Talanta 47 (1998) 509 – 524

Review

Inorganic and methylmercury speciation in environmental

samples
J.E. Sánchez Urı́a *, A. Sanz-Medel
Department of Physical and Analytical Chemistry, Uni6ersity of O6iedo, c/Julián Cla6erı́a 8, 33006 O6iedo, Spain

Received 3 November 1997; received in revised form 25 February 1998; accepted 16 March 1998

Abstract

The different strategies for mercury species analysis in environmentally-related samples are reviewed. After
consideration of the main different steps involved in the speciation of mercury, such steps are discussed with more
extension for mercuric ion and methylmercury. The different approaches for preservation of these mercury species
during the storage of samples are considered. Different ways for the extraction of mercury species from the several
possible environmental compartments and the possibilities for preconcentration of such species after previous
derivatization reactions are discussed. Mercuric ions and methylmercury chromatographic and non-chromatographic
separations along with different techniques used for sensitive and selective detection of mercury are also critically
reviewed. Ranges of published detection limits achievable for such species determination, by using hyphenated
techniques between a chromatographic separation and a specific atomic detector are also given. © 1998 Elsevier
Science B.V. All rights reserved.

Keywords: Mercuric ion; Methylmercury; Speciation; Environmental samples

1. Introduction total mercury emissions to the atmosphere of

Mercury is considered by the Environmental
Protection Agency (EPA) as a highly dangerous
element because its accumulative and persistent
character in the environment and biota. Calcula-
tions of 1973 assessing the antropogenic atmo-
spheric contamination by mercury estimated a
* Corresponding author. Tel.: + 34 8 5103480; fax: + 34 8
5103125.
around 10000 tones. These emissions came from
the calcination of sulphide ores, fossil fuels com-
bustions and heating of other mercury-containing
materials [1]. Similar data were published by
Nriagu [2] 10 years later. Today, both inorganic
and organic mercury compounds are produced in
industrial processes for agriculture, paper indus-
try, pharmaceutical uses, etc. and they are respon-
sible for the vast majority of present antropogenic
contamination of our environment with this toxic
metal.

