Determination of lead in milk and yoghurt

samples by solid phase extraction using a novel

aminothioazole-polymeric resin
Abstract
A preconcentration method was developed by using a new aminothioazole-containing
sulfonamide resin in solid phase extraction for the determination of lead and nickel in milk
and yoghurt samples. The optimisation of experimental conditions was performed. The
optimum parameters for Pb and Ni were found to be 3.5, 30 min and 2.5 mL for pH, contact
time, eluate volume, respectively. After preconcentration step, atomic absorption
spectrometry was used for the determinations. The enhancement of 350- and 50-fold in the
sensitivity of Pb and Ni were achieved by combination of the slotted tube atom trap-atomic
absorption spectrometry with the optimised preconcentration method, respectively. Limits of
detection were found to be 0.15 ng mL1 for Pb and 0.75 ng mL1 for Ni. The lead
concentrations in the studied samples were found to be in the range of 1561 ng Pb mL1 for
milk and 2142 ng Pb g1 for yoghurt samples.

O metod de preconcentrare a fost dezvoltat prin utilizarea unui noua

aminothioazole coninnd rin sulfonamid n extrac ie n faz solid
pentru determinarea plumb i nichel nprobe de lapte i iaurt . A fost
realizat optimizarea condiiilor experimentale. Parametrii optime pentru Pb
i Ni s-au dovedit a fi 3,5, 30 de minute i 2,5 ml pentru pH, timp de
contact, respectiv volum eluat,. Dup primul pas care a fost
preconcentrarea ,a fost folosita spectrometrie de absorb ie atomic pentru
determinri. mbuntirea 350- i 50 ori a sensibilitii Pb si Ni au fost
realizate prin combinarea atom tub-capcan atomic spectrometria de
absorbie cu fante cu metoda de preconcentrare optimizat,
respectiv. Limitele de detectare s-au dovedit a fi 0,15 ng ml-1 pentru Pb i
0,75 ng ml-1 pentru Ni. Concentraiile de plumb n probele studiate au
dovedit a fi n intervalul de 15-61 ng Pb ml-1 pentru lapte i 21-42 ng Pb g1 pentru probele de iaurt.

Highlights
A new modified resin, aminothioazole-containing sulfonamide resin was synthesised.
The synthesised resin was characterised and used for preconcentration. Lead and nickel
levels in milk and yoghurt samples were determined using the resin. The synthesised resin

has high adsorption capacity. The resin can also be examined to remove Pb and Ni from
polluted waters.

Keywords

Lead;
Nickel;
Preconcentration;
Aminothioazole-containing sulfonamide resin;
Milk;
Yoghurt

1. Introduction
There are high interested in the determinations of Pb and Ni in environmental, food and
biological samples due to their extremely toxic for human and animal health. It is known that
lead is health-endangering element for human and its effects include blood enzyme
changes, anaemia, hyperactivity, and neurological disorders (Wang et al., 2009). Lead can
also cause pathophysiological changes in several organ systems including central nervous,
renal, hematopoietic and immune system (Mishra, 2009). Among these damages, one of the
most important issues is that lead can strongly affect intelligence development of children.
Further, inorganic lead compounds are classified as probably carcinogenic to humans (group
2A) by the International Agency for Research on Cancer (IARC, 2006 and Yaman, 2006).
Nickel is a nutritionally essential trace metal for, at least, several animal species, microorganisms and plants (Cempel & Nikel, 2006). It is also known that Ni can cause allergic
reactions and certain Ni compounds have carcinogenic effects (Yaman, 2006). Therefore, its
either deficiency or toxicity symptoms can be occurred when too little or too much Ni is
taken up, respectively. World Health Organization (WHO) established the provisional
tolerable weekly intakes (PTWI) of Pb as 25.0 g kg1 body weight (equivalent to
3.5 g kg1 of body weight per day) for all human groups on the basis that lead is a
cumulative poison, and Ni was updated by WHO, in 2005, as 12 g Ni kg1 of body weight
(Bilandzic et al., 2011, WHO, 1995 and WHO, 2005). As a result, allowable Pb concentrations
in beverages, particularly, step down to the lower values, in recent times. Regarding these
results, the allowable Pb concentration in milk samples was established as 20 g L1 by
authorised committee of Turkish (Turkish Food Codex, 2010). Among lead and nickel sources
in daily life, beverages including milk are very important sources. In case of lead, the major
protein fractions of milk alpha-casein interaction with Pb(II) through cysteine and sulfhydryle
are of strong binding sites as reported by Srinivas, Kaul, and Prakash (2007). On the other
hand, the most affected group from lead toxicity is children because of their incomplete
development. In view of this point, milk is another important food which should be checked
against the determination of toxic metal concentrations, constantly. Briefly, there is a high
interested to determine those metals in milk, yoghurt and similar food samples (Chakraborti

