high separationefficiency and tiny sampling volume are its principal features. Howev-er, its precision, sensitivity and reproducibility for mercury speciationare inferior to those of GC-HPLC related methods. Due to its verysmall sample injection volume, the selection of matched detector

Detector LODa Matrix Ref.

andhSnCl2

CV-AFS 0.01 ng mL−1 Peach leaves [119]

and15 W UVith SnCl2

ETAAS 10 ng L−1 Sea water,fish tissues, hairand wine

[120]

ndNaBH4

IsotopedilutionCV ICP-MS

0.018 ng g−1 Dogfish muscle,oyster, andtuna fish

[122]

Cl22+

CV-AAS/CV-OES

CV-AAS:Hg2+: 5 ng g−1

MeHg+: 0.016 μg g−1

CV-ICP-OESMeHg+: 0.012 μg g−1

Fish [123]

n-lineand

CV-ICP-MS Hg2+: 0.8 μg L−1

MeHg+: 0.08 μg L−1[124]

ryn ofnO4

CV-AAS Hg2+: 0.02 μg g−1

MeHg+: 0.016 μg g−1Biologicalsamples

[29]

2+ by

H4

CV-AFS Hg2+: 8.6 ng L−1

Total Hg: 7.2 ng L−1Biologicalsamples

[28]

g2+

tPVG-AFS Hg2+: 4 ng L−1 Fish muscle,

human hair andwater samples

[130]

withd2+

er atespectively

CV-AAS Hg2+: 60 ng mL−1

MeHg+: 89 ng mL−1Water samples,fish samples

[125]

pecies4, and2+ andomizer

CV-AAS Hg2+: 35 μg L−1

MeHg+: 54 μg L−1Tuna fish [126]

l hg witheeduction4

CV-AFS Hg2+: 1 ng L−1

MeHg+: 3 ng L−1Water samples,cosmetic andseaweed samples

[127]

y HF-LPME ETAAS HF-LLLME:0.1 μg L−1

HF-LPME:0.4 μg L−1

Dogfish muscle,human hairand sludge

[133]

igestedcury in

CV-AAS 6 ng L−1 River water,mineral andsea waters.

[121]

heidationon

CV-AAS/CV-ETAAS

CV AAS: 10 ng L-1

CV-ETAAS:6 ng L−1

Sea food [128]

of MeHg+

andCV-AAS Hg2+: 2.5 ng L−1

MeHg+: 1.7 ng L−1Natural waterand fish samples

[129]

ywith

ETAAS Hg2+: 1 ng g−1

MeHg+: 5 ng g−1Fish [132]

11Y. Gao et al. / Microchemical Journal 103 (2012) 1–14

for CE is very important. An on-line coupled CE cold vapor generationwith electrothermal quartz tube furnace AAS system for mercury spe-ciation has been presented by Deng et al. [113]. The low cost, simplestructure, easy operation, low dead volume, good conductivity, goodgas–liquid separation efficiency and good selectivity of AAS make itattractive as an on-line detector of CE for mercury speciation. Li etal. developed a method for mercury high throughput and rapid speci-ation analysis by short column CE coupled to ICP-MS [114]. A Micro-Mist nebulizer was employed to increase the nebulization efficiencyand a laboratory made removable SC-CE–ICP-MS interface on thebasis of cross design was applied to alleviate buffer contaminationof ICP-MS.

Compared to HPLC and GC-related methods, the application of CEbased hybrid techniques is rare in speciation analysis for mercuryspecies in real samples. But it is promising as a powerful tool to pro-vide useful information on interaction of various Hg mercury specieswith selected biomolecules [112,115].

4.2. Non-chromatographic methods

Non-chromatographic methods based on varied chemical andphysical behaviors of different Hg species open another avenuefor mercury speciation analysis. Recent development of non-chromatographic techniques for mercury speciation analysis issummarized in Table 3. Selective CVG is a promising approach fornon-chromatographic Hg speciation. In many cases, mild reductionreagents such as tin(II) was applied to selective determination ofHg2+, while for the determination of organic Hg species or totalHg, conversion of organic Hg to Hg2+ by UV irradiation, ultrasonictreatment, chemical oxidation, L-cysteine decomposition prior toreduction or strong reductants such as NaBH4/KBH4 are oftenrequired [28,29,119–124].

