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Multivariate optimization for the determination of cadmium and lead in crude palm oil by graphite furnace atomic absorption spectrometry after extraction induced by emulsion breaking
Microchemical Journal xxx (xxxx) xxxx
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Multivariate optimization for the determination of cadmium and lead in crude palm oil by graphite furnace atomic absorption spectrometry after extraction induced by emulsion breaking Gisseli S. Valasquesa, Ana Maria P dos Santosa, Valdinei S. de Souzaa,b, Leonardo S.G. Teixeiraa, ⁎ Juscelia P.S. Alvesc, Mirela de Jesus Santosc, Wagna P.C dos Santosd, Marcos A. Bezerrac, a
Instituto de Química, Universidade Federal da Bahia, Rua Barão de Jeremoabo s/n, Ondina, Salvador 40170-115, Bahia, Brazil Instituto Federal de Educação, Ciência e Tecnologia Baiano, Rodovia BR 116, Santa Inês 45320-000, Bahia, Brazil Departamento de Ciências e Tecnologias, Universidade Estadual do Sudoeste da Bahia, Rua José Moreira Sobrinho s/n, Jequiezinho, Jequié 45206-190, Bahia, Brazil d Instituto Federal de Educação Ciências e Tecnologias, Rua Emídio dos Santos, Barbalho, Salvador 40301-015, Bahia, Brazil b c
ARTICLE INFO
ABSTRACT
Keywords: Crude palm oil Extraction induced by emulsion breaking GF AAS Cadmium Lead Multivariate optimization
The present work reports the development of an analytical method for the determination of trace amounts of Cd and Pb in crude palm oil using graphite furnace atomic absorption spectrometry (GF AAS) after extraction induced by emulsion breaking (EIEB). The method was based on an oil-in-water emulsion preparation to promote a high contact area between the sample drops and the acid extractant solution. Emulsification was assisted by ultrasound energy using HNO3 and Triton X-114 surfactant solutions to increase the efficiency of the extraction. Afterwards, the emulsion was broken by heating, and the acid aqueous phase in the bottom of conical glass tubes was collected for the determination of metals by GF AAS. Cadmium and lead extraction was simultaneously optimized using Doehlert design and desirability function. The best extractions were achieved using a sonication time of 18 min, a HNO3 concentration of 2.0 mol L−1 and a surfactant concentration of 4.4% (v v-1) at a breaking temperature of 90 °C. The developed method presented quantification limits of 0.17 and 0.13 μg kg−1 and precisions (repeatability,%RSD, 10.8 μg kg−1) of 1.8 and 5.4% for Cd and Pb, respectively. Addition/recovery tests gave results between 88.5% and 112%. The amounts of Cd and Pb in the analyzed samples ranged from 1.5–2.5 and 2.53–6.76 μg kg−1, respectively.
1. Introduction Edible oils are foods composed mostly of lipids that are liquids at room temperature and may have animal or vegetable origin. Palm oil currently accounts for approximately 32% of the world's oil production and is used as a raw material in the production of various food products, cosmetics, and biofuels, among others [1–3]. The quality of these oils is affected by the presence of metals, even in trace amounts, which have a strong influence on flavor degradation and oxidative stability [4]. In addition, the determination of toxic metals, such as Cd and Pb, in food is a current demand in society due to growing concern about quality and food safety. Cadmium and Pb are toxic and accumulative metals in the body without known biological functions. These metals impair cell metabolism and have deleterious effects on human health [5–8]. The diffusion of these metals into the environment and,
⁎
consequently, into food originates from anthropogenic activities such as industrial emissions, the application of mineral fertilizers in soils and vehicular emissions due to the burning of fossil fuels [9]. Cadmium accumulates mainly in the liver and kidneys, increasing the risks of cancer, cardiovascular diseases and osteoporosis [10,11]. Efforts have been made to reduce the use of Cd as raw material in the production of pigments, plastics and batteries. Lead accumulates in the skeleton, especially in the bone marrow. Lead is neurotoxic, causes changes in behavior and slows mental and intellectual development. This metal also interferes with calcium and vitamin D metabolism, affects the formation of hemoglobin and causes anemia [4,12,13]. The determination of trace metals, such as Cd and Pb, in samples of edible oils by atomic spectrometry techniques has been a difficult task due to the low concentrations of these analytes and to the complexity of the matrix, which is very viscous and has a high content of organic substances that are difficult to attack and decompose [5,14].
Corresponding author. E-mail address: [email protected] (M. A. Bezerra).
https://doi.org/10.1016/j.microc.2019.104401 Received 6 June 2019; Received in revised form 31 October 2019; Accepted 6 November 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Gisseli S. Valasques, et al., Microchemical Journal, https://doi.org/10.1016/j.microc.2019.104401
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Procedures involving the emulsification of samples for direct introduction into analytical equipment are quite attractive because such procedures allow simpler and faster analysis without a loss of reliability in the generated results [15]. Surfactants are widely used to increase the stability of emulsions. Emulsification procedures offer some advantages in the treatment of edible oil samples, such as simplification of sample treatment; reduction in the load of organic substances and strong inorganic acids introduced into the atomizer; improvements in accuracy and precision; sample stabilization, allowing storage for long periods; and enabling the use of inorganic standards for calibration [16,17]. However, sample dilution can be observed in the emulsification procedure, affecting the sensitivity of the method. For further simplify the matrix introduced into the atomizer for elemental determination, the extraction induced by emulsion breaking (EIEB) procedure has recently been proposed [18]. In this procedure, the emulsion formed by an oily sample is broken by heating or centrifugation. The quantitative transfer of the analytes from a complex oil phase to an aqueous phase, in addition to bringing all the advantages of the emulsification processes, practically decreases the introduction of organic substances into the atomizer, facilitating the pyrolysis step [19]. Furthermore, due to the separation process, preconcentration of the analytes can be observed. Using graphite furnace atomic absorption spectrometry (GF AAS) as an analytical technique, EIEB has been used for the determination of Cr and Mn in edible oils [20], Cu, Fe, Ni and Pb in samples of diesel [16] and Cu, Mn and Ni in biodiesel [21]. In this work, constrained mixture design was applied to optimize a procedure based on emulsification assisted by ultrasound and EIEB for the posterior determination of Cd and Pb in crude palm oil samples by GF AAS.
model 550 heating bath (São Paulo, Brazil) with a capillary thermostat ( ± 2 °C) with a temperature control range of 50 to 120 °C was used to break the emulsions. Total sample digestion was performed in Teflon® decomposition pumps inserted in a steel casing (Parr Instrument Company, Moline, USA). A laboratory stove (Quimis, Model Q317 M12, Diadema, São Paulo, Brazil) formed from Teflon® bombs and a steel casing was used to heat the system. 2.2. Solutions, reagents and samples All reagents used in this work were of analytical purity. Ultrapure water was obtained using a Purelab purification system, model Classic (Elga, High Wycombe, UK). All glassware was immersed in 5% (v v−1) HNO3 solution for decontamination for 12 h, then washed with deionized water and dried in a dust-free environment. Cadmium and Pb solutions were prepared from appropriate dilutions of commercial standards (Merck, Kenilworth, NJ, USA) at concentrations of 1000 mg L−1 in 1% (v v−1) HNO3 solution or from the organic standard WM-21-5X, hydrocarbon oil, at concentrations of 50 μg g−1 (AccuStandard, New Haven, USA). Tungsten, palladium nitrate and magnesium nitrate solutions (1000 mg L−1) (Merck, Kenilworth, NJ, USA) were used as chemical modifiers to stabilize the analytes in the pyrolysis step, allowing a higher temperature to be used. The proportion of Pd/Mg adopted was 5:3 (v v−1). Graphite tubes with integrated platforms were coated with 250 μg of tungsten as a permanent modifier according to a temperature program adapted from the studies of Lima et al. [22] and Freschi et al. [23]. In this procedure, five injections of 50 μL of 1000 mg L−1 solution of the W modifier were introduced onto the surface of the platform with the aid of the autosampler. Triton X-114 (Vetec, Rio de Janeiro, RJ, Brazil) surfactant solutions from 2 to 10% (v v−1) were prepared by dilution of the surfactant in water and solubilization with the aid of the ultrasonic bath. All of the crude palm oil samples analyzed were purchased from markets in the cities of Jequié and Salvador, Bahia, Brazil. The samples were transported to the laboratory and stored at room temperature and were only opened at the moment of analysis.
