Research Article. i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 1 Introduction

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1 Clean 2007, 35 (6), S. Baytak et al. 607 Sıtkı Baytak 1 Abdurrahim Koçyiǧit 2 Ali Rehber Türker 3 1 Harran University, Arts and Science Faculty, Department of Chemistry, Sanliurfa, Turkey. 2 Harran University, Faculty of Medicine, Department of Biochemistry, Sanliurfa, Turkey. 3 Gazi University, Arts and Science Faculty, Department of Chemistry, Ankara, Turkey. Research Article Determination of Lead, Iron and Nickel in Water and Vegetable Samples after Preconcentration with Aspergillus niger Loaded on Silica Gel A method for the determination of Fe(III), Pb(II), and Ni(II) by flame atomic absorption spectrometry (FAAS) after preconcentrating on a column containing Aspergillus niger loaded on silica gel 60 (Biosorbent) is described. The effect of experimental parameters such as ph, flow rate of sample solution, and volume of sample solution were investigated on the recovery of the analytes. The effect of interfering ions on the recovery of the analytes has also been investigated. Recoveries of Fe(III), Pb(II), and Ni(II) were (98 l 2), (98 l 3), (99 l 2)% at the 95% confidence level, respectively. For the analytes, 50-fold preconcentration was obtained. The analytical detection limits for Fe(III), Pb(II), and Ni(II) were 1.7, 5.2, and 1.6 ng/ml, respectively. The proposed method was applied to the determination of trace metals in various water and vegetable samples. The analytes have been determined with relative error lower than 7%. Keywords: Aspergillus niger; Biosorption; Atomic absorption spectrometry; Trace metal; Preconcentration; Water analysis; Received: August 29, 2007; revised: October 17, 2007; accepted: October 17, 2007 DOI: /clen Introduction The importance of the determination of trace metal concentration in natural water and/or in environmental samples is increasing in contamination monitoring studies. Trace metal ions have important roles in a wide spectrum of life functions. As a result, the determination of trace metal ions is becoming increasingly important because of the increased interest in environmental sampling of water, soil, plants, etc. [1]. Some of the trace metals are essential micronutrients for organisms and plants. However, they are toxic at higher levels. Chronic Fe, Pb and Ni poisoning affects the central nervous system. In natural water and biological samples their level is low and previous steps of separation and preconcentration are usually required. For this purpose, many separation/preconcentration procedures for trace metal ions such as liquid-liquid extraction [2], cloud-point extraction [3, 4], liquid membrane [5] and solidphase extraction [6 9] have been widely used. Recently, separation and/or preconcentration of trace metals by adsorption on microorganisms loaded on a support material have been widely used [10 12]. The use of microorganisms as biosorbents for metals has become a good alternative to the other preconcentration methods because of higher recovery, economical advantages, simplicity and environmental protection. In general, microorganisms have the ability to selectively adsorb a specific element without Correspondence: Prof. A. R. Türker, Gazi University, Art and Science Faculty, Chemistry Department, Ankara, Turkey. aturker@gazi.edu.tr preconcentrating the matrix [13]. By changing ph or elution conditions, selectivity can be obtained and this is well demonstrated in the interference effects where the metals are in relatively high concentration. There are many binding sites on the cell wall of microorganisms. Immobilized cell systems can be used in both batch and column experiments. Maquiera et al. [14] have used Saccharomyces cerevisiae immobilizing covalently on controlled pore glass (CPG) for trace metal preconcentration. Godlewska-Zylkiewicz et al. [15] have studied on-line separation of Pt(IV) on columns containing the green algae Chlorella vulgaris immobilized on Cellex-T support. Bag et al. [16] have immobilized Aspergillus niger on sepiolite for use in preconcentration of Fe(II) and Fe(III) in water samples by using a column technique. Likewise, Baytak et al. [17, 18] have used A. niger and Saccharomyces carlsbergensis immobilized on silica gel 60 and Amberlite XAD-4, respectively for the preconcentration of Cr(III), Cu(II), Zn(II) Cd(II) and Mn(II) ions in various samples. In the present work, Fe(III), Pb(II) and Ni(II) have been determined in various water and vegetable samples by FAAS after preconcentration by A. niger loaded on silica gel 60 resin. A. niger is a fungus and one of the most common species of the genus Aspergillus. It grows rapidly on a variety of artificial substrates producing colonies that consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heads. Conidiophores of this species are typically 900 to 1600 lm long, smooth-walled and terminate in pale-brown colored globose vesicles 40 to 60 lm in diameter. It causes a disease called black mold on certain fruits and vegetables, such as grapes, onions, and peanuts, and is a common contaminant of food. Silica gel is a well-known resin and has found widespread application for the separation and preconcentration of trace metal ions, alone or in its functionalized forms.

