Food Chemistry 130 (2012) 209213

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

Analytical Methods

Cloud point extraction for speciation of iron in beer samples by spectrophotometry

Hayati Filik , Derya Giray
Istanbul University, Faculty of Engineering, Department of Chemistry, Avcilar, 34320 Istanbul, Turkey

a r t i c l e

i n f o

Article history:
Received 18 October 2010
Received in revised form 8 June 2011
Accepted 5 July 2011
Available online 14 July 2011
Keywords:
Iron
Speciation
Cloud point extraction
Spectrophotometry
Beer analysis

a b s t r a c t
A new method based on the cloud point extraction (CPE) separation and spectrophotometric detection
was proposed for the determination of iron species. In this method, Fe(II) reacts with 2-(5-bromo-2pyridylazo)-5-diethylaminophenol (5-Br-PADAP) in the presence of EDTA yielding a hydrophobic complex, which then is extracted into surfactant-rich phase. Total iron was determined after the reduction
of Fe(III) to Fe(II) by using ascorbic acid as reducing agent. Variable parameters affecting the CPE efciency were evaluated and optimised. The calibration graph was linear in the range of 5.0112 lg/L (at
742 nm) for both species. Under the optimised conditions, the detection limits of 0.8 lg/L and 1.0 lg/L
and the relative standard deviations of 2.0% and 2.6% (CFe(II) = CFe(III) = 10 lg/L, n = 5) for Fe(II) and Fe(III)
were found, respectively. The proposed method has been applied to the speciation of iron in beer samples
with satisfactory results.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Major, minor and trace metals are important in beer fermentation since they supply the appropriate environment for yeast
growth and inuence yeast metabolism. The major ions of beer
3
2



are Ca, Mg, Na, K, SO2
4 , PO4 , Cl , CO3 , and NO3 /NO2 . The minor
ions of Fe, Cu, Zn, and Mn may be found in trace amounts (Alcazar,
Pablos, Martin, & Gonzalez, 2002; Pohl, 2008). The content of metals is variable and depends on the quality of substrates taken, the
type of beer brewed, and the country of origin of the beer. The
determination of total metal composition of the beer, including
major, minor and trace metals, is of particular interest to brewers
and consumers (Pohl, 2008; Pohl & Prusisz, 2010).
Under normal conditions, the iron content of fermented beer is
below 0.2 mg/L. Large amounts of iron can give a metallic taste to
beer. Iron salts have a negative action at concentrations above
0.2 mg/L during wort production, preventing complete saccharication, resulting in hazy worts, and hampering yeast activity
(Alcazar et al., 2002). The recommended upper limit of iron concentration in brewing liquors and beer is 0.1 mg/L (Pohl, 2008).
Chapon, Louis, and Chapon (1971) reported that beer sample can
reduce a complex of Fe(III) with 2,20 -dipyridyl to the Fe(II) complex.
So, it seems that iron ions exist in the reduced state, Fe(II), in fresh
beer. Therefore, Fe(II) ions dominant in fresh beer are oxidised to
Fe(III) ions during the process of beer oxidation. In the presence
of molecular oxygen, Fe(II) ions are oxidised to Fe(III) ions, the electron being accepted by oxygen during the formation of superoxide
Corresponding author. Tel.: +90 212 473 70 70x17739; fax: +90 212 473 71 80.
E-mail address: lik@istanbul.edu.tr (H. Filik).
0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.07.008