2. Experimental 2.1. Instrumentation Atomic absorption measurements were carried out using an AAnalyst 400 graphite furnace atomic absorption spectrometer (Perkin Elmer, Norwalk, CT, USA) equipped with a background corrector with deuterium arc lamp, HGA 900 (with an integrated platform and covered with pyrolytic graphite) as an atomizer, hollow cathode lamps and an AS 800 autosampler (Perkin Elmer, Norwalk, CT, USA). All measurements were performed at the highest-sensitivity wavelength indicated by the equipment for each element: 228.80 nm (Cd) and 283.31 nm (Pb). High-purity argon gas (99.99%, Itaox, Itabuna, Brazil) was used for the purging and protection of the graphite furnace. The heating programs for the determination of Cd and Pb are shown in Table 1. In all experiments, the autosampler always transferred 20 µL of the sample or the standard solution into the graphite tube and 5.0 µL of modifier solution when the latter was used. A Cristofoli ultrasound bath (Campo Mourão, Brazil) of 42 kHz and 170 W was used to promote the formation of the emulsions. A Fisatom
2.3. Construction of pyrolysis and atomization curves The thermal behavior of Cd and Pb in the aqueous phase obtained after EIEB was evaluated from the pyrolysis and atomization curves. These curves were made under the following conditions: (i) without the use of modifier, (ii) with the use of the Pd/Mg modifier and (iii) with the permanent W modifier impregnated inside the tube (for Cd only). For the construction of the curves, extracts obtained from emulsion breaking of a crude palm oil sample and with metals added to a final concentration of 5.0 μg L−1 were used. For the construction of the pyrolysis curves, for Cd and Pb, a fixed atomization temperature was adopted (1600 °C). For the construction of the atomization curves, the optimum pyrolysis temperature found for each analyte was adopted. All measurements were made using the integrated absorbance as the analytical signal.
Table 1 Graphite furnace heating program for the determination of Cd and Pb by GF AAS in the aqueous phase obtained after extraction induced by emulsion breaking. Steps
Temperature (°C)
Ramp (°C s−1)
Hold (s)
Ar flow rate (mL min−1)
Dry 1 Dry 2 Pyrolysis
100 140 700 (Cd) 900 (Pb) 1500 (Cd) 1900 (Pb) 2600 (Cd) 2600 (Pb)
5 15 10 10 PTAa PTAa 1 1
20 15 20 20 5 5 5 5
250 250 250 250 0 0 250 250
Atomization Clean a
2.4. Optimization of the extraction induced by emulsion breaking procedure The optimization of EIEB for the simultaneous determination of Cd and Pb in crude palm oil samples was performed by multivariate techniques (Doehlert design and desirability function). According to the literature [17], the most influential variables in the extraction process are the concentration of Triton X-114 solution (2 to 8% v v−1, studied at five levels), the concentration of HNO3 solution (0.5 to 2.5 mol L−1, studied at five levels) and the sonication time (10 to 20 min, studied in three levels). A sample, to which the analytes had been added to a final concentration of 20.0 μg L−1, was used in all optimization assays. The desirability function was applied with the objective of finding the best conditions for the simultaneous extraction of both metals. The
PTA: total power during atomization. 2
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Table 2 Doehlert design and responses (integrated absorbance) for the optimization of extraction induced by emulsion breaking of Cd and Pb in crude palm oil. Experiment
Triton X-114 concentration(% (v v−1))
HNO3 concentration (mol L−1)
Sonication time (min)
Integrated Absorbance Pb Cd
1
10
1.5
15
2
8
1.0
10
3
8
1.0
20
4
8
2.0
10
5
8
2.0
20
6
6
0.5
15
7
6
1.5
15
8
6
2.5
15
9
4
1.0
10
10
4
1.0
20
11
4
2.0
20
12
4
2.0
20
13
2
1.5
15
0.225 0.286 0.351 0.343 0.354 0.407 0.430 0.417 0.523 0.485 0.516 0.585 0.557 0.563 0.587 0.572 0.583 0.581 0.603 0.583 0.596 0.621 0.580 0.607 0.629 0.595
0.989 1.004 0.792 0.847 0.766 0.724 0.064 0.062 0.878 0.773 0.681 0.744 0.668 0.576 0.787 0.740 0.643 0.534 0.642 0.617 0.687 0.679 0.688 0.512 0.702 0.643
experiments were performed in duplicate and are presented in Table 2. For all experiments, the formed emulsions were broken at a temperature of 90 °C, and 20 μL of the collected extract was introduced into the graphite furnace for determination. Statistical analyses were performed using Statistica 7.0 software (Statsoft inc., Tulsa, USA) at a confidence level of 95%. 2.5. Extraction induced by emulsion breaking procedure After optimization, the extraction procedure was performed as follows: 5.0 mL of a crude palm oil sample was transferred to a 50-mL capacity conical-bottom glass tube. Then, 0.8 mL of 2.0 mol L−1 nitric acid and 2.2 mL of 4.4% Triton X-114 surfactant (v v−1) were added. The mixture was vigorously shaken manually and sonicated for 18 min to form an emulsion. After obtaining the emulsion, the glass tube was transferred to a water bath at 90 °C for 30 min to promote the emulsion breaking and separation of the aqueous (with the analytes) and oily phases. The aqueous phase was collected and transferred to a container stored in the refrigerator until the determination of metals by GF AAS. Analytical curves were made in the range from 1.0 to 20.0 µg L−1 in three medias: (1) 1.0% HNO3 aqueous solution; (2) 4.4% (v v−1) Triton X-114 with 2.0 mol L−1 HNO3 solution and (3) extract obtained from EIEB processing of a crude palm oil sample under the optimized conditions.
Fig. 1. Pyrolysis and atomization curves for Cd added to the emulsion breaking extract of crude palm oil at a concentration of 5.0 μg L−1. For the construction of the pyrolysis curve, the atomization temperature was fixed at 1600 °C. For the construction of the atomization curves, the pyrolysis temperature was set at 500 °C for the curve without a modifier (■); 700 °C for the curve using the Pd/ Mg modifier ( ); and 550 °C for the curve using W as a permanent modifier (●).
