Mercury Speciation in Urban Landfill Leachate by Cold Vapor Generation Atomic Absorption Spectrometry using Ion Exchange and Amalgamation

Foi desenvolvido um sistema em linha para a especiação de Hg orgânico (Hg org ) e Hg inorgânico (Hg inorg ) em chorume. Clorocomplexos produzidos a partir do Hg inorg são separados em uma coluna trocadora de ânions. O Hg retido na coluna é eluído com NaBH 4 na presença de HCl. O Hg0 produzido é medido por espectrometria de absorção atômica com geração de vapor frio (CV AAS). O Hg org , que não fica retido na coluna, é misturado com NaBH 4 produzindo espécies voláteis de Hg, as quais são amalgamadas numa rede de Au/Pt. O Hg é removido desta coluna por aquecimento a 600 °C e medido por CV AAS. Para a especiação de Hg inorg e Hg org em chorume, as amostras são centrifugadas, filtradas e tratadas com HCl. Os limites de detecção (3 s) são 9 e 12 ng L-1 de Hg para Hg inorg e Hg org , respectivamente. A freqüência de análise é 22 h-1.


Introduction
In several countries, the urban waste is usually discarded in landfills, producing toxic leachate and toxic gases.The rainwater percolation through the residues and its inherent aqueous fraction comprise the leachate, which is characterized by high concentration of organic matter, salts, suspended particles and toxic elements. 1,2Mercury is one of the elements often present in landfill leachate and become an environmental problem. 3norganic Hg (Hg inorg ), mainly as Hg 0 and Hg 2+ , is generally disposed in urban landfills, 4 where it can associate to colloidal particles, complex with organic matter and/or bind to sulfides. 5Under anaerobic conditions a significant fraction of Hg is transformed into more toxic species.Inorganic mercury may be transformed to other toxic species in landfill, such as methyl mercury, ethyl mercury and phenyl mercury. 6However, the concentrations of ethyl mercury and phenyl mercury are very low and usually not detected by conventional techniques.Despite the possibility of formation of many organic and inorganic compounds of Hg in the environment, methyl mercury is usually the most abundant Hg org species found in environmental samples, [7][8][9] whereas Hg 2+ is the main Hg inorg species.
The levels of total Hg (Hg tot ) in the leachate are generally lower than 1 mg L -1 . 4Works related to speciation of Hg in gases emitted from landfills have already been published 7,8 but few dealt with Hg speciation in landfill leachate. 10Mercury speciation in this kind of sample is a challenging task owing to low Hg species concentration.
5][16][17][18] SPE has been frequently used in conjunction with flow injection systems (FI), allowing on-line analyte pre-concentration and matrix separation, which also reduces contamination and loss of analyte.It is worth mentioning that these features are very important when low concentration of a given species is determined.Gas chromatography [19][20][21] or liquid chromatography, 22 combined with element-selective techniques, such as atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS) or inductively coupled plasma mass spectrometry (ICP MS) have been used to measure the concentration of Hg species.
][27] Stable complexes of Hg are produced by reaction of Hg inorg with anions, such as SCN -, CH 3 COO -, I -, Br -and Cl -.The anionic complexes of Hg produced are strongly held by anion exchange resin, while Hg org species typically produce neutral compounds in the presence of these anions and are not retained.Consequently, the separation of Hg inorg and Hg org is possible. 16,17,26,27Anionic chlorocomplexes of Hg (mainly [HgCl 3 ] -, [HgCl 4 ] 2-and [HHgCl 4 ] -) are produced in HCl medium and retained in columns packed with Dowex 1-X8 or IRA 400. 17,26,27Thiourea, L-cysteine, SnCl 2 and NaBH 4 can be used for elution of Hg from the resin. 16,17,27n this work we propose a new approach to speciation of Hg inorg and Hg org , using a sequential and selective retention of chlorocomplexes of Hg inorg by anion-exchange resin and amalgamation of Hg org .NaBH 4 is used to remove the Hg from the anion-exchange resin (in a column), while the amalgamated Hg org (in an Au/Pt gauze) is removed by heating.The speciation and pre-concentration of Hg inorg and Hg org are on-line performed, whereas both Hg org and Hg inorg are determined, allowing better accuracy and precision.The proposed method is applied to the speciation of Hg org and Hg inorg in urban landfill leachate.In this work, all Hg species that do not interact with the anion exchange column and react with sodium tetrahydroborate producing volatile Hg species, which are then amalgamated by the gold gauze, are considered as Hg org .

