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Food Chemistry 124 (2011) 11891193

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Determination of sodium, potassium, calcium, magnesium, zinc and iron in


emulsied chocolate samples by ame atomic absorption spectrometry
C.V.S. Ieggli, D. Bohrer *, P.C. do Nascimento, L.M. de Carvalho
Departamento de Qumica, 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 emulsied chocolate samples by ame 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.
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, nally, triturated until they are
transformed into a liqueur. Primary chocolate categories are dark,
milk and white. The rst 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
eld in food analysis (Reyes & Campos, 2006). However, the accu* Corresponding author. Fax: +55 55 3220 8870.
E-mail address: ndenise@quimica.ufsm.br (D. Bohrer).
0308-8146/$ - see front matter 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 difculties 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; Gldas, 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 (Vias, Pardo-Martnez, & Hernndez-Crdoba, 2000).
Direct emulsication 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 systems homogeneity

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C.V.S. Ieggli et al. / Food Chemistry 124 (2011) 11891193

and stability. In earlier studies, emulsication 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 systems 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
ame was used; the gas ow 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 puried 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 certied 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 ve white chocolate
(brands: Neugebauer, Nestl Classic, Lacta Laka, Neugebauer Dupy,
Garoto), ve milk chocolate (Neugebauer, Nestl Classic, Lacta,
Neugebauer Dupy, Garoto), and seven dark chocolate (Neugebauer,

Table 1
Instrumental parameters for element determination in emulsied 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

C2H2air
N2OC2H2
C2H2air
C2H2air
C2H2air
N2OC2H2

Nestl 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 specic 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
inuence of oil and emulsier amounts on emulsion stability. Their
inuence on emulsion stability was evaluated using a 23 full factorial design. The design required a total of nine experiments per oil/
emulsier 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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C.V.S. Ieggli et al. / Food Chemistry 124 (2011) 11891193

3.1. Formulation studies


Emulsions are classied 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 emulsiers to stabilize emulsions was developed by Grifn and it has been used for about ve decades. According to the
Grifn 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 emulsiers 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% emulsier and 4% oil produced, for
sample amounts varying from 0.2% to 8%, emulsication without
coalescence. In all of the experiments in which the amount of oil
was smaller than the amount of emulsier, the separation of
phases took place immediately after preparation. This shows that
an excess of emulsier 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 nely dispersed oil-in-water emulsion with

long-term stability (Lachman, Lieberman, & Kanig, 2001). In the


initial period of stirring the droplets necessary for the emulsication 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 emulsication, 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 nal 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 dened by the
gures of merit, which should be determined experimentally.
The proposed method was validated for six metals (Na, K, Ca, Mg,
Zn and Fe). The gures 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 emulsication procedure for fatty samples
have been described in the literature. When properly stabilized, the
emulsied 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 t 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 conrmed 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 signicant difference between the values obtained with the proposed method and the certied 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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C.V.S. Ieggli et al. / Food Chemistry 124 (2011) 11891193

Table 3
Regression parameters of the metal analytical curves.

a
b
c

Metal

Rangea (mg L

Potassium
Calcium
Sodium
Zinc
Magnesium
Iron

110
0.58
13
0.050.40
0.54.0
0.12.0

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

LODc (mg L

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 signicant 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 signicant
changes were observed when the cesium/lanthanum chloride buffer
was used for the measurements with acetylene-nitrous oxide ame.

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 certied reference baking chocolate (NIST SRM 2384).
Element

Na
K
Ca
Mg
Fe
Zn
a
b
c
d
e

Concentrations lg g

Emulsion compounds

Oil phase

Surfactant

Certied 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% condence 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

SD, n = 3) in different chocolate samples.

Chocolate type

Brand

Cocoa content (%)

Na

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.

C.V.S. Ieggli et al. / Food Chemistry 124 (2011) 11891193

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 emulsication and subsequent metal determination for FAAS fullled these requisites and proved to be sensitive,
reproducible, simple, and economical.
Acknowledgement
The authors thank CNPq (Conselho Nacional de Desenvolvimento Cientco e Tecnolgico) for scholarships.
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