Cloud Point Extraction of Trace Amounts of Copper and Its Determination by Flow Injection Flame Atomic Absorption Spectrometry

A simple and convenient method based on cloud point extraction was proposed for the determination of copper. The extraction was carried out in the presence of 2-[(2-mercaptophenylimino)methyl]phenol (MPMP) as the chelating ligand and octylphenoxypolyethoxyethanol (Triton X-114) as the non-ionic surfactant. After phase separation, the surfactant rich phase containing the complex was diluted with 1.0 mol L solution of HNO3 in methanol and the analyte concentration was determined by flow injection flame atomic absorption spectrometry. Under the optimum conditions, an enhancement factor of 81 was obtained for the preconcentration of copper with 25 mL sample solution. The calibration graph was linear in the range of 1–150 μg L, the relative standard deviation (RSD) for ten replicate determinations at 80 μg L Cu level was 1.8 %, and the limit of detection (3 s) and quantification (10 s) were 0.15 and 0.50 μg L respectively. The accuracy of method was confirmed by analysis of certified reference materials and recovery experiments. The proposed method was successfully applied to the determination of copper in rice flour and water samples. (doi: 10.5562/cca1803)


INTRODUCTION
2][3] Therefore, the accurate and sensitive procedures for its determination at trace and ultratrace levels are important.
Flame atomic absorption spectrometry (FAAS) is one of the techniques most extensively used for the determination of metal ions.This analytical technique presents some desirable characteristics such as good selectivity, low cost, operational facilities and high analytical frequency.However, FAAS lacks the sensitivity necessary for direct determination of analyte at low levels. 4,5Thus, a preconcentration and separation step is usually needed before measuring.Various separation techniques including solid phase extraction, 6,7 coprecipitation, 8 solvent extraction, 9 dispersive liquidliquid extraction, 10 and cloud point extraction [11][12][13][14][15][16] have been proposed for separation and preconcentration of copper in order to improve the inadequate sensitivity of FAAS and reduce the matrix effect.Among these meth-ods, recently, cloud point extraction has attracted considerable attention mainly because it is in consistence with the "green chemistry" principles. 179][20] This method is based on the property that an aqueous solution of surfactants forms micelles and becomes turbid above a temperature defined as cloud point temperature.Above the cloud point temperature, the original surfactant solution separates into a small volume of surfactant rich phase and a bulk of diluted aqueous phase, in which the concentration of surfactant is close to the critical micellar concentration (CMC).Any analyte solubilized in the hydrophobic core of the micelles will be concentrated into the small volume of the surfactant rich phase which can subsequently be determined by different spectrometric techniques such as flame atomic absorption spectrometry (FAAS), 21 electrothermal atomic absorption spectrometry (ETAAS), 22 inductively coupled plasma mass spectrometry (ICP-MS), 23 inductively coupled plasma optical emission spectrometry (ICP-OES), 24 laser induced-thermal lens spectrometry (LI-TLS), 25 and spectrophotometry. 26-[(2-mercaptophenylimino)methyl]phenol (MPMP) (Figure 1) is a Schiff base and forms stable complexes with some transition metal ions.MPMP has been used as a hydrophobic chelating agent for analytical purposes due to its high stability and its low solubility in aqueous solution.[27][28][29] In this work a novel and convenient cloud point extraction method for the separation and preconcentration of trace copper in aqueous samples prior to its determination by FI-FAAS is described.The method is based on the formation of Cu(II) complex with 2-[(2-mercaptophenylimino)methyl]phenol (MPMP) (Figure 1) followed by its extraction into surfactant-rich phase Triton X-114.

EXPERIMENTAL Apparatus
A Buck Scientific atomic absorption spectrometer (model 210 VCP, East Norwalk, CT, USA), equipped with a copper hollow cathode lamp (current, i = 3.2 mA and slit width, s = 0.7 nm) was used for the determination of copper at wavelength λ = 324.7 nm.A single line flow injection system as described before 30 i.e. is consisted of a peristaltic pump (Ismatic, MS-REGLO/8-100, Zurich, Switzerland) and a rotary injection valve (Rheodyne, Rohnert Park, CA, USA) was used for effective control of the amount of sample and repeatability of the measurements.The pH of solutions was controlled with a Metrohm pH meter (model 691, Herisau, Switzerland) using a combined glass calomel electrode.A thermostatic bath (Shimi Fann, model S.57, Tehran, Iran) and a centrifuge (Hittich, Universal 320, Tuttingen, Germany) were used to accelerate the phase separation process.

