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Improvement of microwave-assisted digestion of milk powder with diluted nitric acid using oxygen as auxiliary reagent
Spectrochimica Acta Part B 66 (2011) 394–398
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Spectrochimica Acta Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s a b
Analytical note
Improvement of microwave-assisted digestion of milk powder with diluted nitric acid using oxygen as auxiliary reagent Cezar A. Bizzi a, e, Juliano S. Barin b, Edivaldo E. Garcia c, Joaquim A. Nóbrega d, Valderi L. Dressler a, e, Erico M.M. Flores a, e,⁎ a
Departamento de Química, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil Departamento de Tecnologia e Ciência dos Alimentos, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil Departamento de Química, Universidade Estadual de Maringá, 87100-900, Maringá, PR, Brazil d Departamento de Química, Universidade Federal de São Carlos, 13565-905, São Carlos, SP, Brazil e Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, SP, Brazil b c
a r t i c l e
i n f o
Available online 7 May 2011 Keywords: Microwave-assisted digestion ICP OES CVG-ICP-MS Trace elements Oxygen pressure
a b s t r a c t The feasibility of using diluted HNO3 solutions under oxygen pressure for decomposition of whole and non-fat milk powders and whey powder samples has been evaluated. Digestion efficiency was evaluated by determining the carbon content in solution (digests) and the determination of Ca, Cd, Cu, Fe, K, Mg, Mn, Mo, Na, Pb and Zn was performed by inductively coupled plasma optical emission spectrometry and Hg by chemical vapor generation coupled to inductively coupled plasma mass spectrometry. Samples (up to 500 mg) were digested using HNO3 solutions (1 to 14 mol L− 1) and the effect of oxygen pressure was evaluated between 2.5 and 20 bar. It was possible to perform the digestion of 500 mg of milk powder using 2 mol L − 1 HNO3 with oxygen pressure ranging from 7.5 to 20 bar with resultant carbon content in digests lower than 1700 mg L− 1. Using optimized conditions, less than 0.86 mL of concentrated nitric acid (14 mol L − 1) was enough to digest 500 mg of sample. The accuracy was evaluated by determination of metal concentrations in certified reference materials, which presented an agreement better than 95% (Student's t test, P b 0.05) for all the analytes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Milk could be considered as a staple food that provides essential nutrients (proteins, lipids, and carbohydrates) and micronutrients (minerals, vitamins, and enzymes) [1,2]. On the other hand, milk can also constitute a source of exposure to toxic elements, especially dangerous for infants, such as Cd, Hg and Pb [3,4]. In this sense, it is important to establish appropriate sample preparation methods for subsequent metal determination in order to assure the quality of final products. In addition, these methods should be developed considering lower reagent consumption and suitable digestion efficiency [5]. Most of conventional sample preparation methods for atomic spectrometric techniques involve sample solubilization with complete or partial matrix decomposition generally using oxidant acids [6]. In previous years, some improvements were proposed towards the development of new procedures based on less conventional approaches to organic matter digestion [7–18], which normally present relatively high efficiency of digestion resulting in low values of residual carbon content. ⁎ Corresponding author at: Departamento de Química, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. Tel./fax: +55 55 3220 9445. E-mail address: [email protected] (E.M.M. Flores). 0584-8547/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2011.04.013
Some procedures based on diluted solutions of nitric acid were also developed for digestion of biological samples [19,20]. The efficiency of diluted nitric acid for oxidation of organic matter can be explained by the regeneration of nitric acid promoted by the combination of the nitrogen oxide species with the oxygen present inside the reaction vessel [20–22]. Based on the reaction mechanism of nitric acid regeneration, which occurs while oxygen is still present in the reaction vessel, digestion procedure has been performed under oxygen pressure [23,24], which allowed digestions with amounts of nitric acid lower than 3 mol L − 1. In the present work, a procedure based on vessels pressurized with oxygen and determination by inductively coupled plasma (ICP)-based spectrometric techniques is proposed for metals determination in milk powder. The determination of essential (Ca, Cu, Fe, K, Mg, Mn, Mo, Na and Zn) and toxic (Cd, Hg and Pb) metals was performed by inductively coupled plasma optical emission spectrometry (ICP OES) and by chemical vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS, only for Hg). Certified reference materials (CRM) were used to check the accuracy. In addition, it was tried to reduce as much as possible the amount of nitric acid needed for the digestion process in order to minimize the blank values and to decrease the consumption of reagents and the consequent generation of laboratory residues.
