Open focused microwave-assisted sample preparation for rapid total and mercury species determination in environmental solid samples

This paper describes rapid, simple microwave-assisted leaching/ digestion procedures for total and mercury species determination in sediment samples and biomaterials. An open focused microwave system allowed the sample preparation time to be dramatically reduced to only 24 min when a power of 40-80 W was applied. Quantitative leaching of methylmercury from sediments by HNO3 solution and complete dissolution of biomaterials by an alkaline solution, such as 25% TMAH solution, were obtained. Methylmercury compounds were kept intact without decomposition or losses by evaporation. Quantitative recoveries of total mercury were achieved with a two-step microwave attack using a combination of HNO3 and H202 solutions as extractant. The whole pretreatment procedure only takes 15 min, which can be further shortened by an automated robust operation with an open focused system. These analytical procedures were validated by the analysis of environmental certified reference materials. The results confirm that the open focused microwave technique is a promising tool for solid sample preparation in analytical and environmental chemistry.


Introduction
Sample preparation is one of the most crucial steps in trace element analysis and frequently controls the quality of the final results obtained [1,2]. Environmental solid samples are generally made into a solution with wet digestion methods and analysed by compatible instrumental techniques, e.g. atomic absorption spectrometry (AAS), inductively coupled plasma coupled to atomic emission spectrometry (ICP-AES) or ICP-mass spectrometry (ICP-MS). Most of the conventional digestion procedures are not only laborious and time-consuming, but also lack sufficient efficiency and reliability. As a well-known example, hot plate digestion techniques with conductive heating, now used widely, easily lead to nonreproducible results. Other extraction methods, such as sonication, distillation or soxhlet extraction, also have the above drawbacks, even though reliable results are usually achieved. In the case of mercury speciation analysis, solid Correspondence to Dr Donard. sample preparation by acid or alkaline extraction with different heating sources (sonication, stream distillation, etc.) requires from 2 to 24 h for complete recovery of the target analytes [3][4][5][6][7][8][9][10]. Innovative techniques such as supercritical fluid extraction (SFE) [1 1-13] and microwave-assisted extraction (MAE) [14][15][16] have been recently developed and are a substantial advance.
However, SFE potentially has technological limitations and shows insufficient extraction efficiency, usually depending on sample matrix and analyte polarity. Moreover, the expensive equipment required increases the cost of the analysis and the extraction step still takes 20-50 min.
The main advantages of the microwave-assisted extraction technique are absence of inertia, rapidity of heating, reduction of extraction time, better reproducibility and reliability, ease of automation, and good ability for selective leaching and total digestion in a wide array of sample matrices [17]. Thus, the application of this technique to sample preparation has been widely investigated in various fields of the environmental and analytical chemistry since it was first applied in 1975 [18]. Two different approaches in microwave extraction procedures are the use of a closed system (pressurized with a closed vessel) or an open system (non-pressurized with an open vessel). They have different characteristics and applications, as shown in table [16]. Nevertheless, for organometallic speciation analysis, open microwave technology based on focused microwaves is preferred to a closed microwave system, because better stability of the target compounds is achieved, due to the milder extraction conditions supplied (20-60W, compared to 1000W typically used in closed system); and better reproducibility is obtained, owing to a perfect control of the microwave energy, precisely focused on the sample. Essential parameters such as extraction medium, applied power, exposure time and sample size must be, however, fully optimized in terms of stability and extraction efficiency of the target analytes to set the optimum extraction conditions for further routine analysis [14-16, 19, 20]. This paper presents microwave-assisted leaching/digestion protocols for total and mercury speciation analysis in environmental solid samples, such as sediments and biological tissues, using an open and low-power focused microwave system (301 PROLABO). Total mercury in sediments was determined by flow injection sample introduction followed by I CP-MS detection, after two-step microwave-assisted acid digestion with concentrated HNO3 and H202. Mercury species, such as methyland inorganic mercury, were analysed in both sediments and biological tissues by an automated on-line system Peak areas were used as the analytical response and mercury concentrations were calculated after normalization of the data to the internal standard signal (ST1) followed by appropriate blank subtraction.
