Talanta 71 (2007) 715721

Determination of Ca, Mg, Fe and Zn partitioning in UHT cow milks by two-column ion exchange and ame atomic absorption spectrometry detection
Pawel Pohl , Bartlomiej Prusisz
Analytical Chemistry Division, Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze Stanislawa Wyspianskiego 27, 50-370 Wroclaw, Poland Received 11 January 2006; received in revised form 21 April 2006; accepted 12 May 2006 Available online 12 June 2006

Abstract An operationally dened fractionation protocol was developed to study the partitioning of Ca, Mg, Fe and Zn in UHT cow milks. The method was based on sorption of distinct metal species by two ion exchange columns, namely strong cation exchanger Dowex 50Wx4 and strong anion exchanger Dowex 1x4, connected in a series. The evaluation of the donation of metal species classes distinguished, that is cationic and anionic fractions, was made after splitting the columns and elution of metal groupings with a 2.0 mol l1 HCl solution, followed by the determination of metal concentrations in the resulting eluates. The amount of third, inert fraction was assessed by measuring metal contents in the efuents obtained after passage of the samples through the columns. The results achieved utilizing two-column ion exchange based procedure were compared with those obtained for the approach in which the columns were considered separately. The fractionation pattern for each metal studied was thoroughly discussed in light of available knowledge relating to the composition of milk. 2006 Elsevier B.V. All rights reserved.
Keywords: UHT milk; Operational fractionation; Column ion exchange; Flame atomic absorption spectrometry

1. Introduction It is well recognized that study of mineral constituents in milk, including such major metals as Ca and Mg, is of special importance due to the retrieval of valuable information on nutritional contribution of that sort of beverages to human diet as well as indication of any alterations occurred during processing and storage of milk and milk products, since both metals have noticeable inuence on the conformation and stability of milk proteins, especially caseins [1]. Measurements of minor and trace metal contents, comprising Al, Cd, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Zn, Tl, is also very helpful in assessment of quality of milk during its manufacturing treatment and production. Indirectly, the distribution of minor and trace metals can be an additional factor valuable for elucidation of possible relationships between animal feeding and environmental conditions [2]. Regardless

Corresponding author. Tel.: +48 71 3203445; fax: +48 71 3284330. E-mail address: pawel.pohl@pwr.wroc.pl (P. Pohl).

the usefulness of determinations of total metal concentrations, formulation of any assumption considering mobility, bioavailability, absorption, accumulation and toxicity of metals present in milk, requires the detection of possible binding forms of metals with the molecules existing in that complex biological uid [3,4]. Typically, for total concentration measurements of metals in milk by means of different spectrometric detection techniques, samples are digested prior to oxidation of organic matrix using a closed-pressurized microwave assisted digestion systems [510] or directly introduced to the instrument after appropriate dilution [4,11,12]. In the case of speciation analysis of metals in whole milk, normally, mild centrifugation or ultra-centrifugation are applied to separate different components of milk, i.e., fat, serum and casein micelles [3,4,7,1013]. The resulted fractions, that is, skimmed milk (without fat) or whey milk (without fat and caseins) are usually further subjected to analysis by size exclusion high performance liquid chromatography (SE-HPLC) in order to nd the distribution of metals between the molecules, mostly proteins, of dened molecular mass range and existing in

