Microchemical Journal 98 (2011) 234239

Contents lists available at ScienceDirect

Microchemical Journal
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c

Fractionation of heavy metals in sediment by using microwave assisted

sequential extraction procedure and determination by inductively
coupled plasma mass spectrometry
M.L. Alonso Castillo, E. Vereda Alonso , M.T. Siles Cordero, J.M. Cano Pavn, A. Garca de Torres
Department of Analytical Chemistry, University of Mlaga, Campus de Teatinos, Mlaga, 29071, Spain

a r t i c l e

i n f o

Article history:
Received 4 August 2010
Received in revised form 12 November 2010
Accepted 6 February 2011
Available online 15 February 2011
Keywords:
Sediment
Sequential extraction
Microwave
Inductively coupled plasma mass
spectrometry

a b s t r a c t
In this work, a fast microwave assisted extraction procedure was developed and optimized for the heavy
metals (Cu, Ni, Cr, Pb and Cd) partitioning in the three-stage sequential extraction procedure proposed by
the European Standards, Measurements and Testing (SM&T) Program, formerly the Community Bureau of
Reference (BCR). The microwave oven procedure was optimized to obtain extraction efciencies similar to the
conventional BCR procedure, in less time, while using smaller volumes of reagents. In the optimization
process, three variables (extraction time, ramping time and microwave power) were considered as factors
and as a response the concentration of different metal ions in each individual BCR fraction. Interactions
between analytical factors and their optimized levels were investigated using a central composite design.
Extractable metals obtained by both comparable methodologies were measured by inductively coupled
plasma mass spectrometry. With the use of optimal microwave conditions, steps 13 of the sequential
extraction (including the hydrogen peroxide digestion in step 3) could be completed between 21 and 22 min.
Detection limits were between 1 and 18 ng l 1. The accuracy of the proposed method was checked with a
certied reference material (CRM) of Lake Sediment BCR 701. Values obtained were in accordance with those
reported for the certied material with only a few exceptions. Different origin sediments (river and marine)
were analyzed by both BCR and MW procedures, and the results obtained were comparable according to the
t-paired-test for a 95% condence level.
2011 Elsevier B.V. All rights reserved.

1. Introduction
The accumulation of heavy metals in environmental samples such
as soils and sediments causes a potential risk to human health due to
the transfer of these elements in aquatic media, their uptake by plants
and their subsequent introduction into the food chain.
Over the last decade, more and more attention has been given to
the determination of species in which a metal is present, since the
toxicity, bioavailability, mobility and other properties depend on the
chemical association of the different components of the sample [1]. In
this sense, in addition to the total concentration of an element in a
sample, fractionation using sequential extraction schemes has been
proposed in order to characterize the chemical forms in which trace
metals are present in soils, sediments and sludges [2,3]. The
sequential extraction schemes proposed by Tessier [4], Campbell
et al. [5] and others [6], have to be mentioned in this context. The use
of fractionation procedures permits, for example, the determination of
how much of the total analyte is available for plants or is accessible to

Corresponding author. Tel.: + 34 95 2131883; fax: + 34 95 2132000.

E-mail address: eivereda@uma.es (E. Vereda Alonso).
0026-265X/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.microc.2011.02.004

the environment [7]. Thus, in all sequential extraction schemes, a

series of reagents is applied to the same sample to sub-divide the total
metal content. The vigor of the treatment generally increases
through the steps of the procedure, from initial mild conditions (e.g.
shaking with water, a salt solution or dilute acetic acid) to the use of
much harsher reagents (e.g. hot mineral acid) so that the successive
fractions obtained correspond to metal association forms with lesser
mobility. The Community Bureau of Reference of the Commission of
the European Communities (BCR) commissioned a research which
led to the development of a harmonized, three-stage, sediment
sequential extraction protocol [8]. However, problems were reported
with irreproducibility of, in particular, step 2 of the original BCR
procedure and, after a thorough revaluation in the late 1990s a
revised protocol was recommended [9]. The BCR method consists of
three successive steps for selective extractions of the following: acidsoluble (step 1), e.g., carbonates, reducible (step 2), e.g., oxides of
ferric and manganese, and oxidizable metals (step 3), e.g., organic
matter and suldes. This method has been widely accepted and
applied to metal fractionation in different environmental samples
[10]. However, conventional extraction methods are tedious and
time-consuming because these methods are batchwise and require a
long shaking time, e.g., 16 h for step 1 of the BCR method, with