et al., 1987, Linsinger, 2005, Maas et al., 2011, Chakraborti et al., 1987 and Orun et al.,
2011).
Due to the allowable low Pb and Ni concentrations in milk samples as well as their low
concentrations in the matrices described above, reliable and sensitive analytical methods
are required for the determinations of these elements. For this purpose, inductively coupled
plasma-atomic emission spectrometry (ICP-AES), electrothermal atomic absorption
spectrometry (ETAAS), voltammetry and inductively coupled plasma-mass spectrometry
(ICP-MS) have been mostly used. Although ETAAS is a more sensitive technique than the
flame atomic absorption spectrometry (FAAS), ETAAS has some disadvantages such as
slowness of procedure, greater proneness to interferences and high cost (Pereira and Arruda,
2003 and Yaman, 2005). For example, a period of 2 or 3 min is sometimes necessary for one
measurement by ETAAS against only a few seconds are sufficient for FAAS. In addition, FAAS
is more economical, and does not require expert operators. On the other hand, direct
determinations of trace level of heavy metals in environmental and food samples by FAAS
are difficult due to low sensitivity of this instrument. Some isolation and/or preconcentration
procedures prior to the determination step can be used to overcome these limitations ( Kaya
et al., 2008b and Yaman, 1998). Due to severe interference effects coming from the matrix
such as salinity, preconcentration/separation procedures for Pb and Ni determinations in
water samples have been used to minimise the matrix effects before ETAAS and ICP-MS
measurements (Chakraborti et al., 1987,Rivas et al., 2009 and Sekhar et al., 2003). Among
preconcentration methods, solid-phase extraction (SPE) has been found a great attention in
recent years (Kaya et al., 2008b and Sekhar et al., 2003). Low amount of waste generated,
high potential for the re-use of solid phase, low matrix effect, high preconcentration factor
and no need to use of toxic solvent are the advantages of SPE when compared with the
other separation and preconcentration methods (Karaaslan et al., 2010, Senkal et al.,
2007,Yaman, 1998, Yaman, 2000 and Yaman, 2001).
It can be said that some of the preconcentration methods are not practical to be used due to
very long preconcentration periods up to 24 h (Venkatesh et al., 2005 and Yavuz et al.,
2009). Rapid and simple preconcentration procedure without using any complexing reagent
can be achieved by using proper adsorbent resin. For this purpose, the use of polymerbonded ligands in preconcentration of metals has been the subject of many research articles
(Karaaslan et al., 2010 and Senkal et al., 2007). Finally, slotted tube atom trap (STAT) has
been used to enhance the sensitivity of FAAS for some elements (Kaya and Yaman,
2008 and Yaman, 2005).
In this study, an aminothioazole-containing sulfonamide resin derived from crosslinked
chlorosulfonated polystyrene (CCPS) was synthesised and characterised. Then, experimental
parameters were optimised for the separation and preconcentration of Pb and Ni at
ng mL1 level by using batch method. The method is based on the SPE of Pb and Ni ions on
the synthesised resin without using any complexing reagent in preconcentration step.
Another purpose of this study was to figure out the lead and nickel contents of milk and
yoghurt samples consumed in Elazig city (with population of 320 000) of Turkey. Due to the
lack of relevant data in this region, the study was focused on the determinations of lead and
nickel in the mentioned beverages. For this purpose, the optimised method was applied to

the digested milk and yoghurt samples for determinations of their Pb and Ni contents. After
the preconcentration procedure, STAT-FAAS was used for improving in the sensitivity of FAAS
for Pb because any increase in the sensitivity of FAAS for Ni was not observed. To check the
reliability of the results, the certified reference material (BCR 151 Skim Milk Powder) was
analysed. Further, the recoveries of Pb and Ni from milk samples fortified with these metals
were found, and some milk samples were also analysed by ICP-MS for their Pb and Ni
contents to the validation the method.