2 608 S. Baytak et al. Clean 2007, 35 (6), Materials and Methods 2.1 Apparatus A Varian GTA-97 SpectrAA Plus model atomic absorption spectrometer equipped with deuterium lamp background correction was used for the determination of analytes. The instrumental parameters were as follows: wavelengths 248.3, 217.0, and nm, bandpass 1.0, 1.0, and 0.2 nm, lamp current 11.2, 5.0, and 4.0 ma, fuel flow rate 0.9, 2.0, and 2.0 L/min, for Fe, Pb and Ni, respectively. All ph measurements were performed with a CRISON 20 model digital ph meter. 2.2 Reagents Doubly distilled deionized water and analytical reagent grade chemicals were used unless otherwise specified. Iron, lead and nickel stock solutions (1000 mg/l) were prepared by dissolving the appropriate amounts of iron powder, Pb(NO 3) 2 (Merck), and Ni(NO 3) 2 N 6H 2O (Merck), respectively. Working solutions were prepared by dilution of the stock solutions immediately prior to their use. Silica gel 60 (Merck mesh) were used as a substrate for the immobilization of A. niger. 2.3 Preparation of Biosorbent A. niger was grown, harvested and prepared according to the procedure given by Bag et al. [15]. In order to prepare the substrate for loading A. niger, commercially available silica gel 60 was washed successively with methanol, water, 1 M HCl and water respectively, to remove organic and inorganic contaminants. Then the loading of A. niger on the substrate was performed as follows: 200 mg of dry A. niger powder was mixed with 2 g of silica gel 60. The mixture was wetted with 2 ml of water and thoroughly mixed. After mixing, the paste was heated in an oven at 1058C for 1 h to dry the mixture. The wetting and drying steps were repeated to maximize the contact between A. niger and silica gel 60, and, therefore, to improve the immobilization efficiency. 2.4 General Preconcentration Procedure 0.2 g of the biosorbent was packed in a glass column (8 mm i.d and 150 mm length). Then, the column was conditioned to the studied ph by passing an aqueous solution having the same ph through the column. An aliquot of a sample solution (100 ml) containing 50 lg Fe(III), 30 lg Pb(II) and 20 lg Ni(II) was taken and the ph was adjusted to the optimum value found experimentally with hydrochloric acid or ammonia. The resulting solution was passed through the column at a flow rate adjusted to the optimum value. Then, the retained metal ions were eluted from the column with 10 ml of 1 mol/l HCl solution. The eluent was aspirated into an air-acetylene flame for the determination of the analytes by AAS. The biosorbent was used repeatedly after washing with 1 mol/l HCl and distilled water. The recovery of analytes was calculated from the ratio of the concentration found by FAAS to that calculated theoretically. 2.5 Dissolution of Tea Leaves (GBW-07605), Green Onion and Carrot Samples Green onion and carrot samples were collected from a garden in Sanliurfa. First, the samples were cleaned with tap water and then double distilled water. Then, the samples were dried at 1108C for 24 h. Approximately 0.5 g of standard reference tea leaves (GBW-07605) and dried vegetable samples were placed separately in 250 ml PTFE beakers. The samples were dissolved according to the method described in the previous study [6]. For dissolution, a minimal volume of 0.05 mol/l nitric acid was added to moisten the sample thoroughly, followed by 10 ml of concentrated nitric acid. The beaker was heated on a hot plate at about 130 l 108C for 3 h. After cooling to room temperature, 3 ml of hydrogen peroxide