0039-9140/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.05.030

716

P. Pohl, B. Prusisz / Talanta 71 (2007) 715721 Table 1 Operating conditions of F-AAS measurements Parameter Ca Mg Fe Zn

the sample metal binding prole [4,911]. Nevertheless, despite the potential applications of the reported approach, the complexity and high costs of the instrumentation as well as experimental difculties, especially with calibration, make that it cannot be considered as routine technique. Usually, the protein identication is performed by comparison of the retention times of standards and samples. In case of an incomplete separation, metals studied cannot be precisely attributed to distinct proteins or metalprotein associations may be recognized incorrectly [11]. Thus, as a rule, metals are only associated with molecular weight of the protein obtained during the column calibration step. Alternatively, a versatile procedure based on use of solid phase extraction and afnity of possible metal forms for sorbents used, namely chelating Chelex-100 and anion exchange Dowex 1, was proposed lately for fractionation of Cu and Mn [14]. Although, the classication of metal species ascertained was operationally dened, it enabled to acquire an insight into the chemical nature of existing metal forms in milk analyzed. Unquestionably, in comparison to the analytical procedures involving HPLC separations, the proposed approach was much cheaper and simpler. More to the point, there is a growing evidence that this approach to fractionation of metals gains in the popularity as it has been applied lately to examine the partitioning of metals in such beverages as beers, wines and tea infusions [1519]. In the present study, the operational fractionation scheme, based on retention of distinct metal species on strong cation and anion exchangers, was proposed and applied to simultaneous discrimination of cationic (sorbed on the cation exchange Dowex 50Wx4 resin), anionic (retained by the anion exchanger Dowex 1x4) and inert (not retained by neither of the two resins used) fractions of Ca, Mg, Fe and Zn in different non-fatted UHT cow milks. In the rst approach devised for the fractionation analysis of metals examined, the samples were treated with ion exchange columns connected in a series. For verication of the reliability of the results achieved, in the second protocol, milk samples analyzed were treated by each ion exchange column separately. The results obtained allowed to make some considerations about the distribution of Ca, Mg, Fe and Zn in milk. 2. Experimental 2.1. Instrumentation A Perkin-Elmer atomic absorption spectrometer, model 1100B, with a deuterium lamp for a background correction was used for metal concentration measurements. A sample introduction assembly comprised a stainless steel concentric nebulizer, a plastic Ryton mixing chamber with a ow spoiler inserted inside, and a siphon interlock with a drain tube for drainage. For atomization, an airacetylene ame was used throughout, operated in a single slot, 10-cm titanium burner with a removable head. The monochromator was an 1800 lines/mm grating working in a Litrow conguration with a focal length of 267 mm and a reciprocal linear dispersion of 1.6 mm/mm. The operating conditions applied for the measurements of Ca, Mg, Fe and Zn using the F-AAS apparatus are given in Table 1.

Analytical wavelength (nm) 422.7 285.2 248.3 213.9 Slit width (nm) 0.7 0.7 0.2 0.7 Air ow rate (l min1 ) 8.0 8.0 8.0 8.0 Acetylene ow rate (l min1 ) 3.4 2.5 2.5 2.5 Lamp current (mA) 10 6 15 6 Linearity range ( g ml1 ) 1.05.0 0.100.50 1.05.0 0.101.0 Detection limit (3) ( g ml1 ) 0.002 0.0004 0.009 0.004

For signal processing a hold signal mode was selected. Readings were integrated at 0.1 s intervals over the integration time of 3 s. Quantitative determinations of metals concentrations in sample digests as well as efuents and eluates collected were made by means of standard addition method. 2.2. Reagents and materials All the reagents employed were of analytical grade. Doubly distilled water was used for the preparation. Stock standard solutions of Ca, Mg, Fe and Zn at concentration of 1000 g ml1 were obtained from Merck (Germany). Other chemicals, including concentrated HNO3 , concentrated HCl, 30% (m/v) solution of H2 O2 , NaOH, KH2 PO4 and EDTA were supplied by POCh (Poland). Two-component (KH2 PO4 NaOH) 0.10 mol l1 buffering solutions giving pH values of 6.0, 6.5 and 7.0 were made according to the description reported by Bower and Bates [21]. The 100-ml working solutions containing mixture of Ca(II), Mg(II), Fe(III) and Zn(II) cations at concentrations of 50, 5.0, 0.20 and 0.20 g ml1 , correspondingly, were prepared by appropriate dilution of the stock solutions. For adjusting the resulting solutions to appropriate pH values, 10 ml portions of the respective buffers were added. The 100-ml working solutions of anionic complexes of Ca, Mg, Fe and Zn (50, 5.0, 0.20 and 0.20 g ml1 , respectively) with EDTA were prepared by addition of 10 ml of proper buffering solutions, followed by 10 ml of a 0.10 mol l1 solution of EDTA. Finally, solutions were made up to the volume with water and mixed well. Two gel-type ion exchange resins, i.e., a strong acidic cation exchanger Dowex 50Wx4 (200400 mesh, sulfonic acid) and a strong basic anion exchanger Dowex 1x4 (100200 mesh, trimethylbenzyl ammonium chloride), purchased from Aldrich (Germany), were used for ion exchange treatments. The resins as received (about 1.2 g) were slurred with water into 10-cm internal diameter glass columns, equipped with frits and stopcocks (Supelco, USA). Pyrex glassware applied in this work as well as solid phase extraction columns were kept in a 10% (m/v) solution of HNO3 for 24 h. Before use, they were washed successively with water and air dried. 2.3. Column operation In order to remove any impurities derived from the manufacturing processes, both resins were initially conditioned. The treatment applied to the Dowex 50Wx4 cation exchanger was as

