Determination of Ultra Trace Amounts of Silver by Gfaas After Dllme Method In Water, Plasma and Urine

Introduction                                   

In the recent years, pollution of the environment by heavy metals has received considerable attention. These elements accumulate in living organisms and are of high toxic potential ¹. For determination of the trace amount of silver, a separation and a pre- concentration step are usually required before their analysis. This is due to their frequent low concentrations and the matrix effects in the environmental samples ². However, liquid–liquid extraction (LLE), as the oldest pre-concentration and separation technique in analytical chemistry, is time-consuming and requires large amount of organic solvents ³. Thus, single drop microextraction (SDME) was developed as an inexpensive alternative, in which sub-mL amounts of organic solvents are used so that there is minimal exposure to toxic organic solvents and has a good enrichment factor ⁴. In order to break up the organic drop and air bubbled formation as the result of fast stirring, the method needs a long time to reach equilibrium and complete extraction. Graphite furnace atomic absorption spectrometry (GFAAS) is a very attractive option for the determination of trace amounts of silver in water and biological samples. However, the direct determination of trace amounts of silver in real samples with GFAAS is usually difficult due to its low level in samples. It was first reported in 2006 by Assadi and co-workers developed a novel microextraction technique, termed DLLME, which is based on a ternary component solvent system like Liquid-liquid extraction ^5-7. This method uses an extracting solvent dissolved in a dispersive solvent, which is miscible with both extraction solvent and water. Methanol, acetone, acetonitrile, etc, have been used as dispersive solvents ⁸ .In this method, the appropriate mixture of extraction solvent and dispersive solvent is injected into the Fine droplets of extraction solvent. After extraction, phase separation is performed by centrifugation and the enriched analyte, in the sediment phase is determined by spectrometry methods. The advantages of the DLLME method are simplicity of operation, rapidity, low cost, high recover, protect the environment against additional Quantities of solvents, and enrichment factors. This method has been applied for the determination of trace organic pollutants and metal ions in the environmental and in biological samples ⁹.The aim of this work is to combine DLLME with GFAAS and develop a new method for the determination of trace Ag in water and biological samples. In this method, PAN, which forms complexes with metal ions and has found numerous applications in trace element separation and pre–concentration by LLE ¹⁰, SPE ¹¹, CPE ¹² and LPME ¹³ methods, was selected as chelating reagent. The factors influencing the efficiency of DLLME extraction and GFAAS

Experimental

Reagents and standards

Used all chemicals in this work, were of analytical reagent grade. Reagent grade cholorobenzene (C6H5Cl), carbon tetrachloride (CCl4) and chloroform (CHCl3), as extraction solvents and methanol, acetone, and acetonitrile, as disperser solvents, and PAN were purchased form Merck chemical company. Doubly distilled deionized water was used throughout. Silver nitrate and nitrate salts of other cations (all from Merck) were of the highest purity available and used without any further purification except for vacuum drying. The stock solution of AgNo₃ (1000 mg L^-1) were prepared by dissolving appropriate amounts of AgNo₃. The chelating agent, 4×10^-6 mol L^-1 PAN solution, was prepared daily by dissolving the appropriate amount of PAN in methanol.

Instrumentation

Shimadzu model AA6300G atomic absorption spectrometer (Kyoto, Japan) with graphite furnace (GFA-EX7i), a silver hallow cathode lamp as radiation source (Hamamatsu photon photonics, Japan) at the 328.1 nm wavelength with a slit width of 0.5 nm, 4 mA current and deuterium background corrector, was used for measurement. An L΄vov total pyrolitic graphite platform tube (shimadzu) was used. The pyrolitic graphite platform was modified according to the procedure described in the Liang and Cao work ¹⁴. Such pre-treatment was found to be effective for at least 200 atomization to cycles of silver determination under the temperature program given in Table 1. The instrumental parameters and temperature program used for graphite atomizer are listed in Table 1. The sample injection volume was 10 µL in all experiments, and calibration solution was pipetted directly into the graphite furnace 25 s after starting the drying step in all experiments. Argon gas with 99.95℅ purity was purchased from Roham Gas Co. (Isfahan, Iran) was used as protected and purge gas. A Behdad Universal Centrifuge (Isfahan, Iran) was used for centrifugation. 2.0 mL syringe (gastight, Hamilton, Reno, NV, USA). Micropipette (Biohit, Finland).

Table. 1. The graphite furnace temperature program for silver determination

Figure.1: Effect of type of extraction solvent on the enrichment factor of Ag in DLLME Extraction conditions: sample volume, 12.00 mL; concentration of Ag, 1.0 ng mL−1, dispersive solvent (methanol), 1.00 ml, concentration of PAN (4×10-6mol L-1). Click here to View figure

Effect of volume of extraction solvent

In order to investigate the effect of the extraction solvent volume, solutions containing different volume of C₆H₅Cl (40, 45, 50, 55, 60 and 65 µL) were subjected to the same DLLME procedure. By increasing the volume of C₆H₅Cl from 40 to 60 µL, the enrichment factor decreased from 255 to 145, because the volume of the sediment phase increased from 25 to 40 µL. therefore, 40 µL of C₆H₅Cl was used as extraction solvent volume in the subsequent experiments. The results illustrated in Fig. 2.

