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Spectrochimica Acta Part B

Analytical note

Determination of methylmercury by electrothermal atomic absorption

spectrometry using headspace single-drop microextraction

with in situ hydride generation

Sandra Gil, Sandra Fragueiro, Isela Lavilla, Carlos Bendicho*

Departamento de Quı́mica Analı́tica y Alimentaria, Area de Quı́mica Analı́tica, Universidad de Vigo, Facultad de Ciencias (Quı́mica),

As Lagoas-Marcosende s/n, 36200 Vigo, Spain

Received 27 July 2004; accepted 26 October 2004

Available online 24 November 2004

Abstract

A new method is proposed for preconcentration and matrix separation of methylmercury prior to its determination by electrothermal

atomic absorption spectrometry (ETAAS). Generation of methylmercury hydride (MeHgH) from a 5-ml solution is carried out in a closed vial

and trapped onto an aqueous single drop (3-Al volume) containing Pd(II) or Pt(IV) (50 and 10 mg/l, respectively). The hydrogen evolved in

the headspace (HS) after decomposition of sodium tetrahydroborate (III) injected for hydride generation caused the formation of finely

dispersed Pd(0) or Pt(0) in the drop, which in turn, were responsible for the sequestration of MeHgH. A preconcentration factor of ca. 40 is

achieved with both noble metals used as trapping agents. The limit of detection of methylmercury was 5 and 4 ng/ml (as Hg) with Pd(II) or

Pt(IV) as trapping agents, and the precision expressed as relative standard deviation was about 7%. The preconcentration system was fully

characterised through optimisation of the following variables: Pd(II) or Pt(IV) concentration in the drop, extraction time, pH of the medium,

temperatures of both sample solution and drop, concentration of salt in the sample solution, sodium tetrahydroborate (III) concentration in the

drop and stirring rate. The method has been successfully validated against two fish certified reference materials (CRM 464 tuna fish and

CRM DORM-2 dogfish muscle) following selective extraction of methylmercury in 2 mol/l HCl medium.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Methylmercury hydride; Headspace single-drop microextraction; Pd(II); Pt(IV) sequestrating ions; ETAAS

1. Introduction

Solid-phase microextraction (SPME) [1] and single-drop

microextraction (SDME) [2] have emerged in last years as

powerful tools for preconcentration and matrix separation

prior to detection. Although originally developed for

organic analytes, their potential for preconcentration of

trace metals and organometals has been recognised [3].

While two sampling modes are available for performing

microextraction techniques (i.e. direct and headspace), the

use of headspace, although requiring volatile or semivolatile

0584-8547/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.sab.2004.10.008

* Corresponding author. Tel.: +34 986 812281; fax: +34 986 812382.

E-mail address: bendicho@uvigo.es (C. Bendicho).

analytes, avoids extraction of potentially interferent non-

volatile compounds [4].

Determination of methylmercury is of paramount impor-

tance owing to the toxicological effects associated with this

Hg species [5]. Methylmercury derivatives are very volatile,

which benefits their separation from the matrix. Methyl-

mercury is commonly determined by several techniques

such as atomic absorption spectrometry (AAS) [6], atomic

emission spectrometry (AES) [7], atomic fluorescence

spectrometry (AFS) [8] and inductively coupled plasma-

mass spectrometry (ICP-MS) [9] after gas chromatography

separation.

Methods involving preconcentration of methylmercury

by SPME prior to its chromatographic separation have

been published. After headspace sampling, gas chroma-

60 (2005) 145–150

Table 1

Thermal program for determination of methylmercury by ETAAS following

headspace single-drop microextraction

Stage Temperature

(8C)Hold time

(s)

Ramp

(8C/s)Gas flow rate

(ml/min)

Drying 120 20 10 300

Ashing 200 (Pd) 10 10 300

400 (Pt) 10 10 300

Atomisation 1400 (Pd) 5 (off) 0

1500 (Pt) 5 (off) 0

Cleaning 2300 3 500 300

S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150146

tography (GC) coupled to AAS [10], MS [11], ICP-MS

[12], AFS [13] and MIP-AES [14] have been used for

detection. Recently, direct couplings between SPME and

an atomic detector without chromatographic interface, as

an efficient way to improve the detection limits of MeHg+

and avoid potential decomposition risk (artefact forma-

tion) existing in chromatographic separations have been

described [15,16].

In a previous work [17], the authors reported for the

first time, a headspace (HS)-SDME technique for precon-

centration of hydride-forming elements such as As, Sb and

Se onto a Pd(II)-containing aqueous drop prior to detection

by electrothermal atomic absorption spectrometry

(ETAAS). The sequestration mechanism proposed lied in

the catalytic decomposition of the hydrides in the Pd(0)

formed on the drop surface. Pd(0) arises as a result of the

reducing action caused by the hydrogen gas that evolves in

the headspace after the sodium tetrahydroborate (III)

decomposition. In this case, Pd fulfils two functions, i.e.

it behaves as both a trapping agent and a matrix modifier

in the furnace.

