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International Journal of Food Science and Technology 2000, 35, 371]375 371

Copper and iron determination in edible vegetable oils by

graphite furnace atomic absorption spectrometry after
extraction with diluted nitric acid

Antonella De Leonardis,* Vincenzo Macciola & Martino De Felice

Dipartimento di Scienze e Tecnologie Agro-Alimentari, Ambientali e Microbiologiche, UniversitaÁ del Molise, Via De Sanctis,
86100 Campobasso, Italy
(Received 8 September 1998; Accepted 18 October 1999)

Summary In this article, a new method for determining copper and iron in edible vegetable oils by using
atomic absorption spectrometry is discussed. The metals were extracted from low quantities of
oil (1]2 g) with a 10% nitric acid solution. The acid solution obtained can be injected directly
into the atomic absorption furnace. The proposed method is simple and allows copper and iron
to be determined in edible vegetable oils with a precision estimated below 15% relative standard
deviation (RSD) for Cu and 10% for Fe.
Keywords Atomic absorption spectrometry, copper, iron.

In the direct methods the oil sample, after being

Introduction
In edible vegetable oils, although copper and iron are
present in very low quantities (ppb) they are very
important mineral elements because they influence
the oxidative processes. The determination of these
metals in the oils needs specific analytical techniques
that are very sensitive and often complicated such as
the neutronic activation (Cichelli et al., 1992),
spectrometry with a plasma torch and atomic
absorption (Castera-Rossignol, 1996).
The most commonly used technique is atomic
absorption spectrometry with atomization in a
graphite furnace, and numerous methods are avail-
able to determine the metals in the oils by this
technique. These essentially vary according to the
procedures of the sample preparation before the
instrumental measurement. In the indirect methods,
the sample is mineralized by using dry-ashing (Evans
et al., 1971; Ooms & Van Pee, 1983), high pressure
bomb digestion, microwave heating or oxygen-
plasma ashing (Sun, 1989). In such cases, the
preparation of the sample is long and laborious.
*Correspondent: Fax: 139 874 404652;
email: Antomac@hpsrv.unimol.it
dissolved in an organic solvent such as methyl]iso-
butyl]ketone (MIBK) or xylene, is injected into the
atomic absorption furnace without other treatments
(McKenzie, 1981; Nash et al., 1983; Casetta &
Aldrighetti, 1986; Farhan et al., 1988; IUPAC,
1988). In this case, the preparation of the sample
was very simple and rapid.
In previous work (De Leonardis et al., 1997), we
compared direct methods of analysis of the oil
sample dissolved in m-xylene with indirect analysis,
by using dry-ashing of the sample and metal recovery
in an acid medium. Both the methods presented
advantages and limits. The direct analysis of the
xylene]oil mixture presented the following instru-
mental problems: the excessive viscosity of the
solvent affected the operative parameters during the
injection phase; during the ashing phase in the
graphite furnace it was essential to use air or oxygen
and this reduced the life of the carbon tube; smoke
was frequently generated and this altered the signal
in the atomization step; the results obtained were not
repeatable.
By using the dry-ashing method, the final solution
obtained was acid and was easily analyzed with the
atomic absorption spectroscopy, but the contamina-

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372 Copper and iron in edible vegetables A. De Leonardis et al.

tion or loss of metals that could occur during the

preparation of the sample exercised a negative
influence on the repeatability of the results.
In this paper we report an investigation on the
feasibility of direct extraction of copper and iron
from vegetable oils using a dilute nitric acid solution
before atomic absorption analysis.
Materials and methods
Instrumentation
The atomic absorption spectrometer was a model
SpectrAA 250 from Varian (Mulgrave, Victoria,
Australia) equipped with a GTA-96 graphite furnace
and a programmable autosampler. The instrumental
parameters and furnace operating conditions are
given in Table 1.
Reagents and containers
All reagents and standard stock solutions used were
from C. Erba (Rodano, Milan, Italy). The concen-
trated nitric acid (HNO3) was superpure, specific for
trace analysis, and was diluted to 10% (v/v)
concentration with deionized water. All containers,
including test tubes with stoppers, were in poly-
propylene and were cleaned with 5% (v/v) hydro-
chloridric acid and the deionized water. The blank
consisted of the 10% dilute nitric acid used for
extraction.
Preparation of standards
The first standard stock solutions had a 1mg ml21
concentration of copper and iron chloride respec-
tively and these were diluted with 10% nitric acid to
give the working solutions. The concentration ranges
of the working solutions were 2]50 ppb for copper
and 50]300 ppb for iron. The procedure of the spiked
standard preparation was the following: 1 ml of 10%
nitric acid containing 50, 25, 12, 6 ppb of copper and
300, 200, 150, 75 ppb of iron respectively, was added
to 2.0 g of an extra virgin olive oil, exactly weighed.
The extraction was performed in accordance with the
published procedure for the preparation of the
sample.
Preparation of sample
The types of oil samples analyzed were: extra virgin,
virgin and refined olive oils, soybean and sunflower
oils. An aliquot (2.0]3.0 g) of every oil was weighed
directly into the test tube and 1 ml of the 10% dilute
nitric acid was added. The oil]acid mixture was
shaken at 40 Hz for 30 s with a test tube mixer until
the layers were completely mixed. The capped test
tube was placed in a shaking water bath at 50 8C for
2 h. After centrifugation at 3000 rpm for 5 min, the
lower acid aqueous layer was withdrawn with a
polypropylene pipette and was loaded directly into
the autosampler of the atomic absorption spectro-
meter.

