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International Journal of Scientific Research in Knowledge, 2(3), pp. 116-123, 2014
Available online at http://www.ijsrpub.com/ijsrk
ISSN: 2322-4541; ©2014 IJSRPUB
http://dx.doi.org/10.12983/ijsrk-2014-p0116-0123
116
Full Length Research Paper
Analysis of Lead(II) in Human Blood Using Dispersive Liquid-Liquid
Microextraction and Graphite Furnace Atomic Absorption Spectrometry
Ali Mazloomifar
Department of Chemistry, Shahre Ray Branch, Islamic Azad University, Tehran, Iran; Email: [email protected]
Received 28 December 2013; Accepted 01 February 2014
Abstract. This paper established a new, rapid and sensitive method for the determination of lead in human blood samples
preconcentrated by dispersive liquid–liquid microextraction (DLLME) prior to graphite furnace atomic absorption
spectrometry. In the proposed approach, diphenylthiocarbazone (dithizone) was used as a chelating agent, and carbon
tetrachloride and ethanol were selected as extraction and dispersive solvents. Important factors that would affect the extraction
efficiency had been investigated including the kind and volume of extraction solvent and dispersive solvent, sample pH, the
amount of chelating agent, extraction time and centrifugation time. The results showed that the coexisting ions contained in
human blood samples had no obvious negative effect on the determination of lead. In the optimum experimental conditions,
the limit of detection and enrichment factor were 0.04 ng mL-1
and 123, respectively. The relative standard deviation (RSD) for
ten replicate determinations of 10 ng mL-1
was 2.87%. The linearity of method was between 0.10-200 ng mL-1
. The method
was successfully applied for the analysis of lead in human blood samples.
Keyword: Lead, Graphite furnace atomic absorption spectrometry, Dispersive liquid-liquid microextraction, human blood
1. INTRODUCTION
Trace levels of heavy metals are widely distributed in
the environment due to soil erosion, waste, volcanic
emission, metal mining, smelting, industrial and
agricultural processes (López-García et al., 2013;
Shah et al., 2012; Silva and Roldan, 2009). Nowadays
the pollution by heavy metals from various
environmental sources has created much more
attention (Zhou et al., 2011). Lead is one of the most
widely distributed toxic heavy metals in the
environment. It is a cumulative poison, affecting the
brain and nervous system and causes a decrease in the
rate of globulin and heme synthesis (Elekofehinti et
al., 2012). Symptoms of lead poisoning include renal
insufficiency, colic, constipation and other
gastrointestinal effects. It also affects the reproductive
system, resulting in sterility, abortions, still birth and
neonatal deaths. The accepted tolerance limit is a
blood lead concentration of 0.4 µgml−1
for adults and
0.1 µgml−1
for children and adolescents (Baghurst et
al., 1992). The threshold between the normal lead
level and the level where physiological effects
become manifest is relatively narrow. It is therefore
desirable to screen exposed populations in order to
identify the danger in time. The lead concentration in
the blood is a measure to the total amount of lead in
the body. A fast, accurate and cheap method for the
determination of lead in blood is therefore needed
(Jaenicke et al., 1998).
The analytical methods of lead usually involves in
using flame atomic absorption spectrometry (FAAS),
electrothermal atomic absorption spectrometry
(ETAAS), graphite furnace atomic absorption
spectrometry (GFAAS), inductively coupled plasma
atomic emission spectrometry (ICP-AES), inductively
coupled plasma mass spectrometry (ICP-MS), and
atomic fluorescence spectrometry (AFS) (Biasino et
al., 2007; Liang and Sang, 2008; Manzoori et al.,
2009; Marchisio et al., 2005; Portugal et al., 2007;
Zhou et al., 2011). Among these techniques, GF AAS
is a very attractive option to determine trace amounts
of lead in complex samples, as it is the most robust
technique for this purpose. However, due to the low
level of lead in many samples its direct determination
with all of the above techniques, including GF AAS,
is on many difficult, and major constituents, such as
organic compounds and inorganic salts, could cause
interferences. Consequently, separation and
preconcentration procedures might be necessary prior
to the GF AAS determination of this element (Carletto
et al., 2011). Various microextraction techniques
including bioabsorption (Liu et al., 2006), the use of
other absorbents (Gama et al., 2006), cloud point
Mazloomifar
Analysis of Lead(II) in Human Blood Using Dispersive Liquid-Liquid Microextraction and Graphite Furnace Atomic
Absorption Spectrometry
117
extraction (CPE) (Candir et al., 2008; Silva and
Roldan, 2009), single drop microextraction (SDME)
(Jiang and Hu, 2008; Liang et al., 2008; Manzoori et
al., 2009; Vidal et al., 2010), liquid phase
microextraction (Nazari, 2008) and hollow fiber liquid
phase microextraction (HF-LPME) (Carletto et al.,
2011), solid phase extraction (SPE) (Karve and
Rajgor, 2007), stir-barsorptive extraction(Kawaguchi
et al., 2006), solid phase microextraction (SPME)
(Lambropoulou et al., 2002; Lemos and Ferreira,
2001) have been proposed for preconcentration of
trace elements. Another promising sample preparation
technique which has attracted considerable attention
in recent years is the dispersive liquid–liquid
microextraction (DLLME).
