Anal Bioanal Chem (2007) 388:837–847
DOI 10.1007/s00216-007-1194-2
ORIGINAL PAPER
Determination of total mercury and methylmercury
in biological samples by photochemical vapor generation
Mariana A. Vieira & Anderson S. Ribeiro &
Adilson J. Curtius & Ralph E. Sturgeon
Received: 16 November 2006 / Revised: 6 February 2007 / Accepted: 8 February 2007 / Published online: 27 February 2007
# Springer-Verlag 2007
Abstract Cold vapor atomic absorption spectrometry (CV-
AAS) based on photochemical reduction by exposure to
UV radiation is described for the determination of methyl-
mercury and total mercury in biological samples. Two
approaches were investigated: (a) tissues were digested in
either formic acid or tetramethylammonium hydroxide
(TMAH), and total mercury was determined following
reduction of both species by exposure of the solution to UV
irradiation; (b) tissues were solubilized in TMAH, diluted
to a final concentration of 0.125% m/v TMAH by addition
of 10% v/v acetic acid and CH3Hg+ was selectively
quantitated, or the initial digests were diluted to 0.125% m/v
TMAH by addition of deionized water, adjusted to pH 0.3
by addition of HCl and CH3Hg+ was selectively quantitated.
For each case, the optimum conditions for photochemical
vapor generation (photo-CVG) were investigated. The
photochemical reduction efficiency was estimated to be
∼95% by comparing the response with traditional SnCl2
chemical reduction. The method was validated by analysis of
several biological Certified Reference Materials, DORM-1,
DORM-2, DOLT-2 and DOLT-3, using calibration against
aqueous solutions of Hg2+; results showed good agreement
with the certified values for total and methylmercury in all
M. A. Vieira : A. S. Ribeiro : A. J. Curtius
Departamento de Química,
Universidade Federal de Santa Catarina,
Florianópolis, SC 88040 900, Brazil
R. E. Sturgeon (*)
Institute for National Measurement Standards,
National Research Council Canada,
Ottawa, ON K1A 0R9, Canada
e-mail: ralph.sturgeon@nrc-cnrc.gc.ca
cases. Limits of detection of 6 ng/g for total mercury using
formic acid, 8 ng/g for total mercury and 10 ng/g for
methylmercury using TMAH were obtained. The proposed
methodology is sensitive, simple and inexpensive, and
promotes “green” chemistry. The potential for application
to other sample types and analytes is evident.
Keywords Mercury . Methylmercury . Speciation .
Photochemical reduction . Vapor generation . Formic acid .
Tetramethylammonium hydroxide
Introduction
Mercury (Hg) is one of the most hazardous of environmental
pollutants, having significant routes of ingestion to humans
through consumption of marine foodstuffs or via industrial,
agricultural and laboratory exposure. Its toxicity depends on
the chemical form, which predominantly includes elemental
mercury (Hg0), inorganic mercury (Hg2+) and methyl-
mercury (CH3Hg+). The accumulation of mercury in fish
tissues is usually as methylmercury, which is more toxic than
inorganic mercury [1].
Interest in, and the need for, determination of Hg in
samples such as water, soil, sediments and biological
materials has led to significant progress in the development
of analytical measurement techniques. The most popular
approach remains cold vapor generation, wherein the mercury
species are typically mineralized to Hg2+ through acidic
attack and then reduced to the element by reaction with
SnCl2 or NaBH4 [2, 3]. A variety of detection techniques is
utilized, most frequently including long-path quartz tube
atomic absorption spectrometry [4, 5], atomic fluorescence
spectrometry [6, 7] and in situ collection of the cold vapor
coupled to an electrothermal atomizer for AAS [8, 9].
838
An alternative to chemical reduction is the use of
photoreduction, based on the exposure of a sample to a
UV field. Early work on the photoassisted generation of
volatile species focused on the production of mercury in the
aquatic environment. Thus, Akagi et al. [10–12] noted the
photochemical alkylation of mercury induced by the action
of sunlight in the presence of several low molecular weight
organic acids (acetic and propionic). These early findings
are today supported by additional observations on the
release of mercury from intertidal sediments [13] as well as
from the aerobic surface waters of lakes [14] in direct
response to sunlight and the presence of dissolved organic
matter of relatively low molecular weight (<5 kDa).
The action of UV light on dissolved organic and inorganic
compounds results in the formation of many intermediate
products [15]. UV irradiation of aqueous systems can
produce OH• and O• radicals, and in the presence of organic
compounds additional radicals such as CO• and R• arise that