was added drop wise. The beaker was heated until complete decomposition of the sample. The resulting solution was transferred into 100 ml volumetric flask by washing the interior surface of the beaker with small portions of 0.05 mol/l nitric acid, and the solution was diluted to the mark with 0.05 mol/l nitric acid. 2.6 Preparation of Water Samples Tap, Balikligöl and waste water samples were collected in the city of Sanliurfa in Turkey. The Balikligöl (natural pool of sacred fish) is a natural aquarium in Sanliurfa. The water samples were filtered first through ordinary filter paper in order to separate coarse particles and suspended matter. Then, they were immediately filtered through a Millipore cellulose nitrate membrane (pore size 0.45 lm), acidified to ph 2 with HNO 3 and stored in previously cleaned polyethylene bottles. 3 Results and Discussion 3.1 Effect of ph on Recovery The ph value plays an important role in the adsorption of ions on microorganisms. Therefore, the retention of metal ions on the column containing the biosorbent was studied as a function of ph. For this purpose, the ph values of synthetic sample solutions prepared as given in Section 2.4 were adjusted to a range of 2 to 10 with HCl or NH 3 solutions while keeping the other parameters constant and the general preconcentration procedure was applied. As shown in Fig. 1, the optimum ph of the sample solution was in the range of 6 to 8 for Fe(III) and Pb(II) and about 8 for Ni(II). At these ph values, the recovery of the analytes is above 95%. The decrease in the recoveries of the analytes at the lower ph values could be due to the competition between protons and the analytes for the adsorption sites of the microorganisms. The decrease in the recoveries at higher ph values may attributed to the formation of anionic hydroxide complexes and to the competition between the ligand of cell wall and the ammonia. It can be seen that all the metal ions studied were recovered poorly at ph a 6orpHA 8. Therefore, a ph of 8 was selected in further experiments. 3.2 Effect of the Amount of Biosorbent The effect of the amount of biosorbent was investigated in the range of 100 to 500 mg. It was found that above 100 mg of biosorbent, the recovery of analytes gradually increased, but reached a plateau at

3 Clean 2007, 35 (6), Biosorption 609 Table 1. The effect of the type and volume of elution solutions on the recovery of analytes. Element Elution solution Volume, ml Recovery,% Figure 1. The effect of ph on the recovery of Fe, Pb and Ni (Fe, 0.5 lg/ml, Pb, 0.3 lg/ml; Ni, 0.2 lg/ml; adsorbent, 0.2 g; sample volume, 100 ml; flow rate, 1 ml/min; elution solution, 10 ml 1 M HCl). about 200 mg of biosorbent. Therefore, 200 mg of biosorbent was found to be optimum and used for further experiments. 3.3 Effect of the Type and Volume of Elution Solutions The type and concentration of eluent is also important for the analytical performance of a solid phase preconcentration system. Because the adsorption of analytes are negligible at ph a 2, various volumes of 1 mol/l HCl and HNO 3 were tested for desorption of retained analytes from the sorbent. As a result of the experiments, 10 ml of 1 mol/l HCl solution was found to be satisfactory for all of the analytes (R A 95%) (see Tab. 1). 