M.L. Alonso Castillo et al. / Microchemical Journal 98 (2011) 234239

separation operations of the extracted solution by ltration at each

step.
Alternatives, such as ultrasonic shaking and microwave (MW)
heating, have been explored to shorten the treatment time [1114]. In
addition to the reduction in analysis time, other advantages of MW over
the conventional hot-plate heating methods include reduced contamination, lower reagent and sample usage and enhanced operator safety.
The MW oven heats the contents to a high temperature very rapidly and
the closed vessel helps in preventing losses due to volatilization of
elements. MW digestion also tends to yield more controlled and
reproducible results than conventional methods. Various MW oven
designs [1214] have been used as energy sources and their mechanical
parameters have been specically optimized in order to obtain the best
positive operation conditions in each case. But until now neither of them
included the treatment with hydrogen peroxide (step 3) inside MW
oven, which lasts approximately three hours.
In developing a MW heating technique for the BCR three-step
sequential extraction procedure one needs to adjust some variables in
order to establish optimum extraction conditions for the determination of heavy metals in each of the different chemical binding fractions
of soils and sediments. A fruitful way to conduct experimental designs
and optimization is according to surface-response methodology
(SRM). This includes a group of mathematicalstatistical techniques
that were designed to optimize the analytical response by producing a
model in which a response function corresponds to several variables
called factors. In this work, three variables, MW power, extraction time
and ramping time were considered as factors in the optimization
process. Interactions between analytical factors and their optimal
levels were investigated using central composite designs (CCDs)
[15,16]. This type of design allows the experimental domain to be
mapped and optimal values for parameters to be deduced, while
reducing the number of experiments compared to a sequential design.
The MW procedure was optimized using a soil with target analyte
concentration values in each step being those obtained by the
application of the revised BCR procedure using a mechanical shaker.
With the use of optimal microwave conditions, steps 13 of the
sequential extraction (including the hydrogen peroxide digestion in
step 3) could be completed between 21 and 22 min. The optimum
conditions established in each step of the BCR procedure were applied
to the determination of copper, nickel, lead, chromium and cadmium
in sediments by inductively coupled plasma mass spectrometry (ICPMS). For the quality control of the analytical performance and the
validation of the newly developed method, the conventional and
optimized alternative extraction methods were compared using the
analysis of BCR 701 Lake Sediment and different origins sediments
(river and marine). The results showed good agreement with the
certied values and were comparable for both methods according to
the t-paired-test for a 95% condence level.
2. Experimental

235

Table 1
Operating conditions for ICP-MS system.
Instrument
Nebulizer gas ow rate (l min 1)
Auxiliary gas ow rate (l min 1)
Plasma gas ow rate (l min 1)
Autolens voltage (V)
Incident power (kW)
Analogical phase voltage (kV)
Digital phase voltage (kV)
Scanning mode
Dwell time (ms)
Number of replicates
Reaction gas, CH4 (ml min 1)

Elan DRC-e (PerkinElmer)

