Pre-Concentration Based on Cloud Point Extraction for Ultra-Trace Monitoring of Lead (II) Using Flame Atomic Absorption Spectrometry

: The cloud point extraction (CPE) method was successfully used for the isolation and pre-concentration of ultra-low concentration of Pb prior to its determination by ﬂame atomic absorption spectrometry (FAAS). Lead(II) reacts with methyl 4,20-diisobutyl-2,5,8,16,19,22- hexaoxo-7,17-dipropyl-3,6,9,15,18,21-hexaaza-1(2,6)-pyridinacyclo-docosaphane-10-carboxylate (DLNL) as chelating agent in the presence of octylphenoxypolyethoxyethanol (Triton X-114) as a nonionic surfactant giving a surfactant-rich phase chelate which could be used for CPE. Factors a ﬀ ecting the CPE such as solution pH, concentrations of the chelating ligand and surfactant, temperature of equilibration, and time were optimized. The e ﬃ cacy features of the proposed protocol such as linear range, lower limit of detection, pre-concentration, and progress factors were evaluated. The method revealed a wide linear range in the range of 7–250 ng / mL of Pb 2 + with a limit of detection of 5 ng / mL using FAAS. Validation of the presented protocol revealed good performance characteristics including high between-batch repeatability, high precision, wide linear range, low limit of detection, and acceptable accuracy. The presented procedure was successfully introduced for the separation and quantiﬁcation of lead (II) in wastewater samples with acceptable results.


Introduction
Heavy metal ion monitoring in different matrices is a significant task because of the harmful effects that such ions have on organisms [1][2][3]. Lead (Pb) is considered as one of the highly hazardous species that affect both the ecological environment and human health in a dangerous way. It can cause cardiovascular effects, renal failure, neurologic damage, and venereal toxicity [4]. Exposure to severe Pb intoxication causes acute failure to the renal and nervous systems, liver, and brain, in addition to sterility, abortion, and neonatal death [5]. The environmental and health problems arise fundamentally from the use of gasoline anti-knock products and paint pigments [6]. As an outcome, the WHO (World Health Organization) has affirmed the ultimate acceptable limit of Pb in drinking water to be 10 ng/mL [7]. For such causes, Pb assessment at low concentration becomes an urgent and

Instrumentation
A GBC (Savant AA, Braeside VIC, Australia) flame atomic absorption spectrometer was used for all AAS measurements. For background correction deuterium-arc was used for non-specific absorption. A hollow cathode lamp for Pb was operated at 7 mA for the determination of lead with 0.7 nm spectral bandwidth. The operating wavelength was 283.3 nm. Aliquots of 20 µL for all samples and calibration solutions were aspirated directly into the flame. Argon 99.996% was used as a protective and purge gas. All pH measurements were made with a Cole palmer pH/mV meter (model 59003-05). A GFL thermostatic bath (Model D-30938) was fixed at the required temperature and used for all subsequent cloud point temperature experiments. Phase separation acceleration was carried out using a Hettich centrifuge (Model EBA 21).

Procedure for CPE
For the pre-concentration procedure, aliquots of 50 mL of either the standard (containing 40 µ g/L Pb 2+ ) or sample solution (six replicate samples of 10 mL of digested acid) were transferred into centrifuge tubes with a glass stopper (25 mL in capacity); the pH of each sample solution was adjusted to pH 6. To this solution, 2.0 mL of (0.08%-0.8% v/v) Triton X-114 solution and 1.0 mL of ligand (DLNL) (50-100 µ M) were added; the pH range (2-10) was adjusted by the addition of 0.1 M HCl/NaOH solution in acetate buffer. The mixture was then introduced to the thermostated water bath and heated at 30-60 °C for 5-20 min. After different time intervals, centrifugation for 10 min at 4000 rpm was done and separation of the two phases was obtained. The contents of tubes were cooled in an ice bath, the surfactant-rich phase became viscous, and the upper aqueous phase was carefully removed by a pipette. To decrease the viscosity of extracts (i.e., the remaining micellar phase (500 µ L)), 0.5 mL acidic ethyl alcohol (0.1 M HNO3) was added to lower its viscosity and make the sample handling easier. The extracted solutions were introduced into the flame by conventional aspiration. The duplicate blanks of both complexing reagents were prepared simultaneously without addition of samples and standards. The recovery is calculated as: where CO represents the lead content in the sample-surfactant mixture of volume VO, CW represents the lead content in the aqueous phase of volume VW, and CS represents the lead content in the surfactant phase of volume VS.