P. Pohl, B. Prusisz / Talanta 71 (2007) 715721

717

follows: the resin beds were washed with 25 ml of water, ushed with 10 ml of a 2.0 mol l1 solution of HCl, then, washed with 25 ml water and nally rinsed with 10 ml of a 1.0 mol l1 solution of NaOH. The excess of NaOH was removed by washing the resin beds with 25 ml portion of water. The beds of the Dowex 1x4 anion exchange resin were washed with 25 ml of water, followed by 10 ml of 2.0 mol l1 HCl solution and rinsed at least using 25 ml of water. Studying the effect of pH of the solutions (at 1.0 ml min1 ) and the ow rate at which the solutions (at pH 6.5) are driven through the columns on the retention efciencies of metals on Dowex 50Wx4 and Dowex 1x4 resins, working solutions containing cations and anions of the metals of interest, respectively, were propelled under the selected conditions through the columns packed with the resins. Each time, 10-ml portions of column efuents were collected and the concentrations of Ca, Mg, Fe and Zn were determined using the F-AAS method. The percentage retention efciencies of Ca, Mg, Fe and Zn were calculated as ratios of the metal quantities measured in the efuents to the metal concentrations present in the initial solutions (mean values for three independent replicates). Solvent elution of Ca, Mg, Fe and Zn from cation and anion exchangers was examined applying 1.0, 2.0 and 4.0 mol l1 solutions of HCl and HNO3 . Each time, after loading the working solutions of Ca, Mg, Fe and Zn (pH 6.5, the ow rate of 1.0 ml min1 ) onto the columns, 10ml portions of the selected eluents were passed through them and the appropriate 10-ml fractions of eluates were collected prior to the analysis by the F-AAS method. The elution ow rate was equal to 1.0 or 2.0 ml min1 . The percentage recoveries of Ca, Mg, Fe and Zn were calculated as ratios of metals concentrations measured in the eluates to the products of the enrichment factor and the concentrations in the solutions loaded onto the columns. In all cases, during loading and elution steps, the solutions were driven through the columns with the aid of a two-channel peristaltic pump. The procedural column blanks were proceeded and taken into consideration in calculations. 2.4. Samples In the present work, three different non-fat polish long life liquid ultra high temperature treated (UHT) cow milks available on the market were chosen for the investigation, i.e., M1 (Laciate, Mlekpol), M2 (Mlekovita, Mlekovita) and M3 (Lowickie, Lowicz). 2.5. Analytical procedures 2.5.1. Determination of total metal concentrations For the determination of the total metal concentrations in analyzed UHT milks, 5.0-ml sample portions were mineralized in the mixture of concentrated HNO3 (5.0 ml) and 30% (m/v) H2 O2 (3.0 ml) using heating on a hot plate in glass beakers covered with watch glasses. After addition of HNO3 , the samples were evaporated to near dryness. After that, the samples were cooled, H2 O2 was added and the heating was repeated to achieve about 0.5-ml sample solutions. The resulting aliquots were transferred quantitatively to 50-ml volumetric asks and brought with water