Different atomization modes for the discrimination of inorganicand MeHg+ by vapor generation-atomic spectrometry was also de-veloped. Typically, Hg2+ can be determined at room temperature aselemental Hg vapor after CVG with low concentration of THB, whiletotal mercury is measured in the presence of different atomizers in-cluding W-coil [125], DBD [126] and flame atomizer [127] after form-ing volatile species with low concentration of THB at the secondsample loading. The total organic mercury concentration was calcu-lated by subtracting the inorganic mercury concentration from thetotal mercury concentration.

Another approach for mercury non-chromatographic speciationanalysis is the use of different types of sorbent material and/or com-plexation reagents for the selective SPE or selective elution of eitherinorganic Hg2+ or MeHg+ or their complexes. Ion-imprinted poly-methacrylic microbeads [121] and [1,5-bis(2pyridyl)-3sulphophenylmethylene] thiocarbonohydrazydel functionalized chelating resin[128] were developed for selective preconcentration of Hg2+. Pre-oxidation of organic mercury before SPE is required for total organicmercury determination. Preconcentration of Hg2+ and MeHg+ on aStaphylococcus loaded Dowex Optipore V-493 micro column, and se-quential elution of retained MeHg+ and Hg2+ with different concen-trations of HCl was also reported for mercury speciation analysis[129]. Additionally, changing the reducing reagent concentrationunder UV irradiation has also been proved to be a successful tech-nique for selective desorption of retained mercury species from coiledreactor (CR) for discrimination of different mercury species [130].

The use of solvent extraction together with acid leaching is also apopular method for the separation of mercury species in solid sam-ples. Saber-Tehrani et al. used toluene to extract MeHg+ after acidleaching and transfer total mercury to organic phase after sample di-gestion by using chelating agent diethyldithiocarbamate (DDTC)[131]. Duarte et al. directly extracted organomercury from KBr andHCl leaching solution of biological samples by using chloroform andback extracted the resultant solution with 1% m/v L-cysteine [132].

Hollow fiber microextraction (HFME) was also developed for the se-lective determination of MeHg+ in biological samples and sludgewith a minimized solvent volume and a high enrichment factor.

5. Conclusions and future trends

Without doubt, mercury speciation analysis is one of the most ac-tive research areas in an analytical chemistry and will continue to be ahot research topic in the near future, as increasing attention is beingpaid to food safety and environmental pollution. The developmentof reliable and rapid sample preparation methods, high efficient andenvironmental friendly sample preconcentration and vapor genera-tion approaches, low energy consumption and miniaturized instru-mentation, and accurate measurement schemes have facilitatedmercury speciation analysis. As a consequence, the quantification ofHg (and its species sometime) in environmental and biological sam-ples as low as nanogram or even pictogram per liter level has been re-alized. Obviously, mercury speciation analysis rather than totalmercury determination offers more useful information to properly as-sess the toxicity and health risks of mercury and further understandbiogeochemical cycling of mercury compounds. The use of non-chromatographic methods for mercury speciation analysis is rapid,low cost, convenient, and can be used as screening or specific toolsfor environmental monitoring, food security and clinical diagnosticsapplication. To obtain a complete picture of the species present insamples, chromatographic based methods are still required. The com-bination of GC with an element-specific detector, such as ICP-MS, is aprimary approach for mercury speciation which offers the possibilityto integrate sample derivatization, clean-up and preconcentrationinto one single step by SPME or purge-and-trap. The LODs and sensi-tivity of GC-related techniques are either comparable or superior tothose achieved by other hyphenated approaches. However, as thefast development of metallomics and proteomics, the combinationof HPLC with ICP-MS will play an important role in understandingof the metabolic pathways, bioavailabilities and toxicological effectsof mercury species in organism as well as quantification of proteins.But the insufficient sensitivity, and the introduction of large amountof organic solvent to detectors from RP separation needs to be settled.Anyway, the preservation of species integrity during the whole ana-lytical procedure is a crucial issue in mercury speciation analysis.The prevention of contamination, minimization of trace analytesloss in during sampling, sample preparation and determinationshould be considered thoroughly. Furthermore, the presence of mer-cury species at extremely low concentration demands more sensitiveand robust analytical techniques. The development of automatic,reagent-free or less reagent consumption, and environmental friendlyanalytical methods is also desired. The miniaturized instrumentationis expected to have attractive applications in field analysis for mercu-ry and mercury species.

Acknowledgment

The authors gratefully thank the National Natural Science Founda-tion of China (No. 20835003) for financial support of this project.

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