Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in Milk, Cheese and Chocolate

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1 Mikrochim. Acta 134, 185±191 (2000) Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in Milk, Cheese and Chocolate I. Karadjova 1;, S. Girousi 2, E. Iliadou 2, and I. Stratis 2 1 Faculty of Chemistry, University of So a, 1126 So a, Bulgaria 2 Analytical Chemistry Laboratory, Chemistry Department, Aristotle University of Thessaloniki, Thessaloniki, Greece Abstract. Combined analytical procedures consisting of wet digestion step followed by instrumental determination ± differential pulse cathodic stripping voltammetry (DPCSV) or electrothermal atomic absorption spectrometry (ETAAS) ± as well as a direct analysis method ± slurry sampling ETAAS ± for the determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in milk, cheese and chocolate are described and compared. Wet digestion using a mixture of HNO 3 -HClO 4 -H 2 O 2 is proposed for complete matrix decomposition prior to trace analyte determination by DPCSV or ETAAS. A mixture of HNO 3 -H 2 O 2 is used for slurry preparation. Optimal instrumental parameters for trace analyte measurements are presented. The reliability of the procedures has been veri ed by analyzing standard reference materials. Results obtained are in good agreement with the certi ed values and the relative standard deviations (for these results) are in the range 5±10 % for wet digestion DPCSV or ETAAS and 3±9 % for slurry sampling ETAAS in the range of 2 mg g 1 (Cd) to 12 mg g 1 (Fe). Key words: Milk products; analysis; slurry; DPCSV; ETAAS. Fast, sensitive, accurate and precise analytical methods are required for daily quality control of milk and milk products. The importance of elements such as Cu, Cr and Fe is related to lipid oxidation involved in storage and processing [1]. Serious attention is paid to the toxicological effects of other heavy metals such as Cd, Co, Ni and Pb in view of the importance of milk To whom correspondence should be addressed and its by-products in the diet of infants and children [2±4]. In the conventional practice, procedures used for the analysis of milk and milk products consist of a sample digestion step and a subsequent instrumental determination of trace elements in the solution obtained. The sample decomposition step is one of the most important points in the analysis of such products with high organic matter content. The choice of the digestion procedure depends on the nature of the organic matrix, on the trace elements to be determined and on the instrumental methods to be used for their determination. Problems encountered in voltammetric measurements are usually associated with the incomplete destruction of organic matter during digestion [5±9]. High pressure decomposition using a mixture of HCl-HNO 3 [10] or HNO 3 -HClO 4 [11,12] as well as low or medium pressure microwave decomposition using HCl-HNO 3 [13,14] have been applied for the analysis of biological materials by differential pulse anodic stripping voltammetry. In the present work the applicability of DPCSV as an analytical method for the determination of Cd, Co, Cu, Ni and Pb in milk and milk products is investigated. A suitable wet digestion mixture and procedure as well as optimal instrumental parameters for DPCSV measurements are presented. 1-phenylpropan-1-pentylsulfonylhydrazon-2-oxime [15] is tested as a selective complexing agent for simultaneous adsorptive voltammetric determination of Cd, Co, Cu, Ni and Pb in the sample digest. Incomplete destruction of organic sample usually does not signi cantly in uence the analyte determinations by ETAAS. Therefore after careful optimisation