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2. Experimental
2.2. Samples, reagents and standards
2.1. Instrumentation
Preliminary experiments were carried out using whole milk powder. After optimization step, the proposed procedure was applied to the digestion of whey, whole and non-fat milk powders, which were purchased in a local market. All samples were dried at 60 °C using an oven (model 400/2ND, Nova Ética, Vargem Grande Paulista, SP, Brazil). Accuracy was evaluated using CRM of skim milk powder (BCR 151, Community Bureau of Reference, Brussels, Belgium) and of non-fat milk powder (SRM NIST 1549, National Institute of Standards and Technology, NIST, Gaithersburg, MD, USA). Samples were weighed using an analytical balance (model AY 220, max. 220 g, 0.1 mg of resolution, Shimadzu, Kyoto, Japan). Distilled-deionized water (Milli-Q, 18.2 MΩ cm, Millipore, Billerica, MA, USA) and analytical-grade nitric acid (Merck, Darmstadt, Germany) were used to prepare samples and standards. Carbon reference solutions used in external calibration for carbon content determination were prepared by dissolution of citric acid (Merck) in water (25 to 500 mg L− 1 of C). Yttrium (1.0 mg L − 1, Spex CertPrep, Metuchen, NJ, USA) was used as internal standard in all samples, blanks and reference solutions for carbon content determination. Metal determination by ICP OES was performed with external calibration using analytical solutions ranging from 1.0 (Cd, Mn, Zn), or 5.0 (Cu, Fe, Mg, Mo), or 10 μg L− 1 (Ca, K, Na, Pb) up to 100 μg L − 1, prepared in 0.7 mol L − 1 HNO3 by appropriate dilution of the multi-element stock solution (SCP33MS, SCP Science, Quebec, Canada). Mercury determination by CVG-ICP-MS was performed with external calibration by dilution of inorganic mercury (Hginorg) standard stock solution Titrisol (1000 mg L − 1, Merck), which was prepared just before use. Sodium tetrahydroborate (0.2% m/v) used for CVG was obtained from Vetec (Duque de Caxias, RJ, Brazil). A 0.1 mol L − 1 KOH (Merck) solution was used for residual acidity determination. Glass and quartz material were soaked in 1.4 mol L − 1 HNO3 for 24 h and further washed with water before use.
A microwave oven (Multiwave 3000 microwave sample preparation system, Anton Paar, Graz, Austria) equipped with eight highpressure quartz vessels was used in the experiments. The internal volume of vessels was 80 mL and the maximum operational temperature and pressure were set at 280 °C and 80 bar, respectively. Analytes (except Hg) were determined by ICP OES using an axial view configuration spectrometer (Spectro Ciros CCD, Spectro Analytical Instruments, Kleve, Germany) using a cross-flow nebulizer coupled to a Scott double pass type nebulization chamber. Plasma operating conditions and selected wavelengths are listed in Table 1, and they were used as recommended by the instrument manufacturer [25]. Chemical vapor generation (CVG) coupled to ICP-MS (PerkinElmer Sciex, Model Elan DRC II, Thornhill, Canada), equipped with a quartz torch with a quartz injector tube (2 mm i.d.), was used for Hg determination. The CVG system consists of a peristaltic pump (Ismatec, Zurich, Switzerland) and a U-type gas–liquid separator [26]. The mixture was pumped to the gas–liquid separator and Hg measurements were performed by ICP-MS. Plasma operating conditions and selected isotope used for Hg determination are also listed in Table 1. Carbon content in digests (related to 500 mg of sample and dilution to 30 mL) was determined by ICP OES [27,28]. In order to remove the volatile carbon compounds before carbon content determination, digests were previously sonicated with an ultrasonic probe [29] (VCX 130 PB, 130 W, 20 kHz, Sonics and Materials Inc., Newton, CT, USA). This procedure was not applied when determining the analytes in final digests, which were just diluted with water and analyzed by ICP OES and CVG-ICP-MS. Argon (99.996%, White Martins-Praxair, São Paulo, SP, Brazil) was used for ICP OES and ICP-MS determination for plasma generation, nebulization, auxiliary gas and also for digestion performed under inert atmosphere. Oxygen (99.9991%, White Martins-Praxair) was used as reagent in digestions performed under oxygen pressure. It is important to mention that all procedures under oxygen pressure were performed employing safety conditions, as recommended by the microwave oven manufacturer [30]. Results for residual acidity were obtained using a titration system (Titrando 836, Metrohm, Herisau, Switzerland) equipped with a magnetic stirrer (module 803 Ti Stand), 20 mL burette (Dosino 800) and pH electrode (LL Electrode plus, model 6.0262.100). Table 1 Operational parameters for determination of C, Ca, Cd, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Pb and Zn. Parameter
ICP OES
ICP-MS
Radio-frequency power (W) Plasma gas flow rate (L min− 1) Auxiliary gas flow rate (L min− 1) Nebulizer gas flow rate (L min− 1) Spray chamber Nebulizer Observation view Analytes C (I) Ca (II) Cd (II) Cu (I) Fe (I) Hg K (I) Mg (I) Mn (II) Mo (II) Na (I) Pb (II) Zn (I)
1600 14.0 1.0 0.85 Double pass, Scott type Crossflow Axial Emission line (nm) 193.091 393.366 226.502 324.752 238.204 – 766.491 285.213 257.610 202.030 589.592 220.353 213.856
1300 15.0 1.2 1.13 * * – Isotope (m/z) – – – – – 202 – – – – – – –
*Direct introduction by CVG, (I) atomic emission and (II) ion emission.