Ar 0.900.98
1.2
15
6.757.50
1.1
2000
1000
Peak-jumping
100
3
0.4

Element

Mass

Cr
Ni
Cu
Cd
Pb

52
60
6365
111
208

reasonably good statistics. Metal concentrations were calculated on

the base of the signal for isotope 111Cd, 52Cr, 63;.65Cu, 60Ni and 208Pb.
During measurements, care was taken to avoid memory effect and
therefore a wash-out time of 1 min was used. CH4 gas was
demonstrated to effectively minimize polyatomic interferences.
For the digestion of samples an Anton Paar (Graz, Austria) Multiwave
3000 microwave oven equipped with a XF-rotor and XF-100 digestion
vessels was used. Unicen 20 centrifuge; speed ranges 04200 rpm, and
a timer of 060 min was used for the complete separation of the
extractants. A Crison pH-meter was employed for pH adjustments of the
reagents.
2.2. Reagents and samples
High-purity reagents were employed in all experiments. Doubly
deionized water with a resistivity of 18.2 M cm, produced by a MilliQ water system (Millipore, Bedford, MA, USA) was used throughout.
Standard solutions of 1000 g ml 1 Ni, Pb, Cu, Cd, and Cr (Fluka,
Buchs, Switzerland) were used. Standards of working strength were
made by appropriate dilution as required, immediately prior to use.
The extracting reagents as listed in Table 2, were prepared from
analytical-grade reagents. Ammonium acetate was purchased from
Sigma (Aldrich, Milwaukee, USA). Acetic acid, hydrochloric acid, nitric
acid, hydrogen peroxide and hydroxylammonium chloride were
analytical-reagent grades from Merck (Darmstadt, Germany).
Once the extraction method was established a standard was
analyzed. The certied Reference Material CRM 701 Lake Sediment
was supplied by The Community Bureau of References Sample (BCR).
All glassware and plastic materials used were previously treated for
48 h in 2 M nitric acid and rinsed with double deionized water. 50 ml
polyethylene vessels were used for storage of extractants.

2.1. Instrumentation
2.3. Sample collection and preparation
The ICP-MS instrument used in all experiments was the Elan DRC-e
model purchased from PerkinElmer SCIEX Instruments, (Concord,
Canada) equipped with quartz torch, nickel sampler and skimmer
cones, a peristaltic pump (maintaining a 1 ml min 1 sample uptake
rate), a cross-ow type pneumatic nebulizer, a double pass Scott-type
spray chamber and an AS-90 autosampler. Instrumental performance
optimization, including the nebulizer gas ow rate, the ion lens
voltage and the alignment of the torch, was carried out following
the manufacturer's instructions. The nebulizer gas ow rate was
adjusted so that the CeO+/Ce+ ratio and Ba++/Ba+ were less than 3%.
Operating conditions are summarized in Table 1. A quantitative
analysis mode (TQM) was used for the data acquisition of the samples.
The scanning of each sample was repeated three times to gather

The river sediment samples were taken from the top 020 cm
depth and were collected in polyethylene containers from three
stations of the Guadalhorce river (Mlaga, Spain) by using a plastic
spoon and from three stations of the Bay of Mlaga. From each station,
610 samples of sediments (12 kg) were collected randomly. On
returning to the laboratory, the samples were spread on the plastic
trays in fume cupboards and allowed to dry at ambient temperature.
The river sediment samples were air-dried for 10 days in a controlled
clean environment while the marine sediments were dried at 105 C
for 24 h. The samples were then ground with an agate pestle and
mortar and sieved with a nylon of 75 m sieve, and homogenized.
The section under the sieve ( b 75 m) was used for the analysis by

236

M.L. Alonso Castillo et al. / Microchemical Journal 98 (2011) 234239

Table 2
Reagents and experimental conditions.
Step

Fraction

1
2

Exchangeable
Reducible

Oxidizable

BCR (sample mass 1.00 g)

MW (sample mass 0.25 g)

Reagent

Time
(h)

Volume of reagent
(ml)

Reagent

Heating time
(s)

Ramping time
(min)

MW power
(w)

Volume of reagent
(ml)

Acetic acid 0.11 M

Hydroxylammonium
chloride 0.5 M pH 1.5
Hydrogen peroxide/
ammonium acetate 1 M

16
16

40
40

Acetic acid 0.11 M

Hydroxylammonium
chloride 0.5 M pH 1.5
Hydrogen peroxide/acetic
acid 5 M

120
105

4.6
6.5

300
400

10
10

120

4.6

300

2/5

ICP-MS. Homogenized sediment samples were stored in polyethylene

bottles in desiccators at room temperature.

The selected factors and their corresponding ranges were determined

after preliminary experiments. A mathematical model CCD can be
described by using second order polynominals, Eq. (1)

