Determination of lead in fish samples by slurry sampling

electrothermal atomic absorption spectrometry

Sue-Jean Huang and Shiuh-Jen Jiang*
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan.
E-mail: sjjiang@mail.nsysu.edu.tw; Fax: 886-7-5253908
Received 2nd May 2000, Accepted 13th June 2000
Published on the Web 21th July 2000

Ultrasonic slurry sampling electrothermal atomic absorption spectrometry (USS-ETAAS) was been applied to the
determination of lead in several fish samples. The influences of instrument operating conditions and slurry
preparation on the signal were examined. Palladium and ammonium nitrate were used as the modifier to improve
the signal. Since the sensitivity to lead in various fish slurries and aqueous solutions was different, the standard
additions method was used for the determination of lead in these fish samples. The method was applied to the
determination of lead in dogfish muscle reference material (DORM-2) and a swordfish muscle sample purchased
from the local market. The analysis results agreed with the reference value. The accuracy was better than 6%. The
precision between sample replicates was better than 16% with the USS-ETAAS method. The detection limit of
lead estimated from standard additions curve was about 0.0530.058 mg g21 in different samples.

Introduction
Electrothermal atomic absorption spectrometry (ETAAS) is a
useful technique for the determination of lead in various
samples and numerous studies have been published.114 Most of
the methods involve prior dissolution of the samples. A variety
of digestion methods for measuring total lead in biological
samples, and both wet and dry ashing, or a combined procedure,
have been reported. All these methods involve the risk of
contamination or loss of lead and it is clear that methods
involving minimal sample handling are required.
In recent years, the direct analysis of solids and slurries by
ETAAS has received much attention in an attempt to eliminate
problems associated with conventional wet oxidation and dryashing sample preparation procedures. Ultrasonic slurry sampling is one of the methods for direct solid sample introduction
that has been successfully used in ETAAS.1517 Compared with
traditional sample preparation methods such as acid digestion
and dry ashing, slurry sampling offers the benefit of reducing
the possibility of analyte loss before analysis. Furthermore,
slurry sampling combines the benefits of solid and liquid
sampling and permits the use of conventional liquid sample
handling apparatus such as an autosampler. Very few studies
using the slurry-based approach to determining lead in food
samples have been published.11
Most of the methods which exist for determining the
concentrations of elements in fish samples require digestion of
the sample with acid before analysis.4,18 In this study, ultrasonic
slurry sampling (USS) ETAAS was used to determine the
concentrations of lead in several fish samples directly. The
influences of instrument operating conditions and slurry
preparation on the signal were investigated. The method was
used for the determination of lead in dogfish muscle reference
material (DORM-2) and a swordfish muscle sample purchased
from the market.

Experimental
Apparatus and conditions
AAS measurements were made on a Perkin-Elmer (Norwalk,
CT, USA) Zeeman/5100 PC atomic absorption spectrometer
equipped with an HGA-6100 graphite furnace atomizer. A lead
DOI: 10.1039/b003484n

hollow cathode lamp (HCL) and pyrolytic graphite-coated

graphite tubes with integrated platforms (Perkin-Elmer, Uberlingen, Germany) were used throughout. The sample introduction system included a Model AS-60 autosampler equipped with
a USS-100 ultrasonic slurry sampler. The USS-100 was set at
25 W (10% power) and a 10 s mix time was used to mix slurries
before injection of 20 ml sample aliquots for analysis. The
optimized instrumental parameters and heating programme are
given in Tables 1 and 2, respectively. The peak area of the
transient signal was used for data handling.
Reagents and slurry preparation
H2O2, NH4NO3 and EDTA were obtained from Merck
(Darmstadt, Germany), trace metal grade HNO3 (70% m/m)
Table 1 Equipment and operating conditions
Wavelength
Slit
Radiation lamp type
Current
Signal mode
Background
Tube type
Sample volume
Modifier volume
USS device power

283.3 nm
0.7 nm
Lead hollow cathode lamp
10 mA
Peak area
Zeeman-effect background correction
THGA
20 ml
20 ml
10% for 10 s

Table 2 Temperature programme

Step

Temperature/C

Ramp/s

Hold/s

Gas flow rate/

ml min21

Drying 1
90
5
10
250
Drying 2
120
5
20
250
Pre-treatment
1200
5
10
250
Drying 1
150
10
20
250
Drying 2
200
10
35
250
5
30
250
Ash
500a, 1100
0
3
0
Atomization
1700a, 2100
Clean
2400
1
10
250
Cooling
20
5
5
250
a Used for selection of modifier and slurry preparation parameters.