2 186 I. Karadjova et al. of the temperature programmes, ETAAS is used for comparative determinations of the trace element, in the sample solutions also used for voltammetric measurements, thus con rming the reliability of the proposed wet digestion DPCSV method. Slurry sampling ETAAS offers potential advantages in analytical speed and convenience compared with wet digestion [16,17]. The main dif culty in this case is to nd (i) the optimal mixture for slurry preparation, (ii) optimal instrumental parameters for accurate ETAAS determination of trace analytes and (iii) a method for calibration. In the present paper slurry sampling ETAAS is investigated as a possibility for trace elements determination in milk and milk products. A mixture of HNO 3 -H 2 O 2 is used for the slurry preparation. The atomisation behaviour of Cd, Co, Cr, Cu, Fe, Ni and Pb with the sample suspensions obtained is studied and optimal instrumental parameters for their accurate ETAAS determination are presented. The reproducibility and accuracy of the analytical methods proposed for determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in milk, cheese and chocolate is investigated by analysis of standard reference materials. Experimental Apparatus A Perkin Elmer Zeeman 3030 atomic absorption spectrometer equipped with a HGA-600 graphite furnace and an AS-60 autosampler were used for atomic absorption measurements. As primary radiation sources an electrodeless discharge lamp for Cd (Perkin Elmer, operated at 5 W in a continuous mode) and hollow cathode lamps for other elements investigated were used. The spectral slit width for all the analytes was 0.7 nm. Pyrolytically coated graphite tubes (part no. B , Perkin Elmer) were used as atomisers. Atomic absorption signals were recorded on an Anadex printer. Only peak areas were used for quanti cation. A Metrohm Herisau E 506 Polarecord equipped with a Metrohm Herisau E 505 voltammetric stand was used for the voltammetric measurements. As working electrode an EA 290 Metrohm Hanging Mercury Drop Electrode with a surface area of 2.22 mm 2 was used; the counter electrode was a platinum wire and the reference electrode was a Ag/AgCl electrode with saturated KCl. Reagents All reagents and solvents were of analytical-reagent grade unless speci ed otherwise. Nitric acid was additionally puri ed by subboiling distillation. Perchloric acid (d ˆ 1.67 g/ml, suprapur 1, E. Merck, Germany), hydrogen peroxide (60%, additionally puri ed by ion exchange using Duolite Strong Cation Exchanger C-20-K), produced in the Laboratory for High-Purity Substances, University of So a and Triton X-100 (Ferak, Berlin, Germany) were used. 1-Phenylpropan-1-pentylsulfonylhydrazon-2-oxime, which was used as a ligand in the voltammetric procedure, was prepared in the Institute for Organic Chemistry, Technische UniversitaÈt Bergakademie, Freiberg Germany. The multielement standard solutions for all the studied elements were prepared from Titrisol 1 (E. Merck, Germany). Doubly distilled water was used throughout. Sample Preparation Procedures Wet digestion in an open vessel. A sample amount of 5 g was accurately weighted into a 100 ml conical ask; 10 ml nitric acid (67 %) were added and the ask was covered with a watch glass. The sample was allowed to stand over night at room temperature in order to reduce the amount of gas produced during the subsequent heating cycle. Then the watch glass was replaced by a glass funnel (5 cm i.d.) inserted into the ask and the sample was heated on a hot-plate at 140 C for about 1 h to gently boil off the nitric acid. After cooling down to room temperature the funnel was taken away, 3 ml perchloric acid (72 %) were added and heating prolonged at 210 C until the liquid turned brown. After cooling down to 25 C, 3 ml hydrogen peroxide (60 %) were added to the sample drop by drop and the content reheated until the liquid turned brown again. The addition of hydrogen peroxide was repeated four times until a colourless solution was obtained and white fumes of perchloric acid were generated. The solution was evaporated to near 1 ml and after cooling was transferred into a 25 ml volumetric ask and made up to the mark with doubly distilled