to the mark. The samples (for each milk three separate samples with a respective blind sample) were subjected to the analysis of metal contents by F-AAS method. 2.5.2. Fractionation analysis For solid phase extraction, 50-ml sample solutions were prepared just by the dilution of 2-ml portions of UHT milks in water (dilution factor of 1 + 25). For partitioning of Ca, Mg, Fe and Zn, resulted milk sample solutions were passed with the ow rate of 2.0 ml min1 through ion exchange columns connected in a series in the following order: the rst strong cation exchanger Dowex 50Wx4 and the second strong anion exchanger Dowex 1x4. Simultaneously, the efuents out-coming from the second column were saved. After loading the sample solutions, the columns were split and distinct metal species retained on both columns were eluted by passing 10 ml of 2.0 mol l1 HCl solution through the resin beds at the ow rate of 2.0 ml min1 . The respective eluates collected (10 ml) along with the efuents gathered (10 ml) were measured using F-AAS method in order to determine the concentrations of metals of interest in classied metal groupings, i.e., broadly meant cationic fraction, anionic fraction and inert fraction (see Fig. 1a). For evaluation of the validity of the results retrieved by the two-column ion exchange fractionation approach described

Fig. 1. Scheme of: (a) two-column ion exchange and (b) single-column ion exchange fractionation approaches.

718

P. Pohl, B. Prusisz / Talanta 71 (2007) 715721

above, sample solutions of analyzed milks were treated with both ion exchangers considering them separately. After passage of the sample solutions through the columns, followed by the elution of metal fractions retained, the donations of cationic and anionic fractions were determined. The third metal fraction (inert species) was distinguished by subtraction of the sum of the concentrations of metals in the cation and the anionic fractions from their total contents in the samples (see Fig. 1b). All the experiments were performed in triplicate. The uncertainty of the results was expressed as a standard deviation. In the case of outputs achieved by subtraction, a propagated standard deviation was given. 3. Results and discussion At rst, the effect of chosen experimental conditions, i.e., pH of the solutions loaded onto the columns, loading ow rate, concentration of acids used for the elution, and ow rate at which the solvents are driven through the columns for the elution, was investigated on sorption and desorption efciencies of Ca, Mg, Fe and Zn for Dowex 50Wx4 and Dowex 1x4 resins using test solutions comprising the metals at the concentration corresponded to 50, 5.0, 0.20 and 0.20 g ml1 , respectively for Ca, Mg, Fe and Zn. Such concentrations of metals under consideration were selected just to correspond to the average content of the analytes in the diluted (1 + 25) milk samples. The pH sets selected for examination was close to the pH of milk samples. 3.1. Retention of metal ions by Dowex 50Wx4 and Dowex 1x4 It was found that strong cation exchanger Dowex 50Wx4 retained quantitatively cations of Ca, Mg, Fe and Zn from the solutions of all pH sets studied, i.e., 6.0, 6.5 and 7.0. The average retention efciencies for metals of interest, determined as mean values of the retention percentages established for the individual pH adjustments, were 100% or very close to that value. Similarly, strong anion exchanger Dowex 1x4 sorbed exhaustively the anionic complexes of Ca, Mg, Fe and Zn in the investigated pH range. For each metal, the average metal retention

efciencies did not differ signicantly from 100%. Such behavior established for the resins used is referred to the fact that both strong exchangers have ion exchange sites active at any pH due to complete dissociation of their functional groups [22]. In consequence, trace metals are pre-concentrated along with simultaneous retention of major metal ions what is valuable feature for the contemporaneous fractionation of Ca, Mg, Fe and Zn undertaken in the present work. It was also assessed that the increase of the ow rate at which the solutions were passed through the columns from 1.0 to 4.0 ml min1 did not result in lessening of the retention efciencies of metals studied either for cation and anion exchangers. For both ne particle size resins, due to very high contact area of the solution with the sorbent, the ion exchange equilibrium is reached very fast and thus, the alterations of the solution ow rate have minor effect on the metal retentions [23]. Hence, for Dowex 50Wx4 resin, the average metal retention percentages, being the mean values of the individual retention efciencies attained under the ow rate sets applied, were equal to 100%, 100%, 99.6% and 100%, respectively for Ca, Mg, Fe and Zn. For Dowex 1x4, the average retention efciencies established for Ca, Mg, Fe and Zn were 100%, 100%, 99.8% and 100%, correspondingly. The standard deviations of single mean retention efciencies estimated for all the experiments upon three replicated measurements, were generally within 3%. 3.2. Elution of metal ions from Dowex 50Wx and Dowex 1x4 Results of percentage recoveries of Ca, Mg, Fe and Zn obtained for Dowex 50Wx4 and Dowex 1x4 using moderately concentrated solutions of HCl and HNO3 as eluent solvents are given in Table 2. As can be seen, the application of 2.0 and 4.0 mol l1 HCl solutions results in exhaustive retrieval of all metals from the strong cation exchanger. For strong anion exchanger Dowex 1x4, it was established that solutions of both acids at concentrations of 2.0 or 4.0 mol l1 are appropriate for the quantitative desorption of the metals studied from the resin, presumably due to the dissociation of their EDTA complexes [14,24].