MeHg+ can be derivatised to form MeHgH upon

reduction with sodium tetrahydroborate (III) [18,19].

According to the mechanism above indicated, this com-

pound should also be efficiently trapped onto a drop

containing a Pt-group element.

In this work, the preconcentration and matrix separa-

tion of MeHg+ by HS-SDME following hydride gener-

ation is proposed. Pd(II) or Pt(IV) are employed as

trapping agents in the aqueous drop. The enriched drop

with mercury is subsequently injected in a graphite tube

for Hg determination by electrothermal atomic absorption

spectrometry (ETAAS). The preconcentration system is

fully characterised through optimisation of the relevant

variables influencing the generation and sequestration of

methylmercury.

2. Experimental

2.1. Apparatus

A Unicam (Cambridge, UK) Model Solaar 939 Spec-

trometer equipped with a GF-90 graphite furnace atomiser

and an FS 90 autosampler was used. An Hg hollow-

cathode lamp was employed as the radiation source.

Integrated absorbance was chosen as the analytical signal.

Atomic absorption measurements were performed at 253.7

nm. The spectral band-pass was 0.5 nm. A deuterium

background corrector was used when necessary. The

thermal program for Hg is shown in Table 1. Pyrolytic

graphite-coated graphite tubes with platform were

employed.

A high precision microsyringe (10 Al) with a plunger

made of polytetrafluoroethylene (PTFE) (Hamilton) was

employed for single-drop microextraction.

Ultrasonic extraction of MeHg+ from fish tissue was

carried out by a Sonics and Materials (Dambury, CT, USA)

Model VC 100 probe ultrasonic processor.

2.2. Reagents

All chemicals were of analytical reagent grade. A stock

solution of MeHg+ (500 mg/l as Hg) was prepared by

dissolving the appropriate amount of MeHgCl (Riedel-de

H7en, Pestanal, Germany) in ultrapure water. Firstly, the

MeHgCl was dissolved in a small amount of propan-2-ol

(Merck, Darmstadt, Germany). The solution was stored at

4 8C prior to use. Diluted working standards were prepared

fresh daily from the stock solution. Sodium tetrahydrobo-

rate (Merck), acetic acid and sodium acetate were used.

CRM BCR 464 tuna fish and CRM NRCC DORM-2

dogfish muscle were used for validation.

The trapping agent solutions were prepared from

Pd(NO3)2d 2H2O (Merck) and H2PtCl6 (Fluka, Steinheim,

Switzerland). L(+) ascorbic acid (Merck) was used to obtain

a reduced Pd matrix modifier for direct determination of

MeHg+ by ETAAS.

2.3. Procedure for headspace single-drop microextraction

of methylmercury

A 5-ml solution in 0.1 mol/l NaOAc/HOAc buffer is

placed into a 40-ml vial closed with a silicone rubber

septum. The septum was pierced by the microsyringe so

that needle tip was located above the surface of the sample

solution. Sampling was carried out by exposing to the

headspace a 3-Al aqueous drop (50 mg/l of Pd(II) or Pt(IV)

in 3% volume/volume HNO3) that is suspended at the

needle tip. Then, 1 ml of sodium tetrahydroborate (III)

(3.5% mass/volume) was injected into the vial while the

solution was being stirred. After allowing trapping of the

MeHgH onto the drop for 2 min, the drop is retracted back

into the microsyringe and subsequently injected in the

graphite furnace. The SDME device is depicted in Fig. 1.

Optimal conditions for HS-SDME of MeHgH were the

following: a 50 mg/l Pd(II) or Pt(IV) concentration in the

drop; a 3.5% mass/volume sodium tetrahydroborate (III) (1

ml injection volume); a 3-min extraction time; medium

composition: a 5-ml sample solution containing 0.1 mol/l

HOAc/NaOAc buffer (pH 5)m1 g NaCl; a 300-rpm stirring

A

B

E

F

G

C D

Fig. 1. Scheme showing the headspace single-drop microextraction device.

(A) Microsyringe (1–10 Al) for suspending the drop; (B) syringe for

injecting the sodium tetrahydroborate (III) solution; (C) 3-Al aqueous dropcontaining Pd(II) or Pt(IV); (D) 40-ml volume vial; (E) sample solution

containing MeHg+; (F) magnetic stirrer; (G) septum.

S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150 147

rate of the sample solution; a 3-Al drop volume; drop and

sample temperature: 20 8C. A 100 ng/ml MeHg+ concen-

tration (as MeHgCl) was used for optimisation.