can promote photoreduction of other species [15, 16]. Guo et
al. [17] were the first to report on the analytical use of photo-
CVG using UV radiation for vapor generation of Se(IV) and
they identified the products of the reaction arising in a
formic acid solution as both SeCO and SeH2. Subsequent
studies by these authors of Se [18], As [19, 20] and Ni [21]
as well as a host of other elements [22, 23] in solutions
containing low molecular weight organic acids (formic,
acetic or propionic) suggested that radical-induced UV
alkylation reactions were leading to the synthesis of volatile
analyte species. More recently, these observations have been
confirmed in other laboratories [24–26].
Recent reports have shown that photocatalytic reduction
of mercury can be achieved using UV irradiation in the
presence of TiO2 as a catalyst and either formic acid [27] or
methanol (as a hole scavenger) [28]. Zheng et al. [29]
undertook photo-CVG in the presence of formic acid to
investigate the speciation of mercury in water by atomic
fluorescence spectrometry. They reported that, when irradi-
ating sample solutions containing formic acid, both Hg2+
and CH3Hg+ are reduced to Hg0, thereby permitting the
quantitation of total mercury. On the other hand, only Hg2+
is reduced (with a comparative efficiency of 10%) to Hg0
using visible light. Li et al. [30] proposed a simple method
of determining mercury in wine or liquor samples by AFS
wherein Hg2+ was reduced to Hg0 by UV irradiation as a
result of the presence of ethanol in the sample matrix. A
limit of detection of 70 pg/mL was achieved. Bendl et al.
[31] reported a method for the determination of Hg in fish
tissues using photo-CVG of Hg0 in acetic acid solutions.
The determination of total mercury and its speciation in
biological samples requires careful consideration of sample
pretreatment. A substantial number of different methods are
currently available for this [1]. Recently, the determination
of mercury in biological materials following their digestion
Anal Bioanal Chem (2007) 388:837–847
with tetramethylammonium hydroxide (TMAH) has been
studied, providing very simple sample preparation proce-
dures [32, 33]. Torres et al. [34] developed a CV-AAS
technique for the speciation of mercury following digestion
of biotissues with TMAH. CVG using NaBH4 permitted
detection of total mercury with a heated quartz tube
atomizer (QTA) and inorganic mercury with the QTA at
room temperature. Formic acid is also an efficient tissue
solubilizer [35] and has been successfully used for the
determination of mercury by CV-AAS [36].
Based on the efficiency with which biological tissues can
be solubilized with either formic acid or TMAH, coupled
with the known capabilities of photo-CVG in formic acid
media for the reduction of mercury, this study was
undertaken to evaluate such a combined procedure for the
speciation of mercury in tissue samples. Although TMAH
has never been examined as a suitable medium for photo-
CVG, this reagent was found to be effective for the direct
determination of not only total mercury, but both inorganic
and methylmercury by performing individual measurements
under optimized conditions.
Experimental
Instrumentation
All measurements were conducted using an AAnalyst 100
atomic absorption spectrometer (Perkin Elmer, Norwalk, CT,
USA) equipped with deuterium arc background correction
and a mercury hollow cathode lamp source. The spectrometer
was operated under the following conditions: wavelength,
253.7 nm; spectral band pass, 0.7 nm; lamp current, 6 mA. A
QTA, mounted in the spectrometer’s flame compartment, was
maintained at room temperature and steady-state absorbance
measurements made in continuous graphics mode. A flow-
through photoreactor was constructed by coiling either quartz
(2.0 mm i.d. ×3 mm o.d. ×200 cm long) or Teflon tubing
(2.5 mm i.d. ×3.5 mm o.d. ×160 cm long) around a low-
pressure UV mercury vapor lamp (254 nm, 15 W, Cole
Parmer, Vernon Hills, IL, USA). The resulting internal
volumes of the coils were comparable at 6 and 8 mL,
respectively. Samples were pumped through the reactor with
the aid of a Minipuls 2 peristaltic pump (Gilson, Middleton,
WI, USA). A 56 mL/min flow of Ar purge gas was
introduced into a gas–liquid separator where it merged with
the effluent from the UV photoreactor, transporting the
volatile species directly to the QTA. Figure 1 schematically
illustrates the system used in this work. Samples were
weighed using an M2P microbalance (Sartorius, Göttingen,
Germany). A Branson 2200 ultrasonic bath (Branson
Ultrasonic, Danbury, CT, USA) was used to facilitate sample
solubilization.
Anal Bioanal Chem (2007) 388:837–847 839