Reagents
All chemicals used throughout this work were of analytical reagent grade.Ultra pure water (18.2MΩ cm) was obtained from a Milli-Q (Millipore, USA) system.The carrier gas used was 99.996% argon from White Martins, Brazil.
Sodium tetrahydroborate (NaBH 4 from Vetec, Brazil) solution was prepared fresh daily by dissolving the reagent in 0.05% (m/v) sodium hydroxide (NaOH from Merck, Germany).Hydrogen peroxide (H 2 O 2 ), hydrochloric (HCl) and nitric (HNO 3 ) acids (all from Merck) were used.These acids were further purified by sub-boiling distillation in quartz still (Milestone, DuoPur, Italy) for the samples and analytical solutions preparation.A 1000 mg L -1 Hg 2+ stock solution (from Merck) was used; solutions ranging from 50 to 1500 ng L -1 Hg 2+ were prepared in 1.0 mol L -1 HCl by serial dilutions of the Hg 2+ stock solution.A Hg org stock solution containing 100 mg L -1 (as Hg) was prepared from CH 3 HgCl (Aldrich, USA).The CH 3 HgCl was firstly dissolved in 1 mL of methanol and then the desired volume was completed by addition of 1.0 mol L -1 HCl.Calibration solutions of Hg org ranging from 50 to 2500 ng L -1 of Hg were prepared in 0.5 mol L -1 HCl mediun, by serial dilutions of the CH 3 HgCl stock solution.Fresh Hg 2+ and CH 3 HgCl calibration solutions were prepared whenever used.
A strongly basic anion resin (Dowex 1-X8) in chloride form was used to separate and pre-concentrate the Hg inorg .The resin particles diameter was in the range of 100 to 200 mesh.A small column was prepared by putting 30 mg of this resin inside a polyethylene tube (10 mm length and 3 mm i.d.), whose ends were sealed with polyurethane foam.This column allowed at least 200 cycles of pre-concentration/ separation.
A small column (from PerkinElmer, Norwalk, USA) containing a gauze of Au/Pt inside was used to retain and pre-concentrate the Hg org .
All the glass and plastic materials used were thoroughly washed with a HNO 3 10% (v/v) solution.After that, they were rinsed with distilled water and left to dry in clean environment.

Instrumental
A flow injection-vapor generation system coupled to an atomic absorption spectrometer was used for on-line separation, pre-concentration and determination of the Hg species.A Vario 6 FL atomic absorption spectrometer (AnalytikJena, Jena, Germany) equipped with a quartz cell (100 mm length and 10 mm i.d.) and deuterium background corrector was used.A Hg hollow cathode lamp operated at 4.0 mA was used as radiation source.The wavelength was set at 253.7 nm and the spectral band pass at 0.8 nm.All measurements were made by integrated absorbance (peak area).
The total concentration of Hg in the solid and liquid phases of the landfill leachate samples was determined by cold vapor generation-atomic absorption spectrometry in batch mode; the hydride generating system HS 5 from AnalytikJena was used.In this case, conditions recommended by the manufacturer of the instrument were used.A microwave oven (Multiwave 3000, Anton Paar, Austria) and a centrifuge (Sigma 3K30, Germany) were used to prepare the samples.

Flow injection-cold vapor generation system
The flow injection-cold vapor generation (FI-CV) system used (Figure 1) consists of a peristaltic pump (Gilson, Minipuls 3, France), four three-way solenoid valves (Cole-Parmer, USA), a gas flow-meter (Key Instruments, USA) and two columns; one contains the ion exchange resin (IEC) and the other contains the gauze of Au/Pt.Tygon tubes with different internal diameters were used to pump the solutions through the FI system.Tubes of polytetrafluorethylene (PTFE), with 0.8 mm i.d., were used to carry the solutions through the FI system.Two home-made gas/liquid separators [GLS 1 (a glass tube, 100 mm length and 10 mm i.d.) and GLS 2 (a glass tube, 40 mm length and 7 mm i.d.)] were used.The GLS 2 was used only for the purpose of retaining drops of water that could reach the column of amalgamation and/or the quartz cell.With the introduction of the second gas-liquid separator, it was not necessary to use any desiccant agent that could retain the analyte and lead to incorrect results.Sequential separation, pre-concentration and determination of Hg org and Hg inorg in the same aliquot of the sample were possible by using the FI system shown in Figure 1.