0039-9140/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved.
PII S0039-9140(98)00116-7
510 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
Moreover, mercuric ion can be converted into
methylmercury. It should be stressed that the
most numerous cases of human poisoning by an
organometal have involved the ingestion of
methylmercury compounds. This organometallic
species is neurotoxic, causes blockage of binding
sites of enzymes, interferes proteine synthesis,
impides thymidine incorporation into DNA, etc.
[3]. Reports of such cases have come from many
areas of the world, but those from Asia have been
the most numerous. Particularly disastrous were
the widespread methylmercuric compound poi-
soning cases of Minamata Bay, Japan, from
which the name ‘Minamata disease’ derived to
describe methylmercury poisoning [4]. These
organomercurials can be consumed in two classes
of foods: fish and shellfish accumulating the poi-
son from waters or sediments; and grain seeds
coated with antifungal methylmercuric prepara-
tions. These dangerous methylmercury species can
be antropogenic, but also inorganic mercury can
be biologically converted into methylmercury in
algae [5], humic substances [6], etc. via methyla-
tion of mercuric derivatives by bacteria [7–10].
Both inorganic and organic mercury tend to be
accumulated in sediments and biota [11,12], par-
ticularly fish and molluscs [13 – 15]. In this way,
neurotoxic methylmercury would eventually reach
our food chain specially from sea food [16]. The
high affinity of methylmercury to sulphydril
groups and lipids of animals would explain its
accumulation in living organisms, particularly in
lipidic tissue of mammals. Conversely, it appears
that sulphide groups in the sediment would be
responsible for the binding and final preconcen-
tration of mercury species [17] in sediments.
Nowadays it seems accepted that the rate and
extent of methylation of Hg(II) in the waters and
sediments depends upon factors such as: the com-
pound of Hg(II) (acetate is easier to methylate
than mercuric chloride), the methylation agent
[18], the chemical composition of the sediment, its
oxygen concentration and the pH [19].
In the waters it is interesting to note that levels
of methylmercury are usually much lower than
those of inorganic mercury. This is due to the
difficulty of methylation reactions in aqueous
phases, from one side, and to the easy decomposi-
tion by solar UV light [20,21] of organomercury
compounds from the other. Recent reports [22]
estimate a total mercury concentration in natural
waters, ranging from 0.2 to 100 ng l − 1, while
methylmercury levels are much lower, around
0.05 ng l − 1 [23]. Of course, higher values can be
found in waters from heavily industrialized areas
[22]. The upperlimit for total mercury concentra-
tion in drinking water recommended by the EC is
1 mg l − 1.
In sediments and biota the levels of methylmer-
cury are higher than in waters because of accumu-
lative phenomena [24]; while methylmercury in
sea water is only found in highly contaminated
areas and amounting only to around 1% of total
element present [25], inorganic mercury and
methylmercury seem to be preconcentrated in sed-
iments and are found at relatively high levels in
fish [14,15,26,27].
Several authors have found the presence of
dimethylmercury in fish [24,28–31]. However, Puk
and Weber have pointed out that most analytical
methods published would not provide reliable re-
sults, or even detect dimethylmercury species, if
present [27]. Studies on the occurrence of other
organic mercury compounds in the environment
are comparatively scarce, even if some recent ref-
erences have been published on the presence of
ethylmercury in water samples [32], soils and sedi-
ments [29,33,34]. Cai et al. attribute this lack of
observations of these species to present analytical
methodologies mainly focused to develop meth-
ods just for methylmercury [34].
Nowadays it is worldwide recognized that the
toxicity of an element (e.g. Hg) is determined by
its particular species occurring in the sample. In
general terms the organic forms of metals (more
hydrophobic) go through biological membranes
quite easily as compared to inorganic forms.
Thus, organomercuric compounds are much more
toxic than inorganic mercury. Therefore, today we
are witnessing a growing interest in analytical
speciation of trace elements [35–37] and of mer-
cury in particular in samples of environmental
origin.
Considering the present knowledge, the main
species or forms of mercury to be identified and
determined in the environment are mercuric ion
 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
(inorganic mercury), methylmercury and, only
occasionally, dimethylmercury. The various re-
views appeared in the last few years about this
topic [27,38 – 41] give evidence of the great scien-
tific interest arisen by mercury speciation.
A possible revival of older disastrous episodes
of 1953 in Minamata [4], and other parts of the
world in the 1960s, could happen now in the
Amazonia as a result of the new ‘gold rush’ of
the so-called ‘garimpeiros’ or gold-diggers. Their
techniques of amalgamation on gold-ore and
further open ‘burning’ of the possibly formed
gold amalgam is responsible for 80% of the to-
tal mercury emission into atmosphere in Brazil
(168 tonnes of the metal according to estima-
tions of 1989). Investigations to evaluate total
mercury contamination in waters, sediments and
biota in Amazonia rivers are already in progress
[26,42,43] and a cooperative project to assess the
extent of methylation of such mercury
(volatilized into atmosphere and then deposited
in soils, flora and rivers) is due to start this year
[44].
In this paper a revision of the different steps
to speciate mercury, particularly inorganic and
methylmercury, in environmental samples is car-
ried out. Precautions to be taken at each step
(extraction, preconcentration, separation and
specific detection), analytical strategies and par-
ticular techniques proposed to tackle this mod-
ern problem of mercury speciation are
thoroughly discussed.
2. The main steps to consider for mercury
speciation in environmental samples
It is quite obvious that the ideal analytical
strategy for speciation of mercury and other
toxic elements would be direct ‘in-situ’ analysis
of the sought species in the desired sample. In
this way the most stringent challenge of specia-
tion, namely preservation of the original nature
of the sought species, would be nearly secured
by avoiding sample manipulations of any type.
Electrochemical and optical (bio)-sensors could
be in the future viable approaches for metal spe-
ciation [45,46]. At present, however, ‘in-situ’ di-
511
rect species-specific techniques are seldom useful
to solve speciation problems in real-life situa-
tions because of lack of the sensitivity. In such
situations previous sample treatments, precon-
centrations, separations, etc., before final detec-
tion are usually needed.
In order to achieve a reliable determination of
the two main environmentally relevant species of
mercury, Hg(II) and CH3Hg + , there are four
different steps to be considered individually:
1. Mercury species ‘extraction’ from the envi-
ronmental sample (e.g. from soil, sediment or
biota) securing the integrity of the sought
species as they are in the sample;
2. Mercury species ‘preconcentration’ from the
obtained extract (again, preserving the iden-
tity of those components) to achieve a final
concentration level matching the detection
limits accessible with the detection technique
selected;
3. Inorganic mercury and methylmercury ‘sepa-
ration’ without disturbing their relative origi-
nal concentration levels;
4. Individual ‘detection’ (determination) of each
of the previously separated species.
Of course, in filtered aqueous samples only
the last three steps should be considered. How-
ever, to speciate a given toxic metal in an
aquatic environment non-dissolved materials
(solids in suspension, sediments and soil) and
biota (plants, fish, shellfish and plankton) should
be dealt with as well.
As a result of the extremely low levels of mer-
cury compounds in such real samples the cou-
pling of the four different steps to develop an
integrated mercury speciation strategy in those
particular samples is almost mandatory. All the
four steps should be ‘tamed’ to such a degree
that risks of contamination, losses, degradations
or changes in the mercury species nature and
their relative concentrations are avoided (or at
least, well assessed to allow for eventual correc-
tions).
Table 1 summarizes the ‘more common tech-
niques’ used to assemble an integrated strategy
made of ‘the four mentioned steps required for
speciation in environmental samples’.
512 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
Table 1
Pretreatment Preconcentration
Homogenization Criogenic trapping
Acidic preservation Column chromatography
Extraction of species Non-chromatographic columns
—Alumina
—Cotton sulphydril
—Dithiocarbamate
—Ditizone
Set-pack cartridges
Electrochemical methods
3. Inorganic and methylmercury extractions
To start with, the environmental sample (water,
sediment or biota) should be made homogeneous
and perhaps preservative agents could be needed
in order to prevent species degradation before
final analysis. It is well known that organometal-
lics (e.g. methylmercury, methylarsenic com-
pounds, tributhyltin) can be degraded in many
ways including microorganisms action, oxidation,
or even UV irradiation. This is why preservation
of CH3Hg + in its original form and relative con-
centration can represent a difficult task, particu-
larly when its extraction from a solid is required;
the matrix of the sample can play an important
role in the stability of the sought compound. For
instance, it has been shown that CH3Hg + in fish
(dry or wet) exhibits a remarkable stability with
time at room temperature, while in shellfish sam-
ples a prolonged storing in the refrigerator, or
several freezing and de-freezing of such samples,
may bring about significant CH3Hg + losses [47].
The stability of mercury species in waters is
rather controversial. There is, however, a sort of
consensus on the positive effects of low pH and
high ionic strength to stabilize mercury species in
solution, particularly when in the solution exists
an oxidizing and complexing environment [48].
Oxidants and complex forming agents appear to
contribute to stabilize Hg(II), while low pH and
high ionic strength prevent cation deposition on
the containers wall. Thus, it is advisable to add
such preservative agents [49] if the analysis is
going to be performed with a delay after sam-
Separation Detection
G.C. CV-AAS
HPLC ETAAS
Non-chromatographic MIP-AES
ICP-AES
ICP-MS
ECD
Electrochemical
AFS
pling. Interestingly, the efficiency of such reagents
to stabilize mercury species seems to be related
with the water matrix (distilled, fresh water or sea
water). Losses of methylmercury in sea water at
the ng l − 1 level have been reported to be caused
by adsorption onto the container walls and con-
version into the inorganic form [50].
Mineral acids are probably the most common
reagents added to water in order to maintain
unaltered mercury species in solution, e.g. solu-
tions with 1–2% of HCl or HNO3 [50,51], or 1%
HNO3 containing 0.05% (m/v) of K2Cr2O7 [52];
other preservative reagents proposed include hu-
mic acids [53], freezing in liquid nitrogen and
further storage at − 80°C [28], Au(III) in diluted
HNO3 acid [54], etc. It has been shown [48] that
the stability of CH3Hg + in waters depends criti-
cally on its concentration level, matrix of the
water, containers material and its pretreatment
for cleaning, temperature, etc. For instance, it
appears that low concentration of CH3Hg +
(lower than 10 ng ml − 1) are very unstable, while
its stability at around 100 ng ml − 1 levels im-
proves substantially [48]. Likewise, the effect of
container material is also stressed in the literature;
PTFE containers are preferred to PVC or glass
made flasks, provided their previous cleaning with
nitric acid [55]. The paper by Leermarkers et al.
[48] is recommended in this respect as it is the
paper by Lepine and Chamberland [56] about the
filtering of water samples immediately after sam-
pling. If a filtration of sample is required, it is
necessary to do it at the very moment of sam-
pling, because freezing and filtration after de-
 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
freezing originates a lost of both, inorganic mer-
cury and methylmercury.
In biota and sediments, immediate freezing of
samples after collection is recommended as a rule
in order to retard microbiol activities [57]; doubts
arise sometimes, though, about the possibility of
species changes if samples are then de-freezed or
dried in the air before speciation measurements
(e.g. in the case of deep sediments originally sur-
rounded by a reducing environment). Liofilization
processes appear to be risky for biota speciation
because some volatile species can be lost and
some metal-bound proteins can degrade [58].
Extracting the sought species of methylmercury
from the solid environmental sample is a delicate
process depending on both, the nature of the
species and of sample matrix. While in water
samples this process is not so troublesome [28,59–
63], in biota or sediments the extraction is proba-
bly the most crucial step of the whole speciation
strategy designed: a complete extraction of Hg(II)
and CH3Hg + should be aimed at without chang-
ing the nature of those species. Of course this is
very difficult and demands a great deal of care in
order to preserve the original speciation. More-
over, the dissolved extracts can be unstable, e.g.
they should be kept out of UV light which could
cleave the bond Hg – C [20,64,65]. As a general
rule, this bond Hg – C should be safe during pre-
treatment or extraction with added reagents. For
example, reagents used for extractions from biota
and sediments should be able to break bonds of
the mercury species with clay materials, humic
substances, sulphides, etc., but the Hg – C bonds
should be left unaltered.
The techniques published for pretreatment ex-
traction of mercury species in environmental sam-
ples can be grouped into three categories.
1. Acid digestion with solvent extraction: this