Results and Discussion
Extraction based on CPE for different analytes has many features such as a high enrichment factor, easy conjunction to automated instruments, simplicity of use, and rapidity. In the presented work, methyl 4,20-diisobutyl-2,5,8,16,19,22-hexaoxo-7,17-dipropyl-3,6,9,15,18,21-hexaaza-1(2,6)-pyridinacyclodocosaphane-10-carboxylate (DLNL) is presented as a chelating agent for Pb 2+ ions forming a stable 1:1 complex. The complex was extracted into surfactant-rich phase containing Triton X-114. To verify the optimum conditions of the complex formation and its extraction of the complex, the performance features such as pH, concentrations of ligand and surfactant, temperature, and time of incubation were evaluated.

Procedure for CPE
For the pre-concentration procedure, aliquots of 50 mL of either the standard (containing 40 µg/L Pb 2+ ) or sample solution (six replicate samples of 10 mL of digested acid) were transferred into centrifuge tubes with a glass stopper (25 mL in capacity); the pH of each sample solution was adjusted to pH 6. To this solution, 2.0 mL of (0.08%-0.8% v/v) Triton X-114 solution and 1.0 mL of ligand (DLNL) (50-100 µM) were added; the pH range (2-10) was adjusted by the addition of 0.1 M HCl/NaOH solution in acetate buffer. The mixture was then introduced to the thermostated water bath and heated at 30-60 • C for 5-20 min. After different time intervals, centrifugation for 10 min at 4000 rpm was done and separation of the two phases was obtained. The contents of tubes were cooled in an ice bath, the surfactant-rich phase became viscous, and the upper aqueous phase was carefully removed by a pipette. To decrease the viscosity of extracts (i.e., the remaining micellar phase (500 µL)), 0.5 mL acidic ethyl alcohol (0.1 M HNO 3 ) was added to lower its viscosity and make the sample handling easier. The extracted solutions were introduced into the flame by conventional aspiration. The duplicate blanks of both complexing reagents were prepared simultaneously without addition of samples and standards. The recovery is calculated as: where C O represents the lead content in the sample-surfactant mixture of volume V O , C W represents the lead content in the aqueous phase of volume V W , and C S represents the lead content in the surfactant phase of volume V S .

Results and Discussion
Extraction based on CPE for different analytes has many features such as a high enrichment factor, easy conjunction to automated instruments, simplicity of use, and rapidity. In the presented work, methyl 4,20-diisobutyl-2,5,8,16,19,22-hexaoxo-7,17-dipropyl-3,6,9,15,18,21-hexaaza-1(2,6)pyridinacyclodoc-osaphane-10-carboxylate (DLNL) is presented as a chelating agent for Pb 2+ ions forming a stable 1:1 complex. The complex was extracted into surfactant-rich phase containing Triton X-114. To verify the optimum conditions of the complex formation and its extraction of the complex, the performance features such as pH, concentrations of ligand and surfactant, temperature, and time of incubation were evaluated.

Effect of pH
Metal-chelate formation and its stability are two critical factors for CPE. pH plays an important role in the formation of metal-chelate complex and occasionally in its extraction [29]. Extraction percentage relies on the pH at which complex formation is carried out. The pH effect was studied within a pH range from 2.0 to 10.0 and the results were presented in Figure 2. At pH >6, the recovery of Pb 2+ ions decreases due to the formation of hydroxide precipitates of lead or plumbate ions. At lower pH, the extraction of Pb decreases as the acidity of the solution increases. This may be attributed to the competition between H 3 O + with Pb 2+ for the reaction with DLNL. Since the maximum recovery was obtained at pH 6, this pH value was selected for all subsequent measurements for lead, resulting in the relative standard deviation (RSD) of 1.9%.
Appl. Sci. 2019, 9, x FOR PEER REVIEW 4 of 10 Metal-chelate formation and its stability are two critical factors for CPE. pH plays an important role in the formation of metal-chelate complex and occasionally in its extraction [29]. Extraction percentage relies on the pH at which complex formation is carried out. The pH effect was studied within a pH range from 2.0 to 10.0 and the results were presented in Figure 2. At pH >6, the recovery of Pb 2+ ions decreases due to the formation of hydroxide precipitates of lead or plumbate ions. At lower pH, the extraction of Pb decreases as the acidity of the solution increases. This may be attributed to the competition between H3O + with Pb 2+ for the reaction with DLNL. Since the maximum recovery was obtained at pH 6, this pH value was selected for all subsequent measurements for lead, resulting in the relative standard deviation (RSD) of 1.9%.

