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Determination of sodium, potassium, calcium, magnesium, zinc and iron in emulsified chocolate samples by flame atomic absorption spectrometry
Food Chemistry 124 (2011) 1189–1193
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Analytical Methods
Determination of sodium, potassium, calcium, magnesium, zinc and iron in emulsified chocolate samples by flame atomic absorption spectrometry C.V.S. Ieggli, D. Bohrer *, P.C. do Nascimento, L.M. de Carvalho Departamento de Química, Avenida Roraima, 1000, Universidade Federal de Santa Maria, 97110-970 Santa Maria, RS, Brazil
a r t i c l e
i n f o
Article history: Received 21 January 2010 Received in revised form 14 July 2010 Accepted 18 July 2010
Keywords: Emulsion Chocolate Flame atomic absorption spectrometry
a b s t r a c t In this study, oil-in-water formulations were optimized to determine sodium, potassium, calcium, magnesium, zinc, and iron in emulsified chocolate samples by flame atomic absorption spectrometry (FAAS). This method is simpler and requires fewer reagents when compared with other sample pre-treatment procedures and allows the calibration to be carried out using aqueous standards. Octyl stearate was used as oily phase. Tween 80 and Triton X100 were tested as surfactants. The optimum type and proportion of formulations were determined and their choice depended on the element studied. The emulsion preparation was performed by a conventional method that involves mixing both phases at 75 ± 5 °C by magnetic stirring and phase inversion to change the water-to-oil ratio by increasing the volume of the surfactant-water external phase and correspondingly decreasing the volume of internal phase. The validation of the method was performed against a baking chocolate standard reference material (SRM 2384) and recoveries ranged from 88.6% for K to 105.5% for Zn. The proposed method allowed the evaluation of the essential metal status of chocolate with minimum sample manipulation and was reproducible and economical. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Chocolate is a product obtained from Theobroma cacao beans. In order to process cacao beans into chocolate or cocoa, they are left to ferment, dried, roasted and, finally, triturated until they are transformed into a liqueur. Primary chocolate categories are dark, milk and white. The first is made by mixing cocoa liqueur, cocoa butter, sugar and vanilla. The second uses the same process, with the increment of milk, and the white does not include the cocoa liqueur, only the cocoa butter, milk and sugar (Afoakwa, Paterson, & Fowler, 2007). Chocolate is consumed all over the world, in all segments of society and by people of all ages. Nowadays, the consumer is more and more concerned with the nutritional status of foodstuff and, considering that chocolate is an extremely rich source of many essential minerals, it can contribute to a healthy diet. Nevertheless, the evaluation of nutrient ingestion is a very complex task (Borchers, Keen, Hannum, & Gershwin, 2000). The available nutritional data are frequently old and incomplete and in many cases unreliable due to lack of description of the analytical procedures (Ribeiro, de Morais, Colugnati, & Sigulem, 2003). The determination of metals in foods has become an important field in food analysis (Reyes & Campos, 2006). However, the accu* Corresponding author. Fax: +55 55 3220 8870. E-mail address: [email protected] (D. Bohrer). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.043
rate determination of metals in chocolate is still an analytical challenge, due to difficulties arising from matrix characteristics. Flame atomic absorption spectrometry (FAAS) is a powerful detection technique for determining elements in the ppm range. The advantages of FAAS include well-characterized interferences, low operator skill required for operation, and comparatively low cost of instrumentation and maintenance (Welz & Sperling, 1999). However, direct chocolate analysis by FAAS is not possible and the determination of metals in this type of matrix necessarily involves sample digestion, considering that it contains a high content of organic compounds. The literature reports methods for chocolate sample treatment involving microwave digestion, wet digestion, and dry ashing (Dahiya, Karpe, Hegde, & Sharma, 2005; Güldasß, 2008; Jalbani et al., 2007; Sepe, Costantini, Ciaralli, Ciprotti, & Giordano, 2001). Acids and peroxides are usually added to improve sample decomposition. All these procedures result in additional steps, which may lead to inconveniences such as contamination and losses during handling (Viñas, Pardo-Martínez, & Hernández-Córdoba, 2000). Direct emulsification with surfactants provides a rapid procedure for sample preparation since this approach does not require any destruction of the organic matrix (Sanz-Medel, de la Campa, Gonzalez, & Fernandez-Sanchez, 1999). It simply reduces the viscosity and the organic content of the sample, making the properties of the chocolate sample close to those capable of being analyzed by FAAS, while maintaining the system’s homogeneity
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and stability. In earlier studies, emulsification of samples was satisfactorily used for the analysis of whole hen eggs measuring Se by graphite furnace atomic absorption spectrometry (GFAAS) and Na, K, Ca, Mg, Zn and Fe by FAAS. The oil-in-water emulsion stabilized the sample, maintaining the system’s homogeneity, and reduced the viscosity and the organic content of the sample, thus allowing egg sample analysis by GFAAS and by FAAS (Ieggli, Bohrer, do Nascimento, & de Carvalho, 2010; Ieggli et al., 2009). The goal of this study was to investigate the use of emulsions as sample preparation for routine determination of Na, K, Ca, Mg, Zn and Fe in chocolate by FAAS. The expected advantages of this procedure are the higher stability and easy handling of sample emulsions, allowing the use of aqueous standards for calibration. The reliability of the procedure was checked by analyzing standard reference material. 2. Experimental 2.1. Instrumentation/procedure All measurements were carried out using an ANALYTIK Jena AG (Jena, Germany) model novAA 300 atomic absorption spectrometer equipped with SpectrAA (Varian, Australia) hollow cathode lamps as the radiation source. An acetylene-air or acetylene-nitrous oxide flame was used; the gas flow rates and the burner height were adjusted in order to obtain the maximum absorbance signal for each element. Other instrumental parameters were set to the values shown in Table 1. 2.2. Reagents and samples All reagents were of analytical grade, and all emulsions were prepared with distilled and deionized water that was further purified by a Milli-Q high purity water device (electrical resistivity of 18.0 MX cm) (Millipore, Bedford, USA). Laboratory glassware was kept overnight in 10% (v/v) HNO3 in ethanol solution and shortly before use was washed with water and dried in a dust free environment. Concentrated nitric acid used in this study was supplied by Merck. Sodium, potassium, calcium, magnesium, zinc, and iron standard solutions (1000 mg L 1) were obtained from the National Institute of Standards and Technology (NIST, USA) and diluted as necessary to obtain working standards. The non-ionic surfactants Triton X100 (Fluka) and Tween 80 (Fluka) were tested for emulsion preparation. Octyl stearate (Galena, Brazil) was used as the oily phase. The certified reference material (CRM) SRM 2384 Baking Chocolate (NIST) was used to check the accuracy of the proposed method. The chocolate samples analyzed in this study were purchased in supermarkets from Santa Maria (Brazil) and the percentage of cocoa varied. The chocolate samples were five white chocolate (brands: Neugebauer, Nestlé Classic, Lacta Laka, Neugebauer Dupy, Garoto), five milk chocolate (Neugebauer, Nestlé Classic, Lacta, Neugebauer Dupy, Garoto), and seven dark chocolate (Neugebauer,