Dispersive liquid–liquid microextraction is a
miniaturized kind of liquid–liquid extraction (LLE) in
which microliter volumes of extraction solvents is
used. An appropriate mixture of the extraction solvent
and the disperser solvent with high miscibility in both
organic and aqueous phases is rapidly injected into the
aqueous solution of sample and a cloudy solution is
then formed as a result of the formation of fine
droplets of the extraction solvent which disperse in the
sample solution. The cloudy solution is centrifuged
and the fine droplets are settled at the bottom of the
conical test tube. The analytes are extracted from the
initial solution and concentrated to a small volume of
the sedimented phase, and the determination of the
analytes in the settled phase can then be performed by
the conventional analytical techniques. In fact,
markedly increases the contact surface between
phases and reduces the extraction time with a
significantly high extraction efficiency (Berijani et al.,
2006).
In the present work, DLLME method has been
used for the preconcentration of lead, after the
formation of a complex with diphenylthiocarbazone
(dithizone), prior to GFAAS determination. The
analytical conditions for the preconcentration of lead
were investigated.
2. MATERIALS AND METHODS
2.1. Reagents and materials
All the reagents and materials were purchased from
Merck (Darmstadt, HE, Germany). A stock solution of
Pb2+
ions (1000 𝜇gmL−1
) was prepared by dissolving
an appropriate amount of Zn(NO3)2⋅6H2O and
diluted with doubly distilled water. Working standard
solution was obtained daily by stepwise dilution of the
standard stock solution. A stock solution containing
dithizone at 10 mol L-1
was prepared in CCl4.
Fig. 1: The effect of pH on the absorbance of the system, conditions: sample volume 5.0 mL containing 10 ng mL
-1 , dispersive
solvent 0.5 mL ethanol, extraction solvent 100 µL CCl4 containing 0.02 mmol L-1
dithizone, extraction time 10 min
2.2. Instruments
A PG Instruments model PG990 atomic absorption
spectrophotometer (Leicestershire, UK) with a
deuterium background correction was used for the
analysis. A lead hollow cathode lamp wads used as
radiation source at 283.3 nm. All measurements were
based on integrated absorbance mode. The furnace
program for determination of lead(II) is shown in
Table 1.
2.3. Dispersive liquid-liquid microextraction
procedure
Aliquots of 5.0 mL sample solution containing Pb (pH
10.0) were placed in a 10-mL screw cap glass test
tube with conic bottom. The amount of 1.0 mL of
ethanol (disperser solvent) and 150µL of carbon
tetrachloride (extraction solvent) containing dithizone
(chelating agent) was injected rapidly into the sample
International Journal of Scientific Research in Knowledge, 2(3), pp. 116-123, 2014
118
solution by using a microsyringe. A cloudy solution
was formed in the test tube. In this step, Pb reacted
with dithizone and was extracted into the fine droplets
of carbon tetrachloride. Then, the solution was
centrifuged at 3000 rpm for 5 min, and the dispersed
fine droplets of carbon tetrachloride were deposited at
the bottom of conical test tube (40 µL). Thirty
microliters of the sediment phase was injected into the
pyrographite furnace of atomic absorption
spectrophotometer for analysis. Calibration was
performed against aqueous standards submitted to the
same DLLME procedure. The enrichment factor was
calculated as the ratio of the analyte concentration in
the sedimented phase (Csed) and the initial
concentration of the analyte (C0) in the aqueous
sample.
EF=Csed/C0
2.4. Human blood analysis
In this experiment, two blood samples were used to
validate the proposed method. Preparation of blood
samples was done by adding deionized water (1:10) to
the collected blood samples from various individuals
who participated in our research and thus, the
detection of lead concentration was done using
dispersive liquid-liquid microextraction coupled with
graphite furnace atomic absorption spectrometry.
3. RESULTS AND DISCUSSION
3.1. Effect of pH
The pH of the sample solution is an important factor
for DLLME procedure. The pH plays a unique role on
metal–chelate formation and subsequent extraction.