water. This solution was used in further measurements by voltammetry and ETAAS. Voltammetric Measurements. An aliquot amount of 10.0 ml of the digest was placed into a 25 ml volumetric ask, neutralized with ammonia of ph 7 and then 0.5 ml of a buffer solution (1M NH 4 OH-NH 4 Cl), and 0.33 ml of the ligand solution (0.001 M solution of 1-phenylpropan-1-pentylsulfonylhydrazon-2- oxime in methanol) were added and the content of the ask made up to the mark with doubly distilled water and then transfered into a polarographic cell; the solution was de-aerated by purging with N 2 gas for 5 min. The nitrogen ow was stopped and after 15 s equilibration time the potential was set to 0.00 V and a new mercury drop was extruded. The voltammogram was recorded using the differential pulse mode (0±1.2 V potential range) with a scan rate of 12.5 mvs 1, and a pulse amplitude of 50 mv. ETAAS Measurements. 20 ml of the digest were introduced into the graphite furnance under the instrumental parameters, given in Table 1. Slurry Preparation. As samples a commercially available dry milk and chocolate were used without additional drying or grinding. An accuratelly weighted sample amount of white or yellow cheese of 5 g was mixed with doubly disstilled water for 20 min in mixer and then freeze dried in Freezer dryer 3, Labconco. Dried powdered samples were ground in an agate ball mill for approximately 45±50 min, producing a ne powder suitable for the preparation of slurries. No sieving is carried out. Certi ed reference materials were used as received without additional grinding. Depending on the concentration of the analyte in the sample, 0.05±0.20 g powder was weighed in a polyethylene container with 2 ml mixture of 1 M HNO 3 and 1.5 M H 2 O 2. After addition of 1 ml 0.1 % v/v aqueous solution of Triton X-100 to the slurries obtained, they were additionally treated for 20 min in an ultrasonic bath. Blanks were run for all methods used.

3 Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in Milk, Cheese and Chocolate 187 Results and Discussion Voltammetric Determination of Cd, Co, Cu, Ni and Pb After Wet Digestion Complete mineralization of the sample is the most serious requirement for accurate voltammetric analysis. In the present work the mixture HNO 3 -HClO 4 - H 2 O 2 was found to be the most suitable for the wet digestion of milk, cheese and chocolate prior to DPCSV measurements. 1-Phenylpropan-1-pentylsulfonylhydrazon-2-oxime forms stable complexes with Cd, Co, Cu, Ni and Pb (ph range 3±10), which can be accumulated by adsorption on the electrode-solution surface and after that stripped off in cathodic form. Experiments performed (variation of peak current and reduction potential as a function of ph) showed that the optimal ph range for a DPCSV measurement of all studied elements is 8±9, so a 1M NH 4 OH-1M NH 4 Cl solution was chosen as buffer electrolyte. The effect of the accumulation potential was evaluated in the range 0±1.2 V. An accumulation at 0.8 V was found to be suitable for obtaining a sensitive and reproducible peak current for all studied elements. Other instrumental parameters e.g. scan rate, pulse amplitude, rest period and drop size were also optimized [15]. The standard addition method is preferred for calibration as then the matrix effects can be largely eliminated (3 additions). ETAAS Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb ETAAS Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb After Wet Digestion. Temperature programmes for accurate ETAAS determination, of Cd, Co, Cr, Cu, Fe, Ni and Pb in the solutions obtained after wet digestion of the samples (milk, white cheese, yellow cheese, chocolate) were optimized with respect to preteratment and atomisation curves prepared for each element and for each of the matrices studied. Results were summarised in Table 1. As it can be expected, after wet digestion no signi cant differences were observed between the matrices investigated. It