digestion type was proposed long ago for
Westöö as a means for the extraction of
methylmercury in foodstuffs with HCl as
acidic medium and benzene as the solvent; the
extraction needs several steps in order to get a
‘clean’ solution of CH3Hg + in benzene
[66,67]. Later on many modifications of West-
öö’s methods were proposed for selective ex-
traction of methylmercury from a mineral
513
acidic medium containing NaCl [68,69], KBr
[62,70,71] and iodoacetic acid [72], generally
using successive extractions with organic sol-
vents such as benzene [73,74], toluene [75,76],
chloroform [62,77] or dichloromethane [28,78].
It seems that benzene is not well suited for
CH3Hg + extractions at low concentrations
down to 0.5 ng l − 1 [63]. Several authors rec-
ommend a back-extraction of the mercury spe-
cies from the benzene or toluene phase to the
aqueous phase, in order to clean or preconcen-
trate the extracted species, using cysteine or
sodium thisulphate [66,67,76]. Problems
derived from solvent background may arise
when toulene is used and the detection is
carried out by microwave-induced plasma-
atomic emission spectrometry (MIP-AES) [79].
In the case of CH3Hg + speciation using chlo-
roform, addition of complexing agents to facil-
itate the extraction of methylmercury to the
chloroformic phase has been proposed [62,77],
while addition of HgCl2 [25,80] or CuCl2 [81]
solutions has been recommended to release the
CH3Hg + from the –SH groups complexing
the mercury species in the solid.
2. Alkaline digestion and extraction: both KOH-
methanol [28,54–56,59,60] and NaOH-Cys-
teine [59] treatments have been proposed to
release methylmercury from sediments while
maintaining original Hg–C bonds. In many
cases, this alkaline extraction appears trouble-
some as compared to acidic ones, because
alkaline solutions are much more difficult to
get in pure form as compared to acidic solu-
tions. Moreover, serious problems encoun-
tered in subsequent steps (preconcentration,
separation or detection) are derived from the
high levels of organic matter, sulphides or
ferric ions co-extracted with the sought
methylmercury species using this sample treat-
ment [81].
3. Acidic volatilization and preconcentration: an
alternative approach is to avoid organic sol-
vent extraction by producing a volatile deriva-
tive of the sought species [72,81]. Vapor
distillation, in a stream of air or nitrogen at
150°C, of a homogeneate of the solid sample
in diluted H2SO4 with excess of NaCl is
514 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
strongly recommended to allow a non-chro-
matographic separation of Hg(II) and
CH3Hg + . The more volatile CH3Hg + Cl −
compound formed is so distilled and is col-
lected in a closed tube. This tube is water-
cooled and stored in the dark in order to keep
extracted methylmercury degradation at a
minimum before its final determination with
the various atomic detectors [53,82,83].
Comparative studies of the performance of the
three extraction approaches cited, from 1 to 3, to
extract methylmercury in the same samples are
scarce. Horvat et al. [81] carried out such type of
studies by applying the three extraction ap-
proaches to two certified sediments. They con-
cluded that distillation of methylmercury from a
8-M H2SO4 solution containing KI, at 145°C in a
nitrogen stream of 60 ml min − 1 was a most
convenient system to isolate and preconcentrate
this mercury species from sediments. In a later
paper [84] the same authors compared both, sol-
vent extraction and distillation, methods for
methylmercury extraction from water samples
concluding that distillation method gives consis-
tent and high recoveries of MeHg in various
samples. However, recent investigations showed
that the distillation procedure used to separate
methylmercury from both water and sediment
samples generates artificially MeHg aided by the
presence of natural organic substances. The mag-
nitude of this artifact appears to be related to the
amount and the type of organic mater present in
the samples [85]. Horvat et al. [81] propose also
the ethylation of the methylmercury before the
final detection of mercury by atomic fluorescence
spectrometry (AFS). It should be noted, however,
that sulphide is the most serious interference for
the ethylation reaction [28]. Moreover artifact for-
mation of CH3Hg + may occur because the ethy-
lation reagent might induce methylmercury
formation from inorganic mercury if it is present
at much higher concentrations [81]. In any case,
the use of HCl alone for this distillation is not
advisable because it may not be able to release
completely the CH3Hg + contained in the sedi-
ment, the amount of final mercury recovered de-
pends on total organic carbon [48] and also on
sulphide content [86,87] of the analyzed sediment.
4. Preconcentration/derivatization
As said before, the low analytical concentra-
tions of organic or methylmercury expected (typi-
cally in the range of pg l − 1) determine very often
the need to carry out preconcentration of such
species in order to allow for concentration levels
measurable by the atomic detector used.
Sometimes liquid–liquid extraction have been
proposed after acid treatments of sediment, biota
or water samples [62,67,70,73,74,80,88,89]. When
the final detection is carried out by gas chro-
matography (GC) with an electron capture detec-
tor (ECD), back-extraction to an aqueous phase
and further re-extraction to the organic solvent is
recommended to clean up the extracts (atomic
detection is less critical and so some of the clean-
ing steps can be disregarded).
Of course, the above mentioned gas–liquid pro-
cedures of volatile mercury species formation and
distillation from environmental samples [28,82,83]
are most appropriate for methylmercury precon-
centration and derivatization by ethylation as a
mean to enhance the separation/sensitivity attain-
able before final specific detection. This concept
has been well developed in papers by Bloom [28],
Rapsomanikis et al. [90,91], Craig et al. [92], etc.
during this decade.
Solid-phase extraction can also be employed for
preconcentration of extracted mercury species.
Lee and Mowrer [63] proposed the use of sul-
phydryl cotton fiber (SCF) packings as sorbents
of methylmercury from an aqueous solution; after
trapping/preconcentration in the minicolumn the
methylmercury is released of the column with 2 M
HCl solution to be then extracted into benzene for
final GC-ECD determination. This system was
later extended to a flow injection analysis configu-
ration with final detection by CV-AFS [93] or
MIP-AES [94]. The group of Jiang [95] applied
these cotton sulphydryl minicolumns to the ‘field
sampling’ of inorganic mercury and methylmer-
cury in waters with interesting results, using a
field sampling kit consisting of an on-line filter
(0.45 mm), a SCF column and a syringe.
Similarly, other groups have investigated the
use of immobilized dithiocarbamates, packed in
minicolumns, for methylmercury preconcentration
 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
[86,96–98]; then, this species is eluted of the
column with an acidic solution of thiourea fol-