Table 1 Instrumental parameters

Fe Cu
and furnace operating condition
lamp current (mA) 5 3
wavelength (nm) 372 324.7
slit width (nm) 0.2 0.5
measurement mode peak height
graphite tube partition pyrolitic coated
gas argon
- flow 3 L m21
sample volumes inject 15 mL
hot inject 60 8C 3 1 s
termic program temperature (8C) time (s)
- Step 1 80 10
- Step 2 100 55
- Step 3 120 10
- Step 4 700 22
- Step 5 2300 8
- Step 6 2700 5

International Journal of Food Science and Technology 2000, 35, 371]375 ã 2000 Blackwell Science Ltd
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Copper and iron in edible vegetables A. De Leonardis et al. 373

Figure 1 Recorded absorption Copper Iron

values of the copper and the iron
600 600
relative to the same oil sample
incubated at 508C fro 2, 3 and 4 500 500

ABS × 1000 (324.7 nm)

ABS × 1000 (372 nm)

hours in the shaking water both.
400 400

300 300

200 200

100 100

0 0
2 hr 3 hr 4 hr

Results and discussion showed that there was a significant difference

Extractions from the same oil sample at different
times demonstrated that incubation in a shaking
water bath for 2 h was sufficient to recover all the
copper and the iron present in the oil sample.
Figure 1 shows that after 2 h the recorded absorbance
values were very low and were below the detection
limit indicated in the technical characteristics of the
instrument.
The analysis of the blank demonstrated that there
was no contamination of copper and iron by the
reagents or polypropylene containers.
In Figs 2 and 3 the calibration curves are reported
for copper and iron respectively. For each element,
two calibration studies were performed: one using the
metal standards in 10% dilute nitric acid and the
other using the standard addition method. The results
between the two slopes for the copper, while the
difference was very slight for the iron. These results
indicated that for copper a standard addition calibra-
tion had to be used, while for iron it was possible to
use the external standard calibration curve.
In Table 2 the average quantities of copper and
iron (expressed in ppb) recovered in four different
trials as regards to the additional standards are
reported for the experimental conditions which we
operated. The accuracy of the results, estimated in a
per cent average of the standard addition recoveries,
was 94 6 23 for copper and 97 6 12 for iron. It is
evident, especially for the copper, that when the
concentration was very low the results obtained were
substantially influenced by the noise and the instru-
mental interference.
The results from the analysis of the different oil

Figure 2 Comparison of an external 1000

y = 15.99 x + 141
calibration acid solutions with a 900 R2 = 0.9972
standard addition curve for copper. 800
700
ABS × 1000

600
500
400
300 y = 9.88 x
200 R2 = 0.9941
100
0
–10 0 10 20 30 40 50
ppb
copper standard in copper standard added to
acid solution extra virgin olive oil

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374 Copper and iron in edible vegetables A. De Leonardis et al.

Figure 3 Comparison of an external 1200

calibration acid solutions with a y = 2.15 x + 317
1000 R2 = 0.9764
standard addition curve for iron.

800

ABS × 1000
600

400 y = 2.15 x
R2 = 0.9904
200

0
–150 –100 –50 0 50 100 150 200 250 300
ppb
iron standard in iron standard added to
acid solution extra virgin olive oil

Table 2 Averages of the standard addition recoveries obtained Table 3 Averages of the copper and iron contents obtained
by four independent determinations from every oil sample by four independent extractions

ppb Cu Fe
Element added ppb Accuracy (%) Sample oil ppb RSD (%) ppb RSD (%)
found

Cu 6 5 ‹ 2 Extra-virgin olive 4 ‹ 0.3 9.3 53 ‹ 2.4 4.6

12 11 ‹ 2 Virgin olive 6 ‹ 0.8 15.0 517 ‹ 15.4 3.0
25 27 ‹ 3 Refined olive 14 ‹ 0.3 1.9 80 ‹ 6.7 8.5
50 49 ‹ 5 94 ‹ 23 Soybean 1 ‹ 0.0 1.6 52 ‹ 5.4 10.3
Sunflower 2 ‹ 0.2 13.1 94 ‹ 3.2 3.4
Fe 75 67 ‹ 13
150 146 ‹ 13
200 201 ‹ 16
300 303 ‹ 14
97 ‹ 12
allows for a reduction in the preparation time and
the manipulation of the samples when compared to
the dry-ashing method. Additionally, the final
solution obtained can be analyzed directly by atomic
samples are reported in Table 3. The reproducibility absorption spectrometry without all the instrumental
obtained with four independent extractions of oil problems that would occur with the direct analysis of
samples was good and the results show that with this the xylene]oil mixture.
method it was possible to determine copper and iron
in edible vegetable oils with a precision estimated to
below 15% RSD for Cu and 10% for Fe. Acknowledgments

This study was financially supported by a University

Conclusion of Molise research fund for the year 1997.

In conclusion we have demonstrated that the

extraction with dilute nitric acid is a reliable method
of sample preparation for the determination of copper
and iron in edible vegetable oils by graphite furnace
atomic absorption spectrometry. It is very important
to use containers of polypropylene because glass
provides a substantial contamination. Even when
using very low quantities of oil samples is it possible
to obtain good results. The proposed procedure
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