Ligand Concentration Effect
One of the key factors affecting the efficiency of extraction is the ligand-chelate concentration. The effect of amount of the chelating ligand DLNL on the recovery of lead ion was examined. Different concentrations of the chelating reagent in the range of 1.0 × 10 −4 to 5.0 × 10 −5 M were used. The results showed that the maximum recovery of lead ions was obtained at 2.0 × 10 −5 M DLNL concentration, as shown in Figure 3. At higher DLNL concentrations, the recovery percentage decreases. This could be explained by the excessive chelating agent being co-extracted into the phase rich with the surfactant, thus decreasing the efficiency of extraction of lead ions.

Ligand Concentration Effect
One of the key factors affecting the efficiency of extraction is the ligand-chelate concentration. The effect of amount of the chelating ligand DLNL on the recovery of lead ion was examined. Different concentrations of the chelating reagent in the range of 1.0 × 10 −4 to 5.0 × 10 −5 M were used. The results showed that the maximum recovery of lead ions was obtained at 2.0 × 10 −5 M DLNL concentration, as shown in Figure 3. At higher DLNL concentrations, the recovery percentage decreases. This could be explained by the excessive chelating agent being co-extracted into the phase rich with the surfactant, thus decreasing the efficiency of extraction of lead ions. Metal-chelate formation and its stability are two critical factors for CPE. pH plays an important role in the formation of metal-chelate complex and occasionally in its extraction [29]. Extraction percentage relies on the pH at which complex formation is carried out. The pH effect was studied within a pH range from 2.0 to 10.0 and the results were presented in Figure 2. At pH >6, the recovery of Pb 2+ ions decreases due to the formation of hydroxide precipitates of lead or plumbate ions. At lower pH, the extraction of Pb decreases as the acidity of the solution increases. This may be attributed to the competition between H3O + with Pb 2+ for the reaction with DLNL. Since the maximum recovery was obtained at pH 6, this pH value was selected for all subsequent measurements for lead, resulting in the relative standard deviation (RSD) of 1.9%.

Ligand Concentration Effect
One of the key factors affecting the efficiency of extraction is the ligand-chelate concentration. The effect of amount of the chelating ligand DLNL on the recovery of lead ion was examined. Different concentrations of the chelating reagent in the range of 1.0 × 10 −4 to 5.0 × 10 −5 M were used. The results showed that the maximum recovery of lead ions was obtained at 2.0 × 10 −5 M DLNL concentration, as shown in Figure 3. At higher DLNL concentrations, the recovery percentage decreases. This could be explained by the excessive chelating agent being co-extracted into the phase rich with the surfactant, thus decreasing the efficiency of extraction of lead ions.

Effect of Triton X-114 Concentration
Triton X-114 is responsible for decreasing the cloud point temperature (CPT) below the room temperature. So, the amount of Triton X-114 not only affects the extraction efficiency but also the volume of the surfactant-rich phase. The phase volume ratio (V org /A queous ) should be minimized to get enhanced extraction efficiency and successful CPE [30]. In this section, the concentration of TX-114 was investigated within the range of 0.08%-0.8% (w/v). The efficiency of extraction towards Pb 2+ increases as Triton X-114 concentration increases from 0.08% to 0.4% (v/v). Above 0.4% concentration, the recovery percentage begins to decline because of the increase in the total Pb 2+ volume and the viscosity of the surfactant phase. So, a concentration of 0.4% (v/v) was selected as an optimal concentration for Triton X-114 to get the highest recovery percentage, as shown in Figure 4.

Effect of Triton X-114 Concentration
Triton X-114 is responsible for decreasing the cloud point temperature (CPT) below the room temperature. So, the amount of Triton X-114 not only affects the extraction efficiency but also the volume of the surfactant-rich phase. The phase volume ratio (Vorg/Aqueous) should be minimized to get enhanced extraction efficiency and successful CPE [30]. In this section, the concentration of TX-114 was investigated within the range of 0.08%-0.8% (w/v). The efficiency of extraction towards Pb 2+ increases as Triton X-114 concentration increases from 0.08% to 0.4% (v/v). Above 0.4% concentration, the recovery percentage begins to decline because of the increase in the total Pb 2+ volume and the viscosity of the surfactant phase. So, a concentration of 0.4% (v/v) was selected as an optimal concentration for Triton X-114 to get the highest recovery percentage, as shown in Figure 4.