2.3. Microwave-assisted acid digestion Samples (up to 500 mg) were transferred to the quartz vessels. Digestion efficiency using nitric acid solutions (6 mL) was evaluated in the following concentrations: 1, 2, 3, 7, and 14 mol L − 1 HNO3. After closing and capping the rotor, vessels were pressurized with 2.5, 5, 7.5, 10, 15 and 20 bar of oxygen. The gases were introduced into the vessels using the valve originally designed for pressure release after conventional acid sample digestion. Then, the rotor was placed inside the oven, and microwave-heating program was started by applying (i) 1000 W with a ramp of 5 min, (ii) 1000 W for 10 min, and (iii) 0 W for 20 min (cooling step) [30]. After digestion, the pressure of each vessel was carefully released. In this work, each run was performed using a minimum of four vessels. The resulting solutions were transferred to 30 mL polypropylene vials and diluted to the mark with water. Cleaning of digestion vessels was carried out with 6 mL of concentrated HNO3 in the microwave oven at 1000 W for 10 min and 0 W for 20 min for cooling. All statistical comparisons were performed using Student's t test, (GraphPad InStat Software Inc., Version 3.00, 1997). A significance level of P b 0.05 was chosen for all comparisons. 3. Results and discussion 3.1. Preliminary evaluation of digestion efficiency Preliminary tests were carried out with air at atmospheric pressure in order to evaluate the effect of oxygen pressure on whole milk powder digestion. Nitric acid concentration was varied in order to achieve a condition of efficient organic matter digestion using as low as possible concentrated acid solution, which was evaluated by the carbon content in digests and residual acidity determination. The same procedure was repeated using oxygen for pressurization of
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reaction vessels. In order to evaluate the effectiveness of digestion procedure, a selected tolerance level lower than 2000 mg L − 1 of carbon content in solution was chosen, which were considered appropriate for subsequent analysis by ICP OES and ICP-MS.
organic matter. This reaction could be effective with diluted nitric acid if digestion vessels were pressurized with oxygen.
3.1.1. Digestion procedure with air Preliminary experiments were performed using 500 mg of whole milk powder to evaluate the minimum nitric acid concentration that was sufficient to obtain suitable values of carbon content in digests. Thus, digestion using different nitric acid concentrations under atmospheric air pressure was tested. When 14 and 7 mol L − 1 HNO3 solutions were used, the carbon content in solution was lower than 532 ± 45 and 720 ± 114 mg L − 1, respectively. However, using 3 mol L − 1 HNO3, final digests presented a yellow color and carbon content values were higher, about 8000 mg L − 1. When 2 mol L − 1 HNO3 was used the digestion was not effective and solid residues remained as suspended particles with a deep brown color (carbon content values as high as 10,500 mg L − 1 were obtained). The residual acidity was also determined in final digests obtained from digestion procedure performed at atmospheric air. When using 7 and 14 mol L − 1 HNO3, which showed good digestion efficiency, the residual acidities were 44 and 64% (m/m), respectively. Although, digestions performed using 3 and 2 mol L − 1 did not present a good digestion efficiency of organic matter, lower residual acidities were obtained in final digests (13% m/m for both, 3 and 2 mol L − 1 HNO3). Final digests obtained using more concentrated nitric acid solutions (7 and 14 mol L − 1), presented a colorless aspect. Best results under these conditions, regarding carbon content in digests and residual acidity were obtained using 7 mol L − 1 HNO3.
For the evaluation of gas phase effect inside the digestion vessel, whole milk powder sample (500 mg) was digested using 2 mol L − 1 HNO3 with oxygen pressures ranging from 20 to 2.5 bar. As it can be seen in Fig. 1, the digestion procedure presented almost the same efficiency (carbon content in digests b2000 mg L − 1) for oxygen pressures from 20 to 7.5 bar. In these conditions, according to the ideal gas behavior the initial amounts of oxygen inside the reaction vessel were 1.94, 1.46, 0.97 and 0.73 g for 258 pressures of 20, 15, 10 and 7.5 bar, respectively. However, the efficiency of digestion decreased for lower oxygen pressure. Higher values of carbon content were observed for 5 and 2.5 bar of oxygen, i.e., 2217 ± 106 and 4528 ± 659 mg L − 1, respectively. In these conditions, the initial oxygen amounts inside reaction vessels were 0.49 and 0.24 g, respectively. The residual acidity was also determined. As expected, the residual acidity in final solutions presented similar values, ranging from 23 to 13% m/m (15 and 2.5 bar, respectively). As previously discussed [23,24], the HNO3 regeneration process was not dependent on the initial pressure. Nevertheless, results obtained in the present study indicate a closer relation between oxygen and the nitric acid amount inside the reaction vessels. When nitric acid concentration was lower than the minimum amount required to obtain an effective organic matter oxidation, the regeneration process could be considered decisive for digestion when oxygen was present inside the reaction vessel in a minimum initial amount of 0.73 g (7.5 bar). When lower amount of oxygen was used, the regeneration process was less effective and final digests showed carbon content in solution higher than 2000 mg L − 1. Therefore, in the range of oxygen pressure evaluated in this work, no significant changes were observed at pressures higher than 7.5 bar based on carbon content and residual acidity values and this pressure of oxygen was selected for further experiments. 3.3. Determination of Ca, Cd, Cu, Fe, Hg, K, Mg Mn, Mo, Na, Pb and Zn in CRM, whey, whole and non-fat milk powder samples