Table 2 Percentage recovery efciencies of Ca, Mg, Fe and Zn for cation exchange and anion exchange resins obtained using HCl and HNO3 solutions at concentration A, 1.0; B, 2.0; C, 4.0 mol l1 Dowex 50Wx4 Ca HCl solutions A 26 2 B 100 1 C 90 2 HNO3 solutions A 44 9 B 104 2 C 97 2 Mg 90 2 101 1 98 2 62 1 109 1 104 2 Fe 18 2 94 3 97 1 21 70 1 84 3 Zn 91 98 2 101 2 51 91 2 96 4 Dowex 1x4 Ca 69 1 100 2 98 1 79 3 100 1 97 2 Mg 89 2 100 1 97 3 88 1 102 2 98 2 Fe 39 2 102 2 95 3 46 1 98 3 98 3 Zn 40 3 99 1 97 2 49 2 100 1 96 3

Mean value S.D. for three independent replicates.

P. Pohl, B. Prusisz / Talanta 71 (2007) 715721 Table 3 Total concentrations (A) and contents of Ca, Mg, Fe and Zn in cationic (B), anionic (C) and inert (D) fractions distinguished using two-column approacha Sample Ca M1 M2 M3 Mg M1 M2 M3 Fe M1 M2 M3 Zn M1 M2 M3
a

719

A 955 14 903 7 1.24 103 0.01 103 191 14 167 4 113 1 2.10 0.08 1.70 0.15 4.10 0.21 1.20 0.05 1.00 0.05 3.79 0.06

B 960 9 903 6 1.18 103 0.03 103 175 6 166 4 104 2 1.05 0.06 1.12 0.10 1.88 0.25 0.40 0.05 0.50 0.04 1.44 0.09

C 2.25 0.66 1.26 0.42 7.13 2.14 0.99 0.10 0.16 0.06 5.30 0.63 1.00 0.07 0.38 0.08 1.79 0.19 <0.02 <0.02 <0.02

D 0.55 0.12 0.50 0.17 29.8 6.2 13.4 2.7 0.06 0.04 1.36 0.31 <0.22 <0.22 <0.22 0.71 0.05 0.42 0.06 2.13 0.18

B + C + D 963 9 905 6 1.22 103 0.03 103 189 6 166 4 111 2 <2.27 0.09 <1.72 0.13 <3.89 0.38 <1.14 0.07 <0.94 0.07 <3.59 0.20

In g ml1 . Propagated standard deviation is given. Information value given by the producer equal to 120 mg per 100 ml of milk.