(Bamforth, Muller, & Walker, 1993). It seems that these metal-catalysed reactions producing active oxygens may occur in beer during
storage and lead to the staling of beer quality. The Fe(II) ions are
oxidised into ferric ions such as free Fe(III), weakly bound Fe(III),
and strong chelates of Fe(III) (non-haeme Fe(III)) during the process
of beer oxidation. The non-haeme Fe(III) ions increase with beer
staling and nally responsible for beer haze (Kaneda, Kano, Koshino, & Ohya-Nishiguchit, 1992; Pohl, 2008).
Several studies of iron concentration in beer sample have been
reported. A standard method for the determination of iron in beer
using conventional spectrophotometry based on complexation to
1,10-phenanthroline or 2,20 -bipyridine (Ampan et al., 2002;
Fernandes, Ranoel, & Lima, 1995; Pohl & Prusisz, 2010). Atomic
spectrometry techniques mainly ame atomic absorption spectrometry (FAAS) (Ampan et al., 2002; Bellido-Milla, Moreno-Perez,
& Hernandez-Artiga, 2000), graphite furnace atomic absorption
spectrometry (GFAAS) (Svendsen & Lund, 2000), inductively coupled plasma spectrometry (ICP-AES/MS) (Alcazar et al., 2002;
Asfaw & Wibetoe, 2005; Wyrzykowska et al., 2001) are extensively employed in the iron determination. Other spectrometric
techniques proposed for measuring the total iron contents in beer
are integrated atom trap FAAS (IAT-FAAS) (Matusiewicz & Kopras,
1997). However, these methods can only detect the total amount of
Fe(II + III) or a certain valence iron. Different approaches are commonly used to differentiate iron species and to quantify both redox
state forms of iron (Pohl & Prusisz, 2006). The most widely used
techniques for the speciation of iron include liquidliquid extraction (LLE) and solid phase extraction (SPE). However, most of the
above-mentioned methods are complicated and time-consuming.
The use of surfactants in analytical chemistry is providing many

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H. Filik, D. Giray / Food Chemistry 130 (2012) 209213

new possibilities separation/preconcentration based on cloud

point extraction which is emerging as an important practical technique. The CPE method is solvent-free and non-polluting. Now, it
has been applied to the separation of environmental and biological
samples with a complicated matrix. For a more detailed discussion
about CPE phenomenon, it is recommended to consult the literature (Mcintire, 1990). To date, several methods for the determination of iron based on CPE procedures have been described (Ghaedi,
Shokrollahi, Niknam, & Soylak, 2009; Giokas, Paleologos, &
Karayannis, 2002; Kandhro et al., 2010; Liang, Sang, & Sun, 2006;
Ohashi, Ito, Kanai, Imura, & Ohashi, 2005; Ortega, Cerutti, Olsina,
Martnez, & Silva, 2004; Paleologos, Giokas, Tzouwara-Karayanni,
& Karayannis, 2002; Shakerian, Dadfarnia, & Shabani, 2009; Silva
Oliveira & Rocha, 2010; Song & Liang, 2010; S
ahin, Tokgz, &
Bektas, 2010). CPE coupled with spectrophotometry for the
speciation of iron in beer sample has also been not reported.
In most cases, the sample must be ashed or digested with acid
to break down the organic matrix prior to analysis. The interests
for analytical procedures that avoid the complete digestion of solid
or liquid samples have been considerably increased in the last
years. Direct spectrometric analysis of complex samples is very
important and challenging. The aim of the present work was to apply CPE as a separation and preconcentration step combined with
UVVis spectrophotometry for the speciation of iron in beer without sample digestion. The experimental parameters affecting the
CPE efciency were investigated in detail. The proposed method
has been applied to the speciation of iron in beer samples with satisfactory results.
2. Experimental
2.1. Instrumentation
Experiments were carried out using a commercially available
miniature bre-optic based spectrometer (HR4000CG-UVVisNIR, Ocean Optics Inc., EW Duiven, Netherlands) which utilises a
small tungsten halogen lamp (Ocean Optics Inc.) as the light source
and a charge-coupled device (CCD, Ocean Optics Inc.) based detector for measurements. A Varian model SpectrAA 220 FS ame
atomic absorption spectrometer (Mulgrave, Vistoria, Australia),
equipped with a deuterium background corrector and automatic
switching of hollow-cathode lamps were used. The iron hollow
cathode lamp was operated with a current of 5.0 mA. The analytical wavelength at 248.3 nm was used with a spectral bandwidth of
0.1 nm. An airacetylene ame was used with an acetylene ow
rate of 2.0 L/min, an air ow rate of 13.5 L/min and a burner height
of 13.5 mm. The pH values of the solutions were measured by a
Hanna HI 221 pH-meter (Midland, ON, Canada) using the full pH
range of 014.