2.4. Sequential extraction BCR method

Y = 0 + j xj + jj xj + jk xj xk
The BCR sequential extraction method [9] was applied, in triplicate,
to 1 g of sample and to the certied reference material BCR 701. The
reagents and operating conditions employed are summarized in
Table 2. The extraction was carried out in 60 ml polyethylene tubes,
which were also used for centrifugation in order to minimize possible
losses of solid sample in the successive extraction steps of the
sequential procedure. After each extraction step, the supernatant
liquid was separated from the solid phase by centrifugation at
3000 rpm for 30 min, decanted into polyethylene containers and
stored at 4 C until analysis. The remaining solid residue was washed
with 20 ml of ultra-pure water by shaking for 15 min and the washings
were discarded after centrifugation.
2.5. Microwave sequential extraction
With the exception of the third fraction, the MW sequential
extraction was carried out using the same reagents as in the revised
BCR procedure, but replacing the magnetic shaking by MW heating
with the aim of reducing the operating time. In addition, in MW
extraction, lower amount of samples was used as compared to the BCR
sequential method. Extractant volumes were also decreased to
maintain a constant solid:solution ratio, since it is known that this
can affect the results obtained. The reagents and operating conditions
employed are summarized in Table 2. In the MW digestion procedure,
0.25 g of the powdered sieved grain size fraction was weighed directly
into each pre-cleaned MW vessel and the reagents for each three
extraction step were added. In the third step (oxidizable fraction)
2 ml of 30% H2O2 was added (for the digestion of the organic matter)
to the soil or sediment residue from the previous step plus 5 ml of
acetic acid extractant (pH of about 2). In all cases the vessels were
placed inside a rotor of the MW system, sealed, tightened using a
torque wrench and nally submitted to MW dissolution program.
After cooling, the supernatant liquid was separated from the solid
phase by centrifugation at 3000 rpm for 30 min. The resulting solution
was evaporated near to dryness to eliminate unreacted reagents [17].
Then, 1 M HNO3 was used to transfer the extracted residue into a
25 ml polyethylene calibrated ask and the sample was diluted to
25 ml, thus the calibrants for the three steps were made in 1 M HNO3.
On the other hand, the remaining solid residue was washed with
10 ml of ultra-pure water by shaking for 15 min and the washings
were discarded after centrifugation. For each set of measures, at least
one blank containing the same amount of reagents as the samples was
also taken through each step. The digests were stored at 4 C until
analysis.
2.6. Optimization strategy
A rotatable uniform central composite design (CCD) [16] was used
to study the empirical relationship between three controlled factors.

where Y is the response of system, xj is the variable of system and 0,

j, jj, jk are the regression coefcient for constant, linear, square and
interaction terms respectively. Regression coefcients are calculated
by tting the value of experimental parameters to the least squares
regression model. A quadratic equation or an equation containing
only signicant terms, results. This can be used to predict the response
of the system at given levels of experimental factors.
The three factor CCD used in this investigation consists of a star
design imposed through the center of a factorial design and comprised a 23 factorial design (8 experiments), a 2 3 star design (6
experiments) and 6 center points. The 20 resulting experiments
were randomly performed with 4 replications (total 80 experiments
and 67 of freedom). The replications were used to estimate the
experimental error due to irreproducibility of the BCR protocol. The
factors and experimental domain levels for CCD experiments appear
in Table 3.
The experimental data were processed by using the STATGRAPHICS program [18]. For CCD, the signicance of the effects was
checked by analysis of the variance (ANOVA) and using p-value
signicance levels. This value represents the probability of the effect
of a factor being due solely to random error. Thus, if the p-value is less
than 5% the effect of the corresponding factor is signicant.
2.7. Analytical procedure for measurement trace element by ICP-MS
Trace elements were determined in the liquid extracts by ICP-MS
using the operation conditions summarized in Table 1. Sediment
samples were analyzed in parallel by both BCR and MW procedures to
ensure the quality of the results obtained. The sample solutions were
quantied by external calibration. Multielement calibrant solutions
were prepared in nitric acid of 1 M. The following analysis sequence
was applied: rst the blank, then the standards and at the end of the
sequence, the samples. The blank intensity was subtracted from both
the standard and the sample intensities.
3. Results and discussion
3.1. Optimization of the microwave sequential extraction
In working with MW, the effective energy depends on the timing
parameters and applied power relative to the total quantity of
material employed in each digestion. The energy of the MW is equally
divided among all of the mass sensitive to it, hence if the quantities
introduced into the oven are varied, as a consequence, the amount of
energy received by each unit of mass will also vary. The objective of
this study is to adjust some variables: MW power, heating time and
ramping time for pre-established amount of the samples (0.25 g) and
the number of samples to be attacked at a time (4 samples). The