Hg separation and pre-concentration procedure
Standard solutions containing 500 ng L -1 Hg (as Hg inorg or Hg org ) were firstly used in order to evaluate the performance of the FI-CV system.The Hg solutions were acidified with HCl (the final concentration of HCl was 1.0 mol L -1 ) and then passed through the IEC column (Figure 1).Only Hgchlorocomplexes are retained by the resin inside the IEC, while the CH 3 HgCl does not interact and passes directly through the column.When exiting this column, the effluent is mixed with a solution of NaBH 4 (at b in Figure 1), producing volatile species of Hg.The volatile Hg species produced are carried by argon to the amalgamation column where they are retained.Thus, Hg 2+ (Hg inorg ) and CH 3 HgCl (Hg org ) are separated and also pre-concentrated.
After a given period (60 s in this work) of preconcentration and separation, the Hg 2+ is removed from the IEC column by a NaBH 4 solution in presence of HCl (the NaBH 4 and HCl solutions are on-line mixed at confluence a in Figure 1).Hg 0 is produced and then transported by argon to the quartz cell (QC in Figure 1) of the atomic absorption spectrometer where the absorbance is measured.Next, the volatile Hg species that were retained in the amalgamation column are removed in the form of Hg 0 by heating to 600 °C.A halogen lamp (HL in Figure 1) placed 1 cm above the column is used for that purpose.The Hg 0 produced is carried to the quartz cell (QC in Figure 1) by Ar where the absorbance is measured.The FI system operation program is summarized in Table 1.

Samples
Landfill leachate samples were collected in 1 L amber glass flasks.After collection, the samples were kept in refrigerator (temperature around 4 °C) until the determination of Hg, which was carried out up to 6 h after collection.The samples were collected in two stabilization ponds that contain the leachate produced by the municipal landfill.Household waste had been disposed in this landfill for 30 years.The stabilization ponds are about 0.5 m depth, while the area comprises 1000 m 2 .The mean values of pH, suspended solids and DQO of the collected samples were 7.90 ± 0.20, 340 ± 62 mg L -1 and 2480 ± 420 mg L -1 O 2 , respectively.

Total Hg determination in landfill leachate
Owing to the presence of suspended solid particles, the leachate was centrifuged for 10 min at 15,000 rpm.The supernatant was removed and filtered through a 0.45 mm pore size-filter.The particles retained by the filter were removed with 6.0 mol L -1 HNO 3 and then added to the solid fraction that had previously been separated by centrifuging.The liquid and solid fractions thus obtained were digested separately in pressurized quartz vessels in microwave oven.
Aliquots of 20 mL of the liquid fraction or the solid fraction were transferred to separate quartz vessels, to which 6 mL of concentrated HNO 3 and 1 mL of H 2 O 2 (30%) were added.For recovery tests, aliquots of the samples were spiked with 50 ng of Hg (in the form of Hg 2+ or CH 3 Hg + ) and then subjected to the same treatment.The quartz vessels were closed and then heated for 10 min at 1000 W and 5 min at 1200 W. Blanks were prepared in the same way.After cooling, the solutions volume was completed to 50 mL with water and then the total concentration of Hg (Hg tot ) was determined by CV-AAS.

Mercury speciation in urban landfill leachate
The speciation of Hg inorg and Hg org was done separately in the liquid and solid fractions of the landfill leachate, following the procedure schematized in Figure 2. In summary, 25 mL of the liquid phase were transferred to a 50 mL polyethylene flask and the volume completed by addition of 2.0 mol L -1 HCl.The final solution was then processed in the FI-CV system coupled to the atomic absorption spectrometer.The solid fraction (about 30 mg) was tansferred to a 50 mL polyethylene flask to which 10 mL of 6.0 mol L -1 HCl were added.The mixture was gently shaken and then heated in microwave oven for 3 min at 60 W. 28,29 After reaching the room temperature, the volume of the mixture was completed to 50 mL with water and then centrifuged at 15,000 rpm for 15 min.Hg org and Hg inorg were measured in the supernatant.Blanks were prepared in the same way as samples.The accuracy was checked by addition of 20 ng of Hg (as CH 3 Hg + or Hg 2+ ) to 30 mL of leachate, which was then subjected to the same treatment above described.It is important to emphasize that there is no appropriate certified reference material available for landfill leachate.Therefore, recovery tests for CH 3 Hg + and Hg 2+ and total Hg determination were used to check the accuracy.

