American Journal of Anal yt ical Chemistry, 2010, 1, 127-134
doi:10.4236/ajac.2010.13016 Published Online November 2010 (http://www.SciRP.org/journal/ajac)
Preconcentration of Cadmium in Environmental Samples
by Cloud Point Extraction and Determination by FAAS
Catalina Bosch Ojeda, Fuensanta Sánchez Rojas*, José Manuel Cano Pavón
Department of An al yt i cal Che mi st r y , Faculty of Sciences, University of Málaga, Málaga, Spain
Received July 21, 2010; revised September 1, 2010; accepted September 3, 2010
Abstract
Cloud point extraction (CPE) has been used for the preconcentration of cadmium, after the formation of a
complex with 1, 5-bis(di-2-pyridylmethylene) thiocarbonohydrazide (DPTH), and further determination by
flame atomic absorption spectrometry (FAAS) using Triton X-114 as surfactant. The main factors affecting
the CPE, such as concentration of Triton X-114 and DPTH, pH, equilibration temperature and incubation
time, were optimized for the best extract efficiency. Under the optimum conditions i.e., pH 5.4, [DPTH] =
6 × 10-3%, [Triton X-114] = 0.25% (v/v), an enhancement factor of 10.5 fold was reached. The lower limit of
detection (LOD) obtained under the optimal conditions was 0.95 μg L−1. The precision for 8 replicate deter-
minations at 20 and 100 μgL−1 Cd were 2.4% and 2% relative standard deviation (R.S.D.). The calibration
graph using the preconcentration method was linear with a correlation coefficient of 0,998 at levels close to
the detection limit up to at least 200 μgL−1. The method was successfully applied to the determination of
cadmium in water, environmental and food samples and in a BCR-176 standard reference material.
Keywords: Cadmium, Flame Atomic Absorption Spectrometry, Cloud Point Extraction, Triton X-114, Water
Samples, Food Samples
1. Introduction
Monitoring the presence of toxic trace elements in di-
verse matrices is an extremely important task to evaluate
occupational and environmental exposure. In this sense,
cadmium is one of the most toxic elements and accumu-
lates in humans mainly in the kidneys and liver and is
classified as a prevalent toxic element with biological
half-life in the range of 10-30 years [1]. One of the
pathways that cadmium enters human body is through
daily intake of food and water, thus the monitoring of
cadmium concentrations in food and water samples is of
significant importance. The maximum contaminant level
allowed by the American Environmental Protection
Agency (US EPA) in standard drinking water is 10 μg
L-1 to provide ample protection of human health.
Monitoring trace element concentrations in biological
materials, particularly biological fluids, might be consid-
ered a difficult analytical task [2,3], mostly due to the
complexity of the matrix and the low concentration of
these elements, which requires sensitive instrumental
techniques and often a preconcentration step. Concretely,
spectrometric techniques for the analysis of trace cad-
mium have developed rapidly due to the increasing need
for accurate measurements at extremely low levels of
this element in diverse matrices. An interesting revision
presented by Ferreira et al. [4] covers separation and
preconcentration procedures, and considers the features
of the application with several spectrometric techniques.
In this way, CPE techniques exploit a peculiar prop-
erty of most non-ionic surfactants that form micelles in
aqueous solution: they become turbid when heated to the
appropriate cloud point temperature. Above the cloud
point temperature, the micellar solution separates into a
small, surfactant rich phase and a larger diluted aqueous
phase. In the aqueous phase, the surfactant concentration
remains near the critical micelle concentration. Any ana-
lyte solubilised in the hydrophobic core of the micelle in
the unheated solution, will be concentrated in the surfac-
tant-rich phase following the cloud point extraction [5,6].
Numerous reports have been published on the precon-
centration of cadmium, alone or in mixtures, by CPE
method prior to its determination using spectrometric
techniques [7-34]. Table 1 lists recent works concerning
with cadmium preconcentration by CPE and determina-
tion by spectrometric techniques.

128 C. B. OJEDA ET AL.
Table 1. Cadmium preconcentration by CPE.
Matrix Reagent/surfactant Pre-concentration factorTechnique Ref.