Analyst, 2000, 125, 14911494

This journal is The Royal Society of Chemistry 2000

1491

from Fisher (Fair Lawn, NJ, USA), Triton X-100 from Sigma
(St. Louis, MO, USA), palladium powder from Kojundo
Chemical Laboratory (Saitama, Japan) and lead element
standard solution (1000 mg ml21) from SPEX (Metuchen, NJ,
USA).
The applicability of the method to real samples was
demonstrated by the analysis of dogfish muscle reference
material DORM-2 (National Research Council of Canada,
Ottawa, Canada) and a swordfish muscle sample purchased
from the local market. The latter sample was cut into small
pieces and dried in an oven at 60 C. The dried fish samples
were then ground at room temperature for 30 min with a Retsch
MM2000 mixer mill and sieved using a Retsch (Haan,
Germany) VE1000 sieving machine. The powders with particle
size < 100 mm were collected for subsequent experiments. In
order to avoid cross-contamination, the mixer mill and sieving
machine were cleaned after each usage. Before weighing for
analysis, the fish samples were dried to constant mass by drying
at reduced pressure at room temperature in a vacuum desiccator
over Mg(ClO4)2 for 24 h.
The slurry was prepared as the following procedure. A 0.2 g
portion of the powder material was transferred into a 10 ml
calibrated flask. Suitable amounts of NH4NO3, H2O2 and Triton
X-100 were added to give a final solution containing 1% m/v
NH4NO3, 1.5% v/v H2O2 and 0.1% v/v Triton X-100. After
various amounts of element standard solutions had been added,
these slurries were diluted to volume with pure water. For
standard addition analysis, the slurries were spiked with various
amounts of lead (0, 1, 2, 5, 10, 20 and 30 ng ml21 in the final
solutions) standard. The slurry was then sonicated for 10 min in
an ultrasonic bath and 1 ml aliquots were removed as needed for
analysis with the use of a pipette while the slurry was being
mixed with a vortex mixer. These aliquots were then deposited
in the autosampler cups for analysis. A reagent blank was
carried through the procedure, as outlined above, to correct for
any analyte in the reagents used for slurry preparation. The
concentration of lead was then determined from the standard
additions calibration curve. For the studies of the effect of
electrothermal atomization conditions and slurry preparation on
the signal, a swordfish slurry sample was prepared according to
the procedure described above.

Results and discussion

Selection of modifier
Modifiers are commonly used in ETAAS analysis. The use of a
modifier would change the chemical or physical characteristics
of the sample and/or the atomizer surface in order to improve
quantification. In this study, several modifiers, including
EDTA, Pd, NH4NO3 and a mixture of Pd and NH4NO3, were
tested for the best signal of lead. The drying temperature was set
at 150 C for 20 s and 200 C for 35 s; the ash temperature was
set at 500 C; and the atomization temperature was set at 1700
C. We found that the absorbance of lead increased and the peak
shape improved significantly when 500 mg ml21 of Pd +1% m/v
NH4NO3 was used as the modifier. After evaluation, a mixture
of Pd and NH4NO3 was selected as the modifier for real sample
analysis in the following experiments. Palladium has been used
as the chemical modifier to improve the signals of some volatile
elements in many ETAAS applications.1923 In the following,
the pyrolytic platforms of the graphite tubes were pre-treated
with palladium by thermal pre-treatment. The temperature
programme used for the thermal pre-treatment of the platform is
given in Table 2. NH4NO3 was chosen as the modifier to be
used for the elimination of some of the possible interferences
and to improve the signals of some selected elements in
previous ETAAS and ETV-ICP-MS applications.2427
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Analyst, 2000, 125, 14911494