6000
100
5000
80
4000 60 3000 40 2000 20
1000 0
2.5
5
7.5
10
15
20
Residual acidity (%)
Accuracy was evaluated by using CRMs of non-fat and skim milk powders. Digestion of CRM, whole milk powder, skim milk powder and whey powder samples was performed using optimized conditions (500 mg of sample mass, 2 mol L − 1 HNO3 and 7.5 bar of O2). Results obtained for Ca, Cd, Cu, Fe, Hg, K, Mg Mn, Mo, Na, Pb and Zn in CRM presented a good agreement (better than 95%, Student's t test, P b 0.05) with certified values as it may be seen in Table 2. It is
Carbon content in digests (mg L-1)
3.1.2. Digestion procedure with oxygen pressure The effect of nitric acid concentration on digestion efficiency was also evaluated under 20 bar of oxygen pressure. Sample masses of 500 mg were used and carbon content was determined in final digests. Digestions were evaluated using nitric acid concentrations ranging from 14 to 1 mol L− 1. A similar effectiveness on organic matter oxidation was observed with solutions ranging from 14 to 2 mol L− 1 HNO3, where was possible to obtain final digests with carbon content values lower than 2000 mg L − 1 (1124 ± 235, 1345 ± 158, 1703 ± 68 and 1450 ± 50 mg L − 1 for solutions of 14, 7, 3 and 2 mol L − 1 HNO3, respectively). The digestion efficiency was low when using 1 mol L− 1 HNO3; final digests presented higher values of carbon content in solution (2345 ± 30 mg L − 1) and slightly brown color with solid residues remaining as suspended particles. The residual acidity in final digests was between 99% m/m (14 mol L − 1 HNO3) and 18% m/m (2 mol L − 1 HNO3). Based on these results the residual acidities obtained for digestions performed under atmospheric pressure were compared with those performed under oxygen pressure. When the same nitric acid concentration was evaluated, the residual acidity for digestion performed under oxygen pressure always presented higher values and the digested solutions were clear. A different behavior was observed in final digests obtained for experiments performed at atmospheric air. This result obtained for digestion performed under oxygen pressure may be due to the absence of NO2 in the gas phase, probably owing to the regeneration process of nitric acid promoted by oxygen, which leads to higher residual acidity values. In this sense, even using an acid solution as diluted as 2 mol L − 1 HNO3 to digest 500 mg of whole milk powder the carbon content values were always lower than 2000 mg L − 1 using 20 bar of oxygen. It results in a reduction in the nitric acid concentration around 8 times without decreasing the digestion efficiency when compared with the use of more concentrated nitric acid solution. As reported in previous studies [23,24], the oxidant action of nitric acid may be improved if a regenerating process occurs, which is mainly dependent on the amount of oxygen available in gas phase during the oxidation of the
3.2. Evaluation of different oxygen pressures
0
Oxygen pressure (bar) Fig. 1. Digestion efficiency of 500 mg of whole milk powder using 2 mol L− 1 HNO3; effect of oxygen pressure (gray bars) on digestion efficiency (horizontal line represents the arbitrarily chosen tolerance level of carbon content in solution = 2000 mg L− 1). Error bars represent the standard deviation (n = 3).
C.A. Bizzi et al. / Spectrochimica Acta Part B 66 (2011) 394–398 Table 2 Elements determined in milk CRMs by ICP OES (mean and confidence interval in μg g− 1, n = 5). Digestion conditions: 2 mol L− 1 HNO3 and 7.5 bar O2. Analyte
Ca Cd Cu Fe Hga K Mg Mn Mo Na Pb Zn a b
397
Table 4 Figures of merit for the proposed digestion procedure based on diluted nitric acid and oxygen pressure.
NIST 1549 (non-fat milk powder)
BCR 151 (skim milk powder)
Parameters
Proposed digestion procedure
Certified
Found
Certified
Found
13 000 ± 500 0.0005 ± 0.0002 0.7 ± 0.1 1.78 ± 0.10 0.0003 ± 0.0002 16 900 ± 300 1200 ± 300 0.26 ± 0.6 0.34b 4970 ± 100 0.019 ± 0.003 46.1 ± 2.2
13 218 ± 1293 b 0.05 0.63 ± 0.11 1.82 ± 0.11 b 0.004 17 101 ± 579 1195 ± 61 0.235 ± 0.051 0.310 ± 0.062 5183 ± 1053 b 1.15 46.9 ± 0.5
– 0.101 ± 0.008 5.23 ± 0.08 50.1 ± 1.3 0.101 ± 0.01 – – – – – 2.002 ± 0.026 –
11 844 ± 127 0.100 ± 0.001 5.24 ± 0.002 48.7 ± 1.9 0.102 ± 0.002 16 390 ± 455 1229 ± 14 0.269 ± 0.015 0.314 ± 0.022 4202 ± 85 1.92 ± 0.11 49.7 ± 3.1
Sample mass, mg Acid concentration, mol L− 1 Oxygen pressure, bar Carbon content in digests, mg L− 1 Residual acidity, % m/m
500 2 7.5 b1700 b18
Determination by CVG-ICP-MS. Non-certified value.