Automated on-line hyphenated system: An automated on-line hypenated D-CT-GC-QFAAS system [14-16, 21, 22] was used for mercury species analysis. This system combines five basic analytical steps (see figure 2): derivatization of mercury species to volatile forms; preconcentration by cryofocusing in liquid nitrogen; gas chromatographic separation during thermal desorption; detection by atomic absorption spectrometry; and data acquisition by a computer. All the steps are controlled through an electronic panel, which is programmed by a computer equipped with BORWIN software [23]. The set-up includes a peristaltic pump, 250-ml reaction vessel, two electronic Teflon-valves, a U-shaped Pyrex column (45 cm length x 5 mm id), Dewar bath, pneumatic pump, adjustable d.c. power supply, flow meter, T-shaped quartz furnace (light path length 20 cm, cm id), atomic absorption spectrometer (Model 5000, Perkin-Elmer) and PC. The operation procedure is automatically and sequentially performed according to a programme previously defined in BORWIN software. First, NaBH4 or NaBEt4 solution is quantitatively transferred from the reaction flask to the reaction vessel by a peristaltic pump. The derivatization and purging steps take place in a 250-ml reaction vessel. The generated volatile Hg species are purged from the reaction vessel and trapped in the column, packed with 2.5g of Chromosorb W HP (60-80mesh) coated with 10% SP2100 (Supelco) and previously silanized with hexamethyldisilazane (Fluka).
During cryofocusing, the column is immersed in a Dewar bath with liquid N (-196C) lifted by a pneumatic pump. In the desorption step, the column, wrapped with 0.5-mm-diameter Nichrome wire, is gradually heated by an adjustable power supply and the volatile mercury species successively elute in order of increasing molecular weight. The flow of the purging/stripping He gas is controlled by a flow meter. Atomization of mercury species occurs in a quartz furnace held at 800C by means of an MHS-20 unit and detected by an atomic absorption spectrometer operated at 253.7 nm with a 0.7 nm slit-width. Data acquisition is finally undertaken by a chromatographic software run on a PC.

Reagents
Analytical grade chemicals and Milli-Qwater were used throughout (unless otherwise stated). A 0.1% mixed solution containing ml of Triton X-100, g of EDTA and ml of NH4OH (25%), diluted to a final volume of with Milli-Q water, was prepared. An approximately 0.01% (m/v) solution of sodium tetraethylborate (NaBEt4) was prepared in a glove-bag, filled with N2, by dissolving the reagent in water. An approximately 4% (m/v) solution of NaBH4 was prepared by dissolving the reagent in water. All vessels were first cleaned with RBS 50 detergent, thoroughly rinsed with tap water, soaked in a 10% HNO3 solution for 24h and finally rinsed with Milli-Q water before use. Standard solutions and certified reference materials A standard stock solution of 1000 lag m1-1 of Hg(II) was prepared by dissolving mercury(II) chloride in 1% HNO3, and that of 1000 lag m1-1 of methylmercury by dissolving methylmercury chloride in methanol. All stock solutions were stored in a refrigerator and protected against light. Working standard solutions were prepared by appropriate dilution in water of the stock solutions and they were stored one week at maximum. Four certified reference sediments, IAEA-356 (International Atomic Energy Agency of Monaco), PASC-1 (National Research Council of Canada), BCR S19 and CRM 580 (Community Bureau of Reference) and three biological reference materials, DORM-1 (Dogfish muscle) and TORT-1 (Lobster hepatopancreas) (National Research Council of Canada) and CRM 463 (Tuna fish muscle) (Community Bureau of Reference), were used to validate the proposed methods.