performing pyrolysis without a modifier, the analytical signal began to decrease drastically from 400 °C. When using the Pd/Mg modifier, the signal remained constant until at least 700 °C, and when using the permanent W modifier, the signal began to decrease at 550 °C. For the atomization curves, when performing pyrolysis at temperatures of 400, 700 and 550 °C, for the curves without a modifier and with the modifier Pd/Mg, the signal reached a maximum at 1500 °C and then began to decrease gradually. When using the W modifier, the absorbance decreased starting at the initial experimental temperature (1450 °C). The pyrolysis and atomization curves for Pb are presented in Fig. 2. In this case, there was a substantial loss of analyte when pyrolysis temperatures higher than 400 °C were used and without using a modifier. In the presence of the Pd/Mg modifier, it was possible to reach temperatures of approximately 900 °C without loss of lead. For
3. Results and discussion 3.1. Pyrolysis and atomization curves To develop a method using GF AAS, the heating program needs to be optimized. The heating program generally includes four steps: drying, pyrolysis, atomization and cleaning. The two steps that are generally more critical for obtaining a high absorbance signal are pyrolysis and atomization. Thus, pyrolysis and atomization curves for Cd and Pb were prepared to study the thermal behavior of the analytes in the absence and presence of modifiers. Fig. 1 shows the pyrolysis and atomization curves for Cd added to the emulsion breaking extract of a crude palm oil sample. When 3
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Table 3 Analysis of variance for the quadratic model fitted to the data obtained after application of the Doehlert design. Source of variation
SS
df
MS
Fcalc
Regression Residual Lack of fit Pure error Total
1.338301 0.230718 0.121896 0.18822 1.569019
9 16 2 14 25
0.148700 0.014920 0.060948 0.007773
9.9665 7.8410
SS: sum of the square; df: degree of freedom; MS: mean of square; R2 = SSR/ SST = 0.8530.
Another method to assess the quality of fitted models is by using the (i) observed value versus predicted value graph and (ii) predicted value versus residual graph. These graphs are shown in Fig. 3. Although there was also a lack of fit, the quadratic model (Fig. 3a and b) can better describe the behavior of the data than the linear model (Fig. 3c and d). Fig. 3a shows a better correlation between the observed values and predicted values than that shown in Fig. 3c. The residuals from the quadratic model (Fig. 3b) are also smaller than the residuals left by the linear model (Fig. 3d). In spite of there are residues that have not been modeled, visual inspection of the responses was used to indicate the experimental conditions that allow the highest extraction. Two of the partial response surfaces generated by the quadratic model are shown in Fig. 4. By analyzing the partial surfaces, it can be observed that the greatest responses were obtained when the following experimental conditions were used: Triton X-114 surfactant concentration of 4.4% (v v−1), 2.0 mol L−1 HNO3 solution and sonication time of 18 min.
Fig. 2. Pyrolysis and atomization curves for Pb added to the emulsion breaking extract of crude palm oil to a concentration of 5.0 μg L−1. For the construction of the pyrolysis curve, the atomization temperature was fixed at 1600 °C, and for the construction of the atomization curves, the pyrolysis temperature was set at 400 °C for the curve without a modifier ( ) and 900 °C for the curve with the Pd/Mg modifier (■).
atomization, the presence of the modifier once again proved to be important. Without a modifier, the signal increased until a temperature of 1700 °C and then decreased. With the Pd/Mg modifier, the atomization temperature that yielded the highest signal was 1900 °C. These results show that the use of Pd/Mg as modifiers allowed a considerable portion of the Cd and Pb content to be stabilized without loss of the analytes by volatilization during the pyrolysis step, permitting the use of higher pyrolysis temperatures. Such high temperatures can contribute to the more efficient burning of the extract obtained after EIEB, which, despite having a lower loading in terms of organic compounds, still has remnants of the original oily matrix and surfactant. Thus, the use of the Pd/Mg modifier is recommended for better performance in the determination of Cd and Pb. The temperatures chosen for pyrolysis and atomization were 700 and 1500 °C for Cd and 900 °C and 1900 °C for Pb, respectively, using the Pd/Mg modifier.
3.3. Matrix effects The extract obtained after the application of EIEB has characteristics that can cause matrix effects from residual oil and surfactant that could be transferred to the aqueous phase. To evaluate whether the extract (obtained using the optimal conditions) presented a matrix effect in the determination of Cd and Pb, calibration curves were constructed in the following media: (i) aqueous, (ii) 10% Triton X-114 (v v−1) and HNO3 2.0 mol L−1 and (iii) EIEB extract obtained from crude palm oil. These analytical curves are presented in Fig. 5, and their parameters are presented in Table 4. To evaluate the solution media and calibration strategies, it was assumed that there was no matrix effect when there was not an intersection between the slopes (confidence level of 95%) of the analytical curves. By observing the confidence intervals of the slopes of the analytical curves for the Cd and Pb, it was noted that the extract obtained from EIEB differs from the other two media, and therefore, it is recommended to use the standard addition calibration technique for accurate determination of these metals.
3.2. Multivariate optimization of the extraction induced by emulsion breaking procedure The three variables that strongly influence EIEB were optimized using a Doehlert design (Table 2). A desirability function was applied to allow the simultaneous optimization of the extraction of both analytes. In this approach, the two data sets (Cd and Pb signals) were transformed into dimensionless scales (individual desirabilities) ranging from 0 to 1 according to a specific mathematical function, which, in this case, aimed at maximization [24–27]. The closer to 1 the response obtained is, the more the response will be maximized. Then, these responses were combined to obtain the overall desirability (D) by calculating the following geometric mean:
D=
3.4. Interferences study The effects of interfering ions on Pb and Cd determination after EIEB were evaluated adding a standard solution (to a final concentration of 10.0 µg L−1 in the sample) of the studied metals. In order to perform this study, Ni2+, Zn2+, Mn2+, Fe3+, Cu2+, Na+, Ca2+, Mg2+ (as nitrate salt), and Cl− and SO42− (as sodic salt) aqueous solutions were added in concentrations in the range from 0.10 to 10 mg L−1 to the sample in the emulsification step. It was considered that exist interference when the analyte signal showed a change of 5% in the signal obtained in the absence of these potential interferences. For Cd determination, Cl−, SO42− Ni2+, Zn2+, Mn2+, Cu2+ did not interfere , until a concentration of 2.0 mg L−1; Fe3+ and Na+ did not interfere until a concentration of 5.0 mg L−1; and Ca2+ and Mg2+ did not interfere until a concentration of 6.5 mg L−1. For Pb determination, Ni2+, Mn2+, Cu2+ did not interfere until a concentration of 2.5 mg L−1; Cl−,
dCd dPb
Thus, by optimizing D, the extraction of both Cd and Pb using EIEB were simultaneously optimized. Table 2 presents the Doehlert design matrix for the optimization of the three variables studied (Triton X-114 concentration, HNO3 concentration and sonication time) and analytical signal responses for the two metals. To describe the behavior of the data obtained by the Doehlert design, linear and quadratic functions were fitted. The fitted models can be evaluated by applying the lack-of-fit test using parameters obtained from analysis of variance (ANOVA). Both the linear (p = 0.00018 < 0.05) and the quadratic (p = 0.005193 < 0.05) fitted models showed a lack of fit at a 95% confidence level. Table 3 presents the analysis of variance obtained by to fit the quadratic model. 4
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Fig. 3. Graphs obtained by fitting mathematical models to the overall desirability. Graphs of observed values versus predicted values for (a) the quadratic model and (c) the linear model and predicted values versus residuals for (b) the quadratic model and (d) the linear model.