Reagents
All chemicals were of analytical grade reagents.Doubly distilled, deionized water was used throughout.The stock solution of copper (1000 mg L -1 ) was prepared by dissolving an appropriate amount of Cu(NO 3 ) 2 •3H 2 O (E.Merck, Darmstadt, Germany) in water and diluting to the mark in a 100 mL volumetric flask.Working standard solutions were prepared daily from the stock solution by appropriate dilution with water.Triton X-114 (volume concentration, φ = 2.5 %) stock solution was prepared by dissolving 2.5 mL of the concentrated solution (Fluka, Chemie AG, Buches, Switzerland) in hot distilled water and diluting to 100 mL.A 0.1 mol L -1 stock buffer solution (pH = 4.5) was prepared by using sodium acetate and hydrochloric acid (E.Merck, Darmstadt, Germany) at appropriate concentrations.2-[(2mercaptophenylimino)methyl]phenol was synthesized and purified according to the literature. 31,32The solution of 1×10 -2 mol L -1 MPMP was prepared by dissolving a proper amount of the reagent in ethanol.

General Procedure
An aliquot of 25 mL of a solution containing Cu(II) (1-150 μg L -1 ), 0.8 mL of solution of Triton X-114 (volume concentration, φ = 2.5 %), 0.25 mL of 1 × 10 -2 mol L -1 of MPMP,and 2 mL of acetate buffer (pH = 4.5) was kept for 5 min in the thermostatic bath at 55 °C.Separation of the phases was achieved by centrifugation for 10 min at 3500 rpm.The mixture was cooled in an ice bath for 5 min which increased the viscosity of the surfactant-rich phase and the aqueous phase was easily decanted by inverting the tube.In order to decrease the viscosity and facilitate sample handling, the surfactant rich phase was diluted to 300 μL with 1.0 mol L -1 solution of HNO 3 in methanol.Finally, 100 µL of the resulting solution was introduced into the FAAS by a single line flow injection system at a flow rate of 2.5 mL min -1 (Figure 2).

Water Preparation
To remove suspended particulate matter, water samples were filtered through a 0.45 µm pore size membrane filter into cleaned polyethylene bottles and were stored in 5 °C before analysis.25 mL of it was treated according to the given procedure.

Rice Flour Preparation
One gram of rice flour was placed in a 100 mL beaker, 7 mL concentrated nitric acid and 4 mL of hydrogen peroxide (ρ = 30 %) was added and the mixture was heated on a hot plate for 10 min.Then the solution was cooled to room temperature, filtered and after adjustment of pH to ~4.5, the mixture was diluted to the final volume of 100 mL with distilled water and was treated according to the given procedure.The recovery experiments was done by spiking 1 g of rice flour with 2 and 5 µg of copper ions, digested and diluted as described above and was analyzed according to the given procedure.

Preparation of Certified Lead Sample
Five milliliter of concentrated nitric acid was added to 0.25 g of a certified lead sample (BCR No. 288).The solution was heated over a water bath for few minutes and 3 mL of hydrogen peroxide (ρ = 30 %) was added.The solution was filtered, diluted with distilled water and the pH was adjusted to ~4.5 with 0.1 mol L -1 ammonia solution.The solution was then transferred into a 100 mL volumetric flask and diluted to the mark with distilled water.25 mL of it was treated according to the given procedure.