Seawater PAN 120 FAAS 7
Waters DDTP/Triton X-114 29 ICP-MS 8
Waters TAN/Triton X-114 58 FAAS 9
Human hair DDTP/Triton X-114 22 FAAS 10
Waste and waters Dithizone/Triton X-114 52 FAAS 11
Seawater DDTC/Triton X-114 52 GFAAS 12
Physiological solution, mineral
and lake waters and tobacco DDTP/Triton X-114 - FAAS 13
Biological materials DDTP/Triton X-114 129 GFAAS 14
Waters PMBP/Triton X-100 23 FAAS 15
Waters Iodide/Triton X-114 55.6 FAAS 16
Water and urine APDC/Triton X-114 13 TS-FF-AAS 17
Waters PONPE 7.5 62 CV-AAS 18
Waters PAN/Triton X-100 50 GFAAS 19
Waters 5-Br-PADAP/Triton X-114 21 GFAAS 20
Water standard reference
material and urine 5-Br-PADAP/PONPE 7.5 22 GFAAS 21
Urine APDC/Triton X-114 15 WCAAS 22
Water and tobacco GBHA/Triton X-114/SDS/NaCl 22 GFAAS 23
Waters Carboxylic acids/OP-10 - FAAS 24
Urine DDTP/Triton X-114 16 GFAAS 25
Drinking waters TAC/Triton X-114 22 FS-FAAS 26
Natural waters DDTP/Triton X-114 - FAAS 27
Waters TAN/Triton X-114 20.3 FAAS 28
Natural waters Schiff base benzylbis thiosemicarbazone/
Triton X-114 140 FAAS 29
Mineral drinking water, river
water and seawater NDDBH/Triton X-114 157 FAAS 30
Waters PAR/Triton X-114 9.4 ICP-OES 31
Environmental samples BIES/Triton X-114 48 FAAS 32
Waters o-phen and eosin/PONPE 7.5 - Molecular fluorescence33
Rice and water Dithizone/Triton X-114 152
93
W-coil ETV-AFS
W-coil GFAAS 34

C. B. OJEDA ET AL.
129
In this work, we report on the results obtained in a
study of the CPE of Cd2+, after the formation of a com-
plex with DPTH using Triton X-114 as surfactant fol-
lowed by analysis by FAAS.
2. Experimental
2.1. Instrumentation
A thermostated bath Model Selecta Precisterm, main-
tained at the desired temperature, was used for the CPE
experiments. Phase separation was achieved with a cen-
trifuge Selecta Centromix in 10mL calibrated conical
tubes.
A Varian Model SpectrAA 50 (Mulgrave, Victoria,
Australia) flame atomic absorption spectrometer was
used for the analysis with the appropriate cadmium hol-
low cathode lamp. The operating parameters were set as
recommended by the manufacturer. Atomic absorption
measurements were carried out in an air-acetylene flame.
The following conditions were used: absorption line Cd:
228.8 nm; slit widths: 0.5 nm; and lamp currents: 4 mA.
2.2. Reagents and Samples
High purity water (resistivity 18 MΩ cm-1) obtained by a
Milli-Q® water purification system (Millipore, Bedford,
MA, USA) was used throughout this work. 1000 mg L-1
stock solutions of cadmium (E. Merck, Darmstadt, Ger-
many). Working standard solution was obtained daily by
stepwise dilution of the standard stock solution. DPTH
solution in DMF was prepared by dissolving solid re-
agent samples prepared and purified by the authors.
Non-ionic surfactant, Triton X-114 stock solution (2%,
v/v) was prepared by dissolving 2 mL of concentrated
solution (Merck, Darmstadt, Germany) in 100 mL hot
deionised water. These reagents were all of analytical
grade or better.
The accuracy of the method for determination of cad-
mium content was checked by analyzing the reference
standard material BCR 176 ‘‘City waste incineration
ash’’; for this the certified cadmium content was 470 ± 9
mg kg-1. The sample was first prepared in accordance
with the instructions on the analysis certificate, after
which an accurately weighed amount (50 mg) was sub-
jected to microwave digestion. The solution obtained
was then adjusted to the optimum pH and, finally, the
sample was diluted to 25 mL with de-ionized water in a
calibrated flask.
