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

Abstract

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 affecting the CPE such as solution pH, concentrations of the chelating ligand and surfactant, temperature of equilibration, and time were optimized. The efficacy 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 Pb²⁺ 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.

1. 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 important concern. Different pre-concentration methods have been presented, such as liquid–liquid microextraction (LLE) [8], coprecipitation [9], ion exchange [10], cloud point extraction( CPE), and solid phase extraction (SPE) [11,12]. CPE is considered now as one of the most efficient separation techniques used for the excellent pre-concentration of very low amounts of different analytes [13,14,15,16,17,18,19]. CPE offers many excellent features such as rapidity, simplicity and environmental compatibility. The process in CPE is phase separation of an aqueous solution containing non-ionic surfactant in addition to the metal–chelate complex. This solution is heated to a certain temperature (i.e., cloud point temperature) after which the solution becomes turbid. The solution produces two phases: one phase which has only the surfactant (surfactant-rich phase), and the other phase which contains a very little amount of the surfactant with critical micellar concentration (CMC) [13]. Several advantages are presented by using CPE, such as high pre-concentration factors, lower cost, fast operation, higher safety and simplicity, no need for large amounts of toxic organic solvent, and ease of coupling to analytical instruments [20,21,22]. The main applications of CPE are the extraction of metal–chelates using a surfactant with posterior quantification using atomic spectrometry techniques.

The most widely used technique is flame atomic absorption spectrometry (FAAS) [23,24,25,26,27]. FAAS offers different features, such as a short time of analysis and high accuracy, but has many limitations in terms of sensitivity. Coupling CPE to FAAS could improve the limit of detection and allow accurate results to be obtained in samples down to ng/mL or even lower levels.

Herein, a new pre-concentration method based on CPE is presented and optimized for the pre-concentration and quantification of Pb²⁺ ions. Pb²⁺ 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) giving a chelate followed by extraction in octylphenoxypolyethoxyethanol (Triton X-114) as a non-ionic surfactant. After optimizing the essential parameters that affect the CPE process, the presented protocol is introduced for the pre-concentration of Pb before its analysis by FAAS.

2. Materials and Methods

2.1. 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).

2.2. Materials and Reagents

Chemicals used in this work were of analytical reagent grade. All working solutions and standards were prepared using deionized water from Milli-Q system (Millipore, Bedford, MA, USA) with a resistivity of 18.2 MΩ cm. Nitric acid (supra-pure nitric acid 65%, Merck) was used without further purification. A 1000 µg/mL Pb²⁺ solution was prepared by dissolving an appropriate amount of pure Pb(NO₃)₂ (Merck, Darmstadt, Germany) in deionized water. Octylphenoxypolyethoxy- ethanol (Triton X-114) was obtained from Merck chemicals (Merck, Darmstadt, Germany).

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) was prepared and characterized as previously described [28]. A 1.0 × 10⁻³ M solution of DLNL chelating agent was prepared by dissolving it in ethanol and placing in 100 mL measuring flask. The solution was then completed to the mark with de-ionized water (Figure 1).

2.3. Procedure for CPE

For the pre-concentration procedure, aliquots of 50 mL of either the standard (containing 40 µg/L Pb²⁺) 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 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:

$$\mathrm{Recovery}(\%)=\frac{{\mathrm{C}}_{\mathrm{S}}{\mathrm{V}}_{\mathrm{S}}}{{\mathrm{C}}_{\mathrm{O}}{\mathrm{V}}_{\mathrm{O}}}\times 100=\frac{{\mathrm{C}}_{\mathrm{O}}{\mathrm{V}}_{\mathrm{O}}-{\mathrm{C}}_{\mathrm{W}}{\mathrm{V}}_{\mathrm{W}}}{{\mathrm{C}}_{\mathrm{O}}{\mathrm{V}}_{\mathrm{O}}}\times 100,$$

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.

3. 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 Pb²⁺ 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.

3.1. 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 Pb²⁺ 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 Pb²⁺ 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%.

3.2. 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.

3.3. 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 Pb²⁺ 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 Pb²⁺ 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.

3.4. 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 Pb²⁺ 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 Pb²⁺ 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.

3.5. 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 Pb²⁺ and the recommended procedure was followed. The recoveries of Pb²⁺ 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 Pb²⁺. The results were presented in

Table 1. Tolerance limit of foreign ions.

