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

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: mazloomifar@yahoo.com
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 liquidliquid 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 (Lpez-Garca 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 gml1 for adults and 0.1 gml1 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

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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 liquidliquid microextraction (DLLME). Dispersive liquidliquid microextraction is a miniaturized kind of liquidliquid 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 gmL1) was prepared by dissolving an appropriate amount of Zn(NO3)26H2O 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 L 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 procedure liquid-liquid microextraction

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 150L of carbon tetrachloride (extraction solvent) containing dithizone (chelating agent) was injected rapidly into the sample

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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 L). 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
Step 1 2 3 4 5 Temperature (oC) 100 250 500 1800 2200

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 metalchelate formation and subsequent extraction. For this propose, the effect of sample pH was investigated systematically in the range of pH 212 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.
Ramp (s) 5 5 10 0 1 Hold(s) 20 20 20 4 2 Ar flow rate (mLmin-1) 250 250 250 0 250

Table 1: Heating program for determination of lead in human blood.

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 Ca2+, Ba2+ , K+, Na+, Zn2+ Ni2+ SO42-, NO3- , Cl-, NO2Fe2+, Cu2+ Tolerance ratio 1000:1 300:1 250:1 1000:1 200:1

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.

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Mazloomifar Analysis of Lead(II) in Human Blood Using Dispersive Liquid-Liquid Microextraction and Graphite Furnace Atomic Absorption Spectrometry Table 4: Determination of lead in human blood
No. sample 1 Spiked (ng mL-1) 0 5 10 0 5 10 Measured (ng mL-1) nd* 4.70 10.2 nd* 4.86 9.89 RSD% (n=5) 4.10 3.25 3.90 2.15 Recovery 95.3 102 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 250L 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 50Lto 150L, 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 150L, 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 L 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 L 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

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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, CCl 4 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 L 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

2) exhibited that there was an excellent linear range over 0.10200 ngmL1 (r2 =0.9982). The precision of this method was 2.87% (RSD, n=10) at the spiked concentration of 10 ngmL1. 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 ngmL1. 3.8. Selectivity

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

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

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Mazloomifar Analysis of Lead(II) in Human Blood Using Dispersive Liquid-Liquid Microextraction and Graphite Furnace Atomic Absorption Spectrometry

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 ngmL1 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 L CCl 4 containing 0.03 mmol L-1dithizone

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 95.3102% 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. REFERENCES Baghurst PA, Mcmichael A, Wigg N, Vimpani G, Robertson (1992). Life-long exposure to environmental lead and childrens intelligence

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aminobenzoic acid sunscreen agents in swimming pool and bathing waters by solidphase microextraction. Journal of Chromatography A, 967: 243-253. Lemos VA, Ferreira SLC (2001). On-line preconcentration system for lead determination in seafood samples by flame atomic absorption spectrometry using polyurethane foam loaded with 2-(2-benzothiazolylazo)-2-p-cresol. Analytica Chimica Acta, 441: 281-289. Liang P, Liu R, Cao J (2008). Single drop microextraction combined with graphite furnace atomic absorption spectrometry for determination of lead in biological samples. Microchimica Acta, 160: 135-139. Liang P, Sang H (2008). Determination of trace lead in biological and water samples with dispersive liquidliquid microextraction preconcentration. Analytical Biochemistry, 380: 21-25. Liu Y, Chang X, Guo Y, Meng S (2006). Biosorption and preconcentration of lead and cadmium on waste Chinese herb Pang Da Hai. Journal of Hazardous Materials, 135: 389-394. Lpez-Garca I, Vicente-Martnez Y, HernndezCrdoba M (2013). Determination of lead and cadmium using an ionic liquid and dispersive liquidliquid microextraction followed by electrothermal atomic absorption spectrometry. Talanta, 110: 46-52.

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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 BuAli Sina university, Hemedan, Iran in 1996. Dr. Mazloomifar has published several scientific articles related to separation field.

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