lowed by its extraction into toluene. There, the
organomercurial is derivatized using the Grig-
nard reagent before its final separation/determi-
nation by several possible techniques,
particularly gas chromatography with mi-
crowave-induced plasma-atomic emission spec-
trometry (GC-MIP-AES) [59,69,86,98].
Non-chromatographic separations can be also
successful as proposed some time ago by Mine-
gawa et al. [97]: using cold vapor generation
these authors described the separation of Hg(II)
(with SnCl2 in KOH solution) from CH3Hg +
(with a mixture of SnCl2 +CdCl2 in KOH solu-
tion) and final determination by CV-AAS.
Cryogenic trapping of both hydride and
ethylide derivatives of mercury species constitute
one of the more successful approaches to mer-
cury compounds preconcentration [24,28,29].
While the use of NaBH4 is more common for
cold vapor and hydride generation, the ethylide
generation reaction is usually achieved by
derivatization ‘in-situ’ (in the solution or acting
on a slurry of the sample) of the Hg(II) and
CH3Hg + at pH= 4.9 with NaB(C2H5)4 to form
volatile diethylmercury and methylethylmercury,
respectively [24,90,91]. It is worth noting that
ethylation reactions of mercury are considerably
slower than the corresponding hydride genera-
tion reactions with NaBH4. However,
NaB(C2H5)4 does not generate the great excess
of hydrogen characterizing NaBH4 decomposi-
tion in acidic solutions. Once the ethylation step
has concluded, the species of derivatized mer-
cury are preconcentrated by cryogenic trapping
with liquid nitrogen using a coiled borosilicate
glass column, previously cleaned and silanized
with a solution of 5% dichlorodimethylsilane in
toluene, then the column is packed with 10%
OV-101 on Chromosorb W AW-DMCS 40–60
mesh. A Nichrom resistance wire surrounding
the column, connected to a variable transformer
coupled to a temperature controller, raised the
temperature of the column (once this is void of
liquid nitrogen) and the ethylated mercury spe-
cies leave the column according to their boiling
points. The sequential detection of mercury spe-
cies was carried out by AAS using a quartz tube
515
as atomization cell.
Modifications proposed by Liang et al. [99]
allow to dispense with the use of cryogenic tem-
peratures for trapping. They proposed to trap
the ethylated derivatives formed in chromato-
graphic columns at room temperature and then
the different ethylated species are separated
there, on heating the column, to be eventually
detected by AFS.
Of course, other less popular approaches have
been published for methylmercury preconcentra-
tion including temperature-controled evaporation
in a vacuum [80], electrochemical preconcentra-
tion [100] or the ‘tandem on-line continuous
separation’ technique [101]. In this last approach
the preconcentration is achieved by continuously
extracting the CH3Hg + of the samples with io-
dide into xylene; the organic extract is continu-
ously merged with a solution of NaBH4 in
dimethylformamide, to form the volatile mercury
species, which are continuously drawn into an
ICP-AES by a stream of argon [101]. In this
way the first liquid–liquid separation operates
the selective extraction of CH3Hg + · I − (while
inorganic mercury remains in the aqueous
phase) and its preconcentration, while the sec-
ond liquid–gas separation step allows for an im-
portant increase in the sensitivity of the specific
mercury detection in the ICP-AES via cold va-
por generation. Once the methylmercury has
been separated, inorganic mercury of the
aqueous phase can be analyzed afterwards by
cold vapor formation from this phase in a flow
system [101].
5. Mercury species separation techniques
As a rule, chromatography is a more powerful
separation technique than non-chromatographic
approaches [102]. Therefore chromatographic
separations are more popular for speciation pur-
poses than non-chromatographic ones. However,
simple non-chromatographic approaches can be
successful to separate adequately one or two
species in a given sample [45]. This is the case
for Hg(II) and CH3Hg + speciation and we will
refer briefly to this particular speciation.
516 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
5.1. Non-chromatographics separations
The described method of ‘tandem on-line con-
tinuous separations’ for methylmercury speciation
[101] constitutes a good example of the possibility
of solving simple speciation problems by resorting
to non-chromatographic separations. The princi-
ple of using selective liquid – liquid extractions to
separate methylmercury from inorganic mercury
by using halogenated acids and organic solvents is
quite common and has been carried out usually in
an ‘off-line’ mode for the final detection of mer-
cury, usually with CV-AAS technology [62].
Flow injection analysis (FIA) systems are the
most adequate for this type of simple separations.
Thus the differential behavior of Hg(II) and
CH3Hg + versus reducing agents (i.e. while Hg(II)
is reduced to Hg0 by SnCl2, the methylmercury
species is not [21,22,103]) can be used to achieve a
simple speciation scheme using CV-AAS detec-
tion. Similarly the minicolumns previously de-
scribed [63,93 – 95] of sulphydril cotton fibers are
also good examples of non-chromatographic ap-
proaches to separate inorganic and methylated
mercury; in this latter FIA system [93], CH3Hg +
seems to be retained in the column while Hg(II)
goes through it. The retained organic mercury is
then released of the column, oxidized to inorganic
mercury (in a flow with acidic KBr/KBrO3), re-
duced to ‘cold vapor’ and detected by AFS [93].
This sulphydryl cotton fibers are not commer-
cially available and reported extraction efficiency
for CH3Hg + in benzene from these columns is
close to 65% [63].
Other non-chromatographic schemes proposed
include the use of photo-oxidation [103,104]:
firstly only inorganic mercury is detected by con-
ventional CV-AAS; secondly, the organic mercury
present is photo-oxidized to inorganic mercury
and the total content of mercury is evaluated; in
this way methylmercury is calculated by substrac-
tion. Recently, Madrid et al. [105] have proposed
the use of microorganisms to achieve this separa-
tion of inorganic and organic monomethylmer-
cury. Using Saccharomices cere6isiae in a
suspension, they showed that CH3Hg + seems to
bind quite selectively to these cells, after incuba-
tion at 37°C during 30 min, while Hg(II) showed
no affinity to S. cere6isiae yeast.
5.2. Chromatographics separations
Admittedly that non-chromatographic simple
technologies of separation may solve particular
problems of speciation [45] it is undebatable that
the most powerful approach to real-life speciation
consists of coupling a chromatographic separation
with a sensitive atomic detection. In fact, the
speciation of mercury in environmental samples is
mainly carried out by the vast majority of work-
ers by resorting to chromatographic separation
techniques, particularly employing GC for this
purpose [25,55,63,66,72,73,90,98,106]. Table 2
shows a schematic of the chromatographic separa-
tion type to be selected depending upon the na-
ture of the species to be separated and quantified
by hybrid speciation techniques [45].