2. Experimental
2.1. Apparatus and reagents
ATI UNICAM Model 929 flame atomic absorption spectrophotometer equipped with ATI
UNICAM hollow cathode lamp (HCl) was used for the determination of Pb and Ni (Unicam
Ltd., Cambridge, England). The optimum conditions for Pb and Ni by FAAS were applied as
follow: wavelength: 217.0 and 232.0 nm, respectively; HCL current: 7.5 mA for both
elements; acetylene and air flow rates: 0.5 and 4.0 L min1 for both elements; slit width: 0.5
and 0.2 nm, respectively. The FT-IR spectroscopy was used for characterisation of the
synthesised resin. To assess the reliability of measurements, some samples were analysed
by PerkinElmer Elan 9000 ICP-MS (Perkin Elmer SCIEX, Concord, Ontario, Canada). The
operation conditions for this instrument were applied as recommended by the
manufacturers.
The STAT described in previous studies (Kaya and Yaman, 2008 and Yaman, 2005) was used
to enhance the Pb sensitivity by FAAS. The pH was measured with (SCHOTT Lab Star pH,
Mainz, Germany) pH meter. In the preconcentration procedure, magnetic stirrers (Velp
Scientific, Milano, Italy) and Hettich EBA III centrifuge (Tutlingen, Germany) were used.
All glass apparatus (Pyrex) were kept permanently in full of 1.0 mol L1 nitric acid when not
in use. Concentrated HNO3 (Merck, Darmstadt, Germany) was used for decomposition of
adsorbed Pb and Ni on the synthesised resin surface. Concentrated hydrogen peroxide
(Merck) and perchloric acid (Merck) together with nitric acid were used for digestion of milk
sample. The diluted standard solutions were prepared from 1000 mg L1 of stock standard
solution of lead and nickel (Merck). Milk powder (BCR 151) as standard reference material
was used to check validation of results.
The buffer solutions in the pH range of 1.56.0 0.1 were prepared by using
0.1 mol L1 citric acid plus 0.1 mol L1 HCl/0.1 mol L1 NaOH solutions. The synthesised resin
was ground and sieved by using a 325-mesh sieve.

ATI UNICAM Modelul 929 Spectrofotometru de absorb ie atomic in

flacara ATI UNICAM Modelul 929 echipat cu lamp cu catod tubular (HCI)
ATI UNICAM a fost utilizat pentru determinarea Pb i Ni (Unicam Ltd.,
Cambridge, Anglia). Condiiile optime pentru Pb i Ni prin FAAS au fost
aplicate dup cum urmeaz: lungime de und: 217,0 i respective 232,0
nm,;HCL curent: 7.5 mA pentru ambele elemente; acetilena i rata debitului

de aer : 0.5 i 4.0 L min-1 pentru ambele elemente; Limea fantei: 0,5 i
respective 0,2 nm,.Spectroscopia FT-IR a fost utilizat pentru caracterizarea
rinii sintetizate. Pentru a evalua fiabilitatea msurtorilor, unele probe au
fost analizate prin Perkin-Elmer Elan 9000 ICP-MS (Perkin Elmer SCIEX,
Concord, Ontario, Canada). Au fost aplicate condiiile de funcionare pentru
acest instrument, conform recomandrilor de ctre productori.
STAT-ul descris n studiile anterioare (Kaya i Yaman, 2008 i Yaman,
2005) a fost utilizat pentru a spori sensibilitatea Pb de FAAS. PH-ul a fost
msurat cu (SCHOTT Lab Steaua pH, Mainz, Germania) pH-metru.
n cadrul procedurii de preconcentrare, au fost utilizate agitatoare
magnetice (Velp tiinific, Milano, Italia) i centrifuga Hettich EBA III
(Tutlingen, Germania)
.Toate aparat de sticl (Pyrex) au fost inute n permanen n acid azotic
de 1,0 mol L-1 acid azotic cnd nu au fost utilizate. HNO3 concentrat
(Merck, Darmstadt, Germania) a fost utilizat pentru descompunerea Pb si
Niabsorbit pe suprafaa rinii sintetizate.Peroxid de hidrogen concentrat
(Merck) i acid percloric (Merck), mpreun cu acid azotic au fost utilizate
pentru digestia eantionelor de lapte. Soluiile standard diluate au fost
preparate din 1000 mg L-1 de soluie etalon stoc de plumb i nichel
(Merck). Lapte praf (BCR 151) ca material standard de referin a fost
folosit pentru a verifica validarea rezultatelor.
Soluiile tampon cu un pH cuprins de 1.5-6.0 0,1 s-au preparat folosind
0,1 mol L-1 acid citric plus 0,1 mol L-1 HCl / 0,1 mol L-1 solu ii de
NaOH. Rina sintetizat a fost mcinat i cernut folosind o sit de 325.