prepared by dissolving an appropriate amount of 5-Br-PADAP in

5 mL ethanol and 2.0 mL concentrated TX-114 to prepare a
1.0  103 mol/L solution. The 5-Br-PADAP reagent has a low solubility in water but this problem was turned over by the addition of
TX-114. The solutions of various cations and anions used for the
interference study were obtained from the respective inorganic
salts (Aldrich) by proper dilution in distilled water. An acetate buffer solution of pH = 5.0 was prepared by mixing appropriate
amounts of a 0.2 mol/L acetic acid solution and 0.2 mol/L sodium
acetate solution then adjusting pH of the resulting solution. Certied standard reference material TMDA-54.4 (trace element fortied calibration standard), obtained from the National Laboratory
for Environmental Testing Research of Canada (Burlington, Canada).
2.3. Sample preparation
The beer samples were purchased randomly from different
markets in Istanbul, Turkey. All samples were obtained with the
same validity date and were stored at ambient temperature in
the market. Four lager beers were tested. As an analytical sample,
beer is a very complex matrix with a relatively high content of different organic compounds originating from brewery processing
(mostly carbohydrates and proteins) and saturated with carbon
dioxide (CO2). The beer was always de-gassed before further sample handling (Pohl, 2008). Aliquots of this solution (5 mL) were
then analysed by both the proposed analytical procedure and the
standard addition method.
2.4. Procedure
An aliquot of a Fe(II) standard solution (5.0112 lg/L) was
transferred to a 10 mL centrifuge tube, 0.1 mL of 1.0  103 mol/L
5-Br-PADAP solution and 2.0 mL acetate buffer solution (pH 5.0)
was added. It was shaken for 2 min. This was followed by the addition of 1.0 mL of 0.1% (w/v) EDTA and 0.5 mL of 5% (v/v) TX-114
solution. The solution was taken up to mark with distilled water.
Subsequently, the sample was shaken and left to stand in a
thermostatted water bath for 5 min at 50 C before centrifugation.
The phase separation was accomplished by centrifuging for 5 min
at 4000 rpm (2700 g). The mixture cooled in an ice bath to increase
the viscosity of the surfactant-rich phase, and the aqueous phase
was easily decanted by simply inverting the tube. The remaining
micellar phase was diluted with ethanol, until a nal volume of
0.5 mL was reached. The absorbance intensity was measured at
742 nm against a reagent blank prepared with distilled water. Total
iron was determined after the reduction of Fe(III) to Fe(II) by using
ascorbic acid as reducing reagent. Then, the concentration of Fe(III)
was calculated by subtracting the concentration of Fe(II) from the
total iron concentration.

2.2. Chemicals and reagents

3. Results and discussion
All reagents were analytical grade and used without further
purication. Triton X-114 (TX-114) (SigmaAldrich, St. Louis, MO)
was used without further purication to prepare a 5% w/v aqueous
solution. Standard stock solutions of Fe(II) and Fe(III) were prepared
by dissolving appropriate amounts of (NH4)2Fe(SO4)2.6H2O and
NH4Fe(SO4)2.12H2O (Merck, Darmstadt, Germany) in 0.1 mol/L
HCl. Working standard solutions in the range of 5112 lg/L were
prepared daily at the appropriate dilution. EDTA 0.10% (w/v) stock
solution was prepared by dissolving the requisite amount of EDTA
(Merck) in distilled water. Ascorbic acid (Merck) 1.0% (w/v) stock
solution was prepared by dissolving 1.0 g of ascorbic acid in
100 mL of distilled water. The solution was freshly prepared every
day. The 5-Br-PADAP (Aldrich, Milwaukee, WI, USA) solution was

The reaction of iron(II) with 5-Br-PADAP in aqueous media has

been studied by several authors, and the experimental conditions
are established (Costa, Ferreira, Andrade, & Lobo, 1993; Ferreira
et al., 2007; Wei, Song, Yin, & Shen, 1983). The absorption spectrum of the Fe(II)5-Br-PADAP complex in surfactant-rich phase
shows a maximum absorbance at 555 and 742 nm. The absorption
at 748 nm is signicantly lower than at 555 nm, however, at
742 nm the method is completely selective for iron(II). Fig. 1 shows
the Uv/Vis absorbance spectra of standard solution (b) and real
beer sample (a) after cloud point extraction. There were no significant differences seen between two analyte absorbance signal
proles.