As can be seen, GC is to be preferred for
species being volatile or able to form easily
volatile derivatives without uncontroled changes
of the compound in the derivatization/separation
processes involved. Thus GC is the more popular
separation technique for mercury and organomer-
cury compounds speciation using stationary
phases consisting of columns of variable length
(15–30 m and 0.3–0.75 cm i.d.) packed with OV
Table 2
Chromatographic-specific (atomic) detection for speciation
analysis
Problem: Volatile, thermostable, neutral species (or able
to produce them by derivatization)
GC “ Interface“ Atomic detector
Problem: Non-volatile/Thermally unstable/charged com-
pounds
HPLC“Interface “Atomic detector
HPLC Characteristics
Direct separation (no derivatization possible)
Integrity of species can be more easily pre-
served
Liquid chromatography is more versatile than
GC
HPLC drawbacks
HPLC provides liquid samples
Atomic detector prefer gaseous samples (less
sensitivity)
HPLC-Atomic detector interfaces with in-
creased sensitivity needed
 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
101, in variable concentration adsorbed on a solid
support of Chromosorb W-HP (80 – 100 mesh)
[107] or silanized glass columns packed with 5%
DEGS-PS on 100 – 120 mesh Supelcoprt (Supelco
Inc.) [108]. GC techniques have been already used
for the speciation of mercury in natural gases and
gas condensates [109].
There is, however, a clear trend today to the
use of open capillary columns, of the non-polar
type, coated with a thin film of phenyl or methyl
silicone [110,116]. It appears that this type of
columns provide more efficient separations and
better resolution, as compared with the above
mentioned Chromosorb-type GC columns. Of
course, the disadvantage of capillary columns is
the general restriction of the small sample volume
accepted (usually around 1 ml of sample). More-
over the more common GC detectors might be
lacking the required selectivity to be used in speci-
ation of mercury in environmental samples. For
instance, the well-known ECD have been pro-
posed for methylmercury speciation. However,
the ECD unselective response determined the
need to resort to laborious cleaning-up processes
of the extract in the organic phase used for the
mercury compounds extraction from the sedi-
ments. Preliminary work, using GC-ECD for mer-
cury speciation [70], used packed columns to
separate the methyl and ethyl mercury halides in
toxicological samples and ill-defined and non-re-
producible chromatographic peaks were reported.
This behavior was ascribed to the polar character
of these mercury halides which would therefore
interact too strongly with the packing material of
the column. A possible way-out to this unfavor-
able situation is the previous column ‘pasivation’
by its pretreatment with a solution of HgCl2 in
benzene or toluene [70,106]. Apart from the risk
of using high concentrations of Hg(II) for this
pasivation (before final analysis of low levels of
mercury compound in the sample) this pretreat-
ment is not durable and must be repeated rather
frequently. Therefore, alternatives to this classical
mercury speciation strategy have been investi-
gated in recent years particularly capillary
columns.
Other alternative approaches involve the use of
pre-column derivatization of mercury species in
517
order to transform them into non-polar di-
alkylderivatives. These latter compounds exhibit
much better chromatographic properties than the
original mercury species to be separated with cap-
illary columns [69,71,111,112]. Capillary columns
were also used for organomercury separation after
the ethylated analyses were preconcentrated on a
wide-bore fused-silica column and then desorbed
by heating of the trap [113]. It should be stressed,
however, that this derivatization approach de-
mands the use of an atomic detector (specific
detector for mercury) instead of the conventional
ECD. Of course, now the halide, which brings
about the measured ECD responses, is no longer
there with the mercury sought compound after
derivatization. Perhaps the most popular specific
detector in this connection has been the Mi-
crowave Induced Plasma in the hybrid technique
GC-MIP-AES [69,72,77,70,86,93,96,98,114]. Mul-
ticapillary columns for GC-MIP-AES, recently
proposed for speciation analysis of butyltin com-
pounds [115] allowing for ultra-rapid (30 s instead
of about 10 min) and more sensitive analysis,
perhaps may offer a good alternative for future
mercury speciation.
In an attempt to overcome the numerous draw-
backs associated with the gas chromatographic
determination of organomercury compounds
[110,111,117] many authors have resorted to alter-
native separation procedures. Particularly HPLC
techniques [75,88,118–126] have been tried with
the final aim of establishing a reliable procedure
for the speciation of such compounds in environ-
mental samples. Of course, HPLC techniques,
usually being less sensitive for detection, are more
suited for polar species and therefore could be
advantageous for organomercury and inorganic
mercury speciation. Moreover, the flexibility of
liquid chromatography, with more separation
mechanisms available, is considerably higher than
that of GC in order to achieve satisfactory separa-
tions of inorganic and organic mercury com-
pounds in complex environmental samples.
Although detection at the exit of the liquid chro-
matographic column has been proposed with dif-
ferent common HPLC detectors including UV-Vis
absorptiometry [75], fluorimetry, electrochemistry,
etc., the most effective detection incorporates an
518 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
atomic/mercury specific detector in the so-called
hybrid approaches which couple the HPLC with
atomic spectrometry. AAS [120,123,124], plasma
detection using photons measurements as in ICP-
AES [101] or MIP-AES [127,128] and mass spec-
trometry with on-line ICP-MS measurements
[121,129,130] have been proposed. The lack of
sensitivity is usually a main limitation [45]. Using
CV-AFS detection better detection limits for mer-
cury speciation can be obtained [28,71,81,84,93,
99,112,131].
There is not doubt that hybrid techniques, us-
ing an atomic detector on-line with the column,
are becoming the most popular approaches to
tackle the modern problem of trace element speci-
ation and this trend is now being noticed in the
case of mercury speciation. The main analytical
task is to couple the separative column (GC or
HPLC) with the atomizer and this operation is
achieved via an adequate ‘interface’. Of course gas
chromatographic columns are compatible with
any on-line atomizer (where atomic vapors are
produced) in a straightforward manner. Thus the
gaseous effluent from the column is directly intro-
duced into the atomizer. The only problem en-
countered can be the possible condensation of
vapors in the interface due to the difference in
temperatures between the column in the chro-
matograph oven, the interface itself and the atom-
izer. Therefore, a heating and thermostating
device to maintain the interface at a temperature
slightly higher than that of condensation of ex-