Table 1 Instrumental parameters for element determination in emulsified chocolate samples. Element
Wavelength (nm)
Slit width (nm)
I (mA)
Integration time (s)
Flame
Potassium Calcium Sodium Zinc Magnesium Iron
766.5 422.7 589.0 213.9 285.2 248.3
0.8 1.2 0.8 0.5 1.2 0.2
4.5 4.0 3.0 6.0 4.0 8.0
3.0 3.0 3.0 3.0 3.0 3.0
C2H2–air N2O–C2H2 C2H2–air C2H2–air C2H2–air N2O–C2H2
Nestlé Classic, Lacta Amaro, Neugebauer Dupy, Garoto, 70% Cocoa Neugebauer and Dark & Soft 50% Cocoa Lacta). 2.3. Emulsion preparation Oil-in-water emulsions were prepared using a specific sequence in order to guarantee their stability. Aliquots of surfactant, oil and chocolate samples or reference material were weighed and placed in an 80 mL beaker, and then heated water (65 °C for Triton X100 and 75 °C for Tween 80, see Section 3.2) was added with continuous agitation until the required volume was reached. Magnetic stirring (3000 rpm) was maintained during 15 min at room temperature (22 °C). The total volume of the system was 50 mL, which was attained by increasing the volume of the surfactant-water external phase and correspondingly decreasing the volume of internal phase. A blank emulsion was prepared in the same way without sample addition. Sample amounts varied between 0.2% and 8.0% (w/v) according to the expected amount of the analyte in the sample. For components present in larger concentrations, such as sodium, potassium, calcium and magnesium, the emulsion was prepared with a maximum sample amount of 0.2%. On the other hand, for smaller amounts of zinc and iron, between 2.0% and 8.0%, were necessary. 2.4. Experimental design and stability study A preliminary factorial design was applied to investigate the influence of oil and emulsifier amounts on emulsion stability. Their influence on emulsion stability was evaluated using a 23 full factorial design. The design required a total of nine experiments per oil/ emulsifier combination. 2.5. Emulsion stability Stability of optimized emulsion was monitored by measuring the extent of gravitational phase separation. The best formulations from experimental design were monitored by the creaming test. For the measurement of physical stability, 10 mL of each prepared chocolate emulsion was poured into a graduated tube and kept at room temperature (25 °C). The volume of the separated cream layer in each tube was recorded after 1, 2, 7, 14, and 21 days of storage. The emulsion stability index (ESI) was calculated as percentage: ESI (%) = (remaining emulsion height/initial emulsion height) 100. The ESI was calculated to show the stability of the emulsions since the larger the ESI value the higher emulsion stability. 3. Results and discussion An emulsion is a thermodynamically stable system composed of water, oil and surfactant (Sinko, 2008). Based on this concept, emulsions have been little exploited as an analytical tool by using all the ideal conditions to produce an adequate and stable emulsion. In order to utilize emulsions as sample preparation for FAAS measurements, some criteria should be taken into account in the formulation planning: the emulsion should contain only the components necessary to stabilize the sample, in other words, sample, surfactant, water and, if necessary, an additional oily component; the emulsion should be stable for a reasonable period, which should last during a time interval long enough to complete the determination procedure; the emulsion should present low viscosity to allow correct sample aspiration; and all components should present low metal contamination and low background during the FAAS measurement.
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3.1. Formulation studies Emulsions are classified as oil-in-water emulsions (O/W) when the oils droplets are dispersed in an aqueous continuous external phase; and water-in-oil emulsions (W/O) when the water droplets are dispersed in an oily continuous external phase. Since the emulsions behave according to their external phase, for analytical purposes, oil-in-water emulsions are the most appropriate option since they allow calibration with aqueous standards, besides presenting characteristics of long-term stability and low viscosity (Remington, 1998). The hydrophilic-lipophilic balance (HLB) system for selecting suitable emulsifiers to stabilize emulsions was developed by Griffin and it has been used for about five decades. According to the Griffin scale, tensoactives with HLB values between 9.6 and 16.7 are considered hydrophilic (water-soluble), and tend to form oilin-water emulsions (Sinko, 2008). Non-ionic emulsifiers such as Tween 80 and Triton X100 satisfy all requirements needed: they have ideal HLB values (13.5 and 15.0, respectively), they do not have foaming properties, avoiding heterogeneous distribution of the oil drops in the emulsion (Sanz-Medel et al., 1999), and moreover, they are inexpensive and readily available in most analytical laboratories. The oil/surfactant ratio was determined by experimental planning. The combination of 4% emulsifier and 4% oil produced, for sample amounts varying from 0.2% to 8%, emulsification without coalescence. In all of the experiments in which the amount of oil was smaller than the amount of emulsifier, the separation of phases took place immediately after preparation. This shows that an excess of emulsifier in relation to oily phase favors emulsion instability. Table 2 present the best formulation from the experimental planning and the metals that could be determined in each formulation. The selection criteria are discussed in Section 3.3. Despite the formulation stability and metal determination capability, it was necessary to analyze the level of contamination by the metals studied in the oils and surfactants chosen. In our previous work, the oil and surfactant metal content was already determined. Octyl stearate presented a relatively low contamination of all the metals investigated. Tween 80 presented high content of sodium and Triton X100 high potassium and calcium content. Thus, for these elements, contamination limited the choice of surfactant and oil (Ieggli et al., 2010).