For this propose, the effect of sample pH was
investigated systematically in the range of pH 2–12
with dithizone as the chelating reagent, which ensured
that lead ion could be extracted as a chelate complex.
The results are shown in Fig. 1. pH value of 10 seems
to be optimum for the complete removal of the Pb(II)
ion concentration by DLLME. Thus, the sample pH
was set at pH 10 in the following experiments.
Table 1: Heating program for determination of lead in human blood.
Step Temperature (oC) Ramp (s) Hold(s) Ar flow rate
(mLmin-1)
1
2
3
4
5
100
250
500
1800
2200
5
5
10
0
1
20
20
20
4
2
250
250
250
0
250
Table 2: Regression and Analytical parameters
Regression equation using DLLME
Linear range
Limit of detection
Preconcentration factor
r2
RSD%(n=10)
A=0.003 + 0.0878C
0.10 – 200 ng mL-1
0.04 ng mL-1
123
0.9982
2.87
Table 3: Influence of foreign ions
Ions Tolerance ratio
Ca2+, Ba2+ , K+, Na+,
Zn2+
Ni2+
SO42-, NO3
- , Cl-, NO2-
1000:1
300:1
250:1
1000:1
Fe2+, Cu2+ 200:1
3. 2. Effect of ligand (dithizone) concentration
The influence of the dithizone concentration on the
DLLME extraction of Pb was evaluated in the
concentration range of 0.001 to 0.1 mmol mL-1
. Figure
2 showed that the signal is maximal when the
concentration of dithizone is in the range of 0.025 to
0.1 mmol L-1
. Therefore, a 0.03 mmol L-1
dithizone
solution was selected as optimal.
Mazloomifar
Analysis of Lead(II) in Human Blood Using Dispersive Liquid-Liquid Microextraction and Graphite Furnace Atomic
Absorption Spectrometry
119
Table 4: Determination of lead in human blood No. sample Spiked (ng mL-1) Measured (ng mL-1) RSD% (n=5) Recovery
1
0
5
10
nd*
4.70
10.2
-
4.10
3.25
-
95.3
102
2
0
5
10
nd*
4.86
9.89
-
3.90
2.15
-
97.2
98.9 *not detected
3.3. Effect of type and volume of extraction solvent
The distribution coefficient and selectivity are the
most important parameters that govern extraction
solvent selection. The selectivity means the ability of
the solvent to pick up the desired component in the
feed as compared to other components. It should have
higher density than water and have extraction
capability of the interested compounds and low
solubility in water. In this experiment, three organic
solvents such as dichloromethane, trichloromethane,
and carbon tetrachloride were checked. The
experimental results showed that the maximal signal
was obtained with carbon tetrachloride as the
extraction solvent. So carbon tetrachloride was used
as the extraction solvent in further experiments.
In this experiment, the volume of carbon
tetrachloride was optimized between 50 and 250µL
and the results were exhibited in Figure 3. As can be
seen, the absorption of lead increased along with the
increase of volume of carbon tetrachloride from
50µLto 150µL, and then decreased when the volume
of carbon tetrachloride further increased. The increase
of absorption was due to the increase of volume of
carbon tetrachloride which can dissolve more lead
complex. But when the volume is over 150µL, some
of carbon tetrachloride could not be dispersed into the
aqueous solution as infinitesimal drops, and existed as
larger drops which decreased the contact area between
lead complex and organic drops, that is, it reduced the
transfer of lead complex into the carbon tetrachloride
phase. Therefore, 150 µL carbon tetrachloride was
selected as volume optimum.
Fig. 2: The effect of ligand (dithizone) concentration on the absorbance of the system, conditions: sample volume 5.0 mL
containing 10 ng mL-1
, dispersive solvent 0.5 mL ethanol, extraction solvent 100 µL CCl4 containing dithizone , extraction
time 10 min.
3.4. Effect of the disperser solvent and its volume
In the DLLME method, dispersive solvent should be
miscible with both water and extraction solvent.
Therefore, acetonitrile, acetone, ethanol, and methanol
were tested as disperser solvent. The results indicate
that there was no significant statistical difference (t
test) between different disperser solvents. Ethanol was
selected for the following experiments due to its less
toxicity.
The volume of dispersive solvent was also an
important factor for achieving good extraction
performance. To obtain the optimized volume of
ethanol, various experiments were performed using
different volumes of ethanol (0.25, 0.50, 0.75, 1. 0,
1.25 and 1.5 mL). The results were exhibited in Figure
4. When the volume of ethanol was small, carbon
tetrachloride could not be dispersed completely and
cloudy solution could not form. At high volume, the
solubility of the complex in water increased by the
International Journal of Scientific Research in Knowledge, 2(3), pp. 116-123, 2014
120
increase of the volume of ethanol. Finally, 1. 0 mL
ethanol was chosen as the optimum volume.