2.2. Collection and digestion of milk samples

The raw and processed cows milk samples were obtained from local farms, shops and
individual farmers. The samples were collected in polyethylene containers which were
washed in 1% nitric acid. A 10-mL sample of animal milk was placed in a flask and a mixture
of 5-mL of concentrated nitric acid and 5-mL of concentrated hydrogen peroxide was added.
After evaporation for 15 min at 100 C, 2-mL of concentrated perchloric acid was also added
and digested at 150 C with stirring, until a clear digest was obtained (approximately 1 h).
After evaporation of reuses up to 5-mL, the clear digest was diluted to 125 mL with water.
The blank digests were carried out in the same way. Skim Milk Powder (BCR 151) was
digested as described elsewhere (Kaya, Akdeniz, & Yaman, 2008a).

2.3. Collection and digestion of yoghurt samples

The yoghurt samples produced from raw and processed milk samples were obtained from
local farms and shops. The samples were collected in polyethylene containers which were
washed in 1% nitric acid. A digestion procedure modified from Kaya et al. (2008a) was
applied to yoghurt samples. A 10-g sample of yoghurt sample was placed in a flask and a
mixture of 5-mL of concentrated nitric acid and 5-mL of concentrated hydrogen peroxide was
added. After evaporation for 15 min at 100 C, 2-mL of concentrated perchloric acid was also
added and digested at 150 C with stirring, until a clear digest was obtained (approximately
1 h). After evaporation of reuses up to 5-mL, the clear digest was diluted to 125 mL with
water. The blank digests were carried out in the same way.

Colectarea i digestia probelor iaurt

Probele de iaurt, produse din probele de lapte crud i prelucrate au fost
obinute de la ferme i magazine locale. Probele au fost colectate n
recipiente de polietilen care au fost splate n 1% acid azotic. O
procedur de dizolvare modificat de la Kaya i colab. (2008a) a fost
aplicata la probele de iaurt. O prob de 10 g de prob iaurt a fost plasat
ntr-un balon i a fost adugat un amestec de 5-ml de acid azotic
concentrat i 5 ml de peroxid de hidrogen concentrat. Dup evaporare la
100 C timp de 15 minute, 2-mL de acid percloric concentrat a fost de
asemenea adugat i dizolvat la 150 C cu agitare, pn cnd o dizolare
clar a fost obinut (aproximativ 1 h). Dup evaporarea reutilizri pn la 5
ml, limpede digest a fost diluat la 125 ml cu ap. Produsele de dizolvare
goale au fost realizate n acelai mod.
2.4. Preparation of the aminothioazole-containing resin
The polymer beads were prepared by the suspension polymerisation of a mixture of styrene
(54 mL, 0.48 mol) and DVB (55% grade, 10 mL, 0.038 mol) in toluene (60 mL), using gumArabic as stabiliser, according to a previously described procedure (Yavuz et al., 2009). The
beads were sieved and the 420590 m size fractions were used for further reactions.
The beaded polymer was chlorosulfonated using chlorosulfonic acid as described elsewhere
(Senkal et al., 2007). The degree of chlorosulfonation was determined by analysis of the
liberation of chloride ions. For these purpose, a polymer (0.2 g.) sample was added to 10%
NaOH (20 ml) and boiled for 4 h. After filtration and neutralisation with HNO 3 (5 M), the
chlorine content was determined by the mercuric-thiocyanate method. This gave a final
chlorosulfonation degree of 4 mmol g1.
Chlorosulfonated resin (10 g) was added portion wise to a stirred solution of 6 g (0.060 mol)
of 2-aminothiazole in 50 ml of 2-methyl-1-pyrrolidone (NMP) at 0 C. The mixture was shaken
with a continuous shaker for 24 h at room temperature. The reaction content was poured
into water (0.5 L), filtered and washed with excess amounts of water and acetone,
respectively. The resin was dried under vacuum at room temperature for 24 h. The yield was
12 g. The reaction mechanism was given in Fig. 1.