Fig. 4. Two partial response surfaces obtained by fitting a quadratic mathematical model to the obtained response (overall desirability), combining the data obtained for Cd and Pb after application of the Doehlert design in EIEB optimization. 5
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Table 5 Analytical characteristics of the developed method for the determination of Cd and Pb in crude palm oil samples after extraction induced by emulsion breaking. Metal −1
LOD (µg kg ) LOQ (µg kg−1) Characteristic mass (pg) Repeatability (%RSD) Analytical sensitivity (Abs/µg kg−1)
Cd
Pb
0.052 0.17 52 2.8 (5.4 µg kg−1) and 1.8 (10.8 µg kg−1) 0.159
0.040 0.13 4.0 2.9 (5.4 µg kg−1) and 5.4 (10.8 µg kg−1) 0.0205
LOD: limit of detection; LOQ: limit of quantification; RSD: relative standard deviation.
can be seen from the LOQs obtained that the method has the capacity to determine the studied metals in the oil palm samples, as will be shown later. The accuracy of the proposed method was determined by performing addition/recovery tests (spike tests). Verifying the accuracy of the method by certified reference materials (CRM) was not possible since no CRM was available. Table 6 shows the measured concentrations and the results of the addition/recovery test for six crude palm oil samples obtained by applying the developed method. The Cd contents in the six analyzed palm oil samples ranged from 1.5 to 2.5 μg kg−1 (Table 6). Recoveries between 85% and 111% for a concentration of 5.4 μg kg−1 of added Cd were achieved. The concentrations of Pb in the six analyzed crude palm oil samples were in the range of 2.5–6.4 μg kg−1. In addition, recoveries between 96% and 113% of the added Pb were obtained. These values show that the method presents enough accuracy for the determination of lead and cadmium in crude palm oil. The Cd and Pb concentrations found in the crude palm oil analyzed in the present work were compatible with some others found in the literature in relation to oil of vegetal origin. Ansari et al. [28] determined Cd, Pb and Zn in different brands of sunflower oil using the acid digestion method and GF AAS, and the levels of Cd and Pb in the samples ranged from 1.70 to 6.18 μg g−1 and 0.79 to 4.4 μg g−1, respectively. Dugo et al. [29] determined the content of Cd, Pb, Cu and Zn in commercial peanuts, sunflower, soy, maize, rice, grapeseed and hazelnut oils using derivative potentiometric stripping analysis (dPSA). Cadmium was found in very low amounts; maize oils presented the highest average concentration of Cd (4.90 μg kg−1) and rice oils presented the lowest (0.71 μg kg−1). Lead was also present in low concentrations, with the highest mean value (55.61 μg kg−1) in nut oils and the lowest (8.60 μg kg−1) in rice oil. Canario and Katskov [30] used a transverse heated filter atomizer (THFA) for the direct determination of Cd and Pb in edible oils using atomic absorption spectrometry. Samples of olive, avocado, grapeseed, sunflower, salad and cooking oils were analyzed. Cadmium concentrations in these samples were between < 0.06 and 0.75 µg L−1, and Pb concentrations were between 2.8 and
Fig. 5. Analytical curves prepared in different media for the study of the matrix effect in the determination of (a) Cd and (b) Pb: (■) aqueous, ( ) 10% Triton X114 (v v−1) and HNO3 2.0 mol L−1 and ( ) EIEB extract obtained from crude palm oil.
SO42− Zn2+ Fe3+ and Na+ did not interfere until a concentration of , , −1 6.0 mg L ; and Ca2+, Mg2+ did not interfere until the concentration of 5.0 mg L−1. 3.5. Analytical characteristics of the method and application The analytical characteristics of the developed method for the determination of Cd and Pb were assessed. The limits of detection (criterion 3 s) and quantification (criterion 10 s), precision (repeatability) and sensitivity were established for the recommended conditions of the extraction procedure. These characteristics are presented in Table 5. It
Table 4 Parameters of the analytical curves obtained in the study of the matrix effect of extraction induced by emulsion breaking (EIEB) extract for the determination of Cd and Pb. Analyte
Media
Calibration strategy
Analytical curveb
R2
Cd
(A) (B) (C)a (A) (B) (C)a
External standardization External standardization Standard addition External Standard addition External Standard addition Standard addition
A = 0.233 ( ± 0.020) CCd + 0.329 A = 0.247 ( ± 0.035) CCd + 0.449 A = 0.171 ( ± 0.020) CCd + 0.693 A = 0.0365 ( ± 0.0025) CPb + 0.335 A = 0.0424 ( ± 0.0020) CPb + 0.329 A = 0.0221 ( ± 0.0021) CPb + 0.387
0.9963 0.9898 0.9928 0.9954 0.9977 0.9916
Pb
Media: (A) aqueous, (B) 10% Triton X-114 (v v−1) and 2.0 mol L−1 HNO3, and (C) EIEB extract obtained from crude palm oil. a Performed in the optimal conditions. b Using a confidence interval of 95%; CCd and CPb: concentration of cadmium and lead, respectively, in µg L−1; R2: coefficient of determination. 6
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Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Grant Number 304582/2018-2) for providing grants, fellowships and financial support.
Table 6 Determination of Cd and Pb in crude palm oil samples by graphite furnace atomic absorption spectrometry after extraction induced by emulsion breaking. Analyte
Sample
Added concentration (µg kg−1)
Found concentration (µg kg−1)
Recovery (%)
Cd
1
0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4
2.01 ± 0.09 7.6 ± 0.2 2.5 ± 0.4 8.5 ± 0.3 1.8 ± 0.2 7.5 ± 0.8 1.81 ± 0.09 6.4 ± 0.9 1.5 ± 0.2 6.8 ± 0.8 1.5 ± 0.2 7.3 ± 0.8 6.4 ± 0.2 12.2 ± 0.8 3.9 ± 0.4 9.6 ± 0.7 5.4 ± 0.1 10.6 ± 0.2 4.1 ± 0.6 10.0 ± 0.8 2.5 ± 0.1 8.6 ± 0.2 4.6 ± 0.5 10.18 ± 0.03
– 109 – 111 – 106 – 85 – 98 – 107 – 107 – 106 – 96 – 109 – 113 – 103
2 3 4 5 6 Pb
1 2 3 4 5 6
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Means and standard deviations found using N = 3.
6.9 µg L−1. López-García, Vicente-Martínez and Hernández-Córdoba [31] used reverse dispersive liquid–liquid microextraction and determined Cd and Pb by electrothermal atomic absorption spectrometry in the analysis of several edible oils. The cadmium and Pb contents varied in the ranges of 12 - 205 ng g−1 and 0.06–5.06 ng g−1, respectively. Mendil et al. [32]determined Cd and Pb, among other metals, in edible oil produced in Turkey and the found concentrations were in the range of 0.09–4.57 µg kg−1 and 0.01–0.03 µg kg−1, respectively. 4. Conclusions The developed allowed the determination of Cd and Pb by GF AAS in crude palm oil after emulsification assisted by ultrasound and extraction induced by emulsion breaking. The Doehlert design approach made it possible to optimize the method quickly and efficiently. The use of the Pd/Mg modifier was essential in obtaining a higher pyrolysis temperature to burn the residues from the original matrix and remaining surfactant in the obtained extract. The method presented a matrix effect in the determination of the two studied metals. Therefore, the determination of these metals requires the construction of analytical curves by the standard addition method using the extract obtained after the extraction procedure. The method validation showed that the proposed method presents adequate analytical parameters for the determination of Cd and Pb in vegetable oil samples, avoiding a timeconsuming or drastic sample pretreatment, such as heating with concentrated acid. Declaration of Competing Interest None. Acknowledgments This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001. The authors are also grateful to Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB), Financiadora de 7
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G. S. Valasques, et al. 76 (2008) 965–977. [28] R. Ansari, T.G. Kazi, M.K. Jamali, M.B. Arain, M.D. Wagan, N. Jalbani, H.I. Afridi, A.Q. Shah, Variation in accumulation of heavy metals in different verities of sunflower seed oil with the aid of multivariate technique, Food Chem. 115 (2009) 318–323. [29] G. Dugo, L. Pera, L.G. Torre, D. Giuffrida, Determination of Cd(II), Cu(II), Pb(II), and Zn(II) content in commercial vegetable oils using derivative potentiometric stripping analysis, Food Chem. 87 (2004) 639–645. [30] M.C. Canário, D.A. Katskov, Direct determination of Cd and Pb in edible oils by
atomic absorption spectrometry with transverse heated filter atomizer, J. Anal. At. Spectrom. 20 (2005) 1386–1388. [31] I.L. García, Y.V. Martínez, M.H. Córdoba, Determination of cadmium and lead in edible oils by electrothermal atomic absorption spectrometry after reverse dispersive liquid–liquid microextraction, Talanta 124 (2014) 106–110. [32] D. Mendil, O.D. Uluözlü, M. Tüzen, M. Soylak, Investigation of the levels of some element in edible oil samples produced in Turkey by atomic absorption spectrometry, J. Hazard. Mater. 165 (2009) 1–3.