3.2. Formation and stabilization of the emulsion Usually emulsions are prepared at temperatures between 70 and 80 °C, and the aqueous phase is heated 5 °C more than the oily phase. However, in this work the best temperatures observed were 65 °C for Triton X100 and 75 °C for Tween 80. Since the behavior of stable systems depends on the preparation conditions, care was taken to assure that the procedure was carried out in exactly the same way for all experiments. Phase inversion, which changes the water-to-oil ratio by increasing the surfactant-water external phase volume and correspondingly decreasing the internal phase volume, provides a finely dispersed oil-in-water emulsion with
long-term stability (Lachman, Lieberman, & Kanig, 2001). In the initial period of stirring the droplets necessary for the emulsification are formed. If the stirring exceeds the necessary period for ideal stability, adhesion can take place due to collision among the droplets. This period of time is usually determined empirically. In this study, the stipulated time was 2 min of manual mechanical stirring to reach emulsification, followed by magnetic stirring at 3000 rpm for 15 min at room temperature for complete stabilization of the system. Creaming test may also be used to determine emulsion stability (Mirhosseini, Tan, Hamid, Yusof, & Chern, 2009). The final appearance of all emulsions was from milky white to slightly brown and all chocolate emulsions presented relatively high values of ESI, since emulsion composition was previously optimized by experimental planning. The octyl stearate/Tween 80/chocolate sample combination presented highest values of ESI (ESI1day = 100%–ESI21days = 96.3%). The octyl stearate/Triton X100/chocolate sample combination presented lower values of ESI (ESI1day = 97.5%–ESI21days = 94.0%). The emulsions presented a precipitate after 24 h. The precipitate was probably formed from the solid content that was not stabilized by the micelles, however homogeneity was re-established by shaking the mixture.
3.3. Analytic applicability study and validation Ideally, a single emulsion should be used for determination of all elements, however this was not possible mainly due to the presence of the analytes as contaminants in both surfactants and oily components (Ieggli et al., 2010) and due to different metal contents in the samples. Based on these criteria, the metals that could be analyzed in the same emulsion are shown in Table 2. The characteristics of an analytical method are defined by the figures of merit, which should be determined experimentally. The proposed method was validated for six metals (Na, K, Ca, Mg, Zn and Fe). The figures of merit presented were linearity of the analytical curves, accuracy and precision. In addition, sensitivity was determined by characteristic concentration (c0). The advantages of the emulsification procedure for fatty samples have been described in the literature. When properly stabilized, the emulsified sample is compatible with most analytical instruments, allowing the use of simple calibration procedures due to the minimization of interferences (Sanz-Medel et al., 1999). In this study, calibration with aqueous standards was possible and the linearity ranges were selected to span the metal concentrations expected in real samples. Analytical curves were constructed by evaluating the relation between response (peak height and absorbance) and concentration by linear regression analysis yielding the results shown in Table 3. In all instances, a linear fit was found to be adequate for the purpose. Table 3 also presents the values found for characteristic concentration for all elements. The accuracy of the method was further confirmed by determining the metals in a baking chocolate CRM. The results are shown in Table 4. Statistical comparison by means of the t-test showed that there was no significant difference between the values obtained with the proposed method and the certified values.
Table 2 Chocolate and emulsion component amounts for metal determination by FAAS. Element
Na K, Ca, Mg Fe Zn
Chocolate sample amount (%)
Emulsion components
White
Milk
Dark
Oil phase
%
Surfactant
%
0.2 0.2 8.0 4.0
0.2 0.2 4.0 2.0
0.2 0.2 2.0 2.0
Octyl Octyl Octyl Octyl
4.0 4.0 4.0 4.0
Triton X100 Tween 80 Triton X100 Tween 80
4.0 4.0 4.0 4.0
stearate stearate stearate stearate
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Table 3 Regression parameters of the metal analytical curves.
a b c
Metal
Rangea (mg L
Potassium Calcium Sodium Zinc Magnesium Iron
1–10 0.5–8 1–3 0.05–0.40 0.5–4.0 0.1–2.0
1
)
Regression equationb
R2
c0 (mg L
A = 0.0619 + 0.1834C A = 0.0039 + 0.0996C A = (0.0087 + 0.2839C)/(1 + 0.0558C) A = 0.0015 + 0.2312C A = (0.0124 + 0.2359C)/(1 + 0.1682C) A = 0.0014 + 0.0228C
0.9938 0.9994 0.9951 0.9958 0.9912 0.9977
0.024 0.044 0.015 0.018 0.019 0.191
1
LODc (mg L
)
1
)
0.05 0.16 0.02 0.02 0.03 0.02
Five standard solutions each curve. Absorbance, C (mg L 1) = concentration of element in standard solution. LOD = limit of detection.
3.4. Application to real samples
The precision of the procedures was determined through repeatability (intra-day precision). Six chocolate emulsions were assayed during the same day, under the same experimental conditions. Intermediate precision (inter-day) was evaluated by assaying freshly prepared emulsions on three different days (n = 3). The repeatability presented good relative standard deviation (RSD) values for all metals. The intermediate precision was evaluated using the RSD and Ftest. The computed F-values were lower than tabulated F-values, indicating no significant difference between the results obtained on different days. All precision data are shown in Table 4. Although FAAS measurement of alkaline and alkaline earth metals may require the presence of an ionization buffer (Welz & Sperling, 1999) for the suppression of analyte ionization, in this study it was not used. Probably, due to the nature of the matrices, no significant changes were observed when the cesium/lanthanum chloride buffer was used for the measurements with acetylene-nitrous oxide flame.
The proposed method was applied to evaluate Na, K, Ca, Mg, Zn, and Fe levels in different chocolate samples. We aimed to contribute to provide a baseline of these elements in different kinds of chocolate (white, milk and dark). The analysis was carried out in triplicate and the results are shown in Table 5. The mineral present in the highest concentration is potassium (K), with amounts varying from 2495 to 6361 lg g 1. Interestingly, the higher the cocoa content the higher the K level. The amount of sodium was found to be directly related to the content of milk and cocoa liqueur in the chocolates, however it also presented great variation among the brands. Chocolates contain amounts of calcium varying from 324 to 4533 lg g 1, where the highest levels are in white chocolates. Chocolate contains levels from 365 to 1834 lg g 1 of Mg, for white and dark chocolate, respectively.
Table 4 Determination of the analytes in a certified reference baking chocolate (NIST SRM 2384). Element
Na K Ca Mg Fe Zn a b c d e
Concentrations lg g
Emulsion compounds
1
Oil phase
%
Surfactant
%
Certified valuea
Foundb
Octyl Octyl Octyl Octyl Octyl Octyl
4.0 4.0 4.0 4.0 4.0 4.0
Triton X100 Tween 80 Tween 80 Tween 80 Triton X100 Tween 80
4.0 4.0 4.0 4.0 4.0 4.0
40 ± 2 8200 ± 500 840 ± 74 2570 ± 150 132 ± 11 36.6 ± 1.7
41 ± 2 7266 ± 182 882 ± 11 2600 ± 192 129 ± 11 38.6 ± 2.7
stearate stearate stearate stearate stearate stearate
Recovery (%)
texpc
103.0 88.6 105.0 101.2 97.5 105.5
0.04 0.09 0.04 1.58 0.01 0.03
Precision Intra-day (RSD)
Inter dayd (RSD/Fexpe)
3.8 2.3 1.0 0.9 4.6 1.2
1.0/0.34 2.3/0.69 0.3/0.14 0.7/0.78 2.6/0.55 0.6/0.88
With 95% confidence limit. Mean value ± standard deviation (n = 3). ttab = 2.31 (p 0.05). Time interval = 3 days. Ftab = 4.26 (p 0.05).