Fig. 1.
The scheme of the reaction mechanism in the synthesis of resin.
Figure options

2.5. Batch preconcentration procedure

The adsorbent resin of 30 mg was added to 60 mL of the model solution containing
20 ng mL1 of Pb and 40 ng mL1 of Ni in flask including the matrix components as mg L1; Ca,
100; Mg, 50; and Fe, 0.5, to represent natural water. This solution containing matrix
components was described as the model solution in the subsequent parts. After pH
adjustment to near the desired value using diluted HCl and NaOH (optimum pH = 3.5 0.1),
10.0 mL of buffer solution was added to this mixture. Then, the pH of this mixture was again
adjusted to the studied pH, if necessary. The mixture was mechanically stirred for 30 min at
room temperature and centrifuged. The residue was dried at 50 C in an oven. After
transferring the residue to a glass beaker by scraping, concentrated HNO 3 of 5.0 mL was
added, and then the mixture was evaporated to near the dryness. After cooling, 3.0 mL of
1.0 mol L1 HNO3 was added, mixed, and centrifuged to take liberated metal to the solution.
The clear solution was measured by using STAT-FAAS for Pb and FAAS for Ni. The blank
solutions were carried out in the same way.One-way Analysis of Variance (ANOVA) was used
for the statistical evaluation of the results.

3. Results and discussion

3.1. The characterisation of new polymeric resin

The major disadvantage of using polymeric resins to remove or preconcentrate toxic metals
from solutions is their hydrolysis by acid and bases in the regeneration step. Sulfonamides
have chemical properties differently from carboxylic amides in that they have reasonable
stability towards acid and base hydrolysis. Therefore, there is no such disadvantage in the
synthesised resin in this study because the sulphonamide group in the resin has resistance
to hydrolysis by acid and bases.
The resin was characterised by FT-IR spectroscopy (Fig. 2). In the sulfonamide resin, NH
stretching vibration occurs at 3400 cm1 and S O stretching vibration occurs at 1320 and
1144 cm1, respectively. The other bands of thiazole group were observed at 1451 cm1 for (C
N) and 668 cm1 for (CS) stretching frequencies.After regeneration process by using
concentrated HNO3 as described in section 2.5, these peaks were observed at the same
values (Fig. 2).

Fig. 2.
FT-IR spectra of aminothioazole containing sulfonamide resin before and after preconcentration procedure.
The line that has the lower transmittance value is before preconcentration procedure.
Figure options

3.2. Optimisation of the method

All of the parameters that may affect the recoveries in the preconcentration step of the
analytical scheme were optimised. These parameters were investigated by using Model
Solutions described above. In the optimisation procedure, other parameters were kept at

optimum values while one parameter was changing for optimisation. The studies on
investigation of each parameter were repeated three times. The pH of the solution and the
contact time are of the most important factors controlling the extractability of metal ions.
The effects of these parameters on recoveries of lead and nickel were examined by batch
method.

3.2.1. Effect of pH on recovery

It is known that hydrogen-ion concentration plays an important role in the preconcentration
of metals by adsorption on the resin because it significantly affects the binding of metal. The
pH values of the model solutions described above were adjusted to appropriate pH in the
range of 1.56, and stirred for 30 min, separately. The obtained results were given in Fig. 3.
The recoveries of Pb and Ni were found to be 90% and 93%, respectively, in the range of pH
3.04.0. It was observed that matrix components were separated from lead and nickel in the
analyte solutions in these pH ranges. Therefore, the pH of solution was selected as 3.5 0.1
in all subsequent studies.

Fig. 3.
Effect of pH on the recovery.
Figure options

Surface of the synthesised resin could be positively charged in strong acid solutions
(pH 2.5) due to the protonation of N atoms, which cannot bind with metal cations.
Because hydroxide compounds of metals in matrix solution can precipitate at high pH
values, lower pH values are considered to be more appropriate for preconcentration step.

3.2.2. Effect of the preconcentration period on recovery

In the optimisation of this parameter, different preconcentration periods (contact times)
were applied to the model solutions. The results showed that the preconcentration period
of 30 min was sufficient for maximum recoveries (up to 90% and 93% for Pb and Ni,
respectively). The obtained contact time is shorter than that of other SPE methods reported
(up to 24 h) in literature (Yavuz et al., 2009). The fast extraction rate indicates that the
sorbent is highly suitable for the preconcentration of trace lead and nickel in aqueous

solution. Further, the shorter extraction period indicates that Pb and Ni ions have a good
accessibility through the chelating sites on the newly synthesised resin, and the binding
constants between the lead and nickel ions and aminothioazole immobilised on the
sulfonamide resin surface are sufficiently high. As a result, 30 min was selected as optimum
contact time and applied throughout the study.

3.2.3. Effect of the buffer volume on recovery

Buffer volume was also optimised to find out the effect of buffer volume on recovery. The
results showed that no obvious variation took place in the extraction yields when more
volume than 10.0 mL of buffer solution was added to 60.0 mL of the sample volume. Hence,
10.0 mL aliquot of buffer solution was added into the mixture in all subsequent experiments,
and this volume was proportionally increased with the sample volume.