M.L. Alonso Castillo et al. / Microchemical Journal 98 (2011) 234239

237

Table 3
Factors and experimental domain levels.
Factor

Ramping time (min)

Heating time (s)
MW power (W)

Step 1

Step 2

Step 3

Low

High

Optimum

Low

High

Optimum

Low

High

Optimum

2.0
50
300

5.0
120
1200

4.5
120
300

2.0
50
300

10.0
140
1200

6.6
105
400

3.0
30
200

6.0
120
600

4.5
120
300

microwave oven procedure was optimized using a soil with target

analyte concentration values in each step being those obtained by the
application of the revised BCR procedure. Thus, the values of heavy
metals obtained from BCR method, were used as reference values for
calculating the percentage of metals recovered by MW sequential
extraction. In all cases, metal recovery was calculated as the following
ratio:% Recovery = (metal extracted by proposed MW extraction/
metal extracted by sequential BCR protocol) 100.
Where interactions exist between the variables, one is unlikely to
nd the true optimum if a conventional univariate optimization is
undertaken manually. For this reason, the optimization of the MW
procedure was carried out by applying the experimental design
methodology. A CCD involving 80 runs was performed in order to
obtain the optimum conditions with the three continuous variables
found to be potentially signicant for this study. The experimental
domain levels for CCD experiments appear in Table 3. Three dimensional representations keeping one of the variables at the optimum
value are presented in Fig. 1. It was observed that in the rst step at a
constant MW power of 300 W better response was obtained when the
heating time and the ramping time were 120 s and 4.5 min,
respectively (Fig. 1a). Similarly, in the second step, for a power of
400 W better response was obtained when the heating time and the
ramping time were 105 s and 6.6 min, respectively, as can be seen
from Fig. 1b. These data indicate that interactions usually occur
between principal factors. This means that the response surfaces in
the factorial space are curved in the domain of the experimental
design. On the other hand, it was not possible to identify a single set of
conditions optimal for all steps, but in nearly all cases in the rst and
third steps, MW power = 300 W, heating time = 120 s and ramping

time = 4.5 min, produced acceptable results. In these steps, if the

microwave power is maintained to a low level of 300350 W, the
ramping time could be varied between 4.5 and 5.0 min and the hold
time between 110 and 130 s. However, in the second step the best
combination of the three factors for simultaneous optimization of
the Ni, Cd, Cr, Pb, and Cu recoveries was MW power = 400 W, heating
time = 105 s and ramping time = 6.6 min. In this step, if the
microwave power is maintained to 400 W the ramping time could
be varied between 6 and 7 min and the hold time between 90 and
120 s. In all cases, high levels of power are not recommended because
the pressure into the digestor is increased too fast and the modern
lab microwave ovens with pressure sensor immediately decrease
the MW power. For latter experiments, the aforementioned
conditions were used for all three extraction steps. The objective
was to obtain a common set of conditions for the ve analytes in each
step to simplify the process and avoiding the need to perform
replicate extractions for each analyte. Thus, in general, in these
optimal conditions, no signicant differences were observed when
comparing the values obtained in each step by the proposed method
and by the BCR method.
On the other hand, it is important to emphasize the problems
which appeared in the third extraction stage due to the H2O2 boiling
under MW heat, initiating a violent reaction of the hydrogen peroxide
with the organic matter which produces a great deal of foam causing
liquid to overow from the extraction vessel. The solution to this
problem was the addition of 5 ml of 1 M ammonium acetate (adjusted
to pH 2 with HNO3) to 2 ml of 30% H2O2 for the third extraction step
and the extraction procedure was then continued as described under
the Experimental section. Utilizing this mixture, no problem with
escaping gases was observed when the vessels cooled, although not all
the hydrogen peroxide was consumed. Centrifugation of the extracts
did not present any problems. However, it was observed that HNO3
caused signicant changes on metal extraction and fractionation
pattern, but when using acetic acid (pH about 2) as extractant, the
metal extraction did not change signicantly, so we select acetic acid
for the experiments to release all metals (Cr, Ni, Pb, Cd and Cu) at
optimum levels as compared to those obtained from BCR procedure.
This treatment signicantly reduced the time required for the
digestion of the organic matter as can be seen in Table 2.
Reagent blanks were negligible and no detectable contamination
was found when aliquots of the sequential extraction reagents were
processed and analyzed with the samples. Detection limits (three times
the standard deviation of the blank, for n = 7 divided by the slope of
the calibration graph) were 0.021 ng ml 1 for Ni, 0.001 ng ml 1 for Cd,
0.003 ng ml 1 for Cr, 0.018 ng ml 1 for Cu and 0.018 ng ml 1 for Pb.
These detection limits were adequate for the analyzed samples being
better than the others found in the bibliography [13,19].
3.2. Analysis of samples

Fig. 1. Response surface obtained from CCD design. a) First and third extraction steps.
b) Second step.