has to be pointed out that complete removal of perchloric acid at the end of the digestion procedure is a matter of great importance [18]. Results obtained during this investigation undoubtedly showed that accurate ETAAS determination of Cd and Pb are possible without using any modi er. Pretreatment temperatures achieved without modi er are 600 C for Cd and 800 C for Pb. These temperatures are high enough to ensure matrix removal during the ashing step and to decrease the values of nonspeci c absorption during the atomisation step. Slurry Sampling ETAAS. It is known that the stability of a slurry depends on the type of the matrix, on the particle size and on the concentration of surfactants e.g. Triton X-100 added for the slurry preparation. Homogeneous suspensions, stable for a long period of time can very easy be prepared from dry milk, skim milk and chocolate and their stability does not depend signi cantly on the particle size. In the case of white and yellow cheese the particle size is very important parameter. Even after increasing the concentration of Triton X-100, a stable suspension can only be produced if the particle size is less than 40 mm. The particle sizes were measured with a laser granulometer mlps-16, PMS (U.S.A.) A general problem encountered when introducing organic slurries directly into the furnace is the production of a carbonaceous residue in the tube. It might be assumed that hydrogen peroxide used for the slurry preparation will assist the decomposition of organic matrix by converting the charring step into an oxidative decomposition process. Experiments provided con rm this suggestion. A concentrations of hydrogen peroxide of 0.1±0.2 M in the slurry was enough to prevent the build up of a carbonaceous residue and also to allow the use of higher ashing temperatures without analyte losses in the case of Cd and Pb. The atomisation behaviour of Cd, Co, Cr, Cu, Fe, Ni and Pb is carefully studied for slurry samples prepared in order to establish the appropriate temperature programmes for their ETAAS determination. The matrix content in the slurry was chosen after taking into account the values of nonspeci c absorption observed for each element in the atomisation step and the background correction capability of the instrument. The possibility for using modi ers was checked in each particular case. The most serious problems were observed with ETAAS determination, of Cd, Co, Ni and Pb and were obviously due to their low concentrations and susceptibility to matrix interferences. The effect of a variation of the pretreatment and atomisation temperatures on the absorbance signals for Cd, Co, Cr, Cu, Ni and Pb in suspensions prepared from dry milk, white cheese and

4 188 I. Karadjova et al. Fig. 1. Pretereatment and atomisation curves for Cd, Co, Cu, Cr, Ni and Pb in the slurries prepared from different samples chocolate as well as their atomisation behaviour in the spiked blank samples is shown in Fig 1. It can be seen that pretreatment temperatures around 700 C could be used for Pb without analyte losses. The attempt to use Pd as modi er to subsequently increase the pretreatment temperature and thus to increase the matrix content in the slurry failed. In the presence of Pd atomisation temperatures around 2300 C are required for a complete atomization of Pb and very high values of nonspeci c absorption were observed. In general, for Pb it is most important to use lower pretreatment temperatures, together with a relatively long pretreatment step (30 s) and to keep the difference between ash and atomisation temperature