Fig. 1 Schematic of photo-

chemical flow-through UV
reactor

Reagents and samples Procedure 2, for the Approximately 5 mL of a

determination of total solution of 25% m/v TMAH
Analytical-reagent-grade materials were used for all of the mercury using TMAH: in methanol were added to a
experiments. All solutions were prepared using 18 MΩ cm 100–300 mg subsample of
deionized, reverse osmosis water (DIW) obtained from a tissue or mussel. The mixture
mixed-bed ion exchange system (NanoPure, model D4744, was left to stand for at least
Barnstead/Thermoline, Dubuque, IA, USA). A standard stock 3 h at room temperature and
solution (1000 mg/L) of inorganic mercury was prepared by then diluted to 50 mL with
dissolution of mercury chloride (Gold Star, Alfa Chemicals, DIW for a final concentration
Ward Hill, MA, USA) in nitric acid. Similarly, a 1000 mg/L of 2.5% m/v TMAH.
stock solution of methylmercury (CH3HgCl) was prepared by
dissolving methylmercury chloride (Alpha Division, Danvers, Procedure 3, for the Samples were prepared as
MA, USA) in propan-2-ol. Formic and glacial acetic acids determination of described above for total
were sourced from Fisher Chemicals (Fair Lawn, NJ, USA); methylmercury using mercury but 1 g subsamples
tetramethylammonium hydroxide pentahydrate was from TMAH: were taken for analysis and
Alfa Aesar (Ward Hill, MA, USA) and antifoam “B” from after the 3 h incubation
Sigma Aldrich (Steinheim, Germany). Plastic and glass period an aliquot of 2.5 mL
containers were washed with tap water and with a diluted of the digest was diluted with
Extran solution followed by immersion in 10% v/v HNO3 for 10% v/v acetic acid to a final
at least 48 h and a thorough rinse with DIW prior to use. volume of 50 mL, yielding a
The following Certified Reference Materials from the final concentration of 0.125%
National Research Council Canada were used to assess the m/v TMAH in the solution.
accuracy of the method: DOLT-2 and DOLT-3 (dogfish liver),
DORM-1 and DORM-2 (dogfish muscle) and TORT-2 Procedure 4, for the Samples were prepared as
(lobster hepatopancreas). determination of described in Procedure 3, and
methylmercury using sufficient HCl was added to
TMAH and a final solution adjust the pH to 0.3 before
Sample preparation pH of 0.3: dilution with 10% v/v acetic
acid to a final concentration
Samples were prepared using four different procedures, as of 0.125% m/v TMAH. The
follows: resulting slurried sample was
Procedure 1, for the Approximately 250 mg subjected to analysis.
determination of total subsamples of tissue were Five drops of the antifoam “B” were added to all solutions
mercury using formic acid: weighed into 50 mL dark glass to eliminate foaming of the sample in the gas-liquid
flasks and 10 mL of formic separator. Corrections for moisture content were undertaken
acid added. The flasks were following the determination of dry mass in accordance with
placed in an ultrasonic bath at the recommendations of the CRM supplier.
50 °C for 6–8 h to complete
solubilization of the tissues, as
described by Scriver et al. [35]. Procedure
After cooling, the samples
were diluted with DIW to a Using the prepared samples, a sufficient volume of each was
final concentration of 10% v/v introduced into the photoreactor by pumping it through
formic acid. either the Teflon or quartz coil at flow rates of 5–12 mL/min
840
to produce steady-state absorbance signals. A number of
parameters were examined (acid or TMAH concentration,
type of acid, flow rate/residence time in the reactor, transport
gas flow rate) in order to optimize response using each of the
three sample preparation procedures and acquire information
on total and speciated mercury. A blank response was
measured before every analytical run using solutions con-
taining only the low molecular weight organic acids or
TMAH.
Results and discussion
Formic acid system
Effect of reagent concentration
Low molecular weight organic acids, such as formic and
acetic acid, are typically employed for photochemical
generation due to their ability to generate volatile species
[15]. The effect of formic acid concentration on the
absorbance by Hg0 derived from photo-CVG of 10 ng/mL
solutions of Hg2+ and CH3Hg+ is shown in Fig. 2a and b.
Both quartz and Teflon coils were investigated using an