blank mean) obtained by ICP OES determination for Ca, Cd, Cu, Fe, K, Mg, Mn, Mo, Na, Pb and Zn was (0.81, 0.05, 0.18, 0.11, 0.59, 0.01, 0.02, 0.44, 0.11, 1.15, 0.07) μg g − 1, respectively. For Hg, a LOQ of 0.004 μg g − 1 was obtained by CVG-ICP-MS. The main analytical figures of merit and optimized conditions of the proposed digestion procedure under oxygen pressure in relation to the conventional wet digestion are shown in Table 4. 4. Conclusions
important to mention that milk powder is a complex matrix to bring into solution by conventional digestion procedures. It is a mixture of proteins, fats and sugars, with different amounts and compositions depending on the milk characteristics. As an example, whole milk powder is composed by 26.3, 26.7 and 38.4% (m/m) of protein, fat and sugar, respectively, while skim milk powder is 36.2, 0.8 and 52.0% (m/ m) of protein, fat and sugar, respectively and, whey powder is 12.9, 1.1 and 74.5% (m/m) of protein, fat and sugar, respectively [2]. However, even with different matrix compositions for different types of milk, the proposed procedure presented quantitative recoveries for Ca, Cd, Cu, Fe, K, Mg Mn, Mo, Na, Pb and Zn by ICP OES determination and for Hg by CVG-ICP-MS determination. In addition, the results obtained for whole milk powder digested by conventional wet digestion (6 mL of 14 mol L − 1 nitric acid, 300 mg of sample mass, 200 °C, 50 bar, polytetrafluorethylene vessels, Model Ethos 1, Milestone system) [31] presented a good agreement (better than 95%, Student's t test, P b 0.05) when compared with results obtained by the proposed procedure. This fact confirms that the proposed procedure, using diluted nitric acid under oxygen pressure was suitable for the determination of these analytes in milk powder samples. The same digestion procedure was applied for Ca, Cd, Cu, Fe, Hg, K, Mg Mn, Mo, Na, Pb and Zn determination in whole milk powder, skim milk powder and whey powder and results are shown in Table 3. The limit of quantification (LOQ, analyte concentration corresponding to the sample blank value plus 10 standard deviations of the
Table 3 Elements determined in commercial milk samples by ICP OES (mean and standard deviation in μg g− 1, n = 5). Digestion conditions for proposed procedure: 6 mL 2 mol L− 1 HNO3 and 7.5 bar O2. Analyte
Ca Cd Cu Fe Hgb K Mg Mn Mo Na Pb Zn
Considering many strategies to organic matter digestion for metal determination, the use of oxygen as auxiliary reagent to decrease the nitric acid amount could be considered an efficient alternative to perform sample digestion. Experimental data suggesting the regeneration of HNO3 could be explained by the oxygen atmosphere that improves the effectiveness of digestion using diluted nitric acid solutions. In addition, it was possible to observe the need of a minimum oxygen amount before digestion procedure in order to promote the regeneration process of nitric acid. Consequently, the use of diluted nitric acid associated with pressurized oxygen atmosphere was proven to be feasible and could be recommended for milk powder digestion, reducing the volume of reagents and the amount of digestion residues. Using digestion vessels under oxygen pressures ranging from 7.5 to 20 bar, it was possible to digest sample masses of up to 500 mg with an amount equivalent to only 0.86 mL of concentrated nitric acid. For digestion performed in the same conditions, but without oxygen pressure, a volume of 7 mL of concentrated nitric acid was necessary to assure the effectiveness of organic matter digestion. These results demonstrate a decrease of more than 8 times of the required volume of concentrated nitric acid. Using optimized conditions the carbon content in digests value was lower than 1700 mg L − 1, which was considered suitable for further analysis. Agreement better than 95% (Student's t test, P b 0.05) was obtained for different CRMs and it was possible to determine Ca, Cd, Cu, Fe, K, Mg Mn, Mo, Na, Pb and Zn by ICP OES and Hg by CVG-ICP-MS in whole milk powder, skim milk powder and whey powder. Another advantage is related to the use of only diluted nitric acid without the need of other auxiliary solutions. It is also important to mention that these developments are in agreement with the recommendations of green chemistry [32,33].