In order to test the accuracy and applicability of the proposed

method to the analysis of soil and sediment samples, a certied
reference material (BCR 701 Lake Sediment), three river sediments
collected from the Guadalhorce river and three sea sediments
collected from the Bay of Mlaga (Spain) were analyzed. The sample
solution was quantied by external calibration, by comparing the net
signals against those of multi-element standards. For this purpose

238

M.L. Alonso Castillo et al. / Microchemical Journal 98 (2011) 234239

Table 4
Determination of metals in BCR-701 Lake Sediment.

First step

Second step

Third step

Metals

Ni

Cd

Cr

Cu

Pb

This work
Certied values
Recovery (%)
This work
Certied values
Recovery (%)
This work
Certied values
Recovery (%)

15.4 0.7
15.4 0.9
100
24.7 0.2
26.6 1.3
93
15.6 0.9
15.3 0.9
102

7.01 0.36
7.34 0.35
96
3.5 0.5
3.77 0.28
93
0.279 0.004
0.27 0.06
103

2.17 0.19
2.26 0.16
96
39 1
45.7 2.0
85
123 2
143 7
86

49 6
49.3 1.7
99
128 5
124 3
103
50.1 0.1
55.2 4.0
91

3.3 1.1
3.18 0.21
103
125.9 0.1
126 3
100
7.65 0.07
9.3 2.0
82

All values reported as mean (n = 4) SD (mg/kg dried basis).

Table 5
Validation of the method.
Samples

River sediment 1

River sediment 2

River sediment 3

Marine sediment 1

Marine sediment 2

Marine sediment 3

Step

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

Ni

Cd

Cr

Cu

Pb

MW

BCR

MW

BCR

MW

BCR

MW

BCR

MW

BCR

4.0 0.7
18.2 2.3
13.2 2.2
3.9 1.2
8.8 0.4
12.3 1.2
17.8 0.3
11.2 0.5
7.0 1.3
4.2 0.3
14.4 1.3
17.0 1.1
4.6 0.1
13.8 1.4
13.6 0.2
4.4 1.2
14.6 0.5
20.3 0.1

4.8 1.1
20.1 1.9
12.2 1.4
5.1 1.3
8.9 2.4
12.0 0.4
12.1 3.3
9.0 1.9
8.2 1.3
3.7 0.2
15.4 2.3
17.1 2.1
3.8 1.3
11.0 2.1
16.3 3.2
4.0 0.3
12.1 2.0
21.1 2.3

0.04 0.03
0.020 0.006
0.005 0.001
0.03 0.01
0.04 0.01
0.007 0.002
0.044 0.001
0.053 0.004
0.015 0.002
0.04 0.01
0.020 0.003
0.008 0.001
0.041 0.004
0.031 0.001
0.031 0.001
0.039 0.001
0.07 0.09
0.073 0.002

0.04 0.01
0.020 0.002
0.010 0.006
0.04 0.01
0.02 0.01
0.010 0.002
0.05 0.01
0.07 0.01
0.03 0.01
0.04 0.01
0.020 0.006
0.010 0.007
0.05 0.01
0.02 0.01
0.03 0.01
0.05 0.02
0.06 0.02
0.07 0.02

0.10 0.02
1.7 0.6
7.2 1.2
0.08 0.01
0.6 0.2
6.3 0.1
0.04 0.01
0.4 0.1
4.9 0.4
0.08 0.02
1.5 0.1
13.2 0.4
0.38 0.02
1.86 0.05
10.7 0.1
0.17 0.01
3.1 0.1
12.87 0.08