irradiation time of 45 s (12 mL/min sample flow rate). The
concentration of formic acid ranged from 0.5 to 50% v/v. In
all cases, no signal was detected in the absence of formic
acid. At concentrations as low as 0.5% v/v formic acid
the signals appear for both species and above 10% v/v the
responses are similar from both species irrespective of the
type of coil used. It was reported previously [18] that less
than 5% of the incident UV radiation is transmitted through
the thin Teflon tube wall. Despite this, the use of a 15 W UV
lamp is more than sufficient to ensure efficient reduction of
inorganic mercury and methylmercury in simple solutions, as
evidenced by the fact that the CH3Hg+ is reduced to the
same extent as the Hg2+ despite the additional need for prior
destruction of the carbon–mercury bond. It is also clear,
however, that it is not possible to differentiate Hg2+ from
CH3Hg+ because both species yield equivalent signals. A
concentration of 10% v/v formic acid was adopted for
subsequent experiments with this reagent. Figure 2c and d
summarize results obtained from the use of acetic acid
during UV irradiation under otherwise similar conditions. In
this case, a concentration of acetic acid less than 5% v/v is
sufficient to achieve maximum response from CH3Hg+ with
no signal from Hg2+; more than 20% v/v acetic acid is
required to promote reduction of Hg2+ from a standard
solution. Without a clear understanding of the mechanism of
the reaction it is not possible to comment further on this
empirical observation. In any case, the use of acetic acid for
photo-CVG reduction of mercury permits direct speciation
of Hg2+ and CH3Hg+. A concentration of 10% v/v acetic
Anal Bioanal Chem (2007) 388:837–847
acid was selected for subsequent experiments with this
reagent. Based on these observations, it is possible that the
results presented by Bendl et al. [31] may correspond not to
measurement of total mercury in the fish tissues they
investigated but only to methylmercury. This would likely
not result in a significant bias in their results, as the major
fraction of the mercury present is methylmercury.
Effect of irradiation time
The residence time of the sample in the flow-through reactor
is an important parameter because the dose of UV radiation
received determines the extent of radical formation and the
efficiency of reduction of mercury. Fixing the length and
volume of the coil, the effect of irradiation time on response
was investigated by changing the sample flow rate using a
solution of 10% v/v formic acid spiked with 10 ng/mL Hg2+
and CH3Hg+. Increasing the pump speed (i.e., decreasing
the irradiation time) produced a linearly proportional
increase in signal intensity for both species. No significant
difference in performance between the Teflon and the
quartz coil was evident, nor between the responses for Hg2+
and CH3Hg+ for residence times varying from 38 to 216 s
(2.5–14 mL/min). An irradiation time of 45 s (sample flow
rate of 12 mL/min) was selected for subsequent experi-
ments using solutions of formic acid. Note that these
conclusions were valid for processing a simple calibration
standard; it will be shown later that only the longer
residence times can be recommended for the processing of
digested fish tissue because of the increased opacity of the
test solution.
Effect of sample pH
The possibility of undertaking speciation measurements was
investigated by examining the effect of sample pH on response
following the addition of NH4OH. The initial pH of 10% v/v
formic acid is 2. Increasing the pH did not improve the
separation in response from the two species, and at a pH
higher than 6 the response decreased for both species.
Adding concentrations of TiO2 in the range 1–4 mg/L as a
photocatalyst did not remediate this situation. When TiO2
was added to the standard solutions, precipitation of the
reagent could be observed in the coils.
Tetramethylammonium hydroxide system
Effect of concentration of TMAH As noted earlier, TMAH
has been successfully used as a solubilizer for biological
tissues and was thus investigated for its efficacy as a
reagent to promote photoassisted reduction of the mercury
species. Figure 3 shows the effect of concentration of
TMAH on the responses from 10 ng/mL of Hg2+ and
Anal Bioanal Chem (2007) 388:837–847 841