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H. Filik, D. Giray / Food Chemistry 130 (2012) 209213

and Fe(III), a pH of 5.0 was selected for subsequent work. This

behaviour is in accordance with the results of previous studies
(Costa et al., 1993; Ferreira et al., 2007).
3.1.2. Effect of 5-Br-PADAP concentration
The effect of 5-Br-PADAP concentration on the analytical performance was studied a Fe(II) solution and various concentrations of
the reagent in the range of 2.0  1062.0  105 mol/L. The results showed that the signal of Fe(II) was increased with increasing
of 5-Br-PADAP concentration up to 5.0  106 mol/L, and then remained constant. As shown in Fig. 3, a nal concentration of
1.0  105 mol/L was sufcient for optimum results. This concentration ensures sufcient excess to compensate for any consumption of the reagent by other metals.

Fig. 1. Absorption spectra of Fe(II) after CPE. (a) Beer sample (concentrated volume
5 mL): 1  105 mol/L 5-Br-PADAP, 0.02% (w/v) EDTA, 0.25% (v/v) TX-114. (b)
Standard sample: 1  105 mol/L 5-Br-PADAP, 0.02% (w/v) EDTA, 0.25% (v/v) TX114, 12.5 lg/L Fe(II).

3.1. Optimisation of cloud point extraction

3.1.1. Effect of pH
The pH of the aqueous solution is an important factor in CPE of
Fe(II) using 5-Br-PADAP, because this parameter is directly related
to the formation of metalligand species. The effect of pH on the
CPE efciency of Fe(II) was studied in the pH range 3.08.0 and
the results are shown in Fig. 2. As can be seen, maximum absorbance was obtained in the pH range 5.06.0. At pH 3.76.0, the
Fe(III)-Br-PADAP complex is completely decomposed in the presence of EDTA. This makes it possible to separate Fe(II) from Fe(III)
through the control of pH of the solution. In order to separate Fe(II)

3.1.3. Effect of surfactant concentration

The variation of extraction efciency upon the surfactant concentration was examined within range: CTX-114 = 0.051.0% (v/v).
The results showed that the absorbance of the solutions increased
by increasing the Triton X-114 concentration up to 0.25% (v/v) and
remained nearly constant between 0.25% and 1.0% (v/v). Therefore,
0.25% (v/v) TX-114 was used in the proposed method. Using more
than 0.25% of surfactant, the analytical sensitivity decreased due to
dilution of the sample by additional surfactant solution.
3.1.4. Effect of ionic strength
The effect of ionic strength at the analytical signal was examined by establishing various concentrations of NaCl in the range
of 00.1 mol/L in the sample solutions. No effect was observed
on the absorbance signal of Fe(II).
3.1.5. Equilibration temperature, equilibration and centrifugation time
To achieve easy phase separation and preconcentration as efcient as possible, optimal incubation time and temperature are
necessary for complete reactions. The dependence of extraction
efciency on equilibration temperature and time were studied in
the range of 2560 C and 510 min, respectively. The results
showed that an equilibration temperature of 50 C and equilibration time of 5 min are adequate to obtain quantitative extraction.
The effect of the centrifugation time on the extraction efciency

0.80

0.80

Fe(II)

0.60

Absorbance

Absorbance

0.60

0.40

0.20

0.40

0.20
Fe(III)

0.00
3

6
pH

0.00

10

15

5-Br-PADAP concentration (x10

Fig. 2. Effect of pH on the CPE of Fe(II) and Fe(III). Extraction conditions:
1  105 mol/L 5-Br-PADAP, 0.02% (w/v) EDTA, 0.25% (v/v) TX-114, 75 lg/L Fe(II)
and 75 lg/L Fe(III) (n = 3).

20
-6

mol/L)

Fig. 3. Effect of 5-Br-PADAP on the CPE of Fe(II). Extraction conditions: 0.02% (w/v)
EDTA, 0.25% (v/v) TX-114, 75 lg/L Fe(II) (n = 3).