3.2.4. Effect of eluent acid concentration and volume on recovery

Acid solutions are widely used for the elution of metal ions from a sorbent due to the
protonation at a chelating site of the sorbent. Among acids, nitric acid replaces the metal
ions from binding sites, and hence is commonly used. Moreover, this acid does not interfere
in the subsequent determination step by FAAS. As a result, nitric acid was chosen as a
striping or back-extraction solution. In addition, suitable acid concentration is another
important factor for elution. Dilute acid solutions (2 and 4 mol L1) were not found to be
enough to complete protonation. On the other hand, high concentration may contaminate
the sample and cause the problem in determination step. So, the back-extraction procedure
was carried out in two steps. In the first step, concentrated nitric acid was used, and then
this acid was evaporated to near the dryness. In the second step, diluted nitric acid solutions
with various concentrations between 0.51.5 mL were used. Related with this step, it was
found that higher recoveries than 90% for Pb and Ni were observed by using
1.0 mol L1 HNO3 in compared to 0.5 and 1.5 mol L1 HNO3. The efficiency of the eluent
volume of 1.0 mol L1 HNO3 was examined by taking its different volumes (1.53.0 mL). It
was observed that 2.5 mL of 1.0 mol L1 HNO3 is sufficient for obtaining the maximum
recoveries (90% for Pb and 93% for Ni). In the first step, concentrated HCl was also used
instead of HNO3. Then, 3.0 mL of 1.0 mol L1 HCl was examined to elution procedure. The
recoveries with 82% and 75% were observed for Pb and Ni, respectively. Consequently,
2.5 mL of 1.0 mol L1 HNO3 in the second step was chosen after using concentrated nitric
acid in the first step to complete desorption of the adsorbed lead and nickel because
maximum recoveries were obtained.

3.2.5. Effect of initial volume of sample on recovery

An important parameter in SPE is the breakthrough volume (initial analyte volume) because
enrichment factor is depending on the volumes of analyte and volume of eluate. In other
words, the breakthrough volume is the maximum solution volume in which the analyte can
be retained within the acceptable recovery range. The optimum initial volume was

experimentally evaluated by using the model solution containing a constant concentration of

20 ng mL1 of Pb and 40 ng mL1 of Ni in different volumes of solutions varying from 25 to
125 mL. It was found that the recoveries were higher than 90% for all studied volumes in
ranges of 25125 mL. So, the initial volume of 125 mL was used for all real samples.

3.3. Analytical performance

The calibration plots were observed to be linear at the concentration range of 1.020.0 ng Pb
mL1 and 6.040.0 ng Ni mL1, by taking of 60 mL initial solution to final volume of 2.5 mL,
due to using 60 ml of model solution in all optimisation steps. In addition, the calibration
curves were also obtained in the concentration ranges of 0.510.0 ng Pb mL1 and 330 ng Ni
mL1 by taking a 125-mL of initial solution to eluate volume of 2.5 mL because Pb and Ni
concentrations in the studied samples may be lower than 1.0 and 6.0 ng mL1, respectively.
Hence, the enrichment factor of 50-times was achieved by using the optimised
preconcentration method. Due to a 7-times sensitivity improvement for Pb by using STAT
(Kaya & Yaman, 2008), a 350-times improving in sensitivity was achieved by using the total
analytical scheme. The obtained calibration plots were linear in the concentration ranges
described above, and the equations of the plots were as follows:
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(0.510.0 ng Pb mL1 after preconcentration-STAT-FAAS)

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(3.030.0 ng Ni mL1 after preconcentration-FAAS)

Relative standard deviations (RSD) were found to be 9% for 4.0 ng Pb mL1 and 10% for
20 ng Ni mL1using 10 replicates of preconcentration procedures. In the statistical method,
the limit of detection (LOD) is defined as three times of the mean values of standard
deviation (SD) of blanks. As for limit of quantification (LOQ), it is defined as ten times of the
mean SD values of blanks. In this study, the levels of Pb and Ni in blank samples were found
to be 0.15 and 0.6 ng mL1 with the SDs of 0.05 and 0.25 ng mL1, respectively. Therefore,
the LODs defined as three times of the SD of the reagent blank were found to be 0.15 and
0.75 ng mL1, respectively. Furthermore, LOQs were found to be 0.5 and 2.5 ng mL1 for lead
and nickel, respectively. The validation of the method was checked by examining the
certified reference material BCR 151 Skim Milk Powder. The results are given in Table 1. It
can be seen that the recovery values were 97% for Pb. For assessing the method
performance, the measured values of the CRMs are compared with the certified values
(from Table 1) following a procedure described by Linsinger (2005). The absolute difference
(m) between the mean measured value (cm) and the certified value (ccrm) was calculated as

m=1.992-2.002=-0.01
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The uncertainty of m is u, was calculated from the uncertainty of the certified value (ucrm)
and the uncertainty of the measurement (um) result (from Table 1) as
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The expanded uncertainty U, corresponding to a confidence level of approximately 95%,

was obtained by multiplication of u by a coverage factor (k) that is usually equal to 2 as
U