It was ascertained as well that changing the elution ow rate from 1.0 to 2.0 ml min1 did not cause any alterations in the recoveries achieved. Taking into account sorption and desorption properties apprised for both resins, sample loading and elution ow rates were used in the fractionation protocol were set to be 2.0 ml min1 . For the recovery of the metals sorbed by both resins, the solution of HCl at concentration of 2.0 mol l1 was taken. 3.3. Fractionation analysis The results of fractionation of Ca, Mg, Fe and Zn in UHT cow milks obtained by means of two-column ion exchange procedure are presented in Table 3. 3.3.1. Calcium and magnesium The concentration of Ca in the cationic fraction distinguished in all UHT milk samples is very high, i.e., it ranges from over 94% to 100% of the total metal concentration. Such relatively large contribution of that fraction can be strictly related to the sample preparation procedure applied, that is dilution of milk. Such treatment causes solubility of colloidal calcium phosphate (CCP), which accounts for almost 67% of the total calcium content, and is closely associated with casein micelles [1,2]. But as can be seen, the dissolution of CCP leads not only to freeing of Ca2+ ions according to the following reaction: Ca3 (PO4)2 + 2H2 O 3Ca2+ + 2HPO4 2 + 2OH . It can also tend to release Ca2+ that is adsorbed on clusters of CCP (e.g., Ca9 (PO4 )6 ), which acquire a positive charge and interact with negatively charged ester phosphate groups of casein leading to stabilization of the casein micelle structure [1,20]. Despite Ca2+ ions originated from casein micelles, other reasonable cationic

species of Ca accounted for cationic fraction assessed can be free Ca2+ cations, originally present in milk, as well as stable cationic complexes with such anions as H2 Citrate and H2 PO4 but also with HCO3 , Cl or even HSO4 [1]. In relation to the total Ca concentration, the donation of anionic and inert fractions separated by the protocol devised is negligible. The contribution of these fractions is within 0.12% and can be attributed to the presence of respective stable anionic (CaCitrate , CaPO4 ) and inert (CaHCitrate, CaHPO4 ) complexes of that metal. Very similar fractionation pattern was observed for Mg, most likely, to its role in the stability of casein system [2,20]. The cationic fraction evaluated for Mg contributes to 9298% of the total concentration of that metal in milk samples analyzed and can be presumably associated with presence of Mg2+ ions released from casein micelles due to the solubility of CCP, as well as originally existing in milk, and stable cationic complexes of Mg(II) with anions of citric and phosphoric acids, e.g., MgH2 Citrate+ and MgH2 PO4 + . The donation of anionic and inert species of Mg amounts only for 0.15% and 0.037%, respectively, of the total metal quantity. Reports concerning the speciation analysis of metals in cow milks are occasional [4]. Nevertheless, the results obtained in the present contribution for Ca and Mg are in good accordance with those achieved in previous studies for human whole milk and milk whey using size exclusion high performance liquid chromatography (SE-HPLC) and indicating that, except for casein micelles, both metals are also bound to non-protein low molecular fraction (<2 kDa) [4,9,10]. The bias between the sum of the fractions distinguished for both major metals and their total concentrations was from 2.6% to +0.3% for Ca and from 2.4% to 0.6% for Mg. It was concluded that there was any contamination or loss problems in the approach applied.

720

P. Pohl, B. Prusisz / Talanta 71 (2007) 715721

3.3.2. Iron and zinc In the case of fractionation of Fe and Zn in UHT milks, noticeable differences between both metals as well as within the samples were found. Iron metal species in UHT milks are distributed almost equally over the cationic and anionic fractions. Presumably, the cationic Fe forms are Fe3+ cations associated with casein micelles. However, as it was formerly ascertained [8], Fe is not associated with CCP but rather linked to amino acids of polypeptide chains of casein. Thus, Fe3+ cations were possibly released from the casein and retained on the strong acidic cation exchanger through the interactions of Fe-casein complexes with SO3 functional groups of Dowex 50Wx4 resin. Other likely cationic complexes present in that fraction are FeHCitrate+ as well as FeHPO4 + and/or FeH2 PO4 2+ . The presence of labile complexes with other relatively low or moderate molecular weight ligands is also possible; because of their instability, they might dissociate on the resin and share the fraction of Fe cationic species. In the case of the anionic fraction, it is difcult to reasonably attach the amount of that class of metal groupings with presence of proper molecules able to complexing Fe. This is mostly due to contradictory information regarding the speciation of Fe in milk and the fact that less attention has been paid in the literature to the speciation of metals in cow milk [3,4]. In binding pattern of Fe in UHT cow milk whey achieved in the experiments with SE-HPLC and reported lately by Rivero Martino et al. [4], only a low molecular fraction (2.7 kDa) was associated with the metal. It tends to the conclusion that Fe can be complexed by lactose or oligosaccharides, which are capable of forming anionic complexes with metal cations [25]. It cannot be excluded, taking into consideration the sample treatment proposed in the present work, that Fe can be also bound to -lactoglobulin and/or -lactalbumin, both observed in a protein prole obtained for untreated UHT milk [4]. Finally, the