Figure.2: Effect of volume of chlorobenzene on the on the enrichment factor of Ag in DLLMEExtraction conditions: sample volume, 12.00 mL; concentration of Ag, 1.0 ng mL−1, extraction solvent (C6H5Cl) volume, 40 μL, dispersive solvent (methanol), 1.00 ml, concentration of PAN (4×10-6mol L-1). Click here to View figure

Selection of dispersive solvent

The disperser solvent most is miscible with both extraction solvent and water sample solution phases. In recent work, different solvents such as, methanol, acetone, and acetonitrile were tested. Several sample solutions were studied using 1 mL of each disperser solvent, which contains 40 µl of chlorobezene and 4×10^-6   mol L^-1 PAN. The obtained enrichment factors form methanol, acetone and acetonitrile, were 253, 215 and 158, respectively. It was found the enrichment factors slightly higher by using methanol, in comparison with the other disperser solvents. Therefore, methanol was used as disperser solvent in all of the subsequent experiments. Results were shown in Fig. 3.

 

Figure. 3. Type of Dispersive solvent. Click here to View figure

Effect of dispersive solvent volume

To acquire the optimized methanol volume, various experiments were conducted using different methanol volumes (0.7, 0.8, 0.9, 1.0, 1.1 and 1.2 mL). These different volumes were subjected to the same DLLME procedure. By increasing the methanol volume to 1.0 mL, the enrichment factors increased. Furthermore, the increase of the methanol volume from 0.7 to 1.2 mL did not considerably affect on the enrichment factors, as shown in Fig. 4. Therefore, an optimal volume of 1.0 ml of methanol was chosen to achieve a better and more stable cloudy solution. The obtained results illustrated in Fig .4.

Figure. 4. Effect of Dispersive solvent volume Click here to View figure

Extraction time effect

Extraction time is one of the most important factors in extraction procedures. The dependence of extraction efficiency upon extraction time was studied within a range of 0-8 min in the constant experimental conditions. In DLLME, extraction times defined as the time that the cloudy solution forms until the start of centrifugation. Fig. 5 shows the enrichment factor of silver versus extraction time. The results show that the extraction time has on significant effect on extraction efficiency. It is revealed that after formation of the cloudy solution, the surface area between extraction solvent and aqueous phase (water sample) is infinitely large. Thereby, complex formation of silver and its transition from aqueous phase (water sample) to extraction solvent is fast. Subsequently, equilibrium state is achieved quickly, so the extraction time is very short. This is the advantage of DLLME technique, low extraction time. In this method, the most time-consuming step is the centrifuging of sample solution in extraction procedure, which is about three min ¹⁶.

Figure. 5. Effect of the extraction time on the enrichment factor of silver Obtained from DLLME Click here to View figure

Effect of PAN concentration

The effect of concentration of PAN in the range of 4×10^-3– 4×10^-8 mol L^-1 on analytical signal were investigated. The influence of PAN concentration was carried out in which the other experimental variables remained constant. The results show that by increasing the concentration of PAN the absorbance increases, as shown in Fig. 6. It seems that slight reduction of extraction in high concentration of PAN is due to the extraction of PAN itself, which can easily saturate the small volume of extraction solvent. Also at high concentration of PAN (4×10^-6mol L^-1) the background absorbance increases. Therefore the amount of considerably PAN was selected to prevent background absorbance and diverse ion interferences.

 

Figure. 6. Effect of the amount of PAN on the enrichment factor of silver obtained from DLLME Click here to View figure

Effect of centrifugation time

Effect of centrifugation time in the range of   2- 5 were investigated. The obtained results show the peak areas gradually constant after 2 min the prolongation of centrifugation time. Thus, 2 min was selected as optimum time of centrifugation.

Effect of pH

pH can play an important role in the producing extractable species from ionic analytes prior to extraction by DLLME. In this study 1-(2-pyridylazo)-2-naphtol (PAN) was used as a complexing agent for Ag ions to produce a neutral oxinate chelate, which is extractable into chlorobenzene. Because the production of oxinate chelate is pH dependent, DLLME was performed at different pH values in the range 2-12. The obtained results show that the pH does not influence the sedimented phase volume, as shown in Fig. 7. The production of oxinate chelate and its extraction is PH independent between PH 8 and 12. In this range, the highest EF and recovery are obtained. Therefore, PH 9.5 was selected for the following studies ¹⁷.

Figure 7. Effect of PH on the absorbance of silver obtained from DLLME Click here to View figure

Effect of foreign ions

In order to study the effect of various cations and anions on the determination of Ag, a fixed amount of silver (1 ng mL^-1), was taken with different amounts of those foreign ions, which are reported to be usually present in different silver real samples ^18-20 and the recommended procedure was followed. A relative error of 5% was considered tolerable. The results are summarized in Table. 2. The results showed that none of the ions examined interfere in extraction and determination of silver, even at an interfering ion to silver ratio of 1000 and higher.

Table. 2. Effect of foreign ions on the pre-concentration and determination of silver (1 ng mL⁻¹)

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