The proposed method was also evaluated by analysis
of cadmium in several spiked food samples. The Cd
concentrations in all the original samples were below the
detection limit. For this purpose, standard solutions con-
taining cadmium were added to 0.2–0.5 g of diverse food
and the resulting materials were mineralized by micro-
wave digestion, adjusted pH and diluted at convenient
volume.
Natural waters were collected in polypropylene bottles
previously cleaned by soaking for 24 h in 10% (v/v) ni-
tric acid and finally rinsed thoroughly with ultrapure
water before use.
2.3. Procedure
10 mL analyte solution containing cadmium, 1mL buffer
solution pH 5.4, DPTH 6 × 10-3 % and 0.25% (v/v) Tri-
ton X-114 was kept in a thermostated bath at 50ºC for 30
min. Phase separation was accelerated by centrifuging
the resultant solution at 3800 rpm for 5 min. The conical
tubes were then immersed in an ice-water mixture for 20
min, allowing ease of removing the supernatant bulk
aqueous phase. A small volume of surfactant rich phase
remained at the bottom of the tube. To decrease the vis-
cosity of the extract and to facilitate sampling, 0.4 mL of
HNO3 0.1M/MeOH was added to surfactant-rich phase.
The cadmium content was determined by flame atomic
absorption at 228.8 nm against a blank solution. Calibra-
tion was carried using different standard solutions of
cadmium submitted to the same preconcentration and
determination procedures. Blank solution was submitted
to the same procedure and measured in parallel to the
samples.
3. Results and Discussion
3.1. Study of the CPE System Variables
In the so-called cloud point extraction, several parame-
ters play a substantial role in the performance and ag-
gregation of the surfactant system, thus entrapping the
analyte species. CPE of metal ions is known to depend
on several factors such as type and amount of reagent
and surfactant, pH of solution, ionic strength, equilibra-
tion temperature and time, etc. We have investigated the
CPE process in order to obtain optimum conditions.
3.1.1. Effec t of pH and Triton X-114 Concentrati o n
The formation of metal complexes and its chemical sta-
bility are the two important influence factors for the CPE,
and the pH plays a unique role on metal chelate forma-
tion and subsequent extraction; in this sense, cadmium(II)
react with DPTH to form intensely coloured complex
and in a previous study, the characteristics of this chelate
were described so cadmium(II) forms a complex with
DPTH in a wide range of pH.
CPE of cadmium was performed in solutions of pH

130 C. B. OJEDA ET AL.
ranging from 3.6 to 5.6. Separation of metal ions by
cloud point method involves the prior formation of a
complex with sufficient hydrophobicity to be extracted in
to the small volume of surfactant-rich phase. Extraction
recovery depends on the pH at which complex formation
occurs.
On the other hand, in CPE, since the temperature cor-
responding to cloud point is correlated with the hydro-
philic property of surfactants, an appropriate surfactant is
important. The surfactants, which have too high or too
low cloud point, are not suitable for the CPE separation/
preconcentration of trace elements. A successful cloud
point extraction should maximize the extraction effi-
ciency by minimizing the phase volume ratio (Vorg/Va-
queous), thus improving its concentration factor. Triton
X-114 was chosen for the formation of surfactant rich
phase due to its recognized physicochemical characteris-
tics: low cloud point temperature, high density of the
surfactant rich phase; which facilitates phase separation
by centrifugation, commercial availability, relatively low
price and low toxicity.
These variables of interest in the CPE process were
optimized with the aid of experimental design. The sig-
nificant factors were optimized by using a factorial de-
sign 32. The pH varied between 3.6 and 5.6, and the Tri-
ton X-114 concentration ranged from 0.1 to 0.4 %. The
experimental results as absorbance are described in Ta-
ble 2. The significance of the effects was checked by
analysis of the variance (ANOVA) and using P-value
significance levels. Also, the ANOVA results produced
the graphs showing the influence of main effects repre-
sented in Figure 2, interaction plot in Figure 3 and
standardized Pareto chart in Figure 4. These data were
fitted into the following function:
Table 2. Factorial design for optimization of the experi-
mental conditions.