5 Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in Milk, Cheese and Chocolate 189 as small as possible. For Cd the use of a modi er was unavoidable. Ashing temperatures around 300 C achieved in the absence of a modi er are too low for matrix mineralisation and removal. The concentration of Pd as modi er was varied from 50±500 mg ml 1 in the slurry solution and it was found that a concentration of 100 mg ml 1 is optimal. This modi er concentration permits the use of a pretreatment temperature around 600 C, which is high enough for matrix removal. At the same time a relatively low atomisation temperature of 1700 C can be used, which is important for the time resolution of the analyte absorbance signal from the signal of nonspeci c absorption. Pretreatment temperatures in the range of 900±1100 C and relatively low atomisation temperatures of 900±2300 C are most suitable for ETAAS determination, of Cr, Cu, Co and Ni. No signi cant differences were observed in the atomisation behaviour of Cd, Co, Ni, and Pb in the suspensions prepared from different type of samples studied. The relatively high content of a Fe and Cu in the matrices investigated permits their determination with ETAAS in slurries with a matrix content of 0.05 mg ml 1 Therefore, no signi cant problems were observed with their determination. The optimal instrumental parameters for ETAAS determination, of Cd, Co, Cr, Cu, Fe, Ni and Pb in the slurries prepared from dry milk, white cheese, yellow cheese and chocolate are presented in Table 1. Calibration. Matrix interferences observed in the proposed analytical procedures/wet digestion/etaas and slurry ETAAS were evaluated according to the slopes of the calibration curves obtained in the presence of the matrix investigated and in the presence of blank solution. Results achieved for dry milk, white cheese and chocolate are reported in Table 2. Unsigni cant differences were observed between the slopes of the calibration curves for Cu and Fe obtained for the blank and sample solution, which favours calibration with aqueous standard solutions for both wet digestion ETAAS and slurry ETAAS. Calibration by standard addition should be used for ETAAS determination, of Cd, Co, Cr, Ni and Pb. However, the slopes of the calibration graphs for different samples (dry milk, white cheese, chocolate) in the case of wet digestion ETAAS do not statistically differ. Subsequently, this analytical procedure requires single standard addition to one of the samples analyzed. Signi cant differences were observed in the atomisation behaviour (appearance time and peak pro les) for all trace elements studied in the slurries prepared compared with aqueous standard solution. Experiments performed, however, showed that under optimal instrumental parameters (Table 1) aqueous standard additions of the elements to the slurry have a similar behaviour as the elements present in the samples. Double peaks were not found for any one of analytes investigated. Therefore, the standard addition method could be used for calibration. As it can be seen from the data presented in Table 2 the Table 1. Recommended conditions for HGA programme for the Zeeman 3030; 20-ml sample aliquot. Parameter Step Temperature/ C 120 variable variable variable Ramp time/s Hold time/s Read ± ± on ± Internal gas ow/ml min Analyte Temperature C Cd (wet digestion) Cd (slurry) Co (wet digestion,slurry) Cr (wet digestion,slurry) Cu (wet digestion,slurry) Fe (wet digestion,slurry) Ni (wet digestion,slurry) Pb (wet digestion) Pb (slurry) ±100 mg ml 1 Pd in the slurry.

6 190 I. Karadjova et al. Table 2. Slopes (Abs. mg 1 ) of the best t linear regression models for standard additions of Cd, Co, Cr, Cu, Fe, Mn, Ni and Pb to the blank sample and samples of milk, white cheese and chocolate Element Slopes (mean s r a )of blank samples Slopes (mean s a r ) of standard additions calibration curves ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ ÐÐ Wet digestion ETAAS Slurry ETAAS Dry milk White cheese Chocolate Dry milk White cheese Chocolate Cd % % % % % % % Co 5.8 3% 5.3 4% 5.2 3% 5.1 3% 5.5 3% 5.0 5% 5.1 4% Cu 6.2 4% 6.1 5% 6.2 4% 6.0 4% 6.2 4% 5.9 5% 6.0 4% Cr % % % % % % % Fe 5.6 7% 5.5 6% 5.5 5% 5.3 5% 5.4 5% 5.2 7% 5.5 6% Ni 4.1 3% 3.7 4% 3.8 4% 3.7 4% 3.9 4% 3.3 6% 3.6 4% Pb 4.5 4% 4.1 3% 3.9 4% 4.0 4% 4.1 5% 3.6 6% 