pected vapors is usually mandatory for GC-
Atomic Detection hybrid approaches [29,40,132].
The interface to couple HPLC columns with the
atomiser can be very simple, via the direct connec-
tion with a teflon tubing, of the exit of the column
with the nebuliser of the AAS [133,134] or plasma
detector [135,136]. Care should be exercised just
to make compatible the flow rate from the HPLC
column with the normal flow rates of the nebu-
liser used [137,138]. Unfortunately, the efficiency
of liquid samples transport attainable using nebu-
lisers is very low (1 – 3% for plasma nebulisers and
5–10% at most for AAS common nebulisers). As
a consequence, the sensitivity observed is limited
particularly using flame-AAS detection where at-
tainable detection limits are too high to approach
real-life speciation problems (demanding detection
limits below 1 mg l − 1). The use of on-line conven-
tional nebuliser AFS or ICP-AES detection would
find similar limitations of the required sensitivity.
A general way-out to this lack of sensitivity of
HPLC separation coupled to atomic detectors is
to resort to on-line derivatization at the exit of the
column to form ‘cold vapor of mercury’ to en-
hance the transport of the metal to the atomic
detector. So, HPLC has been used in connection
with AFS detection for organomercury species
analysis. After the distillation and preconcetration
of the mercury species they were separated onto
the HPLC column, derivatized to mercury cold
vapor and finally detected by AFS [139,140]. The
use of ‘organized media’ in the mobile phase may
help cold vapor formation and so enhance the
sensitivity of the determination [125]. In fact, the
use of vesicles exerts a synergic effect for the
speciation of mercury using AAS or ICP-AES
[141], particularly for MIP-AES detection [128].
Vesicle-mediated HPLC can be coupled on-line
with flame-AAS and particularly with plasma de-
tectors offering both, more flexibility in the mech-
anism of HPLC separation [142] and increased
detectability (because of the beneficial properties
of surfactants to on-line formation of hydrides
and cold vapor of mercury before their introduc-
tion into a plasma). The usefulness of this ap-
proach has been demonstrated for the speciation
of inorganic and methylmercury in sea water
[128,142,143].
Continuous oxidation of mercury in the eluent
and on-line further reduction to cold vapor of
mercury, which is then introduced into the atomic
spectrometric detector, has also been reported
recently [144].
Of course, other derivatization procedures such
as ethylation in aqueous phases [90,91] can be
employed as well.
6. Other separation approaches
There are other separation approaches possible
for mercury speciation. For instance, capillary
electrophoresis (CE), is rapidly becoming popular
for speciation of ionic species and biomolecules,
 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
although its use for chemical speciation and envi-
ronmental analysis is comparatively scarce [145].
Recently, Medina et al. [146] developed a rapid
CE method for speciation of organo-mercury
compounds in biological samples of marine
origin. This technique allows a very convenient
method for mercury speciation in a routine basis
and has been validated for methylmercury deter-
minations in tuna freeze-dried materials [147].
Finally, the possibility of resorting to off-line
couplings of separation techniques and atomic
detection should also be cited here, particularly in
connection with the use of the high sensitive
technique ETAAS to which we will refer later on.
7. Detectors for mercury speciation
As pointed out before, the analytical selectivity
and sensitivity requirements for reliable speciation
of trace and ultratrace amounts of toxic metals in
environmental compartments (atmosphere, water,
soil, sediments and also biota) are extremely high.
Therefore, it appears obvious that detectors used
should exhibit exceptional selectivity and sensitiv-
ity to the element to be speciated. Moreover,
‘hybrid’ techniques are preferred for speciation
and, thus, such detectors should operate ideally
‘on-line’ with the chromatographic column,
providing continuous, real-time information on
the trace element sought for speciation. Most
chromatographic detectors incorporated in com-
mercial instruments are universal or selective but
lacking the required specificity to mercury in envi-
ronmental samples. In many instances those com-
mon chromatographic detectors do not provide
the required sensitivity either. To make matters
worse, typical electrochemical or molecular spec-
troscopic detectors are lacking the necessary abil-
ity to detect organometallics in general or methyl
mercury in particular [137,138].
It is worldwide recognized that in most cases of
real-life analytical speciation we have to resort to
combine a chromatographic separation technique
with an atomic (element-specific) detector. The
case of methylmercury is illustrative in this re-
spect: probably the most common technique for-
merly used for separation and determination of
519
Table 3
Desirable features for an ‘ideal’ detector to be used in a hybrid
technique for trace element speciation
High sensitivity
High specificity
Ability to handle gases (in GC)
Ability to handle liquids (in HPLC and FIA systems)
On-line continuous operation with the separation column
Real-time information on the sought element
Broad linear dynamic range
Multi-element capacity
Multi-isotopic capacity
this compound has been GC with ECD. Problems
encountered using this mercury unselective detec-
tor have been addressed previously. Such prob-
lems favored GC-MIP-AES because of its high
element-specificity towards mercury [69,146,148].
There is no question that the availability of a
commercial instrument for GC-MIP-AES mea-
surements [149] has been decisive to explain the
general acceptance of this ‘hybrid’ technique using
plasma detection for speciation purposes.
Analytical plasmas, both at atmospheric and
reduced pressure [150], offer great analytical po-
tential as ‘element-specific’ detectors. Some of
them, particularly the ICP-MS, are now ap-
proaching many of the main desirable features for
a detector in hybrid chromatographic techniques
(Table 3).
Probably the MIP-AES is the plasma detector
more frequently used for mercury speciation
[69,72,77,86,96,98,114,119,127,128,148,158] as the
direct nebulisation of liquid samples into the ICP-
AES [101,141,151] lacks the required sensitivity
for real-life mercury speciation. However, perhaps
the atomic detector closest to the ‘ideal’ features
for speciation (Table 3) is the ICP-MS whose
importance in environmental analysis has been
rocketing during the last few years [152–155].
Thus, the increasing number of papers devoted to
the use of ICP-MS detection for mercury specia-
tion is not surprising [121,130,156–158]. Table 4
resumes the different atomic (specific) detectors
used for mercury speciation so far. It is apparent
that the detector type more common is still AAS
in its different forms of ‘high sensitivity’ measure-
ments (cold vapor generation in flame quartz
520 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524