Analytical procedures
Analysis of sediments Mercury species analysis: A sample of approximately g of homogenized dry sediment and 10ml of acid solution were placed in an extraction tube and exposed to microwave irradiation at 60 W for 3 min. After irradiation, the sample solution was cooled to room temperature, transferred to a 15ml tube and centrifuged at 5000 rpm for 5 min. The supernatant was poured into a 22-ml Pyrex vial with Teflon cap (Supelco) and finally stored in a refrigerator until analysis. A clean-up procedure was not necessary prior to the analysis after the aqueous phase ethylation method. An aliquot of ml of the extract was analysed by means of the hyphenated Et-CT-GC-QFAAS system. Calibration was performed by the three-point standard-addition method to overcome possible matrix interferences and the sub-sample was subjected to triplicate analysis. Blanks were run after each triplicate analysis to check for the possible memory effects.
Total mercury analysis: A sample of approximately 0.25 g of homogenized dry sediment and 8ml of concentrated nitric acid were placed in an extraction tube and exposed to microwave irradiation at 20 W for 5 min. After extraction, the sample was allowed to cool for about 5 min, followed by the addition of 2ml of H20 and again digested at 20W for further 5 min. After cooling, the extracts were diluted with Milli-Q water and finally stored in a refrigerator until analysis. An aliquot of 0.1 ml of the extract was added to a final solution (5 ml) containing 4 ml of a mixed Triton X-100 solution, which included 0.1% Triton X-100, 0.1% EDTA and 0.1% (v/v) ammonia solution, and an internal standard of thallium (100 ng). The resulting solution was analysed by FI-ICP-MS. The three-point standard-addition method in the same extract, and the addition of an internal standard were used to overcome matrix effects and instabilities of the instrument. Analyses were carried out in duplicate and measured using two isotopes of mercury (Hg, Hg). A blank test was prepared in each set of experiments to check for possible contamination during sample preparation and it was used to calculate the concentration of mercury after appropriate blank subtraction.

Analysis of biotissues
Mercury species analysis: A sample of 0.1-0.5g of pulverized freeze-dried tissue and 5 ml of 25% TMAH alkaline solution were placed in an extraction tube and exposed to microwave irradiation at 60W for 2 min. After irradiation, the sample solution was cooled to room temperature and then diluted with 5ml of methanol. It was then transferred into a 22-ml Pyrex vial with a Teflon cap and stored in a refrigerator until analysis. It is not necessary to have a clean-up stage before analysis by hydride generation. Aliquots of 50-300lal of the extract were directly analysed by the HG-CT-GC-QFAAS hyphenated system. The calibration, reproducibility analysis and blank test carried out in this case are similar to those already described for the analysis of sediments.

Results and discussion
Optimum strategy for microwave-assisted extraction Extraction efficiency is the key to a successful microwaveassisted sample preparation for total and mercury species determination in environmental solution samples. In mercury speciation analysis, the extraction step must provide quantitative speciation of mercury species from the matrix without losses or contamination, and without changes in chemical forms; in total mercury analysis, mercury species must be not only completely liberated from the matrix, but also decomposed to Hg(II) without any loss and contamination. As a result, several variables, such as power applied, exposure time, and concentration and amount of extractant, must be carefully optimized when using an open focused microwave system. Once the optimum extraction agents have been chosen, the two most important variables influencing the extraction efficiency are power applied and exposure time. Figure  3 shows a generic view of extraction efficiency in the power setting versus irradiation time region. The domain of optimum efficiency is located in region B; in region C, above the boundary line of the upper limit, insufficient efficiency is achieved due to degradation or evaporation losses, because of a long time heating or intensive power setting. In region A, below the boundary line of the lower limit, incomplete dissolution or leaching also leads to Table 2. Optimum conditions for the automated on-line D-CT-GC-O_.,FAAS system and FI-ICP-MS system for mercury speciation and total mercury analysis.  The energy focused on the sample can be calculated at each point in the matrix map of the power setting versus irradiation time according to: Q, W x T (where Q, is the energy output in cal, W is power setting in cal/min and T is the exposure time in min). Diagrams like that in figure 3 provide information about the optimum conditions required in each case to get quantitative recoveries during routine work, even though the energy needed to break the carbon-metal bonds remains unknown.