Foreign Ions	Interferent/Pb(II) Ratio
Na+, K+, NH4+	5000
Co2+, Ni2+	3000
Ca2+, Mg2+	4000
Mn2+	2000
Cd2+	2000
Zn2+	1000
Fe2+	800
Cu2+	500
Hg2+	700

. 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 Pb²⁺ ions.

3.6. Figure of Merit and Results

As shown in

Table 2. Analytical characteristics of CPE method using flame atomic absorption spectrometry (FAAS) technique.

Parameter	Analytical Feature
Enrichment factor	50
Limit of detection (ng/mL)	4
Regression equation	Y = 0.0198 C + 0.0360
Correlation coefficient (r2)	0.9981
Linear range (ng/mL)	7–250
%RSD	1.9

, 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. Comparison of the proposed method with other CPE methods for extraction and determination of Pb(II).

Enrichment Method	System	Detection Method	Linear Range, (ng/mL)	RSD, %	Detection Limit, (ng/mL)	Enrichment Factor	Ref.
CPE	APDC DDTC	FAAS	5–20	6.88 8.74	1.14	56 42	[11]
CPE	TAN	FAAS	1.1–160	3.5	1.1	55.6	[23]
CPE	PAN	FAAS	7.5–3500	1.6	5.27	30	[31]
RS-CPE	APDC DDTC	FAAS	Up to 40	4.9	4.3	39	[32]
CPE	1-PTSC	FAAS	0.5–10.0	1.7–4.8	4.8	25	[33]
CPE	TAN	FI-FAAS	50–250	1.6–3.2	4.5	15	[34]
CPE	BCB	FAAS	−	<6.4	7.5	25	[35]
CPE	5-Br-PADAP	GFAAS	0.1–30	2.8	0.08	50	[36]
CPE	Tween 80	FAAS	2–12	≤6	7.2	10	[37]
CPE	PONPE 7.5	Capillary zone electrophoresis	12–400	3.6	11.4	−	[38]
CPE	PAN	FAAS	20–300	2.7	8.0	50	[39]
CPE	DDTP	FAAS	−	−	40 ng/g	18	[40]
CPE	DLNL	FAAS	7–250	1.9	4.0	50	Present work

BCB, brilliant cresyl blue; 1-PTSC, 1-phenylthiosemicarbazide; PAN, 1-(2-pyridylazo)-2-naphthol; DDTP, O,O-diethyldithiophosphate; DDTC, diethyldithiocarbamate; APDC, ammonium pyrrolidinedithiocarbamate; RS-CPE, rapidly synergistic cloud point extraction; TAN, 1-(2-thiazolylazo)-2-naphthol; 5-Br-PADAP, 2-(5-bromo-2-pyridylazo)-5-(diethyl-amino)-phenol; PONPE, 7.5-polyethyleneglycolmono-p-nonylphenylether;DLNL,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.

[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 Pb²⁺ 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(Pb²⁺ 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(Pb²⁺ ng/mL) + 0.0101 (R² = 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 Pb²⁺ samples, after CPE of Pb²⁺ was found to be 1.9%.

3.7. 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. Determination of lead ion (ng mL⁻¹) in river water samples (n = 5).

Sample	Amount of Pb(II), (ng/mL) *		Recovery, %
	Added	Found
River water	−	25.6 ± 0.3	−
	10	35.9 ± 1.1	101.30
	20	45.2 ± 2.2	99.10

* Average of six measurements

display that the recoveries of metal ions were in the range of 95.6%–101.0%.

4. Conclusions

The cloud point extraction behavior of Pb²⁺ using 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 a chelating agent in presence of Triton X-114 was optimized. The extraction recovery was affected by pH, the concentration of the chelating agent, and the surfactant (Triton X-114) concentration. The results obtained showed the utility and validity of the presented method. This method revealed remarkable features such as ease of operation, fast speed, and cost-effectiveness for Pb²⁺ separation and pre-concentration in water samples compared with other common pre-concentration methods. Comparison between the presented method and others reported in literature based on the cloud point extraction of lead are summarized in Table 3. It is interesting to note that a comparison of the proposed method along with those reported before (Table 3) clearly indicated good enhancement in the behavior of the proposed method in terms of the detection limit [31,32,33,34,35,37,38,39,40], linear range [32,33,34,35,36,37,38,40], enrichment factor [31,32,33,34,35,37,40], and the relative standard deviation (RSD) [23,32,33,34,35,36,37,38,39,40].