8
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Multivariate optimization for the determination of cadmium and lead in crude palm oil by graphite furnace atomic absorption spectrometry after extraction induced by emulsion breaking Gisseli S. Valasquesa, Ana Maria P dos Santosa, Valdinei S. de Souzaa,b, Leonardo S.G. Teixeiraa, ⁎ Juscelia P.S. Alvesc, Mirela de Jesus Santosc, Wagna P.C dos Santosd, Marcos A. Bezerrac, a
Instituto de Química, Universidade Federal da Bahia, Rua Barão de Jeremoabo s/n, Ondina, Salvador 40170-115, Bahia, Brazil Instituto Federal de Educação, Ciência e Tecnologia Baiano, Rodovia BR 116, Santa Inês 45320-000, Bahia, Brazil Departamento de Ciências e Tecnologias, Universidade Estadual do Sudoeste da Bahia, Rua José Moreira Sobrinho s/n, Jequiezinho, Jequié 45206-190, Bahia, Brazil d Instituto Federal de Educação Ciências e Tecnologias, Rua Emídio dos Santos, Barbalho, Salvador 40301-015, Bahia, Brazil b c
ARTICLE INFO
ABSTRACT
Keywords: Crude palm oil Extraction induced by emulsion breaking GF AAS Cadmium Lead Multivariate optimization
The present work reports the development of an analytical method for the determination of trace amounts of Cd and Pb in crude palm oil using graphite furnace atomic absorption spectrometry (GF AAS) after extraction induced by emulsion breaking (EIEB). The method was based on an oil-in-water emulsion preparation to promote a high contact area between the sample drops and the acid extractant solution. Emulsification was assisted by ultrasound energy using HNO3 and Triton X-114 surfactant solutions to increase the efficiency of the extraction. Afterwards, the emulsion was broken by heating, and the acid aqueous phase in the bottom of conical glass tubes was collected for the determination of metals by GF AAS. Cadmium and lead extraction was simultaneously optimized using Doehlert design and desirability function. The best extractions were achieved using a sonication time of 18 min, a HNO3 concentration of 2.0 mol L−1 and a surfactant concentration of 4.4% (v v-1) at a breaking temperature of 90 °C. The developed method presented quantification limits of 0.17 and 0.13 μg kg−1 and precisions (repeatability,%RSD, 10.8 μg kg−1) of 1.8 and 5.4% for Cd and Pb, respectively. Addition/recovery tests gave results between 88.5% and 112%. The amounts of Cd and Pb in the analyzed samples ranged from 1.5–2.5 and 2.53–6.76 μg kg−1, respectively.
1. Introduction Edible oils are foods composed mostly of lipids that are liquids at room temperature and may have animal or vegetable origin. Palm oil currently accounts for approximately 32% of the world's oil production and is used as a raw material in the production of various food products, cosmetics, and biofuels, among others [1–3]. The quality of these oils is affected by the presence of metals, even in trace amounts, which have a strong influence on flavor degradation and oxidative stability [4]. In addition, the determination of toxic metals, such as Cd and Pb, in food is a current demand in society due to growing concern about quality and food safety. Cadmium and Pb are toxic and accumulative metals in the body without known biological functions. These metals impair cell metabolism and have deleterious effects on human health [5–8]. The diffusion of these metals into the environment and,
⁎
consequently, into food originates from anthropogenic activities such as industrial emissions, the application of mineral fertilizers in soils and vehicular emissions due to the burning of fossil fuels [9]. Cadmium accumulates mainly in the liver and kidneys, increasing the risks of cancer, cardiovascular diseases and osteoporosis [10,11]. Efforts have been made to reduce the use of Cd as raw material in the production of pigments, plastics and batteries. Lead accumulates in the skeleton, especially in the bone marrow. Lead is neurotoxic, causes changes in behavior and slows mental and intellectual development. This metal also interferes with calcium and vitamin D metabolism, affects the formation of hemoglobin and causes anemia [4,12,13]. The determination of trace metals, such as Cd and Pb, in samples of edible oils by atomic spectrometry techniques has been a difficult task due to the low concentrations of these analytes and to the complexity of the matrix, which is very viscous and has a high content of organic substances that are difficult to attack and decompose [5,14].
Corresponding author. E-mail address: [email protected] (M. A. Bezerra).
https://doi.org/10.1016/j.microc.2019.104401 Received 6 June 2019; Received in revised form 31 October 2019; Accepted 6 November 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Gisseli S. Valasques, et al., Microchemical Journal, https://doi.org/10.1016/j.microc.2019.104401
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Procedures involving the emulsification of samples for direct introduction into analytical equipment are quite attractive because such procedures allow simpler and faster analysis without a loss of reliability in the generated results [15]. Surfactants are widely used to increase the stability of emulsions. Emulsification procedures offer some advantages in the treatment of edible oil samples, such as simplification of sample treatment; reduction in the load of organic substances and strong inorganic acids introduced into the atomizer; improvements in accuracy and precision; sample stabilization, allowing storage for long periods; and enabling the use of inorganic standards for calibration [16,17]. However, sample dilution can be observed in the emulsification procedure, affecting the sensitivity of the method. For further simplify the matrix introduced into the atomizer for elemental determination, the extraction induced by emulsion breaking (EIEB) procedure has recently been proposed [18]. In this procedure, the emulsion formed by an oily sample is broken by heating or centrifugation. The quantitative transfer of the analytes from a complex oil phase to an aqueous phase, in addition to bringing all the advantages of the emulsification processes, practically decreases the introduction of organic substances into the atomizer, facilitating the pyrolysis step [19]. Furthermore, due to the separation process, preconcentration of the analytes can be observed. Using graphite furnace atomic absorption spectrometry (GF AAS) as an analytical technique, EIEB has been used for the determination of Cr and Mn in edible oils [20], Cu, Fe, Ni and Pb in samples of diesel [16] and Cu, Mn and Ni in biodiesel [21]. In this work, constrained mixture design was applied to optimize a procedure based on emulsification assisted by ultrasound and EIEB for the posterior determination of Cd and Pb in crude palm oil samples by GF AAS.