Table 5 Concentration levels of the elements (lg g
1
± SD, n = 3) in different chocolate samples.
Chocolate type
Brand
Cocoa content (%)
Na
K
Ca
Mg
Fe
Zn
White
1 2 3 4 5
n.i. n.i. 27 n.i. n.i.
1033 ± 6 1121 ± 2 1411 ± 7 921.0 ± 3 940.3 ± 1.4
3069 ± 21 3474 ± 19 3952 ± 26 2840 ± 14 2745 ± 14
3990 ± 27 4096 ± 107 4534 ± 92 3404 ± 48 3203 ± 57
365.6 ± 1.6 403.7 ± 1.8 496.8 ± 9.5 325.7 ± 2.5 334.2 ± 1.9
1.2 ± 0.1 1.3 ± 0.2 2.2 ± 0.1 1.7 ± 0.4 3.0 ± 0.2
13.5 ± 0.3 13.5 ± 0.0 13.4 ± 0.3 11.0 ± 0.1 10.3 ± 0.1
Milk
6 7 8 9 10
n.i. n.i. 32 n.i. n.i.
515.2 ± 1.2 450.1 ± 7.2 891.8 ± 3.8 530.8 ± 2.0 932.2 ± 4.4
2661 ± 13 2769 ± 32 3672 ± 8 2596 ± 12 3368 ± 4
1813 ± 47 1546 ± 11 2300 ± 18 1744 ± 19 2523 ± 33
632.7 ± 6.5 736.6 ± 2.8 867.2 ± 1.4 528.6 ± 36.5 714.3 ± 35.6
16.5 ± 0.5 14.7 ± 0.7 24.8 ± 0.7 27.4 ± 0.3 19.4 ± 0.2
7.5 ± 0.1 7.7 ± 0.1 9.4 ± 0.1 9.3 ± 0.1 10.7 ± 0.3
Dark
11 12 13 14 15 16 17
n.i. n.i. 43 n.i. n.i. 50 70
500.3 ± 1.4 127.7 ± 0.1 59.8 ± 0.1 509.8 ± 2.5 93.1 ± 0.8 496.2 ± 3.0 127.7 ± 0.6
3654 ± 34 3849 ± 31 3718 ± 26 3670 ± 11 3746 ± 9 4932 ± 13 6361 ± 34
665.4 ± 31.3 801.6 ± 3.0 324.4 ± 24.0 730.7 ± 23.3 385.7 ± 12.3 2069 ± 55 692.4 ± 28
997.3 ± 11.5 1328 ± 15 1262 ± 7 1036 ± 6 1224 ± 2 1576 ± 37 1834 ± 77
43.3 ± 0.6 36.1 ± 0.5 64.6 ± 1.4 43.0 ± 0.5 35.8 ± 1.1 75.3 ± 1.5 140.8 ± 7.7
12.4 ± 0.1 15.5 ± 0.1 15.4 ± 0.1 12.1 ± 0.1 15.4 ± 0.0 15.8 ± 0.1 23.3 ± 0.1
n.i.: Not informed.
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The minor component contents, Fe and Zn, ranged from 1.2 to 140 lg g 1 and from 8 to 23 lg g 1, respectively. For both metals, the higher the cocoa content the higher the metal content. 4. Conclusion It would ideal if a single emulsion could be used for the determination of all selected elements, however, this was not possible due to the surfactant metal content and the different levels of each metal in the chocolate samples. The main advantages of the proposed method over traditional digestion methods are that it does not require a long sample treatment or large amounts of organic solvents or inorganic acids and it is simple and shows good accuracy and reproducibility. As the challenges and main requirements for emulsion application in atomic spectrometry involve the attainment of stable emulsions with low viscosity, the proposed method including emulsification and subsequent metal determination for FAAS fulfilled these requisites and proved to be sensitive, reproducible, simple, and economical. Acknowledgement The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for scholarships. References Afoakwa, E. O., Paterson, A., & Fowler, M. (2007). Factors influencing rheological and textural qualities in chocolate – a review. Trends in Food Science & Technolology, 18, 290–298. Borchers, A. T., Keen, C. L., Hannum, S. M., & Gershwin, M. E. (2000). Cocoa and chocolate: composition, bioavailability, and health implications. Journal of Medicinal Food, 3, 77–103. Dahiya, S., Karpe, R., Hegde, A. G., & Sharma, R. M. (2005). Lead, cadmium and nickel in chocolates and candies from suburban areas of Mumbai, India. Journal of Food Composition and Analysis, 18, 517–522.