Fig. 2 Effect of concentration 0.6 0.5

of formic (a and b) and acetic a b
acid (c and d) on CV-AAS Quartz coil Teflon coil
response from 10 ng/mL solu-
0.5
tions of: (squares) Hg2+and 0.4
(circles) CH3Hg+. a, c: quartz
coil used; b, d: Teflon coil used
0.4
0.3

0.3

0.2
0.2

0.1
0.1
Absorbance

0.0 0.0
0 10 20 30 40 50 0 10 20 30 40 50

% (v/v) HCOOH
0.5 0.5
c Quartz coil d Teflon coil

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0.0 0.0

0 10 20 30 40 50 0 10 20 30 40 50

% (v/v) CH3COOH

CH3Hg+ in standard solutions using the quartz (Fig. 3a) and
Teflon coil (Fig. 3b) flow-through UV reactor and a
residence time of 45 s. Substantial signals for CH3Hg+
occur in both systems when the concentration of TMAH is
as low as 0.025% m/v. Signals gradually increase for Hg+2
with increasing concentration of TMAH, and above 2.5%
m/v both species are efficiently reduced. This concentration
was adopted for further experiments.
The responses from the two mercury species are clearly
separated according to the data in Fig. 3; when the
concentration of TMAH is 0.125% m/v, CH3Hg+ is prefer-
entially reduced. Samples were thus initially prepared using
2.5% m/v TMAH and then diluted to a final concentration of
0.125% m/v TMAH using DIW. A calibration curve for
CH3Hg+ was prepared in a similar way, but no response
could be detected at concentrations of 0.5, 1.0 and 2.5 ng/mL
due to inadequate detection power. In an attempt to
improve the sensitivities for these low concentration
standards and to determine CH3Hg+ in the samples, acetic
acid was added, in order to achieve an effect evident from
the earlier results shown in Fig. 2. The use of acetic acid
presumably provides for an increase in methyl radicals in
the irradiated solutions, thereby enhancing the rate of
reduction of CH3Hg+ in TMAH. The effect of acetic acid
concentration on the response from 1 and 10 ng/mL Hg2+
and CH3Hg+ in 0.125% m/v TMAH standard solutions
using an irradiation time of 108 s is shown in Fig. 4. For
the 1 ng/mL standard solution of Hg2+ or CH3Hg+, it is not
842
Fig. 3 Effect of TMAH con- 0.5
centration on response from
10 ng/mL (squares) Hg2+or
0.4
(circles) CH3Hg+ using (a) a
quartz and (b) a Teflon coil
flow-through UV reactor 0.3
0.2
0.1
Absorbance
0.0
0 2
0.5
0.4
0.3
0.2
0.1
0.0
0 2
possible to discern separation of the species due to the poor
signal-to-noise ratio (Fig. 4a). However, with the 10 ng/mL
solutions it is clear that such separation can be achieved
(Fig. 4b). Significantly higher absorbance arises from
CH3Hg+ at concentrations of acetic acid above 2.5% v/v
and thus 10% v/v was selected for subsequent study.
Mineral acids such as HCl or H2SO4 were also used to
adjust the sample pH in order to evaluate the ability to
separate the mercury species. Figure 5 shows the effect of
pH on signal intensity for solutions adjusted with HCl.
Samples of DORM-1 were prepared with a final concen-
tration of 0.125% m/v TMAH in 10% v/v acetic acid
spiked with 10 ng/mL CH3Hg+ and Hg2+, and the final pH
was adjusted to fall in the range 0–1.3 using HCl. As noted
by Bendl et al. [31], the addition of HCl caused precipitation
in sample digests, which was removed by centrifugation
before measurements. An initial pH of 2 (without the
addition of HCl) provides for responses from both species
in calibration solutions and in those of digested tissues. By
decreasing the pH, an effective separation of the response
from each species occurs due to selective suppression of the
reduction of Hg2+ and, at pH 0.3, it appears that it is possible
to undertake speciation measurements not only in simple
standards but also in solutions of digested tissue samples. It
is worth noting that, although the use of H2SO4 to adjust the
pH did not give rise to a precipitate, it was not possible to
separate the signals from the two species in this medium,
Anal Bioanal Chem (2007) 388:837–847
a
4 6 8 10
b
4 6 8 10
% (m/v) TMAH
suggesting that it is due to the intervention of the chloride
anion in suppressing the reduction of inorganic mercury.
Experiments were undertaken to discern the effect of added
chloride (as NaCl) on the responses from both Hg2+ and
CH3Hg+. Signal intensity was reduced 50% at 0.5% m/v
added NaCl, completely suppressed for Hg2+ at 1% m/v
NaCl, and 90% suppressed for CH3Hg+ at 2.5% m/v NaCl,
confirming this supposition.
Based on this information, the possibility of investigat-
ing speciation in real samples was tested by adjusting the
pH to 0.3 with HCl. Samples of DORM-1 were solubilized
in TMAH, diluted to a final concentration of 0.125%
TMAH and the solution adjusted to pH 0.3 by the addition
of HCl. Both the supernatant and the residue arising from
subsequent centrifugation were examined separately. The
residue was diluted with 10% v/v acetic acid. By subjecting
each phase to photo-CVG it was found that signals from the
supernatant were low compared to those from the residue
and did not provide satisfactory results for CH3Hg+.
Mercury species are thus retained on the precipitate and
can subsequently be reduced by the UV. As such, analysis
of DORM-1 samples that were not subjected to a prior
centrifugation provided results for CH3Hg+ which were
concordant with the certified values.
Effect of visible light on photoreduction of Hg Zheng et al.
[29] reported on the ability of visible light to photoreduce
Anal Bioanal Chem (2007) 388:837–847 843

Fig. 4 Effect of acetic acid

0.04
concentration on response from
standard solutions containing
0.125% m/v TMAH for (a) 0.03
1 ng/mL and (b) 10 ng/mL
(squares) Hg2+or (circles)
CH3Hg+using a quartz coil 0.02
flow-through UV reactor and an
irradiation time of 108 s
0.01

Absorbance
a
0.00
0 10 20 30 40 50
0.4

0.3

0.2

0.1

0.0 b
0 10 20 30 40 50
% (v/v) CH3COOH

Hg2+ in formic acid, leading to the conclusion that study to evaluate the effect of visible light on the
speciation was possible by alternately exposing a solution photoreduction of Hg2+ and thus the stability of calibration
to UV for the quantitation of total mercury, and visible light solutions and digested samples exposed to ambient labora-
for the selective measurement of inorganic mercury. A tory lighting in the presence of formic acid and TMAH was
Fig. 5 Effect of pH (using HCl 0.35
for adjustment) on response
from (a) a calibration standard
0.28
solution consisting of 0.125%
m/v TMAH and 10%
v/v acetic acid, and (b) a solu- 0.21
bilized sample of DORM-1
containing 0.125% m/v TMAH 0.14
and 10% v/v acetic acid. Both
samples were spiked with 0.07
10 ng/mL (squares) Hg2+and
Absorbance

(circles) CH3Hg+. A quartz coil 0.00 a

flow-through UV reactor was
used and the irradiation time 0.0 0.5 1.0 1.5 2.0
was 108 s
0.28