3.5. Effect of extraction time
In this experiment, extraction time was another
parameter for DLLME. Extraction time is defined as
interval between the injection of the mixture of
disperser solvent (ethanol) and extraction solvent
(CCl4), and before centrifugation. Effect of extraction
time was studied in the range of 5 to 20 min. The
results are shown in figure 5. According to the
obtained results, the absorbance reaches its maximum
value at 15 min and then remains approximately
constant with further increase in time. Thus, 15 min
time was selected as an optimum time.
Fig. 3: Effect of the extraction solvent volume on the analytical responses, conditions: sample volume 5.0 mL containing 10 ng
mL-1
Pb(II), dispersive solvent 0.5 mL ethanol, extraction solvent, CCl4 containing 0.03 mmol L-1
dithizone , extraction time
10 min
Fig. 4: Effect of the dispersive solvent volume on analytical responses, Conditions: sample volume 5.0 mL containing 10 ng
mL-1
Pb(II), extraction solvent 150 µL CCl4 containing 0.03 mmol L-1
, extraction time 10 min
3.6. Effect of ionic strength
Effect adding salt on extraction efficiency of DLLME
was studied with the NaCl concentration in the range
0.0-3.0 (w/v%). No significant impact on the
analytical signal was observed. Hence, NaCl was not
added in all subsequent extraction experiments.
3.7. Analytical performance
The analytical performance of DLLME coupled with
graphite furnace atomic absorption spectrometry for
the preconcentration and determination of lead from
human samples was systematically investigated under
optimized experimental conditions. The results (Table
2) exhibited that there was an excellent linear range
over 0.10–200 ngmL−1
(r2 =0.9982). The precision of
this method was 2.87% (RSD, n=10) at the spiked
concentration of 10 ngmL−1
. And the detection limit
(calculated as the concentration corresponding to three
times the standard deviation of 10 runs of the blank
samples) of proposed method for lead was 0.04
ngmL−1
.
3.8. Selectivity
Many metal ions existing in real samples would form
stable chelating complexes with dithizone within a
wide pH range and may be co-extracted along with
the analytes. Moreover, this competitive chelating
Mazloomifar
Analysis of Lead(II) in Human Blood Using Dispersive Liquid-Liquid Microextraction and Graphite Furnace Atomic
Absorption Spectrometry
121
effect coupled with co-extracted effect would
influence the chelating degree between lead and
dithizone and make the extraction efficiency of lead
decrease. Therefore, a series of experiments have been
designed using a standard solution of 10 ngmL−1
lead
under the above optimized conditions. The tolerance
limit was defined as the concentration of added
species caused less than ± 5 % relative error. The
results are given in table 3.
Fig. 5: Effect of the extraction time on the analytical responses, conditions: sample volume 5.0 mL containing 10 ng mL
-1
Pb(II) , dispersive solvent 1.0 mL ethanol, extraction solvent 150 µL CCl4 containing 0.03 mmol L-1
dithizone
3.9. Application
In order to demonstrate the applicability and
reliability of the proposed method for real samples,
several blood samples were collected. The recovery
experiments of different amounts of Pb were carried
out, and the results are shown in Table 4. The results
indicate that the recoveries in the range of 95.3–102%
are reasonably well for trace analysis.
4. CONCLUSION
A new method of DLLME combined with GFAAS
has been proposed for the determination of Pb in
human blood samples. The experimental results
demonstrated that proposed method had many merits
such as excellent enrichment performance, simplicity,
sensitivity, easy to operate, cost-effective and low
consumption of organic solvents. The results indicated
that proposed method had high tolerance to coexisting
ions and perfect analytical performance and proved
that proposed method was a good alternative for the
determination of lead in human blood samples.
ACKNOWLEDGMENT
The author thanks the research council at the
University of Islamic Azad, Shahre Ray Branch for
financial support.
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Mazloomifar
Analysis of Lead(II) in Human Blood Using Dispersive Liquid-Liquid Microextraction and Graphite Furnace Atomic
Absorption Spectrometry
123
Dr. Ali Mazloomifar is a assistant Professor in analytical chemistry at Shahre Rey Branch, Islamic
Azad university, Iran. Dr. Mazloomifar received his Ph.D in analytical chemistry (chromatography)
from Islamic Azad university, Iran in 2002. He obtained degree in Master of science in analytical
chemistry in 1998 from Tabriz university. He received his first degree in applied chemistry from Bu-
Ali Sina university, Hemedan, Iran in 1996. Dr. Mazloomifar has published several scientific articles
related to separation field.