model 550 heating bath (São Paulo, Brazil) with a capillary thermostat ( ± 2 °C) with a temperature control range of 50 to 120 °C was used to break the emulsions. Total sample digestion was performed in Teflon® decomposition pumps inserted in a steel casing (Parr Instrument Company, Moline, USA). A laboratory stove (Quimis, Model Q317 M12, Diadema, São Paulo, Brazil) formed from Teflon® bombs and a steel casing was used to heat the system. 2.2. Solutions, reagents and samples All reagents used in this work were of analytical purity. Ultrapure water was obtained using a Purelab purification system, model Classic (Elga, High Wycombe, UK). All glassware was immersed in 5% (v v−1) HNO3 solution for decontamination for 12 h, then washed with deionized water and dried in a dust-free environment. Cadmium and Pb solutions were prepared from appropriate dilutions of commercial standards (Merck, Kenilworth, NJ, USA) at concentrations of 1000 mg L−1 in 1% (v v−1) HNO3 solution or from the organic standard WM-21-5X, hydrocarbon oil, at concentrations of 50 μg g−1 (AccuStandard, New Haven, USA). Tungsten, palladium nitrate and magnesium nitrate solutions (1000 mg L−1) (Merck, Kenilworth, NJ, USA) were used as chemical modifiers to stabilize the analytes in the pyrolysis step, allowing a higher temperature to be used. The proportion of Pd/Mg adopted was 5:3 (v v−1). Graphite tubes with integrated platforms were coated with 250 μg of tungsten as a permanent modifier according to a temperature program adapted from the studies of Lima et al. [22] and Freschi et al. [23]. In this procedure, five injections of 50 μL of 1000 mg L−1 solution of the W modifier were introduced onto the surface of the platform with the aid of the autosampler. Triton X-114 (Vetec, Rio de Janeiro, RJ, Brazil) surfactant solutions from 2 to 10% (v v−1) were prepared by dilution of the surfactant in water and solubilization with the aid of the ultrasonic bath. All of the crude palm oil samples analyzed were purchased from markets in the cities of Jequié and Salvador, Bahia, Brazil. The samples were transported to the laboratory and stored at room temperature and were only opened at the moment of analysis.
2. Experimental 2.1. Instrumentation Atomic absorption measurements were carried out using an AAnalyst 400 graphite furnace atomic absorption spectrometer (Perkin Elmer, Norwalk, CT, USA) equipped with a background corrector with deuterium arc lamp, HGA 900 (with an integrated platform and covered with pyrolytic graphite) as an atomizer, hollow cathode lamps and an AS 800 autosampler (Perkin Elmer, Norwalk, CT, USA). All measurements were performed at the highest-sensitivity wavelength indicated by the equipment for each element: 228.80 nm (Cd) and 283.31 nm (Pb). High-purity argon gas (99.99%, Itaox, Itabuna, Brazil) was used for the purging and protection of the graphite furnace. The heating programs for the determination of Cd and Pb are shown in Table 1. In all experiments, the autosampler always transferred 20 µL of the sample or the standard solution into the graphite tube and 5.0 µL of modifier solution when the latter was used. A Cristofoli ultrasound bath (Campo Mourão, Brazil) of 42 kHz and 170 W was used to promote the formation of the emulsions. A Fisatom
2.3. Construction of pyrolysis and atomization curves The thermal behavior of Cd and Pb in the aqueous phase obtained after EIEB was evaluated from the pyrolysis and atomization curves. These curves were made under the following conditions: (i) without the use of modifier, (ii) with the use of the Pd/Mg modifier and (iii) with the permanent W modifier impregnated inside the tube (for Cd only). For the construction of the curves, extracts obtained from emulsion breaking of a crude palm oil sample and with metals added to a final concentration of 5.0 μg L−1 were used. For the construction of the pyrolysis curves, for Cd and Pb, a fixed atomization temperature was adopted (1600 °C). For the construction of the atomization curves, the optimum pyrolysis temperature found for each analyte was adopted. All measurements were made using the integrated absorbance as the analytical signal.
Table 1 Graphite furnace heating program for the determination of Cd and Pb by GF AAS in the aqueous phase obtained after extraction induced by emulsion breaking. Steps
Temperature (°C)
Ramp (°C s−1)
Hold (s)
Ar flow rate (mL min−1)
Dry 1 Dry 2 Pyrolysis
100 140 700 (Cd) 900 (Pb) 1500 (Cd) 1900 (Pb) 2600 (Cd) 2600 (Pb)
5 15 10 10 PTAa PTAa 1 1
20 15 20 20 5 5 5 5
250 250 250 250 0 0 250 250
Atomization Clean a
2.4. Optimization of the extraction induced by emulsion breaking procedure The optimization of EIEB for the simultaneous determination of Cd and Pb in crude palm oil samples was performed by multivariate techniques (Doehlert design and desirability function). According to the literature [17], the most influential variables in the extraction process are the concentration of Triton X-114 solution (2 to 8% v v−1, studied at five levels), the concentration of HNO3 solution (0.5 to 2.5 mol L−1, studied at five levels) and the sonication time (10 to 20 min, studied in three levels). A sample, to which the analytes had been added to a final concentration of 20.0 μg L−1, was used in all optimization assays. The desirability function was applied with the objective of finding the best conditions for the simultaneous extraction of both metals. The
PTA: total power during atomization. 2
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Table 2 Doehlert design and responses (integrated absorbance) for the optimization of extraction induced by emulsion breaking of Cd and Pb in crude palm oil. Experiment
Triton X-114 concentration(% (v v−1))
HNO3 concentration (mol L−1)
Sonication time (min)
Integrated Absorbance Pb Cd
1
10
1.5
15
2
8
1.0
10
3
8
1.0
20
4
8
2.0
10
5
8
2.0
20
6
6
0.5
15
7
6
1.5
15
8
6
2.5
15
9
4
1.0
10
10
4
1.0
20
11
4
2.0
20
12
4
2.0
20
13
2
1.5
15
0.225 0.286 0.351 0.343 0.354 0.407 0.430 0.417 0.523 0.485 0.516 0.585 0.557 0.563 0.587 0.572 0.583 0.581 0.603 0.583 0.596 0.621 0.580 0.607 0.629 0.595
0.989 1.004 0.792 0.847 0.766 0.724 0.064 0.062 0.878 0.773 0.681 0.744 0.668 0.576 0.787 0.740 0.643 0.534 0.642 0.617 0.687 0.679 0.688 0.512 0.702 0.643
experiments were performed in duplicate and are presented in Table 2. For all experiments, the formed emulsions were broken at a temperature of 90 °C, and 20 μL of the collected extract was introduced into the graphite furnace for determination. Statistical analyses were performed using Statistica 7.0 software (Statsoft inc., Tulsa, USA) at a confidence level of 95%. 2.5. Extraction induced by emulsion breaking procedure After optimization, the extraction procedure was performed as follows: 5.0 mL of a crude palm oil sample was transferred to a 50-mL capacity conical-bottom glass tube. Then, 0.8 mL of 2.0 mol L−1 nitric acid and 2.2 mL of 4.4% Triton X-114 surfactant (v v−1) were added. The mixture was vigorously shaken manually and sonicated for 18 min to form an emulsion. After obtaining the emulsion, the glass tube was transferred to a water bath at 90 °C for 30 min to promote the emulsion breaking and separation of the aqueous (with the analytes) and oily phases. The aqueous phase was collected and transferred to a container stored in the refrigerator until the determination of metals by GF AAS. Analytical curves were made in the range from 1.0 to 20.0 µg L−1 in three medias: (1) 1.0% HNO3 aqueous solution; (2) 4.4% (v v−1) Triton X-114 with 2.0 mol L−1 HNO3 solution and (3) extract obtained from EIEB processing of a crude palm oil sample under the optimized conditions.