212

H. Filik, D. Giray / Food Chemistry 130 (2012) 209213

Table 1
Determination of iron species in beer samples (n = 5).

a
*

FAAS (lg/L)

Found (lg/L)

Total Fe

Fe(II)

104.6 0.07

102.1 0.7

Bottled
330 mL

99.2 0.08

Marmara

Canned
500 mL

Carlsberg

Bootle
330 mL

Beer samples

Packing

Efes Pilsen

Canned
500 mL

Becks

tcala

Spiked (lg/L)

Found (lg/L)

Fe(III)

Fe(II)

Fe(III)

Fe(II)

Fe(III)

Fe(II)

Fe(III)

nd*

25

25

127.4 0.3
102.2 0.6

24.6 0.4

101

98

1.65
1.26

97.4 1.0

nd*

25

25

122.8 0.7
97.4 0.9

24.3 0.7

102

97

1.07
1.35

107.3 0.05

108.2 0.8

nd*

25

25

133.5 0.8
108.4 0.3

24.2 0.6

101

97

1.92
1.45

94.8 0.02

93.1 0.5

nd*

25

25

118.2 0.9
93.1 0.5

25.1 0.9

100

100

0.54
1.05

Recovery (%)

Theoretical value for t = 2.77 (P = 0.05).

nd: not detected,

was also studied within a range of 110 min. A centrifugation time

of 5 min at 4000 rpm was selected as optimum, since complete
separation occurred for this time and no appreciable improvements were observed for longer times.
3.2. Other conditions
3.2.1. Effect of EDTA
The Fe (II)-5-Br-PADAP complex is more stable than Fe(III)5-Br-PADAP complex at pH 37 (Oszwaldowski & Okada, 1999)
and even EDTA cannot remove Fe(II) from Fe(II)5-Br-PADAP
complex. However, Fe(III)-5-Br-PADAP complex is decomposed
totally in presence of EDTA (Costa et al., 1993). Fe(II)5-Br-PADAP
chelate is electrically neutral (as Fe(II)L20); whereas the Fe(III)(5-Br-PADAP) chelate is positively charged (as Fe(III)L2+) and the
Fe(III) chelate is prone to the formation of the ion-pairing species
(Oszwaldowski & Okada, 1999; Oszwaldowski & Pikus, 2002). Using
EDTA as masking agent increased the selectivity of the CPE procedure. The effect of the EDTA concentration on the absorbance intensity of Fe(II) was studied. The EDTA concentration was varied in the
range 01.0  103 mol/L in order to maximise the absorbance
signal. The maximum colour intensity was obtained in the concentration range 2.7  105 and 1.0  103 mol/L EDTA. Therefore, a
concentration of 5.4  104 mol/L was chosen to account for other
extractable species that might potentially interference with the
assaying of Fe(II). The lowest signal intensity was observed in the
absence of EDTA. EDTA can be tolerated up to 1.0  103 mol/L.
3.2.2. Effect of the order of addition of reagents
The best order of reagent addition is as follows: Fe(II), chromogenic reagent (5-Br-PADAP), buffer solution (pH = 5.0), EDTA and
TX-114. EDTA can be added after 2 min complex formation. EDTA
solution must be added after the formation of Fe(II)5-Br-PADAP
complex, in order to guarantee the stability and reproducibility
of the CPE system. Other sequences gave lower absorbance values
compared with the above sequence. It was observed that the ascorbic acid behaves as a reductant, thus it must always be added before the buffer addition as the acid medium increases Fe(III)
reduction. Ascorbic acid was selected as a reducing agent because
it is efcient and friendlier to the environment. In order to determine the optimal concentration of the ascorbic acid, concentrations between 0.01% and 0.1% (w/v) were studied. Fe(III)
reduction was complete above 0.02% ascorbic acid and remained
constant up to 0.1% (w/v). A concentration of 0.05% (w/v) ascorbic
acid was chosen for subsequent experiments.
3.2.3. Effect of diluting agent
The surfactant-rich phase was very viscous; ethanol was added
to the surfactant-rich phase after CPE to facilitate its transfer into