Fig. 1 shows the effect of the amount of Pd modifiers on the

integrated peak area of lead. As shown, the signal increased
with increase in Pd concentration and reached a maximum when
the concentration was 700 mg ml21. In subsequent experiments,
a Pd concentration of 700 mg ml21 was selected. Fig. 2 shows
the effect of the concentration of NH4NO3 in the prepared slurry
on the integrated peak area of lead. As shown, the signal
increased with increase in NH4NO3 concentration and reached
a maximum when the NH4NO3 concentration was about 1%
m/v. In subsequent experiments, an NH4NO3 concentration of
1% m/v was selected.
Effect of slurry preparation on ion signal
ETAAS has been successfully applied to the analysis of
slurries.1517 Certain factors such as particle size, analyte
partitioning, maximum slurry concentration and slurry homogeneity were important for the success of the analysis of slurries
by ETAAS.15,16 The effects of several parameters of the slurry
preparation on the ion signals were investigated in this work as
described below.
An important factor in the slurry technique is the slurry
concentration. However, dilution of the slurry can only be
carried out within a limited range. The effect of dilution factor
on lead signal was studied and the result is shown in Fig. 3. As
shown, the sensitivity (absorbance concentration) of lead

Fig. 1 Effect of Pd concentration on Pb signal. The slurry solution

contained 2% m/v swordfish sample and 1% m/v NH4NO3. Each data point
represents the mean of five measurements. All data are relative to the first
point.

Fig. 2 Effect of NH4NO3 concentration on Pb signal. The slurry solution

contained 2% m/v swordfish sample and was spiked with various amounts
of NH4NO3. The platform was thermally pre-treated with 20 ml of 700
mg ml21 Pd. Each data point represents the mean of five measurements. All
data are relative to the first point.

increased with increase in dilution factor. This could be due to

the alleviation of non-spectroscopic effects when the dilution
factor was increased. In order to balance sample homogeneity,
analyte signal and the complete vaporization of the introduced
sample, a dilution factor of 50 was used in subsequent
experiments.
The concentration of acid in the slurry solution could affect
the rate of the extraction of the metal ions and the precision of
signal measurement.17 Table 3 shows the effect of various
additives on the absorbance of lead. As shown, the addition of
HCl and HNO3 reduced the signal of lead. In subsequent
experiments, no acid was used in slurry preparation. However,
it is interesting to see that the addition of 1% v/v H2O2 increased
the lead absorbance slightly. Further, the precision was
improved when 1% m/v H2O2 was added. In USS-ETAAS
analysis, the direct introduction of suspensions could be
problematic beacuse of high background values or the build-up
of carbonaceous residue, both of which reduce sensitivity and
precision. The addition of H2O2 to biological samples has
proved very effective in preventing carbonaceous residues
building up inside the tube.28 The effect of H2O2 concentration
in the slurry sample on the lead signal was also studied in this
work, and the result is shown in Fig. 4. As shown, the lead signal
increased with increase in H2O2 concentration and reached a
maximum when the H2O2 concentration was 1.5% v/v.
Furthermore, as shown in Fig. 5, a more symmetrical and sharp
peak could be obtained when 1.5% v/v H2O2 was added. In the
following experiments, a H2O2 concentration of 1.5% m/v was
used in all slurry preparations. The use of oxidant did not
produce premature deterioration of the pyrolytic atomizer.29
In another experiment, we found that the concentration of
surfactant did not affect the lead signal significantly. However,
a more symmetrical and sharp peak could be obtained when a
small amount of Triton X-100 was added. Since the sample
homogeneity could affect the repeatability of the signal
determined, for better precision in subsequent experiments
0.1% v/v Triton X-100 was used in all slurry preparations.

Selection of ash and atomization temperatures

Fig. 6 shows the effect of ash temperature and atomization
temperature on the lead signal. As shown, the signal of lead
increased slightly with increase in ash temperature and reached
a maximum when the temperature was about 1100 C. This
could be due to the removal of more volatile matrix during the
ash stage and alleviation of the non-spectroscopic interference
in the atomization stage. However, the signal of lead decreased
rapidly when the ash temperature was > 1100 C. This could be
due to the vaporization of lead. In subsequent experiments, the
ash temperature was set at 1100 C.
As shown in Fig. 6, the signal of lead did not change
significantly until the atomization temperature was > 2100 C.
A more symmetrical and sharper peak could be obtained when
a higher atomization temperature was used. In subsequent
experiments, 2100 C was selected as the atomization temperature. A summary of the electrothermal atomization temperature programme is given in Table 2.