Fig. 1. Pyrolysis and atomization curves for Cd added to the emulsion breaking extract of crude palm oil at a concentration of 5.0 μg L−1. For the construction of the pyrolysis curve, the atomization temperature was fixed at 1600 °C. For the construction of the atomization curves, the pyrolysis temperature was set at 500 °C for the curve without a modifier (■); 700 °C for the curve using the Pd/ Mg modifier ( ); and 550 °C for the curve using W as a permanent modifier (●).
performing pyrolysis without a modifier, the analytical signal began to decrease drastically from 400 °C. When using the Pd/Mg modifier, the signal remained constant until at least 700 °C, and when using the permanent W modifier, the signal began to decrease at 550 °C. For the atomization curves, when performing pyrolysis at temperatures of 400, 700 and 550 °C, for the curves without a modifier and with the modifier Pd/Mg, the signal reached a maximum at 1500 °C and then began to decrease gradually. When using the W modifier, the absorbance decreased starting at the initial experimental temperature (1450 °C). The pyrolysis and atomization curves for Pb are presented in Fig. 2. In this case, there was a substantial loss of analyte when pyrolysis temperatures higher than 400 °C were used and without using a modifier. In the presence of the Pd/Mg modifier, it was possible to reach temperatures of approximately 900 °C without loss of lead. For
3. Results and discussion 3.1. Pyrolysis and atomization curves To develop a method using GF AAS, the heating program needs to be optimized. The heating program generally includes four steps: drying, pyrolysis, atomization and cleaning. The two steps that are generally more critical for obtaining a high absorbance signal are pyrolysis and atomization. Thus, pyrolysis and atomization curves for Cd and Pb were prepared to study the thermal behavior of the analytes in the absence and presence of modifiers. Fig. 1 shows the pyrolysis and atomization curves for Cd added to the emulsion breaking extract of a crude palm oil sample. When 3
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Table 3 Analysis of variance for the quadratic model fitted to the data obtained after application of the Doehlert design. Source of variation
SS
df
MS
Fcalc
Regression Residual Lack of fit Pure error Total
1.338301 0.230718 0.121896 0.18822 1.569019
9 16 2 14 25
0.148700 0.014920 0.060948 0.007773
9.9665 7.8410
SS: sum of the square; df: degree of freedom; MS: mean of square; R2 = SSR/ SST = 0.8530.
Another method to assess the quality of fitted models is by using the (i) observed value versus predicted value graph and (ii) predicted value versus residual graph. These graphs are shown in Fig. 3. Although there was also a lack of fit, the quadratic model (Fig. 3a and b) can better describe the behavior of the data than the linear model (Fig. 3c and d). Fig. 3a shows a better correlation between the observed values and predicted values than that shown in Fig. 3c. The residuals from the quadratic model (Fig. 3b) are also smaller than the residuals left by the linear model (Fig. 3d). In spite of there are residues that have not been modeled, visual inspection of the responses was used to indicate the experimental conditions that allow the highest extraction. Two of the partial response surfaces generated by the quadratic model are shown in Fig. 4. By analyzing the partial surfaces, it can be observed that the greatest responses were obtained when the following experimental conditions were used: Triton X-114 surfactant concentration of 4.4% (v v−1), 2.0 mol L−1 HNO3 solution and sonication time of 18 min.
Fig. 2. Pyrolysis and atomization curves for Pb added to the emulsion breaking extract of crude palm oil to a concentration of 5.0 μg L−1. For the construction of the pyrolysis curve, the atomization temperature was fixed at 1600 °C, and for the construction of the atomization curves, the pyrolysis temperature was set at 400 °C for the curve without a modifier ( ) and 900 °C for the curve with the Pd/Mg modifier (■).
atomization, the presence of the modifier once again proved to be important. Without a modifier, the signal increased until a temperature of 1700 °C and then decreased. With the Pd/Mg modifier, the atomization temperature that yielded the highest signal was 1900 °C. These results show that the use of Pd/Mg as modifiers allowed a considerable portion of the Cd and Pb content to be stabilized without loss of the analytes by volatilization during the pyrolysis step, permitting the use of higher pyrolysis temperatures. Such high temperatures can contribute to the more efficient burning of the extract obtained after EIEB, which, despite having a lower loading in terms of organic compounds, still has remnants of the original oily matrix and surfactant. Thus, the use of the Pd/Mg modifier is recommended for better performance in the determination of Cd and Pb. The temperatures chosen for pyrolysis and atomization were 700 and 1500 °C for Cd and 900 °C and 1900 °C for Pb, respectively, using the Pd/Mg modifier.
3.3. Matrix effects The extract obtained after the application of EIEB has characteristics that can cause matrix effects from residual oil and surfactant that could be transferred to the aqueous phase. To evaluate whether the extract (obtained using the optimal conditions) presented a matrix effect in the determination of Cd and Pb, calibration curves were constructed in the following media: (i) aqueous, (ii) 10% Triton X-114 (v v−1) and HNO3 2.0 mol L−1 and (iii) EIEB extract obtained from crude palm oil. These analytical curves are presented in Fig. 5, and their parameters are presented in Table 4. To evaluate the solution media and calibration strategies, it was assumed that there was no matrix effect when there was not an intersection between the slopes (confidence level of 95%) of the analytical curves. By observing the confidence intervals of the slopes of the analytical curves for the Cd and Pb, it was noted that the extract obtained from EIEB differs from the other two media, and therefore, it is recommended to use the standard addition calibration technique for accurate determination of these metals.
3.2. Multivariate optimization of the extraction induced by emulsion breaking procedure The three variables that strongly influence EIEB were optimized using a Doehlert design (Table 2). A desirability function was applied to allow the simultaneous optimization of the extraction of both analytes. In this approach, the two data sets (Cd and Pb signals) were transformed into dimensionless scales (individual desirabilities) ranging from 0 to 1 according to a specific mathematical function, which, in this case, aimed at maximization [24–27]. The closer to 1 the response obtained is, the more the response will be maximized. Then, these responses were combined to obtain the overall desirability (D) by calculating the following geometric mean:
D=
3.4. Interferences study The effects of interfering ions on Pb and Cd determination after EIEB were evaluated adding a standard solution (to a final concentration of 10.0 µg L−1 in the sample) of the studied metals. In order to perform this study, Ni2+, Zn2+, Mn2+, Fe3+, Cu2+, Na+, Ca2+, Mg2+ (as nitrate salt), and Cl− and SO42− (as sodic salt) aqueous solutions were added in concentrations in the range from 0.10 to 10 mg L−1 to the sample in the emulsification step. It was considered that exist interference when the analyte signal showed a change of 5% in the signal obtained in the absence of these potential interferences. For Cd determination, Cl−, SO42− Ni2+, Zn2+, Mn2+, Cu2+ did not interfere , until a concentration of 2.0 mg L−1; Fe3+ and Na+ did not interfere until a concentration of 5.0 mg L−1; and Ca2+ and Mg2+ did not interfere until a concentration of 6.5 mg L−1. For Pb determination, Ni2+, Mn2+, Cu2+ did not interfere until a concentration of 2.5 mg L−1; Cl−,
dCd dPb
Thus, by optimizing D, the extraction of both Cd and Pb using EIEB were simultaneously optimized. Table 2 presents the Doehlert design matrix for the optimization of the three variables studied (Triton X-114 concentration, HNO3 concentration and sonication time) and analytical signal responses for the two metals. To describe the behavior of the data obtained by the Doehlert design, linear and quadratic functions were fitted. The fitted models can be evaluated by applying the lack-of-fit test using parameters obtained from analysis of variance (ANOVA). Both the linear (p = 0.00018 < 0.05) and the quadratic (p = 0.005193 < 0.05) fitted models showed a lack of fit at a 95% confidence level. Table 3 presents the analysis of variance obtained by to fit the quadratic model. 4
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Fig. 3. Graphs obtained by fitting mathematical models to the overall desirability. Graphs of observed values versus predicted values for (a) the quadratic model and (c) the linear model and predicted values versus residuals for (b) the quadratic model and (d) the linear model.