spectrophotometric cell. The amount of 0.3 mL ethanol (i.e. nal

volume of 0.5 mL) was chosen to have an appropriate amount of
sample for transferring and measuring the sample absorbance. Because the pH of the nal ethanolic solution was already approximately 5, no extra pH-adjustment of the nal solution was
required. The pH range should be between 5.0 and 6.0. At pH values below 5.0 or above 6.0 the absorbance signal decreased
signicantly.
3.3. Analytical performance
A calibration curve was constructed by preconcentration of
10 mL of sample standard solutions with TX-114. From measurements made under the optimum conditions described above, the
calibration graph was linear in the range of 5.0112 lg/L (at
742 nm) for both species. The calibration equation is A =
0.00895C + 0.018 with a correlation coefcient of 0.9987 (n = 5),
where A is the absorbance and C is the concentration of Fe(II) in
the initial sample solution in lg/L. The limit of detection (LOD), dened as CL = 3SB/m (where CL, SB, and m are the limit of detection,
standard deviation of the blank, and slope of the calibration graph,
respectively). The LOD values of 0.8 and 1.0 lg/L were obtained for
Fe(II) and Fe(III), respectively. The relative standard deviations
(RSD) resulting from the analysis of ve replicates of 10 mL solution
containing 10 lg/L Fe(II) and Fe(III) were 2.0% and 2.6%, respectively. The preconcentration factor, calculated as the ratio between
the volume of the aqueous phase and the nal volume (0.5 mL) of
the surfactant-rich phase, was 20.
3.4. Interferences
The effects of representative potential interfering species were
tested. In these experiments, solutions of 50 lg/L of Fe(II) and
the added interfering ions were treated according to the recommended procedure. Metal ions as Ca(II), Mg(II), Cu(II) and Mn(II)
and others, which also react with 5-Br-PADAP and that could be
present in beer samples do not interfere because the analytical
measure is done at 742 nm and the 5-Br-PADAP complexes of these
metal ions have maximum absorption at 555 nm. To increase the
tolerance limits (i.e. increases the selectivity), the addition of EDTA
as masking agent clearly improves the situation.
3.5. Analysis of real samples
The accuracy of the proposed method, a certied reference
water sample (TMDA 54.4) was analysed and the analytical result
(379 2 lg/L) was in good agreement with the certied value
(382 5 lg/L).

H. Filik, D. Giray / Food Chemistry 130 (2012) 209213

The proposed method was applied for the speciation of Fe(II)

and Fe(III) in different beer samples. However, the total iron content of the beer was also determined (i.e. FAAS) after acid decomposition of the beer (Bellido-Milla et al., 2000; Pohl & Prusisz,
2007). In addition, the recovery experiments of different amounts
of Fe(II) and Fe(III) were carried out, and the results are shown in
Table 1. Recoveries for Fe(III) and Fe(II) were found in the range
of 97102%, indicating that the method enables the determination
of both species in the analysed samples without matrix effects. A
comparison using t-test at 95% condence interval demonstrates,
that there is no signicant difference amongst the achieved results
using the proposed method and the FAAS method (Table 1). The
present method is able to detect both Fe(II) and Fe(III) ions, it
was found that only Fe(II) ion was detected in beer samples by
means of the present method. The Fe(II) concentrations obtained
are always higher than the Fe(III) contents. It can be explained considering the reductive character of the beers.
4. Conclusion
To simplify and improve the speed of beer analysis, the acid
digestion step was eliminated. The method proposed is opportune,
considering the analytical problem that is the Fe(II) determination
in the presence of Fe(III). The proposed CPE preconcentration
method exhibits good precision, accuracy, sensitivity and selectivity which allows iron determination in beer samples at lg/L levels.
From the results, it can be considered that the recommended procedure is a suitable technique for routine analysis of different species of iron in aqueous samples. The limit of detection of the
method (LOD) is better or comparable to some of the previously reported CPE methods (Ghaedi et al., 2009; Giokas et al., 2002; Kandhro et al., 2010; Paleologos et al., 2002; Shakerian et al., 2009;
Silva et al., 2010; Song & Liang, 2010).
Acknowledgement
The authors gratefully acknowledge the nancial support from
_
the Scientic and Technical Research Council of Turkey (TBITAK
Grand No: 109T856).
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