Table 4
Hyphenated techniques with element ‘specific’ detection for mercury speciation

Separation Atomic spectrometric detector References Detection limits (range)

GC (CV)-AAS [47,83,90 – 92,118] 5–167 pg

HPLC (CV)-AAS [120,142,143] 4–16 mg l−1
GC ETAAS [73] 0.04 ng
GC (CV)-AFS [71,81,84,85,93,99,158] 0.01–6 ng l−1; 0.6–1.3 pg
HPLC (CV)-AFS [33,34,139,140] 0.015–0.1 mg
GC MIP-AES [69,72,77,86,93,96,98,114,127] 0.04–10 ng l−1
HPLC (CV)-MIP-AES [128] 0.35 ng ml−1
GC ICP-AES [113,151] 3 pg, 0.6 ng l−1
HPLC ICP-AES [142] 0.1 ng ml−1
GC ICP-MS [19,129] 0.12–1 pg
HPLC ICP-MS [121,126,130,156] 16–400 ng l−1

GC, Gas Chromatography;

HPLC, High Performance Liquid Chromatography;
CV, Cold Vapor;
AAS, Atomic Absorption Spectrometry;
ETAAS, Electrothermal Absorption Atomic Spectrometry;
AFS, Atomic Fluorescence Spectrometry;
MIP-AES, Microwave Induced Plasma Atomic Emission Spectrometry;
ICP-AES, Inductively Coupled Plasma Atomic Emission Spectrometry;
ICP-MS, Inductively Coupled Plasma Mass Spectrometry.

tubes (CVAAS) and electrothermal vaporization
in a graphite furnace (ETAAS)) while AFS detec-
tion of mercury is also rather popular.
As said previously, any of these atomic detec-
tors will need an appropriate interface to connect
the exit of the column with the atomization cell.
When using HPLC as a separation technique, the
low efficiency of nebulisers demands usually a
derivatization technique, on-line with the flow of
the mobile phase, to enhance the efficiency of the
transport of mercury species to the atomiser. For
this purpose the use of SnCl2 and NaBH4 to
generate hydrides and the cold vapor of mercury
are the most popular derivatization reactions to
form volatile species. Recently, the use of
NaB(C2H5)4 for metal ethylation from an aqueous
phase has also been proposed [90,91] and can also
be most useful to increase the sensitivity by form-
ing a volatile compound.
Of course, this volatile species formation, par-
ticularly of Hg0, has proved most useful for the
preconcentration step of mercury and methylmer-
cury by amalgamation [109] or in adequate traps
[29,120]. As Fig. 1 represents schematically, the
final detection to provide the ‘speciation result’ is
only a final step in mercury speciation in environ-
mental materials, where the extremely low levels
of the element and the complexity of the matrix of
the sample render previous preconcentration and
separation stages virtually mandatory.
Sensitivity enhancements of atomic detection by
resorting to volatile species generation is not re-
stricted to AAS measurements. The extremely
high sensitivity of AFS coupled to ‘cold vapor’
generation provides one of the more sensitive and
selective atomic detectors for mercury [159], with
instruments providing excellent features [160,161]
available today in the market.
ETAAS detection constitutes a different pic-
ture. The very low limits of detection affordable
by ETAAS for mercury (in the low mg l − 1 level)
and the high selectivity of today’s ETAAS tech-
nology favor the use of this AAS methodology for
mercury speciation [73]. However, the discontinu-
ous character of ETAAS determinations (drying,
ashing, atomization and cleaning-up) should be
stressed in this respect. In other words, the use of
this particular detector is limited because of its
‘off-line’ obliged operation. Notwithstanding that
drawback, there are good examples of speciation
 J.E. Sánchez Urı́a, A. Sanz-Medel / Talanta 47 (1998) 509–524
Fig. 1. Schematically representation of several steps involved in mercury speciation in environmental samples.
by collecting one or various (species) coming out
of the column and then carrying those separated
sample fractions to the corresponding ETAAS
‘off-line’ analysis for mercury [73]. In any case,
the discontinuous nature of ETAAS operations
make this detector more appropriate for the anal-
ysis ‘off-line’ of fractions separated by discontinu-
ous (non-chromatographic) techniques
particularly with the help of FIA strategies [162].
8. Conclusion
To conclude, the need of several analytical im-
portant steps (Table 1) for reliable mercury speci-
ation should be stressed. Errors can occur in any
of those basic steps. Therefore, each step (extrac-
tion, pretreatments, preconcentration, separation
and detection) should be validated adequately,
whichever the final strategy selected for mercury
speciation in environmental samples. The present
need for quality assurance and quality control of
all speciation results in general [45] and of mer-
cury speciation data in environmental matrices in
particular [147] cannot be overemphasized and
should be extensively addressed in the near future
521
by analytical scientists developing in the field of
chemical speciation of trace elements in environ-
mental materials.
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