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Güldasß, M. (2008). Comparison of digestion methods and trace elements determination in chocolates with pistachio using atomic absorption spectrometry. Journal of Food Nutrition Research, 47, 92–99. Ieggli, C. V. S., Bohrer, D., Noremberg, S., do Nascimento, P. C., de Carvalho, L. M., Vieira, S. L., et al. (2009). Surfactant/oil/water system for the determination of selenium in eggs by graphite furnace atomic absorption spectrometry. Spectrochimica Acta Part B, 64, 605. Ieggli, C. V. S., Bohrer, D., do Nascimento, P. C., & de Carvalho, L. M. (2010). Determination of sodium, potassium, calcium, magnesium, zinc, and iron in emulsified egg samples by flame atomic absorption spectrometry. Talanta, 80, 1282–1286. Jalbani, N., Kazi, T. G., Jamali, M. K., Arain, M. B., Afridi, H. I., Sheerazi, S. T., et al. (2007). Application of fractional design and Doehlert matrix in the optimization of experimental variables associated with the ultrasonic-assisted digestion of chocolate samples for aluminum determination by atomic absorption spectrometry. Journal of AOAC International, 90, 1682–1688. Lachman, L., Lieberman, H.A., & Kanig, J.L. (2001). Teoria e Prática na Indústria Farmacêutica, v. II, Ed. Fundação Calouste Guebenkian, Lisboa. Mirhosseini, H., Tan, C. P., Hamid, N. S. A., Yusof, S., & Chern, B. H. (2009). Characterization of the influence of main emulsion components on the physicochemical properties of orange beverage emulsion using response surface methodology. Food Hydrocolloids, 23, 271. Remington, Farmacia (1998). Tomo 1, 19ª ed., Editorial Médica Panamericana, Buenos Aires. Reyes, M. N. M., & Campos, R. C. (2006). Determination of copper and nickel in vegetable oils by direct sampling graphite furnace atomic absorption spectrometry. Talanta, 70, 929–932. Ribeiro, P., de Morais, T. B., Colugnati, F. A. B., & Sigulem, D. M. (2003). Tabelas de composição química de alimentos: análise comparativa com resultados laboratoriais. Revista de Saúde Pública, 37, 216–225. Sanz-Medel, A., de la Campa, M. R. F., Gonzalez, E. B., & Fernandez-Sanchez, M. L. (1999). Organised surfactant assemblies in analytical atomic spectrometry. Spectrochimica Acta Part B, 54, 251–287. Sepe, A., Costantini, S., Ciaralli, L., Ciprotti, M., & Giordano, R. (2001). Evaluation of aluminium concentrations in samples of chocolate and beverages by electrothermal atomic absorption spectrometry. Food Additives & Contaminants, 18, 788–796. Sinko, P.J. (2008). Martin: físico-farmácia e ciências farmacêuticas, 5ª ed., Ed. Artmed, Porto Alegre. Viñas, P., Pardo-Martínez, M., & Hernández-Córdoba, M. (2000). Rapid determination of selenium, lead and cadmium in baby food samples using electrothermal atomic absorption spectrometry and slurry atomization. Analytical Chimica Acta, 412, 121–130. Welz, B., & Sperling, M. (1999). Atomic Absorption Spectrometry (3rd ed.). Weinheim: Wiley-VCH.
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Analytical Methods
Determination of sodium, potassium, calcium, magnesium, zinc and iron in emulsified chocolate samples by flame atomic absorption spectrometry C.V.S. Ieggli, D. Bohrer *, P.C. do Nascimento, L.M. de Carvalho Departamento de Química, Avenida Roraima, 1000, Universidade Federal de Santa Maria, 97110-970 Santa Maria, RS, Brazil
a r t i c l e
i n f o
Article history: Received 21 January 2010 Received in revised form 14 July 2010 Accepted 18 July 2010
Keywords: Emulsion Chocolate Flame atomic absorption spectrometry
a b s t r a c t In this study, oil-in-water formulations were optimized to determine sodium, potassium, calcium, magnesium, zinc, and iron in emulsified chocolate samples by flame atomic absorption spectrometry (FAAS). This method is simpler and requires fewer reagents when compared with other sample pre-treatment procedures and allows the calibration to be carried out using aqueous standards. Octyl stearate was used as oily phase. Tween 80 and Triton X100 were tested as surfactants. The optimum type and proportion of formulations were determined and their choice depended on the element studied. The emulsion preparation was performed by a conventional method that involves mixing both phases at 75 ± 5 °C by magnetic stirring and phase inversion to change the water-to-oil ratio by increasing the volume of the surfactant-water external phase and correspondingly decreasing the volume of internal phase. The validation of the method was performed against a baking chocolate standard reference material (SRM 2384) and recoveries ranged from 88.6% for K to 105.5% for Zn. The proposed method allowed the evaluation of the essential metal status of chocolate with minimum sample manipulation and was reproducible and economical. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Chocolate is a product obtained from Theobroma cacao beans. In order to process cacao beans into chocolate or cocoa, they are left to ferment, dried, roasted and, finally, triturated until they are transformed into a liqueur. Primary chocolate categories are dark, milk and white. The first is made by mixing cocoa liqueur, cocoa butter, sugar and vanilla. The second uses the same process, with the increment of milk, and the white does not include the cocoa liqueur, only the cocoa butter, milk and sugar (Afoakwa, Paterson, & Fowler, 2007). Chocolate is consumed all over the world, in all segments of society and by people of all ages. Nowadays, the consumer is more and more concerned with the nutritional status of foodstuff and, considering that chocolate is an extremely rich source of many essential minerals, it can contribute to a healthy diet. Nevertheless, the evaluation of nutrient ingestion is a very complex task (Borchers, Keen, Hannum, & Gershwin, 2000). The available nutritional data are frequently old and incomplete and in many cases unreliable due to lack of description of the analytical procedures (Ribeiro, de Morais, Colugnati, & Sigulem, 2003). The determination of metals in foods has become an important field in food analysis (Reyes & Campos, 2006). However, the accu* Corresponding author. Fax: +55 55 3220 8870. E-mail address: [email protected] (D. Bohrer). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.043
rate determination of metals in chocolate is still an analytical challenge, due to difficulties arising from matrix characteristics. Flame atomic absorption spectrometry (FAAS) is a powerful detection technique for determining elements in the ppm range. The advantages of FAAS include well-characterized interferences, low operator skill required for operation, and comparatively low cost of instrumentation and maintenance (Welz & Sperling, 1999). However, direct chocolate analysis by FAAS is not possible and the determination of metals in this type of matrix necessarily involves sample digestion, considering that it contains a high content of organic compounds. The literature reports methods for chocolate sample treatment involving microwave digestion, wet digestion, and dry ashing (Dahiya, Karpe, Hegde, & Sharma, 2005; Güldasß, 2008; Jalbani et al., 2007; Sepe, Costantini, Ciaralli, Ciprotti, & Giordano, 2001). Acids and peroxides are usually added to improve sample decomposition. All these procedures result in additional steps, which may lead to inconveniences such as contamination and losses during handling (Viñas, Pardo-Martínez, & Hernández-Córdoba, 2000). Direct emulsification with surfactants provides a rapid procedure for sample preparation since this approach does not require any destruction of the organic matrix (Sanz-Medel, de la Campa, Gonzalez, & Fernandez-Sanchez, 1999). It simply reduces the viscosity and the organic content of the sample, making the properties of the chocolate sample close to those capable of being analyzed by FAAS, while maintaining the system’s homogeneity