3.4 Effect of Flow Rates of Sample Solutions The retention of an element on a sorbent also depends upon the flow rate of the sample solution. Therefore, it was examined under optimum conditions (ph, eluent type, etc.) by applying the general procedure. The solution was passed through the column with the flow rates adjusted in a range of 1 to 6 ml/min. The optimum flow rates was found to be 3 ml/min for Fe(III) and Pb(II), and 4 ml/min for Ni(II) ions. Therefore, a flow rate of 3 ml/min was selected in further experiments for all of the analytes. The flow rate of elution solution was 1 ml/min. 3.5 Effect of the Volume of Sample Solutions In order to determine the maximum applicable volume of sample solution (or minimum applicable concentration of the analytes), the effect of the volume of sample solution passed through the column on the recovery of the analytes was investigated. For this purpose, 50, 100, 250, 500, 750, and 1000 ml of sample solutions containing fixed amount of analytes, corresponding to 1.0, 0.50, 0.2, 0.1, 0.066, and 0.05 lg/ml of Fe(III), 0.60, 0.30, 0.12, 0.06, 0.04, and 0.03 lg/ml of Pb(II) and 0.40, 0.20, 0.08, 0.04, 0.027, and 0.02 lg/ml of Ni(II) respectively, were passed through the column under the optimum conditions determined experimentally. It was found that the analytes in up to 500 ml of sample solution could be recovered quantitatively. At higher sample volumes, the recoveries decreased gradually with increasing volume of sample solution. Because the Fe HCl HNO Pb HCl HNO Ni HCl HNO Mean of three determinations. elution volume was 10 ml, a preconcentration factor of 50 was obtained for all of the analytes studied. It can be concluded that 0.1 lg/ml Fe(III), 0.06 lg/ml Pb(II) and 0.04 lg/ml Ni(II) could be determined by this method for 500 ml of sample volume. However, these concentrations cannot be determined directly by FAAS with sufficient accuracy. 3.6 Analytical Parameters For an estimation of the precision of the method, successive retention and elution cycles were performed under the optimum conditions and the relative standard deviations of the recoveries were calculated. The recoveries are 98 l 2, 98 l 3, and 99 l 2% for Fe, Pb, and Ni, respectively as the mean of five determinations at the 95% confidence level with a relative standard deviation of 2%. The calibration graphs were linear up to 5 lg/ml for Ni and Pb, and 10 lg/ml for Fe. The instrumental detection limits based on mean of blank values plus three times the standard deviation of the blank values were found to be 86, 260 and 80 lg/l for Fe(III), Pb(II), and Ni(II) (N = 20), respectively. The analytical detection limits for Fe(III), Pb(II), and Ni(II), calculated by dividing the instrumental detection limit by the preconcentration factor (50), were 1.7, 5.2 and 1.6 lg/l. 3.7 Effect of Interfering Ions Interferences from alkali metal ions [Na(I) and K(I)] and alkaline earth elements [Ca(II) and Mg(II)], which are generally found in high concentrations in water samples, on the recovery of analytes were examined. The interference effect of the analytes on each other was also studied. In order to investigate the effect of the interfering ions, alkaline and alkaline earth elements in a range of 0.5 to 1000 lg/ml were added to the synthetic sample containing fixed amounts of the analytes (0.5 lg/ml Fe(III), 0.3 lg/ml Pb(II), and 0.2 lg/ml Ni(II)). As can be seen from Tab. 2, Na and K do not interfere with the determination up to 500 lg/ml. On the other hand, Ca and Mg interfere with the determination at concentrations above 40 and 5 lg/ml, respectively. The last value corresponds to about 5 8 times the analyte concentrations. Therefore, it can be concluded that the analytes cannot be determined accurately in water samples containing more than 5 lg/ml magnesium.