Incubation Time and Equilibrium Temperature Effects
Time of incubation and temperature at which equilibrium is attained were also optimized. The temperature was investigated within the range 20-80 °C. The obtained results clarified that the recovery of Pb 2+ ions reaches its optimized value in the range of 50-70 °C. Above 70 °C, the extraction efficiency decreases. This is probably due to instability of the complex formed between Pb 2+ ions and DLNL. This could decrease the efficiency of extraction. So, 70 °C was chosen as an optimized temperature for all subsequent measurements. Investigation of the incubation time within the range 5-30 min was carried out, in which the analytical signal was completely based on the incubation time. Incubation for 10 min was selected as an adequate time for all subsequent measurements.

Effect of Diverse Ions
Flame atomic absorption spectrometry (FAAS) provided high selectivity towards analyte determination. Interferences from other cations may be attributed to the pre-concentration step, in which these metal ions can form stable complexes when they react with the ligand. This could lead to a decrease in extraction efficiency. To study the effect of interfering ions, different amounts of foreign ions were added to the standard solution of 10 µ g/L of Pb 2+ and the recommended procedure was followed. The recoveries of Pb 2+ ions in these studies were higher than 95%. The tolerable limit was defined as the largest amount of foreign ions that produced an error not exceeding ±5% in the determination of Pb 2+ . The results were presented in Table 1. From the results obtained, we can conclude that the existence of high amounts of interfering ions that can be commonly present in water samples have no remarkable effect on the CPE of Pb 2+ ions.

Incubation Time and Equilibrium Temperature Effects
Time of incubation and temperature at which equilibrium is attained were also optimized. The temperature was investigated within the range 20-80 • C. The obtained results clarified that the recovery of Pb 2+ ions reaches its optimized value in the range of 50-70 • C. Above 70 • C, the extraction efficiency decreases. This is probably due to instability of the complex formed between Pb 2+ ions and DLNL. This could decrease the efficiency of extraction. So, 70 • C was chosen as an optimized temperature for all subsequent measurements. Investigation of the incubation time within the range 5-30 min was carried out, in which the analytical signal was completely based on the incubation time. Incubation for 10 min was selected as an adequate time for all subsequent measurements.

Effect of Diverse Ions
Flame atomic absorption spectrometry (FAAS) provided high selectivity towards analyte determination. Interferences from other cations may be attributed to the pre-concentration step, in which these metal ions can form stable complexes when they react with the ligand. This could lead to a decrease in extraction efficiency. To study the effect of interfering ions, different amounts of foreign ions were added to the standard solution of 10 µg/L of Pb 2+ and the recommended procedure was followed. The recoveries of Pb 2+ ions in these studies were higher than 95%. The tolerable limit was defined as the largest amount of foreign ions that produced an error not exceeding ±5% in the determination of Pb 2+ . The results were presented in Table 1. From the results obtained, we can conclude that the existence of high amounts of interfering ions that can be commonly present in water samples have no remarkable effect on the CPE of Pb 2+ ions.

Figure of Merit and Results
As shown in Table 2, the analytical features of the proposed method revealed linearity over the range of 7-250 ng/mL with a detection limit of 4 ng/mL. The relative standard deviation (RSD) obtained was 1.9% for seven samples of 10 ng/mL Pb(II). A comparison between the proposed method and others reported in literature based on the cloud point extraction of lead are summarized in Table 3 [31][32][33][34][35][36][37][38][39][40]. The table presents a good enrichment factor, high sensitivity, and good linearity range of this presented method. The calibration graph for the pre-concentration of Pb 2+ with DLNL was linear with a correlation coefficient of 0.9981 at the range of 10-250 ng/mL. The obtained regression equation was Abs = 6.3123(Pb 2+ ng/mL) + 0.0523. In order to determine the enhancement factor (EF), calibration curves were prepared without CPE. The calibration equation obtained was Abs = 0.1262(Pb 2+ ng/mL) + 0.0101 (R 2 = 0.9995). The experimental enhancement factor calculated as the ratio of slopes of the calibration graphs with and without pre-concentration was 50. The detection limit (LOD) and quantification limit (LOQ) were calculated as under 3 and 10 s/m, respectively, where s is the standard deviation (n = 10) of the blank and m is slope of the calibration graph. The LOD and LOQ were calculated as 4.0 and 7.0 ng/mL, respectively. The analytical characteristics, i.e., precision of methods, were expressed as the % relative standard deviation (%RSD) of a minimum six independent analyses of standard Pb 2+ samples, after CPE of Pb 2+ was found to be 1.9%.

Analysis of Natural Samples
The presented protocol has been introduced to real river water samples to evaluate the feasibility of the method. The water samples were collected from different zones in the river Nile, filtrated, and then transferred to a 50 mL centrifuge tube to do the CPE procedure. Meanwhile, the spiked river water was also determined. The results in Table 4 display that the recoveries of metal ions were in the range of 95.6%-101.0%.