3.9 5% S r a Relative standard deviation of the slope. Slopes (Abs. ng 1 ). slopes of calibration curves for different type, of slurry samples are statistically different and a standard addition for each type of samples is recommended. Accuracy and Precision To check the over-all precision of the procedures i.e. wet digestion/etaas, wet digestion/dpcsv and slurry ETAAS, sample, of dry milk and white cheese were analysed for Cd, Co, Cr, Cu, Fe, Ni and Pb. The results obtained are presented in Table 3. As it can be expected wet digestion/dpcsv and wet digestion/ ETAAS have very similar RSD values, determined by the wet digestion procedure used. The simple sample preparation step in slurry ETAAS lets expect a better reproducibility. This is true in the case of milk and chocolate samples, but for cheese samples the RSD values are higher, which is probably due to some inhomogeneity of slurries prepared. Detection limits obtained depend on the amount of sample used for the analysis. The methods were applied to standard reference materials. Results achieved and listed in Table 4 are in good agreement (ANOVA, Student's test, 95 % Table 3. Analysis of dry milk and white cheese by using wet digestion DPCSV, wet digestion ETAAS and slurry sampling ETAAS (three paralel determinations) Element Wet digestion DPCSV Wet digestion ETAAS Slurry ETAAS Mean, RSD, % DL, Mean, RSD, % DL Mean, Dry milk Cd Co < DL ± 20 < DL ± 20 Cr ± ± ± Cu Fe ± ± ± Ni Pb < D.L ± White cheese Cd Co < DL ± 20.0 < DL ± 20 Cr ± ± ± Cu Fe ± ± ± Ni Pb criteria. RSD, % DL

7 Determination of Cd, Co, Cr, Cu, Fe, Ni and Pb in Milk, Cheese and Chocolate 191 Table 4. Comparative results for Cd, Co, Cr, Cu, Fe, and Pb in certi ed reference materials (three parallel determinations) SRM Milk powder A-11 (mean s) Element Certi ed value Wet digestion DPCSV Wet digestion ETAAS Slurry ETAAS Cd ( ) < DL Co ( ) < 5 < 5 Cr ( ) ± Cu (m g 1 ) Fe (mg g 1 ) ± Pb ( ) CRM ± 150, skim milk powder (mean s) Element Certi ed value Wet digestion DPSPV Wet digestion ETAAS Slurry ETAAS Cd ( ) Cu (mg g 1 ) Fe(mg g 1 ) ± Pb(mg g 1 ) con dence level) with the certi ed values. This indicates that the method are unbiased and proved their validity and versatility. Acknowledgements. The authors wish to thank the Greek Ministry of Education for the scholarship provided to Dr. I. Karadjova, research fellow from the University of So a in Bulgaria References [1] H. Jonsson, Milchwissenschaft 1976, 31, 210. [2] D. Snodin, J. Ass. Publ. Analyst 1973, 11, 112. [3] J. Koops, D. Westerbeek, Neth. Milk Dairy J. 1978, 32, 149. [4] E. Merian, Metals and Their Compounds in the Environment, Occurrence. Analysis and Biological Relevance VCH. Weinheim, [5] M. WuÈrfels, E. Jackwerth, Fresenius Z. Anal. Chem. 1985, 322, 354. [6] M. WuÈrfels, E. Jackwerth, M. Stoeppler, Fresenius Z. Anal. Chem. 1988, 329, 459. [7] M. WuÈrfels, E. Jackwerth, M. Stoeppler, Fresenius Z. Anal. Chem. 1988, 330, 160. [8] G. Knapp, Mikrochim Acta II 1991, 445. [9] K. Pratt, H. Kingston, W. MacCrehan, W. Foch, Anal. Chem. 1988, 60, [10] E. Sucman, M. Sucmanova, O. Celechovska, S. Zima, 6th Colloquium Atomspektrometrische Spurenanalytik. UÈ berlingen, 1991, p [11] P. Schramel, S. Hasse, G. Knapp, Fresenius Z. Anal. Chem. 1987, 326, 142. [12] P. Schramel, S. Hasse, Fresenius Z. Anal. Chem. 1993, 346, 794. [13] E. Stryjewska, S Rubel, I. Skowron, Chem. Anal. (Warsaw) 1994, 39, 609. [14] E. Stryjewska, S Rubel, I. Szynkarczuk, Fresenius J. Anal. Chem. 1996, 354, 128. [15] E. Iliadou, S. Girousi, U. Dietze, M. Otto, A. Voulgaropoulos, C. Papadopoulos, Analyst 1997, 122, 597. [16] C. Bendicho, M.T.C. de Loos-Vollebregt, J. Anal. Atom. Spectrometry 1991, 6, 353. [17] Z. de Benzo, M. Velosa, C. Ceccardi, M Guardia, A. Salvador, Fresenius Z. Anal. Chem. 1991, 339, 235. [18] J. Martinsen, F. Langmyhr, Anal. Chem. Acta 1982, 135, 137. Received August 24, Revision January 20, 2000.

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