Whey powder
Non-fat milk powder
Whole milk powder
Proposed procedure
Proposed procedure
Proposed procedure
Wet digestiona
Acknowledgements
8720 ± 197 b 0.05 0.276 ± 0.089 4.77 ± 0.17 0.006 ± 0.001 11 484 ± 53 931 ± 13 0.481 ± 0.010 0.357 ± 0.014 3990 ± 25 b 1.15 23.6 ± 0.1
12 763 ± 717 b 0.05 1.07 ± 0.12 3.57 ± 0.02 0.004 ± 0.0002 15 392 ± 389 1186 ± 95 0.326 ± 0.001 0.191 ± 0.013 5844 ± 136 b 1.15 41.9 ± 0.5
8773 ± 88 b 0.05 0.314 ± 0.003 3.01 ± 0.07 b 0.004 11 257 ± 108 825 ± 15 0.226 ± 0.006 0.167 ± 0.009 3869 ± 132 b 1.15 32.5 ± 0.2
8598 ± 344 b0.05 0.323 ± 0.010 3.10 ± 0.03 b0.004 11 032 ± 221 817 ± 33 0.219 ± 0.011 0.174 ± 0.003 3946 ± 158 b1.15 31.9 ± 1.0
The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Proc. Nr. 573672/2008-3 and Instituto Nacional de Ciências e Tecnologias Analíticas Avançadas Proc. Nr. 573894/2008-6), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Process 2006/59083-9) for grants and fellowships. Appendix A. Supplementary data
a
Conventional wet digestion in closed system using concentrated HNO3 (details in Ref. [31]). b Determination by CVG-ICP-MS.
Supplementary data to this article can be found online at doi:10.1016/j.sab.2011.04.013.
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[17] G.C.L. Araujo, A.R.A. Nogueira, J.A. Nobrega, Microwave single vessel acid-vapor extraction: effect of experimental parameters on Co and Fe determination in biological samples, Microchim. Acta 144 (2004) 81–85. [18] D.M. Santos, M.M. Pedroso, L.M. Costa, A.R.A. Nogueira, J.A. Nobrega, A new procedure for bovine milk digestion in a focused microwave oven: gradual sample addition to pre-heated acid, Talanta 65 (2005) 505–510. [19] G.C.L. Araujo, M.H. Gonzalez, A.G. Ferreira, A.R.A. Nogueira, J.A. Nobrega, Effect of acid concentration on closed-vessel microwave-assisted digestion of plant materials, Spectrochim. Acta Part B 57 (2002) 2121–2132. [20] M.H. Gonzalez, G.B. Souza, R.V. Oliveira, L.A. Forato, J.A. Nobrega, A.R.A. Nogueira, Microwave-assisted digestion procedures for biological samples with diluted nitric acid: identification of reaction products, Talanta 79 (2009) 396–401. [21] G. Knapp, A.S. Panholzer, P. Kettisch, Pressure-controlled microwave-assisted wet digestion systems, in: H.M. Kingston, S.J. Haswell (Eds.), Microwave-enhanced Chemistry, Fundamentals, Sample Preparation and Applications, American Chemical Society, Washington, 1988, pp. 423–451. [22] J.T. Castro, E.C. Santos, W.P.C. Santos, L.M. Costa, J.A. Nobrega, M.G.A. Korn, A critical evaluation of digestion procedures for coffee samples using diluted nitric acid in closed vessels for inductively coupled plasma optical emission spectrometry, Talanta 78 (2009) 1378–1382. [23] C.A. Bizzi, E.M.M. Flores, R.S. Picoloto, J.S. Barin, J.A. Nobrega, Microwave-assisted digestion in closed vessels: effect of pressurization with oxygen on digestion process with diluted nitric acid, Anal. Methods 2 (2010) 734–738. [24] C.A. Bizzi, J.S. Barin, E.I. Müller, L. Schmidt, J.A. Nobrega, E.M.M. Flores, Evaluation of oxygen pressurized microwave-assisted digestion of botanical materials using diluted nitric acid, Talanta 83 (2010) 1324–1328. [25] Spectro Ciros CCD, software version 01/March 2003, Spectro Analytical Instruments GmbH & Co. KG: Kleve, Germany, 2003. [26] L.E. Kaercher, F. Goldschmidt, J.N.G. Paniz, E.M.M. Flores, V.L. Dressler, Determination of inorganic and total mercury by vapor generation atomic absorption spectrometry using different temperatures of the measurement cell, Spectrochim. Acta Part B 60 (2005) 705–710. [27] M.F. Mesko, J.S.F. Pereira, D.P. Moraes, J.S. Barin, P.A. Mello, J.N.G. Paniz, J.A. Nóbrega, M.G.A. Korn, E.M.M. Flores, Focused microwave-induced combustion: a new technique for sample digestion, Anal. Chem. 82 (2010) 2155–2160. [28] S.T. Gouveia, F.V. Silva, L.M. Costa, A.R.A. Nogueira, J.A. Nobrega, Determination of residual carbon by inductively-coupled plasma optical emission spectrometry with axial and radial view configurations, Anal. Chim. Acta 445 (2001) 269–275. [29] E.M.M. Flores, M.F. Mesko, D.P. Moraes, J.S.F. Pereira, P.A. Mello, J.S. Barin, G. Knapp, Determination of halogens in coal after digestion using the microwaveinduced combustion technique, Anal. Chem. 80 (2008) 1865–1870. [30] Anton Paar GmbH, Multiwave 3000®, Microwave Sample Preparation System, Software Version v1.27-Synt, Graz, Austria, 2003. [31] Milestone SRL, Ethos 1, Advanced in Sample Preparation, Software Version 1, Sorisole, Itália, 2005. [32] F.R.P. Rocha, L.S.G. Teixeira, J.A. Nobrega, Green strategies in trace analysis: a glimpse of simple alternatives for sample pretreatment and analyte determination, Spectrosc. Lett. 42 (2009) 418–429. [33] M. de la Guardia, Green analytical chemistry, Trends Anal. Chem. 29 (2010) 577.