3. Results and discussion

3.1. Optimisation of the HS-SDME method

Sequestration of methylmercury hydride onto the drop

containing the reduced noble metal on its surface could be

explained through the catalytic decomposition mechanism,

as proposed in a previous paper [17]. Unlike recent work

dealing with SDME of organometals [20,21], an aqueous

drop containing Pd(II) or Pt(IV) ions is employed here

instead of an organic solvent drop. Other trapping agents

tried in this work, which are based on the affinity of

mercury for binding thiol groups, such as l-cysteine or

diethyldithiocarbamate, did not provide efficient trapping

of methylmercury hydride. These compounds act as strong

complexing agents for mercury ions in solution, but are

unable to sequestrate MeHgH from the headspace.

Fig. 2 shows the effect of the noble metal concentration in

the drop. Trapping is equally effective with Pd(II) and Pt(IV).

As can be observed, maximum preconcentration was

obtained with a concentration in the drop about 50 mg/l of

Pd or 10 mg/l of Pt. These concentrations were similar to that

found as optimal for sequestrating volatile covalent hydrides

such as AsH3, SbH3 and SeH2 [17]. Despite HS-SDME being

an equilibrium-based technique, optimisation of the extrac-

tion time is necessary to achieve an efficient sequestration.

Fig. 3 shows the effect of the extraction time in the range 15–

300 s. For both Pt and Pd, increasing extraction occurs up to a

ca. 180 s extraction time. This time is much shorter than that

found for the use of HS-SPME [16] using the same

derivatisation procedure. As in both cases, the mass transfer

in the headspace is assumed to be identical, a faster mass

transfer in the drop must occur in comparison with the SPME

fiber coating. This could be an important advantage of SDME

in comparison with SPME approaches for sample preparation

prior to determination of methylmercury.

Similar performance is observed for Pt and Pd. Pd was

chosen for optimisation of the remaining variables.

The use of an HOAc/NaOAc buffer has been recom-

mended for generation of MeHgH [10]. In this study, both

the buffer concentration and the pH achieved were

optimised. The pH was studied in the range 2–9. While

poor performance is observed at acid pH, a pH between 5

and 9 yielded similar results. The buffer concentration was

studied in the range 0.1–1 mol/l. The higher the buffer

concentration, the less stable the drop becomes at the tip of

the needle. This effect was thought to be caused by the

increased pressure reached inside the vial as the buffer

concentration increased. A 0.1 mol/l buffer at pH 5 was

considered as adequate for efficient MeHgH generation.

The salting-out effect was studied by addition of NaCl.

Additions of NaCl masses in the range 0–5 g to a 5-ml

sample volume were performed. The extraction increased

slightly up to 1 g NaCl and remained constant from that

NaCl mass. A 1 g mass of NaCl was added to the sample

solution for further experiments.

The effect of both the sample solution and drop temper-

ature was also tested. The optimisation curve for the sample

solution temperature showed that extraction increased from 5

to 20 8C, and levelled off from 20 8C. On the contrary, whenthe drop temperature was studied, a steady extraction is

observed in the range 10–30 8C, but extraction diminished

from a 30 8C drop temperature. A 20 8C temperature was

chosen as optimal for both sample solution and drop. Other

variables optimised did not display any influence in the

intervals studied. Thus, a constant trapping efficiency was

observed when the stirring rate of the sample solution was

varied in the range 100–900 rpm. Likewise, the sodium

tetrahydroborate (III) concentration did not show any

influence in the range 0.5–6% mass/volume.

Sequestration experiments performed with Hg(II) salts

under the optimal generation and trapping conditions

established for MeHg+ showed that the trapping efficiency

0,000

0,050

0,100

0,150

0,200

0,250

0,300

0,350

0 50 100 150 200 250 300 350

Trapping agent concentration (mg/l)

Inte

grat

ed a

bsor

banc

e (s

)

PdPt

Fig. 2. Effect of the trapping agent concentration on Hg absorbance. Uncertainty intervals for N=3 replicate measurements are shown.

S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150148

was at least five times less for Hg(II) in comparison with

MeHg+. Some trapping observed for Hg(0) generated upon

reaction between Hg(II) and sodium tetrahydroborate (III)

could be due to the ability of Hg(0) to form amalgams with

noble metals.

Finally, optimisation of the method was accomplished by

studying the effect of the drop volume on extraction. As

expected, a larger drop surface exposed to the headspace

brought about an improvement in the extraction efficiency.

The maximum allowable drop volume was 3 Al. Larger dropvolumes caused the detachment of the drop from the

microsyringe tip during sampling.

3.2. Analytical characteristics

Analytical characteristics for the sequestration of MeHg+

onto a Pd(II) or Pt(IV)-containing drop were established.