RESULTS AND DISCUSSION
Separation of metal ions by CPE often involves the formation of a hydrophobic complex to be extracted into the surfactant-rich phase.Preliminary experiments confirmed that MPMP forms a stable complex with copper ions, which is extractable into surfactant-rich phase of Triton X-114.MPMP has one oxygen, one sulfur and one nitrogen donating group in its structure and acts as three dentated ligand.The ligand forms a stable 1 :1 complex with copper(II). 32In order to establish the best conditions for the formation and extraction of complex, the effective parameters such as pH, concentrations of ligand and surfactant, temperature, and incubation time were optimized by univariable method.
The pH of sample solution is one of the influencing factors in the cloud point extraction of metal ions, since the extraction yield depends on the pH at which complex formation occurs.The influence of pH on the extraction of Cu 2+ was evaluated in the pH range of 1-11. Figure 3 shows that the absorbance is nearly constant in the pH range of 4-8.The possibility of extraction in a wide pH range can be mentioned as an advantage of this method.The decrease in absorbance at pH greater than 8 is probably due to the precipitation of copper as copper hydroxide, whereas the signal decrease at pH < 4 may be due to the competition between hydronium and analyte for reaction with MPMP.Hence, a pH of 4.5 was chosen for further extractions.
The variation of absorbance as a function of the MPMP concentration was investigated for copper complex formation during the cloud point extraction procedure.Concentration of MPMP was varied in the range of 0.1-1.7 × 10 -4 mol L -1 .Figure 4 shows the absorbance increases by increasing ligand concentration up to a concentration of 9 × 10 -5 mol L -1 of ligand and thereafter reaches a plateau.Thus, a concentration of 1×10 -4 mol L -1 of MPMP was chosen for further experiments.
Triton X-114 was selected for cloud point extraction of copper complex because of its commercial availability, low cloud point temperature and high density. 33,34Another factor which has unique role in CPE is surfactant concentration as it determines the narrow range in which maximum extraction efficiency and analytical signal are achieved.The effect of Triton X-114 volume concentration, φ on copper extraction was investigated in the range of 0.01-0.15%.As can be seen from Figure 5, the absorbance increased by increasing Triton X-114 volume concentration, φ up to 0.08 % and then leveled off at higher concentration.The  progressive decrease in the absorbance at lower concentrations of Triton X-114 is due to inadequacy of the surfactant assemblies to entrap the hydrophobic complex quantitatively.Triton X-114 volume concentration, φ of 0.08 % was chosen as the optimum concentration.
Equilibrium temperature and the incubation time were also optimized.The effect of equilibrium temperature was investigated in the range of t = 25-85 °C.As Figure 6 shows the absorbance of the analyte reach maximum in 50-70 °C.The decrease in absorbance at temperature higher than 70 °C is probably due to the decomposition of the complex which reduces the extraction efficiency.So, an equilibrium temperature of 55 °C was selected as optimum temperature for further studies.The dependence of analytical signal upon incubation time was also investigated in the range of 1-10 min.It was observed that an incubation time of 5 min is adequate for quantitative extraction.
The influence of ionic strength on the analytical signal of copper (80 µg L -1 ) was studied.The results showed that addition of NaCl in the interval of 0.1-0.7 mol L -1 has no significant effect on the sensitivity and cloud point extraction efficiency.Furthermore, the effect of centrifugation time on the extraction efficiency was investigated in the range of 1-20 min at 3500 rpm.It was found that a centrifugation time of 10 min was enough for complete phase separation.

Interferences
The effect of selected cations and anions on the extraction and determination of 20 μg L -1 copper by the proposed method was investigated.A relative error of less than  5 % was considered to be within the range of experimental error.The results are shown in Table 1.It can be seen that the presence of other cations and anions at the given mole ratio has no significant influence on CPE of Cu under the optimum conditions.

Analytical Figures of Merit
Calibration graph for the sample volume of 25 mL gave good linearity over the concentration range of 1-150 µg L -1 for Cu.Table 2 summarizes the analytical character-istic of the proposed method.The relative standard deviation (RSD) for 10 replicates measurements of 80 µg L -1 of copper was  1.8 %.The limit of detection and quantification defined as the concentration equivalent to three times and ten times of the standard deviation of the blank divided by the slope of the calibration graph were 0.15 μg L -1 and 0.50 μg L -1 , respectively.The enhancement factor calculated as the ratio of the slopes of the calibration graphs with (2.532 × 10 -3 ) and without (3.126× 10 -5 ) preconcentration, was 81.

Application
The proposed method has been applied to the determination of Cu in water samples and rice flour.The reliability of method was checked by recovery experiments.As can be seen from Table 3, in all samples, the copper recovery is almost quantitative (95.6-99.9%), which demonstrates the applicability of the proposed method for the sample type examined.

CONCLUSION
The results of this work demonstrate the possibility of using MPMP as an effective chelating agent for the cloud point extraction and separation of copper prior to its determination by FI-FAAS.The method can be successfully applied to the determination of copper in rice flour and water samples.The method proved to be convenient, rapid, and sensitive for determination of copper in real samples with limited interferences.Furthermore, the proposed method was compared with other reported methods using CPE and FAAS (Table 4).As it is demonstrated, the proposed method presents superior or comparable analytical figures of merit to reported methods.

Figure 2 .
Figure 2. Flow injection system coupled to flame atomic absorption spectrometry for the injection and determination of copper.
in % (n = 10) 1.8(a)  C expressed in μg L -1 (a)  Mean and standard deviation related to three determinations.

Table 2 .
Figures of merit for

Table 3 .
Determination of copper in rice flour and water samples

Table 4 .
Comparison of analytical characteristics of the present method and some of those reported previously for the CPE of Cu and its determination by FAAS