Analysis of Lead(II) in Human Blood Using Dispersive ... · amount of chelating agent, ... to the...

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

mailto:[email protected]

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


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



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


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



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



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.

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