The equation of the linear range of the calibration curves

were the following:

Pd(II): Y=0.0025 [MeHg+]–0.0236; r2=0.995

Pt(IV): Y=0.0029 [MeHg+]–0.0693; r2=0.996

where Y is integrated absorbance, and [MeHg+] is the

concentration of methylmercury (ng/ml).

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0 50 100 150

Extractio

Inte

grat

ed a

bsor

banc

e (s

)

Fig. 3. Effect of the extraction time on Hg absorbance using Pd(II) or Pt(IV) as

shown.

The calibration curves were linear up to 300 ng/ml.

Detection limits (LODs) (3r criterion) were 5 and 4 ng/ml

for trapping with Pd(II) and Pt(IV), respectively. Quantifi-

cation limits (10r criterion) were 18 and 14 ng/ml with both

trapping agents, respectively. RSDs, calculated from 10

replicates, were about 7% with both sequestrating agents.

The LOD of MeHg+, using the same instrument under

optimal furnace conditions and without preconcentration,

was 190 ng/ml for a 3-Al injection volume, which means

that a preconcentration factor of ca. 40 is achieved. It is

important to emphasize that direct determination (i.e.

without preconcentration) of MeHg+ by ETAAS required

the use of a reduced Pd modifier (0.25 Ag PdF0.5 Agascorbic acid) so that this species was thermally stabilised.

Without the reduced Pd modifier, the LOD of MeHg+ was

about 20 times worse. In the HS-SDME method proposed

here, a reduced Pd is already achieved during sampling of

the headspace as a result of the hydrogen evolved.

The LOD obtained by HS-SDME–ETAAS is comparable

to those obtained with HS-SPME–GC–ICPMS and HS-

SPME–GC–AAS (Table 2). Nevertheless, an improved

LOD is obtained by direct couplings between micro-

extraction and a detector such as SPME–ICPMS and

SPME–QF–AAS. A disadvantage of SPME is the limited

fiber lifetime and impaired precision with prolonged usage.

200 250 300 350

n time (s)

PdPt

trapping agents. Uncertainty intervals for N=3 replicate measurements are

Table 2

Comparison of LODs found in the literature for determination of

methylmercury after microextraction using headspace sampling

Analytical technique LOD (ng/ml) RSD (%) Ref.

SPME–GC–AAS 2.6 9 [10]

SPME–HG–QF–AAS 0.4 7 [16]

SPME–ICP-MS 0.2 2.4 [15]

SPME–GC–ICP-MS 3.7 17 [12]

SPME–GC–MS 1.3 6 [23]

SPME–GC–AFS 3 9 [22]

SPME–GC–MIP–AES 0.1 – [14]

SDME–ETAAS 4 7 This work

S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150 149

The SDME–ETAAS technique is fast, simple and cost-

effective as compared with more sophisticated couplings for

determination of methylmercury. Precision (expressed as

RSD) for the headspace sampling approaches reported in

Table 2 is typically in the range 6–9%.

3.3. Method validation

The method has been validated against CRM BCR 464

Tuna fish (certified MeHg+: 5.5F0.17 Ag/g) and CRM

NRCC DORM-2 (certified MeHg+: 4.47F0.32 Ag/g).Marine biological tissues can contain both inorganic

mercury and methylmercury. A separation of both species

is needed so that the developed method can be applied to

determination of methylmercury. The approach used here

lies in the different sulphur binding strengths of both species

(MeHg+bHg2+), which allows their separation in acidic

media with variable HCl concentration. Selective extraction

of MeHg+ is carried out according to the method established

by Rio-Segade and Bendicho [24]. The standard addition

method was used for calibration. The found values were

5.45F0.43 and 4.39F0.35 Ag/g (N=4) for CRM 464 and

CRM DORM-2, respectively. These values was in excellent

agreement with the certified ones, no significant differences

being observed (t-test, P=0.05). A recovery study at the 50

ng/ml analyte level was also performed with CRM 464. For

this purpose, the solid sample was spiked prior to extraction

with an MeHg+ amount in order to reach that final

concentration in the extract. The average recovery was

94F8% (N=3).

4. Conclusions

An effective sequestration of methylmercury hydride

onto a Pd(II) or Pt(IV)-containing aqueous drop (3 Al) is

demonstrated. This sampling technique combined with

ETAAS constitutes an attractive alternative to sophisticated

couplings employed for methylmercury determination,

being fast, simple and cost-effective. In contrast to other

SDME methods, no toxic organic solvents are required,

since the sequestration mechanism lies in the catalytic

decomposition of the methylmercury hydride onto an

aqueous drop containing Pd or Pt. The method is well

suited to determination of this Hg species in fish tissue by

ETAAS provided that a selective extraction is applied as

first sample pretreatment.

Acknowledgments

This work has been financially supported by the Galician

government (Xunta de Galicia) in the framework of Project

PGIDT01PX13101PR.

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