0.10 0.01
1.0 0.7
4.9 1.3
0.10 0.06
0.80 0.02
5.0 0.2
0.03 0.01
0.4 0.1
4.7 0.8
0.10 0.08
2.0 0.3
10.0 2.9
0.6 0.2
31
91
0.16 0.05
4.0 1.2
10.2 1.1

0.4 0.1
2.7 0.6
2.1 0.4
0.5 0.1
3.5 0.5
9.1 1.3
0.45 0.04
3.2 0.1
14.8 2.3
0.4 0.2
5.0 0.3
7.0 0.3
0.94 0.03
7.2 0.2
6.95 0.03
0.24 0.01
7.0 0.4
9.43 0.06

0.6 0.1
2.0 0.5
2.9 0.6
0.8 0.2
3.0 0.3
10.1 1.8
0.6 0.3
3.1 0.1
15.2 1.9
0.4 0.1
4.0 1.3
6.9 1.2
1.1 0.1
5.2 1.1
9.2 2.9
0.4 0.2
6.3 0.8
11.2 2.3

0.34 0.01
1.8 0.6
0.4 0.1
0.3 0.1
2.2 0.5
0.50 0.01
0.3 0.1
6.4 0.3
4.2 1.3
0.5 0.1
8.5 0.4
2.48 0.03
0.564 0.004
11.7 1.2
1.91 0.01
0.76 0.01
15.7 0.2
2.4 0.01

0.20 0.04
1.9 0.1
0.40 0.06
0.20 0.01
2.2 0.4
0.4 0.04
0.10 0.05
7.2 1.4
4.0 0.4
0.40 0.04
9.1 1.9
2.4 0.1
0.41 0.05
12.8 0.9
2.3 0.8
0.8 0.2
16.3 1.1
2.3 0.6

All values reported as mean (n = 4) SD (mg/kg dried basis).

calibration plots were constructed from eight aqueous standard

solutions within the concentration ranges of 30300 ng ml 1 for Ni,
0.05100 ng ml 1 for Cd, 0.31500 ng ml 1 for Cr, 31500 ng ml 1
for Cu and 11500 ng ml 1 for Pb. Correlation coefcients of 0.998 or
better were obtained for all graphs, indicating good linearity for the
working ranges aforementioned. The results, as the average of four
separate determinations, are shown in Tables 4 and 5. In all cases data
are reported as mean values (mg kg 1) standard deviation (n = 4).
In the case of certicate sample, no signicant differences were
observed, for p = 0.05 when comparing the values obtained by the
proposed method and the certied values (t-test), with only a few
exceptions (Ni in step 2, Cr in steps 2 and 3, and Pb and Cu in step 3).
The results obtained for the analyses of extracts of sediments from
different origins (river and marine) produced by both BCR and MW
procedures were comparable, according to the t-paired-test for a 95%
condence interval.
4. Conclusions
In developing a MW heating technique for the BCR three-step
sequential extraction procedure, one needs to adjust some variables in
order to establish optimum extraction conditions for the determination of heavy metals in each of the different chemical binding fractions
of soils and sediments. In this study, by using a CCD design we have
been able to establish the optimum conditions for the proposed MW
procedure. The optimum conditions established in each step of the
BCR procedure were applied to the determination of copper, nickel,
lead, chromium and cadmium in sediments of different origins by ICPMS. The tests carried out provide evidence that the proposed method
is a viable alternative to other methods described in the literature. In