less abounded inert fraction of Fe (below 0.22 g ml1 ) can be ascribed to the presence of FeCitrate and FePO4 complexes. In the case of Zn, similarly as for Ca and Mg [8], the cationic fraction of that metal, accounted for 3350% of its total content, probably contributes to the presence of Zn2+ cations released from casein micelles via the dissolution of CCP. Other cationic species might be for example ZnH2 PO4 + or labile metal complexes with low molecular weight inorganic and organic ligands. The anionic fraction is likely to comprise some stable anionic complexes of Zn, e.g., ZnCitrate and/or ZnPO4 , but the concentration of that class of metal species is very low (below 0.02 g ml1 ). The most abounded fraction of Zn (from 42% to 59% of the total metal concentration) is that of inert species. It can likely contain complexes of Zn with low molecular weight ligands (for example ZnHCitrate or ZnHPO4 ) as well as species being metal bound to immunoglobulins or casein residues, which are the main group of proteins separated by the SE-HPLC in UHT milk [4]. For both metals, sum of the fractions separated is well compared to the total concentrations determined. The bias (in %) was assessed to be from 5.1% to +8.1% for Fe and from 6.0% to 5.0% for Zn. 3.4. Verication of results For verication and critical evaluation of the results attained by the two-column ion exchange fractionation approach proposed in the present study, UHT milk samples analyzed were treated with the columns separately. Respectively, the cationic and anionic fractions were obtained by passing the samples through the single columns, followed by the recovery of the species retained on the exchangers by plain elution. The third fraction was established by difference of the sum of metal concentrations in the cationic and anionic fractions distinguished

Table 4 Total concentrations (A) and contents of Ca, Mg, Fe and Zn in cationic (B), anionic (C) and inert (D) fractions distinguished using single-column approacha Sample Ca M1 M2 M3 Mg M1 M2 M3 Fe M1 M2 M3 Zn M1 M2 M3
a

A 955 14 903 7 1.24 103 0.01 103 191 14 167 4 113 1 2.10 0.08 1.70 0.15 4.10 0.21 1.20 0.05 1.00 0.05 3.79 0.06

B 954 4 901 9 1.21 103 0.01 103 182 4 167 4 112 1 1.20 0.14 1.19 0.09 2.09 0.25 0.44 0.09 0.50 0.10 1.13 0.09

C 3.25 0.56 1.50 0.42 10.0 1.2 0.41 0.09 0.29 0.09 9.60 2.28 0.94 0.27 0.44 0.27 1.56 0.15 <0.11 <0.11 <0.11

D = A (B + C) 0.50 11.41 20.0 14.2 8.59 14.56 0.07 0.32 0.45 0.36 >0.65 0.10 >0.39 0.11 >2.55 0.11

In g ml1 . Propagated standard deviation is given. Information value given by the producer equal to 120 mg per 100 ml of milk.

P. Pohl, B. Prusisz / Talanta 71 (2007) 715721

721

and the total metal contents measured. The results achieved for that fractionation approach are in Table 4. As can be seen, the concentrations of metals in distinct fractions achieved for that protocol are strictly reliable to those found for the approach with two ion exchange columns linked in a series what indicates the reliability and the soundness of the fractionation scheme described. The accurateness of the results of the total concentration determinations was evaluated by the analysis of spike samples of milk. The recovered concentrations of Ca, Mg, Fe and Zn were close to 100% of the expected values. A good accuracy of the procedure demonstrates as well the determined value of the total Ca content in milk M3 (124 1 mg per 100 ml of milk) being very well comparable with the information value given by the producer (120 mg of Ca per 100 ml of milk). 4. Conclusions The paper contributes to the eld of fractionation analysis of metals in cow milks for which the experimental evidence of metal partitioning is occasionally reported in the literature. In terms of prediction of metal bioavailability to human organisms, the proposed operationally dened fractionation procedure, based on solid phase extraction on ion exchange resins, i.e., Dowex 50Wx4 and Dowex 1x4, facilitates obtaining relevant information about existing groupings of Ca, Mg, Fe and Zn in milk, helpful for more comprehensive understanding of the speciation of metals in milk. Although, the two-column ion exchange procedure developed provides the classication of metal species due to their charge and chemical behavior, the acquired outcomes are well corresponded to the results previously published in the literature and referred to the elucidation of metal associations with ligands of different molecular masses by means of size exclusion high performance liquid chromatography. In comparison to that method, the presented approach is inexpensive, easy in operation, and does not require a sophisticated equipment.