0.21

0.14

0.07

0.00 b
0.0 0.5 1.0 1.5 2.0
pH
844 Anal Bioanal Chem (2007) 388:837–847

1.2 flasks. A control solution was prepared and stored in the

a
dark for 2 h. A fresh standard solution was prepared prior to
1.0
the actual measurement sequence and used to normalize the
0.8 responses from all other samples following their processing
through the UV photoreactor.
0.6 In the formic acid system, exposure of the sample to
0.4 visible light did not result in any statistically significant loss
Normalized absorbance

of mercury species from the solution, as shown by the

0.2 results in Fig. 6a. This contrasts with the results of Zheng et
al. [29] for which a reduction of approximately 10% was
0.0 achieved and used for analytical purposes. For the TMAH
, ,
0/0 1/0 2/0 0/2 0 /0 system (Fig. 7b), exposure of standard solutions to visible
light resulted in significant losses of Hg2+, whereas losses
1.0 b
of CH3Hg+ were minor. Moreover, significant losses of
0.8
Hg2+ were also measurable for solutions kept in the dark
for 2 h, suggesting an intrinsic ability of TMAH to reduce
0.6 Hg2+ at room temperature in pure solutions of the standard.
This has significant implications for the analysis of biotissues
0.4 solubilized with TMAH—inorganic mercury appears unsta-
ble in calibration standards and methylmercury is thus
0.2 recommended for the preparation of such solutions.
An additional study was performed to evaluate the
0.0
, , stability of mercury in solutions of DOLT-2 tissue samples
0/0 1/0 2/0 0/2 0 /0
digested with formic acid or TMAH to which a 10 ng/mL
Exposure time, h spike of Hg2+ was added. The samples were purged with a
Fig. 6 Effect of visible and ambient laboratory fluorescent light on 65 mL/min flow of Ar for 4 min prior to processing them
photo-CVG of mercury using 10 ng/mL (light bars) Hg2+or (dark through the UV photoreactor for measurement of residual
bars) CH3Hg+. (0/0) freshly prepared solution; (1/0) 1 h exposure to
visible light; (2/0) 2 h exposure; (0/2) 2 h exposure to ambient mercury content. Figure 7a and b display results for Hg2+,
fluorescent laboratory light and (0’/0’) 2 h in the dark. a, 10% v/v from which it is evident that no losses have occurred.
formic acid; b, 2.5% m/v TMAH Chemical reduction of inorganic mercury (noted from the
results of Fig. 6a) either does not occur in such digested
undertaken. Standard solutions in 10% v/v formic acid or tissue samples because of the complexity of the matrix
2.5% m/v TMAH spiked with 10 ng/mL of Hg2+ or (possible inhibition of the formation of radicals or reducing
CH3Hg+ and contained in glass volumetric flasks were gases) or the presence of partially digested or solubilized
exposed for one and two hours to light from a 50 W high amino acids and proteins in the solution stabilizes any Hg0
intensity tungsten lamp (from a slide projector), and for two formed. This suggests that quantitation of Hg2+ be
hours to ambient laboratory (fluorescent) lighting. Care was undertaken in such digests using the method of additions.
taken to ensure there was no increase in the temperature of A third study was undertaken using standard solutions
the solutions during exposure by forced air cooling of the containing 50 ng/mL Hg2+ or CH3Hg+. In addition to 10% v/v

Fig. 7 Influence of visible light

1.0 a 1.0 b
Normalized absorbance

on the photo-CVG response

from a 10 ng/mL Hg2+spike to
digested DOLT-2 in (a) 10% v/v 0.8 0.8
formic acid and (b) 2.5% m/v
TMAH. Conditions: (0/0) 0.6 0.6
freshly prepared sample; (1/0)
1 h exposure to visible light; 0.4 0.4
(2/0) 2 h exposure; (0/2) 2 h
exposure to ambient fluorescent
0.2 0.2
laboratory light and (0’/0’) 2 h
in the dark. Each sample was
purged for 4 min at 65 mL/min 0.0 0.0
0/0 1/0 2/0 0/2
, ,
0 /0 , ,
Ar prior to measurement of 0/0 1/0 2/0 0/2 0 /0
residual mercury
Exposure time, h
Anal Bioanal Chem (2007) 388:837–847 845

Fig. 8 Effect of purge time 1.2

(65 mL/min Ar) on the photo- a 1.0 b

Normalized absorbance
CVG response from 50 ng/mL 1.0
(light bars) Hg2+or (dark bars) 0.8
CH3Hg+in: (a) 10% v/v formic 0.8
acid and (b) 2.5% m/v TMAH
0.6
(for Hg2+) or 0.125% m/v 0.6
TMAH plus 10% v/v acetic acid
(for CH3Hg+) 0.4
0.4