Fig. 4. Two partial response surfaces obtained by fitting a quadratic mathematical model to the obtained response (overall desirability), combining the data obtained for Cd and Pb after application of the Doehlert design in EIEB optimization. 5
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Table 5 Analytical characteristics of the developed method for the determination of Cd and Pb in crude palm oil samples after extraction induced by emulsion breaking. Metal −1
LOD (µg kg ) LOQ (µg kg−1) Characteristic mass (pg) Repeatability (%RSD) Analytical sensitivity (Abs/µg kg−1)
Cd
Pb
0.052 0.17 52 2.8 (5.4 µg kg−1) and 1.8 (10.8 µg kg−1) 0.159
0.040 0.13 4.0 2.9 (5.4 µg kg−1) and 5.4 (10.8 µg kg−1) 0.0205
LOD: limit of detection; LOQ: limit of quantification; RSD: relative standard deviation.
can be seen from the LOQs obtained that the method has the capacity to determine the studied metals in the oil palm samples, as will be shown later. The accuracy of the proposed method was determined by performing addition/recovery tests (spike tests). Verifying the accuracy of the method by certified reference materials (CRM) was not possible since no CRM was available. Table 6 shows the measured concentrations and the results of the addition/recovery test for six crude palm oil samples obtained by applying the developed method. The Cd contents in the six analyzed palm oil samples ranged from 1.5 to 2.5 μg kg−1 (Table 6). Recoveries between 85% and 111% for a concentration of 5.4 μg kg−1 of added Cd were achieved. The concentrations of Pb in the six analyzed crude palm oil samples were in the range of 2.5–6.4 μg kg−1. In addition, recoveries between 96% and 113% of the added Pb were obtained. These values show that the method presents enough accuracy for the determination of lead and cadmium in crude palm oil. The Cd and Pb concentrations found in the crude palm oil analyzed in the present work were compatible with some others found in the literature in relation to oil of vegetal origin. Ansari et al. [28] determined Cd, Pb and Zn in different brands of sunflower oil using the acid digestion method and GF AAS, and the levels of Cd and Pb in the samples ranged from 1.70 to 6.18 μg g−1 and 0.79 to 4.4 μg g−1, respectively. Dugo et al. [29] determined the content of Cd, Pb, Cu and Zn in commercial peanuts, sunflower, soy, maize, rice, grapeseed and hazelnut oils using derivative potentiometric stripping analysis (dPSA). Cadmium was found in very low amounts; maize oils presented the highest average concentration of Cd (4.90 μg kg−1) and rice oils presented the lowest (0.71 μg kg−1). Lead was also present in low concentrations, with the highest mean value (55.61 μg kg−1) in nut oils and the lowest (8.60 μg kg−1) in rice oil. Canario and Katskov [30] used a transverse heated filter atomizer (THFA) for the direct determination of Cd and Pb in edible oils using atomic absorption spectrometry. Samples of olive, avocado, grapeseed, sunflower, salad and cooking oils were analyzed. Cadmium concentrations in these samples were between < 0.06 and 0.75 µg L−1, and Pb concentrations were between 2.8 and
Fig. 5. Analytical curves prepared in different media for the study of the matrix effect in the determination of (a) Cd and (b) Pb: (■) aqueous, ( ) 10% Triton X114 (v v−1) and HNO3 2.0 mol L−1 and ( ) EIEB extract obtained from crude palm oil.
SO42− Zn2+ Fe3+ and Na+ did not interfere until a concentration of , , −1 6.0 mg L ; and Ca2+, Mg2+ did not interfere until the concentration of 5.0 mg L−1. 3.5. Analytical characteristics of the method and application The analytical characteristics of the developed method for the determination of Cd and Pb were assessed. The limits of detection (criterion 3 s) and quantification (criterion 10 s), precision (repeatability) and sensitivity were established for the recommended conditions of the extraction procedure. These characteristics are presented in Table 5. It
Table 4 Parameters of the analytical curves obtained in the study of the matrix effect of extraction induced by emulsion breaking (EIEB) extract for the determination of Cd and Pb. Analyte
Media
Calibration strategy
Analytical curveb
R2
Cd
(A) (B) (C)a (A) (B) (C)a
External standardization External standardization Standard addition External Standard addition External Standard addition Standard addition
A = 0.233 ( ± 0.020) CCd + 0.329 A = 0.247 ( ± 0.035) CCd + 0.449 A = 0.171 ( ± 0.020) CCd + 0.693 A = 0.0365 ( ± 0.0025) CPb + 0.335 A = 0.0424 ( ± 0.0020) CPb + 0.329 A = 0.0221 ( ± 0.0021) CPb + 0.387
0.9963 0.9898 0.9928 0.9954 0.9977 0.9916
Pb
Media: (A) aqueous, (B) 10% Triton X-114 (v v−1) and 2.0 mol L−1 HNO3, and (C) EIEB extract obtained from crude palm oil. a Performed in the optimal conditions. b Using a confidence interval of 95%; CCd and CPb: concentration of cadmium and lead, respectively, in µg L−1; R2: coefficient of determination. 6
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Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Grant Number 304582/2018-2) for providing grants, fellowships and financial support.
Table 6 Determination of Cd and Pb in crude palm oil samples by graphite furnace atomic absorption spectrometry after extraction induced by emulsion breaking. Analyte
Sample
Added concentration (µg kg−1)
Found concentration (µg kg−1)
Recovery (%)
Cd
1
0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4 0 5.4
2.01 ± 0.09 7.6 ± 0.2 2.5 ± 0.4 8.5 ± 0.3 1.8 ± 0.2 7.5 ± 0.8 1.81 ± 0.09 6.4 ± 0.9 1.5 ± 0.2 6.8 ± 0.8 1.5 ± 0.2 7.3 ± 0.8 6.4 ± 0.2 12.2 ± 0.8 3.9 ± 0.4 9.6 ± 0.7 5.4 ± 0.1 10.6 ± 0.2 4.1 ± 0.6 10.0 ± 0.8 2.5 ± 0.1 8.6 ± 0.2 4.6 ± 0.5 10.18 ± 0.03
– 109 – 111 – 106 – 85 – 98 – 107 – 107 – 106 – 96 – 109 – 113 – 103
2 3 4 5 6 Pb
1 2 3 4 5 6
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Means and standard deviations found using N = 3.
6.9 µg L−1. López-García, Vicente-Martínez and Hernández-Córdoba [31] used reverse dispersive liquid–liquid microextraction and determined Cd and Pb by electrothermal atomic absorption spectrometry in the analysis of several edible oils. The cadmium and Pb contents varied in the ranges of 12 - 205 ng g−1 and 0.06–5.06 ng g−1, respectively. Mendil et al. [32]determined Cd and Pb, among other metals, in edible oil produced in Turkey and the found concentrations were in the range of 0.09–4.57 µg kg−1 and 0.01–0.03 µg kg−1, respectively. 4. Conclusions The developed allowed the determination of Cd and Pb by GF AAS in crude palm oil after emulsification assisted by ultrasound and extraction induced by emulsion breaking. The Doehlert design approach made it possible to optimize the method quickly and efficiently. The use of the Pd/Mg modifier was essential in obtaining a higher pyrolysis temperature to burn the residues from the original matrix and remaining surfactant in the obtained extract. The method presented a matrix effect in the determination of the two studied metals. Therefore, the determination of these metals requires the construction of analytical curves by the standard addition method using the extract obtained after the extraction procedure. The method validation showed that the proposed method presents adequate analytical parameters for the determination of Cd and Pb in vegetable oil samples, avoiding a timeconsuming or drastic sample pretreatment, such as heating with concentrated acid. Declaration of Competing Interest None. Acknowledgments This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001. The authors are also grateful to Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB), Financiadora de 7
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