addition, it is important to emphasize that in the third step of the

proposed method, the H2O2 treatment for the digestion of organic
matter was carried out simultaneously with the extraction of all
metals using acetic acid as extractant inside the microwave oven and
consequently the total operation time of this step was reduced from
3 h to only 6.6 min.
Acknowledgements
The authors thank the Regional Government of Andaluca (project
no. P07-RNM-02502) and the Spanish Ministerio de Ciencia y
Tecnologa (MCyT) for supporting this study, (project no. CTQ
200600735) and also FEDER funds.
References
[1] M. Tuzen, H. Sari, M. Soylak, Microwave and wet digestion procedures for atomic
absorption spectrometric determination of trace metals contents of sediment
samples, Anal. Lett. 37 (2004) 19251936.
[2] E.D. Hullebusch, S. Utomo, M.H. Zandvoort, P.N.L. Lens, Comparison of three
sequential extraction procedures to describe metal fractionation in anaerobic
granular sludges, Talanta 65 (2005) 549558.
[3] A. Fuentes, M. Liorens, J. Saez, M.J. Aguilar, J.F. Ortuno, V.F. Meseguer, Comparative
study of six different sludges by sequential speciation of heavy metals, Bioresour.
Technol. 99 (2008) 517525.
[4] A. Tessier, P.G.C. Campbell, M. Bisson, Sequential extraction procedure for the
speciation of particulate trace metals, Anal. Chem. 51 (7) (1979) 844851.
[5] P.G.C. Campbell, A.G. Lewis, P.M. Chapman, A.A. Crowder, W.K. Fletcher, B. Inber, S.N.
Luoma, P.M. Stokes, M. Winfrey, Panel on Biologically Available Metals in Sediments,
Pub. No. NRcc27694, National Research Council of Canada, Otawa, 1988, p. 190.
[6] C. Gleyzes, S. Tellier, M. Astruc, Fractionation studies of trace elements in
contaminated soils and sediments: a review of sequential extraction procedures,
Trac-Trend Anal. Chem. 21 (2002) 451467.

M.L. Alonso Castillo et al. / Microchemical Journal 98 (2011) 234239

[7] Ph. Quevauviller, Operationally dened extraction procedures for soil and
sediment analysis. I. Standardization, Trac-Trend, Anal. Chem. 17 (1998) 289298.
[8] Ph. Quevauviller, G. Rauret, H. Muntau, A.M. Ure, R. Rubio, J.F. Lpez Snchez,
H.D. Fiedler, B. Griepink, Evaluation of a sequential extraction procedure for
the determination of extractable contents in sediment, Fresenius' J. Anal. Chem.
349 (1994) 808814.
[9] G. Rauret, J.F. Lpez Snchez, A. Sahuquillo, R. Rubio, C. Davidson, A. Ure, Ph.
Quevauviller, Improvement of the BCR three step sequential extraction procedure
prior to the certication of new sediment and soil reference materials, J. Environ.
Monit. 1 (1999) 5761.
[10] J.R. Bacon, C.M. Davidson, Is there a future for sequential chemical extraction?
Analyst 133 (2008) 2546.
[11] C.M. Davidson, G. Delevoye, Effect of ultrasonic agitation on the release of copper,
iron, manganese and zinc from soil and sediment using the BCR three stage
sequential extraction, J. Environ. Monit. 4 (2002) 541548.
[12] C. Real, R. Barreiro, A. Carballeira, The application of microwave heating in
sequential extractions of heavy metals in estuarine sediments, Sci. Total Environ.
152 (1994) 135142.

239

[13] M.K. Jamali, T.G. Kazi, M.B. Arain, H.I. Alfridi, N. Jalbani, G.A. Kandhro, A.Q. Shah, J.A.
Baig, Speciation of heavy metals in untreated sewage sludge by using microwave
assisted sequential extraction procedure, J. Hazard. Mater. 163 (2009) 11571164.
[14] B. Prez Cid, I. Lavilla, C. Bendicho, Application of microwave extraction for
partitioning of heavy metals in sewage sludge, Anal. Chim. Acta 378 (1999)
201210.
[15] G. Sado, M.C. Sado, Les Plans d'Expriences, Afnor Techniques, Paris, 1991, p. 100.
[16] J. Gouply, Plans d'Expriences pour Surfaces de Rsponse, Dunod, Paris, 1991,
p. 253.
[17] M.B. Arain, T.G. Kazi, M.K. Jamali, H.I. Alfridi, R.A. Sarfraz, J.A. Baig, G.A. Kandhro,
M.A. Memo, Time saving modied BCR sequential extraction procedure for the
fraction of Cd, Cr, Cu, Ni, Pb and Zn in sediment samples of polluted lake, J. Hazard.
Mater. 160 (2008) 235239.
[18] Statgraphics Plus Version 6.0 Reference Manual, Manugistics, Rockville, MD, 1992.
[19] C.M. Davidson, A.L. Duncan, D. Littlejohn, A.M. Ure, L.M. Garden, A critical
evaluation of the three-stage BCR sequential extraction procedure to assess the
potential mobility and toxicity of heavy metals in industrially-contaminated land,
Anal. Chim. Acta 363 (1998) 4555.