Hg inorg preconcentration
The retention of Hg inorg in the IEC column is influenced by the concentration of HCl in the sample solution, because the HCI is required to form the main anionic chlorocomplexes of Hg that are retained in the column.These complexes have high affinity with anionic resins such as Dowex 1-X8, feature that allows the separation of Hg inorg and Hg org .According to Figure 3, the Hg 2+ absorbance increases with the HCl concentration increasing up to 0.1 mol L -1 and then remains constant.However, it was observed that the accuracy was improved when the concentration of HCl was in the range of 0.5 to 1.0 mol L -1 .Moreover, formation of precipitate was observed in HCl medium if the acid concentration was 2.0 mol L -1 or higher than this, probably due to the presence of organic compounds.Therefore, the concentration of HCl in the sample and solutions of Hg 2+ was fixed in 1.0 mol L -1 .
Regarding to the sample flow rate, it was observed that the Hg 2+ signal was nearly the same in the range of 2.5 to 15.0 mL min -1 (Figure 3).However, if the sample flow rate was higher than 10 mL min -1 the precision was worse (RSD higher than 10%).

Hg inorg elution
Mercury elution from the Dowex 1-X8 resin is difficult because of the strong interaction between the Hg chlorocomplexes and the resin.Few reagents are suitable for the elution; only those who have high affinity with the Hg inorg or are able to convert the Hg inorg chlorocomplexes into other species of Hg are feasible.L-cystein or mixture of SnCl 2 and HCl have been proposed 16,17,26 for removing Hg from the Dowex 1-X8 resin.
A mixture of SnCl 2 (10% m/v) and HCl (2.0 mol L -1 ) was investigated in the present work.Unfortunately, it took more than 180 s to finish the Hg elution from the IEC column.NaBH 4 and HCl were then investigated, whose solutions were on-line mixed in the FI system (confluence a in Figure 1).In this case, the elution of Hg inorg was completed within 60 s, but it was necessary to adjust the concentration of NaBH 4 because of hydrogen production, which deteriorated the sensitivity and stability of the FI-CV system.In order to overcome this drawback, the NaBH 4 and HCl solutions concentrations were investigated individually.Sensitivity, time for analyte elution and stability of the FI-CV system were taken into account.According to Figure 4, the highest sensitivity is observed for HCl when its concentration is about 3.0 mol L -1 .The NaBH 4 concentration influence on Hg inorg is also depicted in Figure 4, where we can note that the highest signal is obtained when the concentration of NaBH 4 is 0.1% (m/v).The analyte signal decreasing as a function of reductand increasing may be due to the greater quantity of hydrogen produced, which dilutes the Hg vapor cloud in the quartz cell.
The influences of HCl and NaBH 4 solutions flow rate (mixed in confluence a in Figure 1) to elute Hg from the IEC column were investigated.It was observed that the absorbance was almost the same when the flow rates ranged from 1 to 6 mL min -1 .The argon flow rate was also investigated and the best performance was observed for a flow rate of 100 mL min -1 .Small drops of water were transported to the quartz cell at higher argon flow rate, hampering the correct determination of Hg.Moreover, tailed peaks were observed if the gas flow rate was less than 50 mL min -1 .All established conditions are summarized in the Table 2.