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and stability. In earlier studies, emulsification of samples was satisfactorily used for the analysis of whole hen eggs measuring Se by graphite furnace atomic absorption spectrometry (GFAAS) and Na, K, Ca, Mg, Zn and Fe by FAAS. The oil-in-water emulsion stabilized the sample, maintaining the system’s homogeneity, and reduced the viscosity and the organic content of the sample, thus allowing egg sample analysis by GFAAS and by FAAS (Ieggli, Bohrer, do Nascimento, & de Carvalho, 2010; Ieggli et al., 2009). The goal of this study was to investigate the use of emulsions as sample preparation for routine determination of Na, K, Ca, Mg, Zn and Fe in chocolate by FAAS. The expected advantages of this procedure are the higher stability and easy handling of sample emulsions, allowing the use of aqueous standards for calibration. The reliability of the procedure was checked by analyzing standard reference material. 2. Experimental 2.1. Instrumentation/procedure All measurements were carried out using an ANALYTIK Jena AG (Jena, Germany) model novAA 300 atomic absorption spectrometer equipped with SpectrAA (Varian, Australia) hollow cathode lamps as the radiation source. An acetylene-air or acetylene-nitrous oxide flame was used; the gas flow rates and the burner height were adjusted in order to obtain the maximum absorbance signal for each element. Other instrumental parameters were set to the values shown in Table 1. 2.2. Reagents and samples All reagents were of analytical grade, and all emulsions were prepared with distilled and deionized water that was further purified by a Milli-Q high purity water device (electrical resistivity of 18.0 MX cm) (Millipore, Bedford, USA). Laboratory glassware was kept overnight in 10% (v/v) HNO3 in ethanol solution and shortly before use was washed with water and dried in a dust free environment. Concentrated nitric acid used in this study was supplied by Merck. Sodium, potassium, calcium, magnesium, zinc, and iron standard solutions (1000 mg L 1) were obtained from the National Institute of Standards and Technology (NIST, USA) and diluted as necessary to obtain working standards. The non-ionic surfactants Triton X100 (Fluka) and Tween 80 (Fluka) were tested for emulsion preparation. Octyl stearate (Galena, Brazil) was used as the oily phase. The certified reference material (CRM) SRM 2384 Baking Chocolate (NIST) was used to check the accuracy of the proposed method. The chocolate samples analyzed in this study were purchased in supermarkets from Santa Maria (Brazil) and the percentage of cocoa varied. The chocolate samples were five white chocolate (brands: Neugebauer, Nestlé Classic, Lacta Laka, Neugebauer Dupy, Garoto), five milk chocolate (Neugebauer, Nestlé Classic, Lacta, Neugebauer Dupy, Garoto), and seven dark chocolate (Neugebauer,
Table 1 Instrumental parameters for element determination in emulsified chocolate samples. Element
Wavelength (nm)
Slit width (nm)
I (mA)
Integration time (s)
Flame
Potassium Calcium Sodium Zinc Magnesium Iron
766.5 422.7 589.0 213.9 285.2 248.3
0.8 1.2 0.8 0.5 1.2 0.2
4.5 4.0 3.0 6.0 4.0 8.0
3.0 3.0 3.0 3.0 3.0 3.0
C2H2–air N2O–C2H2 C2H2–air C2H2–air C2H2–air N2O–C2H2
Nestlé Classic, Lacta Amaro, Neugebauer Dupy, Garoto, 70% Cocoa Neugebauer and Dark & Soft 50% Cocoa Lacta). 2.3. Emulsion preparation Oil-in-water emulsions were prepared using a specific sequence in order to guarantee their stability. Aliquots of surfactant, oil and chocolate samples or reference material were weighed and placed in an 80 mL beaker, and then heated water (65 °C for Triton X100 and 75 °C for Tween 80, see Section 3.2) was added with continuous agitation until the required volume was reached. Magnetic stirring (3000 rpm) was maintained during 15 min at room temperature (22 °C). The total volume of the system was 50 mL, which was attained by increasing the volume of the surfactant-water external phase and correspondingly decreasing the volume of internal phase. A blank emulsion was prepared in the same way without sample addition. Sample amounts varied between 0.2% and 8.0% (w/v) according to the expected amount of the analyte in the sample. For components present in larger concentrations, such as sodium, potassium, calcium and magnesium, the emulsion was prepared with a maximum sample amount of 0.2%. On the other hand, for smaller amounts of zinc and iron, between 2.0% and 8.0%, were necessary. 2.4. Experimental design and stability study A preliminary factorial design was applied to investigate the influence of oil and emulsifier amounts on emulsion stability. Their influence on emulsion stability was evaluated using a 23 full factorial design. The design required a total of nine experiments per oil/ emulsifier combination. 2.5. Emulsion stability Stability of optimized emulsion was monitored by measuring the extent of gravitational phase separation. The best formulations from experimental design were monitored by the creaming test. For the measurement of physical stability, 10 mL of each prepared chocolate emulsion was poured into a graduated tube and kept at room temperature (25 °C). The volume of the separated cream layer in each tube was recorded after 1, 2, 7, 14, and 21 days of storage. The emulsion stability index (ESI) was calculated as percentage: ESI (%) = (remaining emulsion height/initial emulsion height) 100. The ESI was calculated to show the stability of the emulsions since the larger the ESI value the higher emulsion stability. 3. Results and discussion An emulsion is a thermodynamically stable system composed of water, oil and surfactant (Sinko, 2008). Based on this concept, emulsions have been little exploited as an analytical tool by using all the ideal conditions to produce an adequate and stable emulsion. In order to utilize emulsions as sample preparation for FAAS measurements, some criteria should be taken into account in the formulation planning: the emulsion should contain only the components necessary to stabilize the sample, in other words, sample, surfactant, water and, if necessary, an additional oily component; the emulsion should be stable for a reasonable period, which should last during a time interval long enough to complete the determination procedure; the emulsion should present low viscosity to allow correct sample aspiration; and all components should present low metal contamination and low background during the FAAS measurement.
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3.1. Formulation studies Emulsions are classified as oil-in-water emulsions (O/W) when the oils droplets are dispersed in an aqueous continuous external phase; and water-in-oil emulsions (W/O) when the water droplets are dispersed in an oily continuous external phase. Since the emulsions behave according to their external phase, for analytical purposes, oil-in-water emulsions are the most appropriate option since they allow calibration with aqueous standards, besides presenting characteristics of long-term stability and low viscosity (Remington, 1998). The hydrophilic-lipophilic balance (HLB) system for selecting suitable emulsifiers to stabilize emulsions was developed by Griffin and it has been used for about five decades. According to the Griffin scale, tensoactives with HLB values between 9.6 and 16.7 are considered hydrophilic (water-soluble), and tend to form oilin-water emulsions (Sinko, 2008). Non-ionic emulsifiers such as Tween 80 and Triton X100 satisfy all requirements needed: they have ideal HLB values (13.5 and 15.0, respectively), they do not have foaming properties, avoiding heterogeneous distribution of the oil drops in the emulsion (Sanz-Medel et al., 1999), and moreover, they are inexpensive and readily available in most analytical laboratories. The oil/surfactant ratio was determined by experimental planning. The combination of 4% emulsifier and 4% oil produced, for sample amounts varying from 0.2% to 8%, emulsification without coalescence. In all of the experiments in which the amount of oil was smaller than the amount of emulsifier, the separation of phases took place immediately after preparation. This shows that an excess of emulsifier in relation to oily phase favors emulsion instability. Table 2 present the best formulation from the experimental planning and the metals that could be determined in each formulation. The selection criteria are discussed in Section 3.3. Despite the formulation stability and metal determination capability, it was necessary to analyze the level of contamination by the metals studied in the oils and surfactants chosen. In our previous work, the oil and surfactant metal content was already determined. Octyl stearate presented a relatively low contamination of all the metals investigated. Tween 80 presented high content of sodium and Triton X100 high potassium and calcium content. Thus, for these elements, contamination limited the choice of surfactant and oil (Ieggli et al., 2010).