4 610 S. Baytak et al. Clean 2007, 35 (6), Table 2. Effect of other ions on the recovery of analytes. Concentrations of Fe(III), Pb(II) and Ni(II) are 0.5, 0.3, and 0.2 lg/ml, respectively, and the sample volume is 100 ml. Interfering ions Concentration, lg/ml 3.8 Reusability of Sorbent The stability and potential reusability of the sorbent was assessed by monitoring the change in the recoveries of analytes through several adsorption-elution cycles. The procedure was carried out five times in one day and five more runs were performed one day later. The sorbents were always stored in water. The sorbent seems to be relatively stable up to 18 runs for Fe and Pb, 20 runs for Ni. 3.9 Validation of the Method Recovery a,% Fe Pb Ni Na K Ca Mg Fe Pb Ni Mean of three determinations. Table 3. Determination of analytes in Certified Reference Material (GBW ). Element Certified, lg/g Found b), lg/g Relative error, % Fe Pb Ni The composition of the tea leaves powder (GBW-07605) was Fe 264, Ni 4.6, Cu 17.3, Pb 4.4, Zn 26.3, Cd 0.057, Cr 0.80, Co 0.18, Sb 0.056, and Bi lg/g. p b) Mean of five determinations at 95% confidence level (x l ts= ffiffiffi N ). In order to evaluate the accuracy of the proposed method, a certified reference material (Tea leaves GBW-07605) was analyzed. The results were compared with the certified values using the t-test at the 95% confidence level (see Tab. 3). The results were in good agreement with the certified values. The difference between the certified Table 4. Determination of analytes in green onion and carrot samples. Sample Element Added, lg/g Foundp, lg/g x l ts= ffiffiffi N Green onion Fe 122 l l 5 5 Pb 19 l l 3 6 Ni l 3 3 Carrot Fe 96 l l 4 5 Pb 13 l l 3 6 Ni 12 l l 3 6 Mean of five determinations at 95% confidence level. values and measured values are less than the uncertainty, resulting in random errors at the 95% confidence level. The results also indicate that the developed preconcentration method for the analytes is not affected by potential interferences from the major matrix elements of the analyzed plant and water samples Application Relative error, % Table 5. Determination of analytes in water samples (sample volume 250 ml for Fe(III), 500 ml for Pb(II) and Ni(II)). Sample Element Added (lg/l) Found (lg/l) Relative error (%) Tap water Fe 74 l l 5 3 Pb 18 l l 3 6 Ni 8 l l 3 7 Balikligöl Fe 140 l 6 water l 7 3 Pb 22 l l 3 6 Ni 12 l l 3 6 Waste water Fe 216 l l 28 6 Pb 46 l l 8 4 Ni 32 l l 5 7 p Mean of five determinations at 95% confidence level (x l ts= ffiffiffi N ). The proposed method was applied to the determination of the analytes in water and vegetable samples mentioned in Section 2.4. Water samples of 250 ml for Fe(III) and 500 ml for the Pb(II) and Ni(II) and the prepared solutions of the vegetables were adjusted to the optimum ph of 8 and subjected to the recommended column procedure for the preconcentration and determination of the analytes. The accuracy of the determination was also checked by analyzing the spiked samples. The results were shown in Tabs. 4 and 5. By using A. niger loaded on silica gel 60, the analytes were determined in the samples with an acceptable accuracy (relative error a7%) and precision for trace determination.

5 Clean 2007, 35 (6), Biosorption 611 Table 6. Comparative data on the biosorption of analytes on microorganisms immobilized on a support material. Elements Sample Sorbent Detection technique PF LOD (lg/l) Ref. Cu(II), Pb(II), Zn(II), Water, dust and A. fumigatus immobilized on Diaion FAAS [7] Fe(III), Ni(II), Co(II) black tea HP-2MG, column technique Cu(II), Pb(II), Fe(III), Tea, mushroom Bacillus sphaericus immobilized on FAAS [11] Co(II) wheat, rice, soil Diaion SP-850, column technique Fe(II), Fe(III) Water A. niger immobilized on sepiolite, FAAS (for Fe(II)) [16] column technique Fe(III), Co(II), Mn(II), Alloy and water A. tumefacients immobilized on FAAS [19] Cr(III) Amberlite XAD-4, column technique Pb(II), Cd(II) Tap and river Seeds of Sterculia lychnophera Hance FAAS [20] waters Fe(III), Ni(II), Pb(II) Waters and vegetables A. Niger immobilized on silica gel, column technique FAAS This work FAAS: Flame atomic absorption spectrometry, PF: Preconcentration factor, LOD: Limit of detection. 4 Conclusions A simple, rapid, precise and accurate method was developed for the separation and preconcentration of Fe(III), Pb(II), and Ni(II) by the use of A. niger loaded on silica gel 60. The analytes were quantitatively recovered from the column packed with the biosorbent with high precision. The results obtained are comparable to those of other studies on biosorption in the literature (see Tab. 6). This method is very economical, only 0.2 g of sorbent is needed and repeated use is possible. The preconcentration technique used in this work is not only convenient, but it also is simple and the duration of the preconcentration step is about 45 min, including regeneration of sorbent for 100 ml of sample solution. 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