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Spectrochimica Acta Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s a b
Analytical note
Improvement of microwave-assisted digestion of milk powder with diluted nitric acid using oxygen as auxiliary reagent Cezar A. Bizzi a, e, Juliano S. Barin b, Edivaldo E. Garcia c, Joaquim A. Nóbrega d, Valderi L. Dressler a, e, Erico M.M. Flores a, e,⁎ a
Departamento de Química, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil Departamento de Tecnologia e Ciência dos Alimentos, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil Departamento de Química, Universidade Estadual de Maringá, 87100-900, Maringá, PR, Brazil d Departamento de Química, Universidade Federal de São Carlos, 13565-905, São Carlos, SP, Brazil e Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, SP, Brazil b c
a r t i c l e
i n f o
Available online 7 May 2011 Keywords: Microwave-assisted digestion ICP OES CVG-ICP-MS Trace elements Oxygen pressure
a b s t r a c t The feasibility of using diluted HNO3 solutions under oxygen pressure for decomposition of whole and non-fat milk powders and whey powder samples has been evaluated. Digestion efficiency was evaluated by determining the carbon content in solution (digests) and the determination of Ca, Cd, Cu, Fe, K, Mg, Mn, Mo, Na, Pb and Zn was performed by inductively coupled plasma optical emission spectrometry and Hg by chemical vapor generation coupled to inductively coupled plasma mass spectrometry. Samples (up to 500 mg) were digested using HNO3 solutions (1 to 14 mol L− 1) and the effect of oxygen pressure was evaluated between 2.5 and 20 bar. It was possible to perform the digestion of 500 mg of milk powder using 2 mol L − 1 HNO3 with oxygen pressure ranging from 7.5 to 20 bar with resultant carbon content in digests lower than 1700 mg L− 1. Using optimized conditions, less than 0.86 mL of concentrated nitric acid (14 mol L − 1) was enough to digest 500 mg of sample. The accuracy was evaluated by determination of metal concentrations in certified reference materials, which presented an agreement better than 95% (Student's t test, P b 0.05) for all the analytes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Milk could be considered as a staple food that provides essential nutrients (proteins, lipids, and carbohydrates) and micronutrients (minerals, vitamins, and enzymes) [1,2]. On the other hand, milk can also constitute a source of exposure to toxic elements, especially dangerous for infants, such as Cd, Hg and Pb [3,4]. In this sense, it is important to establish appropriate sample preparation methods for subsequent metal determination in order to assure the quality of final products. In addition, these methods should be developed considering lower reagent consumption and suitable digestion efficiency [5]. Most of conventional sample preparation methods for atomic spectrometric techniques involve sample solubilization with complete or partial matrix decomposition generally using oxidant acids [6]. In previous years, some improvements were proposed towards the development of new procedures based on less conventional approaches to organic matter digestion [7–18], which normally present relatively high efficiency of digestion resulting in low values of residual carbon content. ⁎ Corresponding author at: Departamento de Química, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. Tel./fax: +55 55 3220 9445. E-mail address: [email protected] (E.M.M. Flores). 0584-8547/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2011.04.013
Some procedures based on diluted solutions of nitric acid were also developed for digestion of biological samples [19,20]. The efficiency of diluted nitric acid for oxidation of organic matter can be explained by the regeneration of nitric acid promoted by the combination of the nitrogen oxide species with the oxygen present inside the reaction vessel [20–22]. Based on the reaction mechanism of nitric acid regeneration, which occurs while oxygen is still present in the reaction vessel, digestion procedure has been performed under oxygen pressure [23,24], which allowed digestions with amounts of nitric acid lower than 3 mol L − 1. In the present work, a procedure based on vessels pressurized with oxygen and determination by inductively coupled plasma (ICP)-based spectrometric techniques is proposed for metals determination in milk powder. The determination of essential (Ca, Cu, Fe, K, Mg, Mn, Mo, Na and Zn) and toxic (Cd, Hg and Pb) metals was performed by inductively coupled plasma optical emission spectrometry (ICP OES) and by chemical vapor generation inductively coupled plasma mass spectrometry (CVG-ICP-MS, only for Hg). Certified reference materials (CRM) were used to check the accuracy. In addition, it was tried to reduce as much as possible the amount of nitric acid needed for the digestion process in order to minimize the blank values and to decrease the consumption of reagents and the consequent generation of laboratory residues.