0.2 0.2

0.0 0.0
0 2 4 6 8 10 0 2 4 6 8 10
Time, min

formic acid, a medium optimized for speciation of methyl-
mercury consisting of 0.125% m/v TMAH and 10% v/v
acetic acid or 2.5% m/v TMAH for total Hg was also
examined. A 65 mL/min flow of Ar was passed through the
solutions for varying periods of time while they were
maintained in the dark. Results are presented in Fig. 8a
and b. Inorganic mercury is unaffected by the flow of gas
whereas losses of CH3Hg+ occur which increase with
increasing purge time in the formic acid solution. For the
TMAH/acetic acid system (Fig. 8b), both species suffer
losses with increasing Ar purge time, consistent with the
conclusions drawn from Fig. 6b, but more pronounced
because of the mechanical stripping of the Hg0 from solution
induced by the flow of Ar.
Analytical applications Using optimized experimental con-
ditions, the accuracy of the proposed methodology was
evaluated by analyzing several Certified Reference Materials.
Total mercury was determined in samples that had been
solubilized in formic acid and diluted to a final concentration
of 10% v/v. An external calibration curve was constructed
using the same formic acid medium spiked with Hg2+.
Samples and standards were subjected to photo-CVG by
pumping the solutions through a quartz coil in the photo-
reactor at a rate of 12 mL/min (45 s irradiation time). At this
flow rate the results were not concordant with the certified
values. By reducing the flow rate to 5 mL/min (108 s
irradiation time), satisfactory results were achieved, as
summarized in Table 1. Relative standard deviations were
below 5% in most cases, except for TORT-2 (15% RSD),
which most likely arose because the analyte concentration is
so low in this sample. Samples of DOLT-2, DOLT-3 and
TORT-2 produced dark solutions when digested with formic
acid, whereas that for DORM-1 was yellow. This may be the
reason why poor results were obtained when a high sample
flow rate was used; penetration of UV into the solution is
more efficient for clear and colorless samples than for the
more opaque ones. When a Teflon coil was used in the UV
photoreactor, results were satisfactory only for DORM-1.
Clearly, the efficiency of the process is dependent on the
dose of UV radiation received which, in turn, is influenced
by not only the residence time of the sample in the UV field,
but also the absorptivity of the sample solution and the
transmission efficiency of the tubing.
For the determination of total mercury following
digestion with TMAH (procedure 2), external calibration
curves were prepared by spiking Hg2+ into 2.5% m/v
TMAH. Sample processing was initially undertaken using a
quartz coil at a flow rate of 12 mL/min (residence time of
45 s). As for the formic acid system, the results were
discordant with the certified values for total mercury for
DOLT-2, DOLT-3 and TORT-2 and, as such, the samples
were re-run at a flow rate of 5 mL/min to increase exposure
time to the UV field to 108 s. Under these conditions,
results were satisfactory, as evidenced by data summarized
in Table 1. When the Teflon coil was used it was only

Table 1 Determination of total mercury in biological materials by CV-AAS following photo-CVG*

CRM Certified value, mg/kg Solubilized in 10% v/v formic acid Solubilized in 2.5% m/v TMAH

Determined**, mg/kg RSD, % Determined**, mg/kg RSD, %

DORM-1 0.798±0.074 0.75±0.01 1.5 0.78±0.02 2.7

DOLT-2 2.14±0.28 2.00±0.10 5.0 2.20±0.05 2.3
DOLT-3 3.37±0.14 3.27±0.03 0.9 3.38±0.06 1.8
TORT-2 0.27±0.06 0.27±0.04 14.8 0.30±0.02 6.7