Experiment Triton (%) pH Absorbance
1 0.4 3.6 0.349
2 0.1 5.6 0.517
3 0.1 4.8 0.53
4 0.4 4.8 0.471
5 0.4 5.6 0.546
6 0.2 5.6 0.485
7 0.1 3.6 0.182
8 0.2 3.6 0.325
9 0.2 4.8 0.477
Figure 1. Scheme of the CPE procedure.

C. B. OJEDA ET AL.
131
Figure 2. Influence of main effects on the absorbance.
Figure 3. Interaction plots.
Figure 4. Pareto’s chart.
Absorbance = –1,85484 + 1,25039(Triton) + 0,825714
(pH) – 0,205556(Triton)2 – 0,213299(Triton) (pH) –
0,0718056 (pH)2
This equation shows a maximum condition of absorb-
ance for a Triton X-114 concentration of 0.25 % and pH
5.4.
3.1.2. Effect of DPTH Concentration
The variation of the analytical signal as a function of the
concentration of DPTH in the range of 5 × 10-3 – 2 ×
10-2% (w/v) was studied, and the experimental results in
Figure 5 demonstrated that the signal intensity of the
analyte was accentuated by DPTH at concentrations up
to about 6 × 10-3% (w/v). The maximum signal intensity
achieved with this concentration remained practically
constant up to the highest amount studied. A 6 × 10-3%
(w/v) DPTH was selected for further research.
3.1.3. Effect of Ionic Strength
The influence of ionic strength was examined by study-
Figure 5. Influence of DPTH concentration.
ing the extraction efficiency for NaCl concentration in
the range 0.5-3%. Ionic strength had no significant effect
upon percent recovery and sensitivity.
3.1.4. Effects of the Equilibration Temperature and
Time
It is desirable to employ the shortest incubation time and
the lowest possible equilibrium temperature, which
comprise completion of the reaction and efficient separa-
tion of the phases. As mentioned above, after evaluating
the incubation time in the range 10-40 min, it was kept
for 30 min, which is sufficient for the completion of the
complex reaction and also for the clouding process. It
was also observed that a temperature of 50ºC is sufficient
for maximum recovery of the cadmium.
In general, centrifugation time hardly ever affects mi-
celle formation but accelerates phase separation in the
same sense as in conventional separations of a precipitate
from its original aqueous environment. Therefore, a cen-
trifugation time of 5 min at 3800 rpm was selected as
optimum, since complete separation occurred for this
time and no appreciable improvements were observed for
long time.
In the phase separation step, the surfactant-rich phase
with high viscosity was settled. The addition of a diluent,
such as 0.4 mL of HNO3 0.1 M/methanol mixture re-
duces the surfactant phase viscosity and facilitates its
transfer into the flame. The optimized conditions of CPE
are summarized in Table 3.
3.2. Analytical Characteristics
Table 4 summarizes the analytical characteristics of the
optimized method, including regression equation deter-
mining before and after cloud point extraction method,
linear range, limit of detection and reproducibility of Cd
after CPE. The limit of detection, defined as CL = 3SB/m

132 C. B. OJEDA ET AL.
Table 3. The optimized conditions for Cd determination using CPE method.
Optimum CPE conditions
pH 5.4 Equilibrium temperature (ºC) 50
DPTH (%) 6 × 10-3 Equilibrium time (min) 30
Buffer: Acetic acid/Sodium acetate (M) 0.2 Centrifuge time (min) 5
Triton X-114 (%, v/v) 0.25 Diluent (mL): HNO3 0.1M/methanol 0.4
Table 4. Analytical characteristics of the proposed method.
Regression equation after CPE A = 0.0021[Cd(II)] + 0.0025; R2 = 0,9985
Regression equation before CPE A = 0.0002[Cd(II)] + 0,0005; R2 = 0,9906
Linear range (ng mL-1) 10-200
Limit of detection (ng mL-1) 0.95
Limit of determination (ng mL-1) 4.3
Reproducibility (R.S.D.%) n = 8 2.4, [Cd(II)] = 20 ng mL-1; 2.0, [Cd(II)] = 100 ng mL-1
Improvement factor 10.5
(where CL, SB, and m are the limit of detection, standard
deviation of the blank, and slope of the calibration equa-
tion, respectively), was 0.95 ng mL-1. The improvement
factor, defined as the slope ratio of the calibration graph
of the CPE method to that of the calibration graph with-
out preconcentration, was 10.5.