3.2. Formation and stabilization of the emulsion Usually emulsions are prepared at temperatures between 70 and 80 °C, and the aqueous phase is heated 5 °C more than the oily phase. However, in this work the best temperatures observed were 65 °C for Triton X100 and 75 °C for Tween 80. Since the behavior of stable systems depends on the preparation conditions, care was taken to assure that the procedure was carried out in exactly the same way for all experiments. Phase inversion, which changes the water-to-oil ratio by increasing the surfactant-water external phase volume and correspondingly decreasing the internal phase volume, provides a finely dispersed oil-in-water emulsion with
long-term stability (Lachman, Lieberman, & Kanig, 2001). In the initial period of stirring the droplets necessary for the emulsification are formed. If the stirring exceeds the necessary period for ideal stability, adhesion can take place due to collision among the droplets. This period of time is usually determined empirically. In this study, the stipulated time was 2 min of manual mechanical stirring to reach emulsification, followed by magnetic stirring at 3000 rpm for 15 min at room temperature for complete stabilization of the system. Creaming test may also be used to determine emulsion stability (Mirhosseini, Tan, Hamid, Yusof, & Chern, 2009). The final appearance of all emulsions was from milky white to slightly brown and all chocolate emulsions presented relatively high values of ESI, since emulsion composition was previously optimized by experimental planning. The octyl stearate/Tween 80/chocolate sample combination presented highest values of ESI (ESI1day = 100%–ESI21days = 96.3%). The octyl stearate/Triton X100/chocolate sample combination presented lower values of ESI (ESI1day = 97.5%–ESI21days = 94.0%). The emulsions presented a precipitate after 24 h. The precipitate was probably formed from the solid content that was not stabilized by the micelles, however homogeneity was re-established by shaking the mixture.
3.3. Analytic applicability study and validation Ideally, a single emulsion should be used for determination of all elements, however this was not possible mainly due to the presence of the analytes as contaminants in both surfactants and oily components (Ieggli et al., 2010) and due to different metal contents in the samples. Based on these criteria, the metals that could be analyzed in the same emulsion are shown in Table 2. The characteristics of an analytical method are defined by the figures of merit, which should be determined experimentally. The proposed method was validated for six metals (Na, K, Ca, Mg, Zn and Fe). The figures of merit presented were linearity of the analytical curves, accuracy and precision. In addition, sensitivity was determined by characteristic concentration (c0). The advantages of the emulsification procedure for fatty samples have been described in the literature. When properly stabilized, the emulsified sample is compatible with most analytical instruments, allowing the use of simple calibration procedures due to the minimization of interferences (Sanz-Medel et al., 1999). In this study, calibration with aqueous standards was possible and the linearity ranges were selected to span the metal concentrations expected in real samples. Analytical curves were constructed by evaluating the relation between response (peak height and absorbance) and concentration by linear regression analysis yielding the results shown in Table 3. In all instances, a linear fit was found to be adequate for the purpose. Table 3 also presents the values found for characteristic concentration for all elements. The accuracy of the method was further confirmed by determining the metals in a baking chocolate CRM. The results are shown in Table 4. Statistical comparison by means of the t-test showed that there was no significant difference between the values obtained with the proposed method and the certified values.
Table 2 Chocolate and emulsion component amounts for metal determination by FAAS. Element
Na K, Ca, Mg Fe Zn
Chocolate sample amount (%)
Emulsion components
White
Milk
Dark
Oil phase
%
Surfactant
%
0.2 0.2 8.0 4.0
0.2 0.2 4.0 2.0
0.2 0.2 2.0 2.0
Octyl Octyl Octyl Octyl
4.0 4.0 4.0 4.0
Triton X100 Tween 80 Triton X100 Tween 80
4.0 4.0 4.0 4.0
stearate stearate stearate stearate
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Table 3 Regression parameters of the metal analytical curves.
a b c
Metal
Rangea (mg L
Potassium Calcium Sodium Zinc Magnesium Iron
1–10 0.5–8 1–3 0.05–0.40 0.5–4.0 0.1–2.0
1
)
Regression equationb
R2
c0 (mg L
A = 0.0619 + 0.1834C A = 0.0039 + 0.0996C A = (0.0087 + 0.2839C)/(1 + 0.0558C) A = 0.0015 + 0.2312C A = (0.0124 + 0.2359C)/(1 + 0.1682C) A = 0.0014 + 0.0228C
0.9938 0.9994 0.9951 0.9958 0.9912 0.9977
0.024 0.044 0.015 0.018 0.019 0.191
1
LODc (mg L
)
1
)
0.05 0.16 0.02 0.02 0.03 0.02
Five standard solutions each curve. Absorbance, C (mg L 1) = concentration of element in standard solution. LOD = limit of detection.
3.4. Application to real samples
The precision of the procedures was determined through repeatability (intra-day precision). Six chocolate emulsions were assayed during the same day, under the same experimental conditions. Intermediate precision (inter-day) was evaluated by assaying freshly prepared emulsions on three different days (n = 3). The repeatability presented good relative standard deviation (RSD) values for all metals. The intermediate precision was evaluated using the RSD and Ftest. The computed F-values were lower than tabulated F-values, indicating no significant difference between the results obtained on different days. All precision data are shown in Table 4. Although FAAS measurement of alkaline and alkaline earth metals may require the presence of an ionization buffer (Welz & Sperling, 1999) for the suppression of analyte ionization, in this study it was not used. Probably, due to the nature of the matrices, no significant changes were observed when the cesium/lanthanum chloride buffer was used for the measurements with acetylene-nitrous oxide flame.