=2u=2x0.030=0.060.
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Table 1.
Mean Pb and Ni concentrations (ng mL1) determined for milk and yoghurt samples in this study. The values
are mean SD, n = 3.
Sample type

Pb

Ni

Raw milk from Farm 1

15 2

bdl

Raw milk from Farm 2

19 2

bdl

Raw milk from Farm 3

27 3

bdl

Raw milk from Farm 4

35 4

bdl

Packed Milk 5

29 3

bdl

Packed Milk 6

42 5

bdl

Packed Milk 7

37 4

bdl

Packed Milk 8

61 6

Yoghurt 1

22 4(ng g )

bdl

Yoghurt 2

26 5(ng g )

bdl

Yoghurt 3

21 4(ng g )

bdl

Yoghurt 4

32 5(ng g )

bdl

Yoghurt 5

27 4(ng g1)

bdl

Yoghurt 6

42 6(ng g1)

bdl

Raw milk from farm 1 + 10 ng mL1 Pb and Ni

24 3
Recovery: 93%

9.0 1.0
Recovery: 90%

CRM milk powder (BCR 151)

Certified: 2.002 0.026 ng/g

Found: 1.992 0.015
Recovery: 99%

Raw milk from Farm 1

This method: 15 2
ICP-MS: 14 2
Recovery: 93%

This method: bdl

ICP-MS: 8 0.7
Recovery:

bdl
1
1
1
1

bdl: Below detection limit.

p < 0.05 With respect to the ICP-MS results.

Table options

After comparing m (0.01) with U (0.06) (because of m < U), it was concluded that there is
no significant difference between the measurement result and the certified value, at a
confidence level of about 95%.

In addition, the accuracy of the method was also studied by examining the recoveries of Pb
and Ni from milk samples fortified with these metals. The results in Table 1 show that, at
least, up to 95% of the lead and nickel added to these samples were recovered. Finally,
some samples were also analysed by ICP-MS to determine the accuracy of results. It was
found that the differences between the results obtained by the developed preconcentration
method and ICP-MS were not meaningful (Table 1). In other words, the differences were
originated from unknown sources. For this purpose, One-way Analysis of Variance (ANOVA) is
conducted to test the equality of group means. One of the pairwise comparisons test, Tukey
HSD, was carried out to find which of group means are different from each other. SPSS
(version: 13) statistical program was used for all statistical computations. Statistical findings
are given in Table 1.

3.4. Effect of interfering ions on recovery

In view of the high selectivity provided by FAAS, the interference effects of some cations and
anions were examined using the preconcentration procedure. The hypothesis was initially
that the interferents might decrease the extraction efficiency of the analyte. The interfering
cations and anions were selected depending on their major natural abundance in milk and
yoghurt. A binary mixture of 125 mL of Pb and Ni, and each interfering ion, i.e., Na +, K+, Mg2+,
Ca2+, Cl and SO42 in three concentration levels (100, 200 and 500 mg L1) was extracted by
the batch method. Nitrate or sodium forms were used as ionic salts of compounds. From the
results obtained, the overall percent recoveries of Pb and Ni were found to be higher than
90%. These results indicated that the ions mentioned above do not interfere the Pb and Ni
determination at the optimised conditions.