Microwave-assisted extraction of sediments Mercury speciation analysis: The choice of extraction medium is the first step towards understanding the behaviour and extraction efficiency of methylmercury from sediments under mild microwave irradiation [14-16, 19, 20]. Acid solutions have commonly been used in the extraction of organomercury compounds from sediments [9,[24][25][26][27][28]. Thus, four different acid solutions, nitric (2 mol dm-3), hydrochloric (2 mol dm-3), sulphuric (1 mol dm -) and acetic acids (100%), were selected to check the stability of MeHg + and to investigate the MeHg + extraction efficiency from reference sediments in a microwave field. Each of the extractants was spiked with an amount of MeHg + and exposed to a microwave field during varying heating time at 60W. The results obtained after up to 8 min heating show good stability of MeHg + in HNO3 and HC1 solutions, but only 80-90% of averaged MeHg + recoveries in H2SO4 and CHCOOH solvents. MeHg + losses are probably due to evaporation of extractant during vigorous heating. In another set of experiments, reference sediments suspended in the acid solutions mentioned above were exposed to microwaves at 60 W for 3 min. Quantitative recoveries were obtained by 2 M nitric and hydrochloric acids. Overall recoveries of about 85% and 55% for pure acetic acid and mol dm -3 sulphuric acid were observed, respectively. These low recoveries are mainly due to incomplete recovery from sediments by CH3COOH and partial adsorption on fine organic particles in the case of H2SO4. Additionally, interference problems were achieved in the determination step when analysing HC1, HSO4 and CHCOOH leachates. This was not the case, however, for HNO3 leachates. Taking into account all the facts mentioned above, nitric acid solution is an excellent extractant for methylmercury leaching from sediments, in terms of extraction efficiency and matrix interference. MeHg + microwave-assisted leaching from sediments with 2moldm -HNO was, therefore, optimized by constructing the corresponding response surface of power applied versus time irradiation using BCR S19 and CRM N580 reference materials [ Figure 4(a)]. The optimum yields (100%) were obtained in 1-7 min in the 100-20 W range. Extreme conditions (longer time heating and/or higher power setting) result in evaporation losses or in rapid boiling out of the extractant, even though quantitative recovery may be achieved. Summarizing, 2-4 min heating versus 60-40W power conditions are recommended as the optimum condition for microwave-assisted leaching of methylmercury from sediments using 2 moldm -HNO3 as extractant. In a further recovery study, the effect of changing HNO3 concentration was investigated. Quantitative and non-destructive MeHg + recovery was obtained by extraction with 2 mol dm -3 up to 10moldm -HNO, at 60W for 3 min irradiation; moldm -HNO3 led to insufficient recovery and degradation of MeHg + for concentrated HNO. HNO spiked with MeHg + confirms that the analysis is not affected by matrix effects. No clean-up procedure is necessary, but the extract must be centrifuged after microwave irradiation prior to the analysis, in order to prevent readsorption on suspended matter. The chromatogram in figure 6(a) was obtained for the analysis of the reference CRM N580 sediment using 6moldm -3 HNO3 solution as extractant. The peak at 1.3 min corresponds to Hg and is due to reduction of Hg 2+ during the determination step. High content of Hg 2+ in sediments is responsible for the appearance of such a peak for Hg .   Total mercury analysis: The choice of the best extraction agent for microwave-assisted digestion and of the most appropriate analytical technique for the final determination are of crucial importance in total mercury analysis [29]. Mercury species are easily and strongly bound to organic matter, e.g. thiol groups, humic substances and amino acids. Thus, oxidizing agents such as concentrated HNO3 and H202 solution were chosen to liberate the mercury species from the organic matrix and to fully oxidize them to Hg(II). The use of sulphuric acid as extractant is not recommended. The simple digestion procedure proposed here is as follows: the sediment (about 0.25 g) is decomposed by a two-step attack with (1) concentrated HNO at 20W power for 5min, followed by cooling the mixture about 5 min; and (2) a subsequent extraction with 30% H202 at 20 W power for   (table 4). Obtained recoveries ranged from 95 to 105% and the reproducibility was better than 10% in a mercury concentration range between 4 and 100 lag g -. The addition of Triton X-100 as surfactant and ETDA as complexing agent is necessary to obtain linear calibration curves in order to eliminate memory effects and improve efficiency during sample transport [29]. The use of an FI system prior to I CP-MS detection, standard addition method and complexation of mercury by EDTA improves reliability of the results, compared to conventional ICP-MS methods using direct calibration. Detection limits, calculated as three times the standard deviation of the blank divided by the slope of the calibration curve, are 10 pg g-1 and 1.0 ng g-1 for solutions and dry sediment samples, respectively.