3.3. Effect of Foreign Ions
The effects of representative potential interfering species
were tested. To check these effects, a standard solution
containing cadmium and other ions was prepared and
cadmium was determined using the procedure proposed.
The results obtained (Table 5) showed that at the tested
concentrations, the other ions do not interfere in the pro-
cedure proposed.
3.4. Applications
In order to evaluate the analytical applicability of the
proposed method, it was applied to the determination of
cadmium in several samples.
A reference material (BCR-176 “City Waste Incinera-
tion Ash”) certified for its content in cadmium was ana-
lyzed for method validation. Recovery experiments (Ta-
ble 6) were conducted as well.
In view of the application of the method to the deter-
mination of cadmium in food samples, the ability to re-
cover cadmium from different samples spiked with cad-
mium was investigated. All samples were arbitrarily se-
lected and acquired from a local superstore. For this
purpose, standard solutions containing different quanti-
ties of cadmium were added to samples and the resulting
material was prepared as described under Experimental.
Standard additions method was used in all instances and
the results were obtained by extrapolation. The results of
these analyses are summarised in Table 6, and indicated
excellent recoveries in all instances.
In the laboratory, before the preconcentration proce-
dure, all the water samples were filtered through a 0.45
µm pore-size membrane filter to remove suspended par-
ticulate matter and were stored at 4ºC. The optimized
methodology was applied for the determination of Cd in
different water samples and the analytical results along
with the recovery are given in Table 6.
As can be seen, good recoveries were obtained in the
spiked real samples analysis.
Table 5. Tolerance ratio of diverse ions on the determina-
tion of cadmium (100 ng mL-1).
Ions Tolerance ratio (m/m)
K+, I-, Pb2+, Mg2+, HCO3-,  100
Ca2+, Cr3+, Al3+, Cu2+, Fe3+,
Mn2+, Ba2+, SO4=, F- 50

C. B. OJEDA ET AL.
133
Table 6. Determination of cadmium in real samples.
Sample Added (ng mL-1)Found (ng mL-1)a Recovery (%)
Tap water 20 19.9  0.1 99.5
Sea water 20 21.3  1.0 106.5
Certified sea water
20
40
60
20.9  0.8
39.3  0.8
61.4 ± 1.2
104.5
98.3
102.3
Added (g g-1) Found (g g-1)
Apple 2.46 2.45 ± 0.29 99.6
Lettuce 4.88 4.94 ± 0.5 101.2
Liver 3.98 3.68 ± 0.52 92.4
Chick-pea 2.33 2.36 ± 0.18 101.3
Fish 2.37 2.39 ± 0.18 100.8
Bignonia leaves 4.17 4.18 ± 0.8 100.2
Pinus leaves 4.47 4.33  0.5 96.7
Soil 5.23 5.63  0.4 107.6
Certified value
(mg kg-1)
Found value
(mg kg-1)
BCR 176 470  9 475 ± 25 101.1
amean ± standard deviation (n = 3)
4. Conclusions
The reagent DPTH was successfully employed in a CPE
procedure for the determination of cadmium in environ-
mental and food samples by FAAS. The method signifi-
cantly inproved the performance of the FASS detection
for cadmium. With the low cost and easily available ac-
cessories, the detectability of a traditional FAAS instru-
ment can be comparable to that of more sophisticated
instruments, such as ET-AAS. Also, CPE offers many
advantages over traditional liquid-liquid extraction, such
as elimination of handling large volumes of volatile,
toxic and flamable organic solvents. The method has
been validated by the analysis of standard reference ma-
terial.
5. Acknowledgements
The authors thank to the Ministerio de Ciencia e In-
novación for supporting this study (Projects CTQ2009-
07858) and also the Junta de Andalucia.
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