*with quartz coil; no significant difference with Teflon coil

**mean and standard deviation (n=3), dry mass corrected.
846 Anal Bioanal Chem (2007) 388:837–847

possible to determine total Hg in samples of DORM-1 at Table 3 Figures of merit (in ng/mL)
this flow rate because of the poor penetration of the already Slope−1±std dev R2 LOD LOD (ng/g)
reduced intensity of UV through the opaque sample
solutions. It is possible that longer residence times for Hg2+
irradiation could alleviate this problem, but this was not (a) 0.032±0.004 0.9999 0.03 6
(b) 0.026±0.006 0.9999 0.04 8
investigated.
CH3Hg+
For the determination of CH3Hg+ in biological samples
(c) 0.018±0.002 0.9994 0.05 10
solubilized with TMAH (procedure 3), an external calibra- SnCl2
tion curve was prepared in a mixture of 0.125% m/v (d) 0.033±0.003 0.9999 0.02 –
TMAH and 10% v/v acetic acid by spiking with CH3Hg+.
With the exception of TORT-2, results summarized in Calibration curves prepared in: (a) 10% v/v formic acid; (b) 2.5% m/v
TMAH with Hg2+ standard; (c) 0.125% m/v TMAH+10% v/v acetic
Table 2 show good agreement with the certified values. For acid with CH3Hg+ standard and (d) following SnCl2 reduction (see
TORT-2, the concentration of CH3Hg+ is lower than the text for optimized conditions).
limit of quantitation. Relative standard deviations of
replicate measurements were better than 6.4% for measure-
ments of both total and organic mercury using a TMAH Calibration curves based on standard solutions of Hg2+ or
sample preparation. Although speciation analysis is difficult CH3Hg+ in the range 0.5–25 ng/mL were generated using
because of the substantial dilution of the sample to achieve formic acid and TMAH systems optimized for the
a concentration of 0.125% m/v TMAH, this impediment determination of mercury in samples of digested biological
could be readily overcome by using a more powerful tissue (i.e., 108 s residence time). Correlation coefficients
detection technique, such as AFS or ICP-MS. were higher than 0.999. Estimated limits of detection
For the determination of CH3Hg+ in biological samples (LOD), defined as three times the standard deviation of
which were solubilized with TMAH diluted with acetic acid ten replicate measurements of the blank divided by the
and adjusted to pH 0.3 with HCl (procedure 4), an external slope of the calibration curve, were calculated for each
calibration curve was prepared using the same medium as system and also for samples. The data in Table 3 illustrate
the samples by spiking with CH3Hg+. The results presented that the LODs obtained using photo-CVG based on UV
in Table 2 also show good agreement with the certified irradiation of formic acid or TMAH for the determination of
values for methylmercury, demonstrating yet another photo- total Hg are similar to those obtained using a conventional
CVG approach to the speciation of mercury. SnCl2 system to reduce Hg2+ to Hg0. Procedural LODs for
Results reported in Tables 1 and 2 for the determination the samples are generally adequate for the analysis of these
of both total mercury and methylmercury in tissue samples CRMs, and the use of more powerful instrumentation for
using photo-CVG were subjected to a t-test to ensure that detection should extend the lower range of concentrations
there was no statistical difference from the certified value of which can be addressed.
the measurand at the 95% level of confidence. It is of interest to determine the efficiency of generation,
which is defined by the convolution of the efficiency of
Figures of merit Figures of merit for the determinations of formation of the volatile species with those of its gas–liquid
total mercury and methylmercury in biological tissues using separation and transport to the detector. Generation effi-
a quartz coil flow-through UV reactor are shown in Table 3. ciency was estimated from a comparison of CV-AAS

Table 2 Determination of methylmercury in biological materials by CV-AAS following photo-CVG*

CRM Certified value, mg/kg A B

Determined**, mg/kg RSD, % Determined**, mg/kg RSD, %

DORM-1 0.731±0.060 0.78±0.05 6.4 0.76±0.02 2.6

DORM-2 4.47±0.32 4.25±0.07 1.6 4.19±0.07 1.7
DOLT-2 0.693±0.053 0.68±0.04 5.9 0.66±0.02 3.0
DOLT-3 1.7 1.63±0.10 6.1 1.41±0.08 5.7
TORT-2 0.152±0.013 <LOD ND ND ND

(A) solubilization of tissues with 2.5% m/v TMAH and diluted to 0.125% m/v with 10% v/v acetic acid
(B) solubilization with TMAH and diluted to 0.125% m/v with acetic acid and adjusted to pH 0.3 using HCl
*with quartz coil; no significant difference with Teflon coil
**mean and standard deviation (n=3), dry mass corrected
ND: not determined
Anal Bioanal Chem (2007) 388:837–847
response using a conventional SnCl2 chemical reduction
method with the results obtained using photo-CVG. The
reduction conditions were optimized for the tin chloride
system (4% v/v HCl; 0.5%, m/v SnCl2 in 3% v/v HCl; gas
flow rate of 50 mL/min and 60 cm length of reaction coil)
and the flow rates of reductant and sample set to 2.5 mL/min
to match the flow rates in the photo-CVG system in order to
ensure delivery of identical fluxes of Hg2+ to the gas–liquid
separator. From a comparison of the slopes of the
calibration curves, the efficiency of photo-CVG with UV
irradiation was determined to be approximately 95% of that
obtained by conventional chemical-CVG.
Conclusions
Photo-CVG with UV irradiation combined with CV-AAS
detection for analysis of samples solubilized with formic acid
or TMAH has been successfully used to determine total
mercury and methylmercury in biological tissues. Both
reagents provide stable solutions of the analyte and promote
reduction of Hg2+ and CH3Hg+ to Hg0 following exposure to
the UV field. The methodology does not require extensive
sample preparation, it eliminates the need to use (unstable
and expensive) reducing agents, and it provides limits of
detection that are comparable to conventional methods for
the determination of mercury by vapor generation.
Acknowledgement M. A. Vieira and A.J. Curtius are grateful to the
Conselho Nacional de Pesquisas e Desenvolvimento Tecnológico
(CNPq) for a research scholarship.
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