Microwave-assisted extraction of biomaterials Mercury speciation analysis: To obtain quantitative recoveries of mercury species incorporated in the biological matrix, complete dissolution of the biological tissue is necessary [15,16,30]. Two candidate approaches to obtain good solubilization of biotissues are acid and alkaline hydrolysis procedures. In this study, five extraction agents, HNO3, HC1, CH,3COOH, TMAH and methanolic-KOH solutions, were investigated to understand the stability and extraction efficiency of methylmercury in simple solutions and reference biomaterials.
MeHg + stability in simple solutions was tested in the same way as for the sediments (see earlier). MeHg + was spiked in each extraction medium, followed by irradiation at a preset power for varying heating times. Quantitative recoveries were obtained for TMAH after 6 figure 4(b). Similar to the study of MeHg + optimization in sediments, the zone of quantitative yields (100%) is located at 1-6min and 100-20W. Special care must be taken, however, to avoid evaporation losses or rapid boiling out of the extractant in the case of long time heating and high power setting. As a result, irradiation for 2-4 min at 60-40 W using 25% TMAH solution as extractant is recommended as the optimum condition for microwave-assisted alkaline digestion of tissues in an open focused microwave system. In a further recovery study, the effect of various TMAH concentrations was investigated. Quantitative MeHg + recovery was obtained by extraction with 10-25% TMAH solution for 0.2-0.5 g of dry biotissue at 60W for 2min irradiation. These conditions allowed simultaneous quantitative extraction of methyl-and inorganic mercury from biomaterials.
Analytical figures of merit." The biotissue extract after microwave digestion can be analysed without any clean-up step [see figure 5]. The proposed analytical procedure was validated by analysing (HG-CT-QFAAS) three different reference biomaterials, CRM 463, DORM-1 and DORT-1, after 2min/60W microwaveassisted digestion of 0.1-0.5g of tissue with 5ml of 25% TMAH solution. The results obtained for methylmercury are in good agreement with the certified values, as shown in table 5. Inorganic mercury can also be simultaneously extracted and determined by this method. The sums of the concentrations of both mercury species present in the tissues also match certified total lnercury content in the biotissues (table 5). A reproducibility of 4-10% was obtained in the determination of both mercury species. The detection limits for both Hg +2 and MeHg + were calculated as 50ngg-I for 0.2 g of pulverized dry sample and 0.05 ml of extract.

Conclusions
Simple, rapid, efficient and quantitative sample leaching/ digestion protocols based on a microwave-assisted technique have been developed for the determination of total and mercury species in environmental solid samples such as sediments and biomaterials. The use of an open focused microwave system offers reproducible and quantitative recovery of the analytes and keeps the organomercury species intact. The appropriate extractants, a combination of HNO3/H202, HNO3 and 25% TMAH solutions, were chosen for total and mercury species determination in sediments and biotissues after careful evaluation of the stability and extraction efficiency of methylmercury in a microwave field. Optimum extraction conditions of 2-4min irradiation and 40-60W power were selected for mercury speciation analysis following a matrix approach. Sample throughput can be controlled by instrumental analysis time, rather than by sample preparation step. A drastic reduction of time is achieved in sample preparation when microwave technology is used, compared to other currently available methods [15,16,31]. Microwave-assisted techniques for total and mercury speciation analysis offer advantages in terms of simplicity, reliability and analysis time and cost. This technique might be extended to provide similar sample preparation protocols for other metal and metalloids in environmental metrices [31][32][33].