References
[1] P.F. Fox, P.L.H. McSweeney, Diary Chemistry and Biochemistry, Chapman and Hall, New York, 1998. [2] N.P. Wong, R. Jenness, M. Keeney, E.H. Marth (Eds.), Fundamentals of Diary Chemistry, Chapman and Hall, New York, 1999. [3] A. Sanz Medel, M. Montes Bayon, M.L. Fernandez Sanchez, Anal. Bioanal. Chem. 377 (2003) 236. [4] F.A. Rivero Martino, M.L. Fernandez Sanchez, A. Sanz Medel, J. Anal. Atom. Spectrom. 17 (2002) 1271. [5] P. Licata, D. Trombetta, M. Cristani, F. Giofre, D. Martino, M. Calo, F. Naccari, Environ. Int. 30 (2004) 1. [6] A. Lante, G. Lomolino, M. Cagnin, P. Spetolli, Food Control 17 (2006) 229. [7] F.A. Rivero Martino, M.L. Fernandez Sanchez, A. Sanz Medel, Anal. Chim. Acta 442 (2001) 191. [8] F.V. Silva, G.S. Lopes, J.A. Nobrega, G.B. Souza, A.R.A. Nogueira, Spectrochim. Acta Part B 56 (2001) 1909. [9] E. Coni, B. Bocca, B. Galoppi, A. Alimonti, S. Caroli, Microchem. J. 67 (2000) 187. [10] B. Bocca, A. Alimonti, E. Coni, M. Di Pasquale, L. Giglio, A. Piccioli Bocca, S. Caroli, Talanta 53 (2000) 295. [11] P. Bermejo, E. Pena, R. Dominguez, A. Bermejo, J.M. Fraga, J.A. Cocho, Talanta 50 (2000) 1211. [12] P. Bermejo, R. Dominguez, A. Bermejo, Talanta 45 (1997) 325. [13] P. Bermejo, R. Dominguez, A. Bermejo, Frezenius J. Anal. Chem. 357 (1997) 457. [14] O. Abollino, M. Aceto, M.C. Bruzzoniti, E. Mentasti, C. Sarzanini, Anal. Chim. Acta 375 (1998) 299. [15] P. Pohl, B. Prusisz, Anal. Chim. Acta 502 (2004) 83. [16] I. Karadjova, B. Izgi, S. Gucer, Spectrochim. Acta Part B 57 (2002) 581. [17] R. Svendsen, W. Lund, Analyst 125 (2000) 1933. [18] S.B. Erdemoglu, K. Pyrzynska, S. Gucer, Anal. Chim. Acta 411 (2000) 81. [19] K.E. Odegard, W. Lund, J. Anal. Atom. Spectrom. 12 (1997) 403. [20] M. Philippe, Y. Le Graet, F. Gaucheron, Food Chem. 90 (2005) 673. [21] V.E. Bower, R.G. Bates, J. Res. Natl. Bur. Stand. 55 (1955) 197. [22] V. Camel, Spectrochim. Acta Part B 58 (2003) 1177. [23] J. Korkisch, Handbook of Ion Exchange Resins: Their Application of Inorganic Analytical Chemistry, CRC Press, Boca Raton, 1989. [24] J. Inczedy, Analytical Applications of Complex Equilibria, Ellis Horwood, Chichester, 1976. [25] S.J.J. Debon, R.F. Tester, Food Chem. 73 (2001) 401.