The proposed method was applied to evaluate Na, K, Ca, Mg, Zn, and Fe levels in different chocolate samples. We aimed to contribute to provide a baseline of these elements in different kinds of chocolate (white, milk and dark). The analysis was carried out in triplicate and the results are shown in Table 5. The mineral present in the highest concentration is potassium (K), with amounts varying from 2495 to 6361 lg g 1. Interestingly, the higher the cocoa content the higher the K level. The amount of sodium was found to be directly related to the content of milk and cocoa liqueur in the chocolates, however it also presented great variation among the brands. Chocolates contain amounts of calcium varying from 324 to 4533 lg g 1, where the highest levels are in white chocolates. Chocolate contains levels from 365 to 1834 lg g 1 of Mg, for white and dark chocolate, respectively.
Table 4 Determination of the analytes in a certified reference baking chocolate (NIST SRM 2384). Element
Na K Ca Mg Fe Zn a b c d e
Concentrations lg g
Emulsion compounds
1
Oil phase
%
Surfactant
%
Certified valuea
Foundb
Octyl Octyl Octyl Octyl Octyl Octyl
4.0 4.0 4.0 4.0 4.0 4.0
Triton X100 Tween 80 Tween 80 Tween 80 Triton X100 Tween 80
4.0 4.0 4.0 4.0 4.0 4.0
40 ± 2 8200 ± 500 840 ± 74 2570 ± 150 132 ± 11 36.6 ± 1.7
41 ± 2 7266 ± 182 882 ± 11 2600 ± 192 129 ± 11 38.6 ± 2.7
stearate stearate stearate stearate stearate stearate
Recovery (%)
texpc
103.0 88.6 105.0 101.2 97.5 105.5
0.04 0.09 0.04 1.58 0.01 0.03
Precision Intra-day (RSD)
Inter dayd (RSD/Fexpe)
3.8 2.3 1.0 0.9 4.6 1.2
1.0/0.34 2.3/0.69 0.3/0.14 0.7/0.78 2.6/0.55 0.6/0.88
With 95% confidence limit. Mean value ± standard deviation (n = 3). ttab = 2.31 (p 0.05). Time interval = 3 days. Ftab = 4.26 (p 0.05).
Table 5 Concentration levels of the elements (lg g
1
± SD, n = 3) in different chocolate samples.
Chocolate type
Brand
Cocoa content (%)
Na
K
Ca
Mg
Fe
Zn
White
1 2 3 4 5
n.i. n.i. 27 n.i. n.i.
1033 ± 6 1121 ± 2 1411 ± 7 921.0 ± 3 940.3 ± 1.4
3069 ± 21 3474 ± 19 3952 ± 26 2840 ± 14 2745 ± 14
3990 ± 27 4096 ± 107 4534 ± 92 3404 ± 48 3203 ± 57
365.6 ± 1.6 403.7 ± 1.8 496.8 ± 9.5 325.7 ± 2.5 334.2 ± 1.9
1.2 ± 0.1 1.3 ± 0.2 2.2 ± 0.1 1.7 ± 0.4 3.0 ± 0.2
13.5 ± 0.3 13.5 ± 0.0 13.4 ± 0.3 11.0 ± 0.1 10.3 ± 0.1
Milk
6 7 8 9 10
n.i. n.i. 32 n.i. n.i.
515.2 ± 1.2 450.1 ± 7.2 891.8 ± 3.8 530.8 ± 2.0 932.2 ± 4.4
2661 ± 13 2769 ± 32 3672 ± 8 2596 ± 12 3368 ± 4
1813 ± 47 1546 ± 11 2300 ± 18 1744 ± 19 2523 ± 33
632.7 ± 6.5 736.6 ± 2.8 867.2 ± 1.4 528.6 ± 36.5 714.3 ± 35.6
16.5 ± 0.5 14.7 ± 0.7 24.8 ± 0.7 27.4 ± 0.3 19.4 ± 0.2
7.5 ± 0.1 7.7 ± 0.1 9.4 ± 0.1 9.3 ± 0.1 10.7 ± 0.3
Dark
11 12 13 14 15 16 17
n.i. n.i. 43 n.i. n.i. 50 70
500.3 ± 1.4 127.7 ± 0.1 59.8 ± 0.1 509.8 ± 2.5 93.1 ± 0.8 496.2 ± 3.0 127.7 ± 0.6
3654 ± 34 3849 ± 31 3718 ± 26 3670 ± 11 3746 ± 9 4932 ± 13 6361 ± 34
665.4 ± 31.3 801.6 ± 3.0 324.4 ± 24.0 730.7 ± 23.3 385.7 ± 12.3 2069 ± 55 692.4 ± 28
997.3 ± 11.5 1328 ± 15 1262 ± 7 1036 ± 6 1224 ± 2 1576 ± 37 1834 ± 77
43.3 ± 0.6 36.1 ± 0.5 64.6 ± 1.4 43.0 ± 0.5 35.8 ± 1.1 75.3 ± 1.5 140.8 ± 7.7
12.4 ± 0.1 15.5 ± 0.1 15.4 ± 0.1 12.1 ± 0.1 15.4 ± 0.0 15.8 ± 0.1 23.3 ± 0.1
n.i.: Not informed.
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The minor component contents, Fe and Zn, ranged from 1.2 to 140 lg g 1 and from 8 to 23 lg g 1, respectively. For both metals, the higher the cocoa content the higher the metal content. 4. Conclusion It would ideal if a single emulsion could be used for the determination of all selected elements, however, this was not possible due to the surfactant metal content and the different levels of each metal in the chocolate samples. The main advantages of the proposed method over traditional digestion methods are that it does not require a long sample treatment or large amounts of organic solvents or inorganic acids and it is simple and shows good accuracy and reproducibility. As the challenges and main requirements for emulsion application in atomic spectrometry involve the attainment of stable emulsions with low viscosity, the proposed method including emulsification and subsequent metal determination for FAAS fulfilled these requisites and proved to be sensitive, reproducible, simple, and economical. Acknowledgement The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for scholarships. References Afoakwa, E. O., Paterson, A., & Fowler, M. (2007). Factors influencing rheological and textural qualities in chocolate – a review. Trends in Food Science & Technolology, 18, 290–298. Borchers, A. T., Keen, C. L., Hannum, S. M., & Gershwin, M. E. (2000). Cocoa and chocolate: composition, bioavailability, and health implications. Journal of Medicinal Food, 3, 77–103. Dahiya, S., Karpe, R., Hegde, A. G., & Sharma, R. M. (2005). Lead, cadmium and nickel in chocolates and candies from suburban areas of Mumbai, India. Journal of Food Composition and Analysis, 18, 517–522.
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