3.5. Applications to real samples

All of the optimum values determined were applied in the preconcentration procedure for
milk and yoghurt samples prepared/digested as described in experimental section. After
applying the optimised method, the results obtained were given in Table 1. The values given
are the mean of three different portions of the same sample. Concentrations of Pb in the
milk samples were found to be in range of 1535 ng mL1 for raw milk and 2961 ng mL1 for
commercially packed milk. Nickel concentrations in all studied milk samples could not be
detected due to its levels lower than the LOQ. The obtained lead concentrations except two
for raw milk are higher than the maximum allowable levels (20 ng Pb mL1) restricted by
interested authority in our country (Turkish Food Codex, 2010). Lead and Ni concentrations
in yoghurt samples were found to be in the range of 2142 ng mL1 and bdl,
respectively. Venkatesh et al. (2005) applied a solid phase extraction method by using 2,3hydroxypyridine loaded Amberlite XAD-2 for preconcentration of trace metal ions in water,
milk and vitamin samples. Shan, Tie, and Xie (1988) determined trace elements in water,
sea-water and biological samples by ICP-AES after preconcentration with ammonium
pyrrolidinedithiocarbamate precipitation. The observed optimum conditions in this study are
found better than in those studies regarding time, pH and sensitivity. Related with reported
values for Pb and Ni concentrations in animal milk and yoghurt in the literature, Ayar, Sert,

and Akin (2009) found 103 ng Pb mL1 for Cows milk and 93 ng Pb mL1 for Cows yoghurt,
by using ICPAES (Ayar et al., 2009). Qin, Wang, Li, Tong, and Tong (2009) and Bilandzic et al.
(2011) determined lead concentrations in ranges of 1235 ng mL1 for different kinds of milk
and 3659 ng mL1 for Cows milk by using GFAAS, respectively (Qin et al.,
2009 and Bilandzic et al., 2011). Giri, Singh, Jha, and Tripathi (2011) found 480 ng Ni mL1 in
Cows milk taken from U mining area while lead was below detection limit, by using AAS ( Giri
et al., 2011). Al-Othman (2010)) reported Pb concentrations in fresh milk and yoghurt
samples as 6.0 and 25.0 ng mL1using ICPAES, respectively (Al-Othman, 2010). Enb, Abou
Donia, Abd-Rabou, Abou-Arab, and El-Senaity (2009) found Pb and Ni concentrations in
ranges of 4062 and 23 ng mL1 in milk and 3960 and 12 ng mL1 in yoghurt samples,
respectively (Enb, Abou Donia, Abd-Rabou, Abou-Arab, and El-Senaity, 2009). The other
reported levels of Pb and Ni are as follows: 11244 ng Pb mL1 in Cows milk (De Castro et al.,
2010), 43 ng Pb mL1 and 20 ng Ni mL1 in Cows milk (Kazi et al., 2009), 18 ng Pb mL1 in
Cows milk (Yaman, 1998), 911 ng Pb mL1 in Cows milk using ICP-MS (Frazzoli & Bocca,
2008) and 19126 ng Pb mL1 in yoghurt (Kaya et al., 2008a). From those results, Pb and Ni
levels in milk and yoghurt samples change in the range of below detection limit (bdl)-244 g
Pb L1 and 1480 g Ni L1 for milk samples, and 19126 g Pb kg1 and 12 g Ni kg1 for
yoghurt samples, respectively. The observed Pb and Ni concentrations in this study are in
the ranges of those results.

4. Conclusions
The optimised preconcentration method in this study has some advantages than the other
reported methods in this field in view of sensitivity and no need to use of ligand at the
preconcentration step because the ligand is imprinted to the resin at the beginning of the
analytical procedure. The developed method is applicable for the determination of ultratrace
amounts of Pb and Ni in a variety of milk and yoghurt samples with low LOD, high accuracy
(recovery >95%) and high precision (RSD <9%). The optimised method together with the
STAT has high sensitivity, selectivity and reliability for Pb. The sensitivity of FAAS was
increased up to 50-times by using the optimised SPE conditions. A 350-fold improvement in
the sensitivity of Pb by FAAS was achieved by the combination of STAT and preconcentration
method because the STAT method can reduce the potential interferences by dilution. So, the
ng mL1 levels of Pb and Ni in both milk and yoghurt samples could be determined by this
method. It can be concluded that the developed preconcentration method combined with
STAT-FAAS can be compete with ET-AAS method taking into consideration the sensitivity,
interferences and cost. The analysis procedure of this method is not complex compared to
the previous similar works taking into consideration LOD, operation conditions such as
column procedure, reagent species and resin amount or the required time. This method can
also be applied to more complex samples such as soil and biological samples. The obtained
Pb levels in most of the milk samples were higher than the restricted concentrations by the
authorised committees. Although Ni concentrations in the studied milk and yoghurt samples
were found to be lower than the LOQ and the maximum allowable levels, the developed
method can be applied to nickel analysis in different types of samples.

Finally, it was concluded that the synthesised resin can be examined to remediation of
polluted environmental matrices such as soil and water by using more than one use. The IR
spectra have supported this conclusion.