Hg org preconcentration
As previously discussed, organic Hg species produce neutral compounds in presence of HCl and do not interact with anion exchanger resins.In the present work, after passing through the IEC column, the Hg org was transformed into volatile species and then amalgamated by the Au/Pt gauze (Figure 1).Parameters that influence on the generation of volatile Hg org species, their amalgamation, releasing from the Au/Pt gauze and transportation to the quartz cell were investigated.The best conditions were then established (Table 2).At the conditions shown in Table 2, the effluent of the IEC column containing Hg org and HCl (1.0 mol L -1 ) was merged with a 0.1% (m/v) NaBH 4 solution at a flow rate  of 4.0 mL min -1 .The volatile Hg org species produced were then transported to the Au/Pt column by argon at a flow rate of 100 mL min -1 .Next, the retained Hg was removed from the gauze by heating at 600 °C.Under catalytic influence of the noble metals (Au and Pt) the organic gaseous mercury species are decomposed and then amalgamated. 29Thus, prior decomposition of the volatile Hg species for Hg 0 production and subsequent amalgamation is not required.
The influence of NaBH 4 concentration was also investigated for CH 3 Hg + (not shown in Figure 4) and it was observed that the analyte signal increased in presence of up to 0.1% (m/v) NaBH 4 , remaining constant at higher concentrations.In previous studies 28,30 it was observed the CH 3 Hg + signal was negligible at low NaBH 4 concentration but reached a maximum in presence of 0.75% (m/v) NaBH 4 .However, in those works the NaBH 4 had to be sufficient to transform the entire CH 3 Hg + to Hg 0 in order to be detected by AAS.In the present work, the volatile Hg species (mainly CH 3 HgH) are previously reduced to Hg 0 by the noble metals (Au/Pt in the amalgamation column) 29 prior to the determination by AAS.As a consequence, a lower amount of NaBH 4 is needed.In addition, the same solution of NaBH 4 (fixed in 0.1% m/v) can be used for both Hg inorg and Hg org determination.

Influence of Hg species concentration
Organic and inorganic Hg species are present in different concentrations in the sample and each one can influence the separation of the other.The influence of the concentration of each species on the separation was investigated by setting up the concentration of Hg 2+ in 100 ng L -1 and increasing the concentration of CH 3 Hg + and vice versa.According to Figure 5, one Hg species do not affect the separation of the other for concentrations of 1500 ng L -1 Hg inorg or Hg org .It was also observed that the capacity of the IEC and amalgamation columns used were limited to 40 ng of Hg 2+ and 65 ng of Hg (in the form of CH 3 HgCl), respectively.

Determination of Hg in landfill leachate
For Hg inorg and Hg org speciation, the liquid fraction of the samples was acidified with HCl, while the solid fraction was acidified with HCl and heated in microwave oven 30 in order to extract the Hg species, as summarized in Figure 2. The concentrations of Hg inorg and Hg org determined in both fractions are shown in Table 3.We can see that the sum of Hg inorg and Hg org determined by using the proposed speciation method is in agreement with the concentration of Hg tot determined by CV AAS.Moreover, the recoveries of the spiked Hg 2+ and CH 3 HgCl are in the range of 98 to 105%.These results demonstrate the reliability of the proposed method for Hg speciation.It is also important to note in Table 3 that the ratio of H org to Hg tot in both fractions of the sample is expressive, showing that the Hg inorg may be converted to more toxic species of Hg in municipal landfills.The concentrations of methyl mercury found in the liquid fraction of the leachate samples analyzed in the  In (A) the Hg 2+ concentration was fixed in 100 ng L -1 while that of CH 3 Hg + was varied.In (B) the CH 3 Hg + was fixed in 100 ng L -1 (as Hg) and that of Hg 2+ was varied.All solutions were processed at the established conditions shown in Table 2.

Table 1 .Figure 1 .
Figure 1.FI-CV system used for Hg org and Hg inorg speciation.P: peristaltic pump; V 1 , V 2 , V 3 , and V 4 : three-way solenoid valves; IEC: ion-exchange column; Au: Au/Pt amalgamation column; Ar: argon; GLS 1 and GLS 2 : gas/liquid separators; HL: halogen lamp; QC: quartz cell; R: sample or reagent solution recycling; W: waste; x: 10 mm; y: 10 mm; z: 100 mm; a and b: confluences.Numbers in parenthesis indicate the solutions flow rate, in mL min -1 .Dotted line means the valve is closed in that direction.

Figure 2 .
Figure 2. Sample preparation procedure for Hg org and Hg inorg speciation in urban landfill leachate.

Figure 5 .
Figure5.Influence of CH 3 Hg + concentration on Hg 2+ separation (A) and Hg 2+ on CH 3 Hg + separation (B).In (A) the Hg 2+ concentration was fixed in 100 ng L -1 while that of CH 3 Hg + was varied.In (B) the CH 3 Hg + was fixed in 100 ng L -1 (as Hg) and that of Hg 2+ was varied.All solutions were processed at the established conditions shown in Table2.

Table 3 .
Mercury concentration in liquid and solid fractions of landfill leachate.Values are the mean and standard deviation of 3 sample replicates Dry weight at 105 °C; b Determined by CV AAS; c Determined by the proposed FI-CV AAS method; d Calculated from the mean values of Hg tot and the sum of Hg inorg + Hg org a