Fig. 4 Effect of H2O2 concentration on Pb signal. The slurry solution

contained 2% m/v swordfish sample and 1% m/v NH4NO3 and was spiked
with various amounts of H2O2. Each data point represents the mean of five
measurements. All data are relative to the first point.

Fig. 3 Effect of dilution factor on Pb signal. The slurry solution contained

various amount of fish sample and 1% m/v NH4NO3. Each data point
represents the mean of five measurements.

Table 3 Effect of various additives on lead signala (n = 7)

Additive

Absorbance

RSD/(%)

No other additive
0.036 0.002
5.55
0.038 0.002
5.26
1% v/v HNO3
1% v/v HCl
0.040 0.002
5.00
1% v/v H2O2
0.048 0.001
2.08
a Values are means of seven measurements standard deviation. The slurry
solution contained 2% m/v swordfish sample in 1% m/v NH4NO3 and was
spiked with various additives.

Fig. 5 Effect of H2O2 additive on Pb peak shape: (a) without H2O2 and (b)
with 1.5% v/v H2O2. The slurry solution contained 2% m/v swordfish
sample, 1% m/v NH4NO3 and various amounts of H2O2.

Analyst, 2000, 125, 14911494

1493

The detection limit of lead in different samples was

determined from the standard additions curve for lead. It was
based on the usual definition of the concentration of the analyte
yielding a signal equivalent to three times the standard deviation
of the reagent blank signal (n = 7). The detection limit
estimated from the standard addition curve was in the range
0.0530.058 mg g21 for lead in different samples. A better
detection limit is to be expected with more purified reagents.

Acknowledgement

Fig. 6 Effect of ash temperature and atomization temperature on Pb

signal. The slurry solution contained 2% m/v swordfish sample, 1% m/v
NH4NO3, 1.5% v/v H2O2 and 0.1% m/v Triton X-100. All data are relative
to the first point.

This research was supported by a grant from the National

Science Council of the Republic of China under Contract NSC
89-2113-M-110-018.

References
Table 4 Determination of lead in fish samples by USS-ETAASa (n =
3)
Concentration/mg g21
Sample

Founda

1
2
3

Reference value

DORM-2
0.069 0.011
0.065 0.007b
Swordfish
1.25 0.04
1.25 0.02c
a Results are means of three measurements standard deviation. b NRCC
certified values. c Determined by ICP-MS after digestion.

4
5
6
7
8

Determination of lead in fish samples by USS-ETAAS

In order to validate the USS-ETAAS method, the concentrations of lead were determined in the dogfish muscle reference
material DORM-2 and a swordfish sample purchased from the
local market. In order to evaluate the possibility of using an
external calibration method for the determination of lead in fish
samples by USS-ETAAS, a recovery test experiment was
performed by spiking the DORM-2 slurry solution with 5 ng
ml21 of lead and then applying the USS-ETAAS method with
external calibration. The determined value was calculated
against the theoretical value. The recovery of lead was about
67% in fish slurry. The sensitivity to lead was different in the
aqueous solution and in the slurry solution. This could be due to
the different thermal behaviour and/or non-spectroscopic interference between slurries and aqueous solution. Therefore, the
external calibration method could not be used for the quantification of lead in these samples and the standard additions method
was used. The results are given in Table 4. The concentration
of lead in the DORM-2 dogfish muscle reference sample
determined using the standard addition method was in good
agreement with the certified value. The accuracy was better than
6%. The precision between sample replicates was better than
16% with the USS-ETAAS method. The result for the swordfish
for which no certified value was available was also found to be
in good agreement with the reference value. The reference value
for the swordfish sample was obtained by digesting the fish
sample with a microwave digestion system (CEM MDS 2000)
and then analysing the solution by ICP-MS with pneumatic
nebulization. This experiment indicated that lead could be
readily quantified by USS-ETAAS.

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