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2. Experimental
2.2. Samples, reagents and standards
2.1. Instrumentation
Preliminary experiments were carried out using whole milk powder. After optimization step, the proposed procedure was applied to the digestion of whey, whole and non-fat milk powders, which were purchased in a local market. All samples were dried at 60 °C using an oven (model 400/2ND, Nova Ética, Vargem Grande Paulista, SP, Brazil). Accuracy was evaluated using CRM of skim milk powder (BCR 151, Community Bureau of Reference, Brussels, Belgium) and of non-fat milk powder (SRM NIST 1549, National Institute of Standards and Technology, NIST, Gaithersburg, MD, USA). Samples were weighed using an analytical balance (model AY 220, max. 220 g, 0.1 mg of resolution, Shimadzu, Kyoto, Japan). Distilled-deionized water (Milli-Q, 18.2 MΩ cm, Millipore, Billerica, MA, USA) and analytical-grade nitric acid (Merck, Darmstadt, Germany) were used to prepare samples and standards. Carbon reference solutions used in external calibration for carbon content determination were prepared by dissolution of citric acid (Merck) in water (25 to 500 mg L− 1 of C). Yttrium (1.0 mg L − 1, Spex CertPrep, Metuchen, NJ, USA) was used as internal standard in all samples, blanks and reference solutions for carbon content determination. Metal determination by ICP OES was performed with external calibration using analytical solutions ranging from 1.0 (Cd, Mn, Zn), or 5.0 (Cu, Fe, Mg, Mo), or 10 μg L− 1 (Ca, K, Na, Pb) up to 100 μg L − 1, prepared in 0.7 mol L − 1 HNO3 by appropriate dilution of the multi-element stock solution (SCP33MS, SCP Science, Quebec, Canada). Mercury determination by CVG-ICP-MS was performed with external calibration by dilution of inorganic mercury (Hginorg) standard stock solution Titrisol (1000 mg L − 1, Merck), which was prepared just before use. Sodium tetrahydroborate (0.2% m/v) used for CVG was obtained from Vetec (Duque de Caxias, RJ, Brazil). A 0.1 mol L − 1 KOH (Merck) solution was used for residual acidity determination. Glass and quartz material were soaked in 1.4 mol L − 1 HNO3 for 24 h and further washed with water before use.
A microwave oven (Multiwave 3000 microwave sample preparation system, Anton Paar, Graz, Austria) equipped with eight highpressure quartz vessels was used in the experiments. The internal volume of vessels was 80 mL and the maximum operational temperature and pressure were set at 280 °C and 80 bar, respectively. Analytes (except Hg) were determined by ICP OES using an axial view configuration spectrometer (Spectro Ciros CCD, Spectro Analytical Instruments, Kleve, Germany) using a cross-flow nebulizer coupled to a Scott double pass type nebulization chamber. Plasma operating conditions and selected wavelengths are listed in Table 1, and they were used as recommended by the instrument manufacturer [25]. Chemical vapor generation (CVG) coupled to ICP-MS (PerkinElmer Sciex, Model Elan DRC II, Thornhill, Canada), equipped with a quartz torch with a quartz injector tube (2 mm i.d.), was used for Hg determination. The CVG system consists of a peristaltic pump (Ismatec, Zurich, Switzerland) and a U-type gas–liquid separator [26]. The mixture was pumped to the gas–liquid separator and Hg measurements were performed by ICP-MS. Plasma operating conditions and selected isotope used for Hg determination are also listed in Table 1. Carbon content in digests (related to 500 mg of sample and dilution to 30 mL) was determined by ICP OES [27,28]. In order to remove the volatile carbon compounds before carbon content determination, digests were previously sonicated with an ultrasonic probe [29] (VCX 130 PB, 130 W, 20 kHz, Sonics and Materials Inc., Newton, CT, USA). This procedure was not applied when determining the analytes in final digests, which were just diluted with water and analyzed by ICP OES and CVG-ICP-MS. Argon (99.996%, White Martins-Praxair, São Paulo, SP, Brazil) was used for ICP OES and ICP-MS determination for plasma generation, nebulization, auxiliary gas and also for digestion performed under inert atmosphere. Oxygen (99.9991%, White Martins-Praxair) was used as reagent in digestions performed under oxygen pressure. It is important to mention that all procedures under oxygen pressure were performed employing safety conditions, as recommended by the microwave oven manufacturer [30]. Results for residual acidity were obtained using a titration system (Titrando 836, Metrohm, Herisau, Switzerland) equipped with a magnetic stirrer (module 803 Ti Stand), 20 mL burette (Dosino 800) and pH electrode (LL Electrode plus, model 6.0262.100). Table 1 Operational parameters for determination of C, Ca, Cd, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Pb and Zn. Parameter
ICP OES
ICP-MS
Radio-frequency power (W) Plasma gas flow rate (L min− 1) Auxiliary gas flow rate (L min− 1) Nebulizer gas flow rate (L min− 1) Spray chamber Nebulizer Observation view Analytes C (I) Ca (II) Cd (II) Cu (I) Fe (I) Hg K (I) Mg (I) Mn (II) Mo (II) Na (I) Pb (II) Zn (I)
1600 14.0 1.0 0.85 Double pass, Scott type Crossflow Axial Emission line (nm) 193.091 393.366 226.502 324.752 238.204 – 766.491 285.213 257.610 202.030 589.592 220.353 213.856
1300 15.0 1.2 1.13 * * – Isotope (m/z) – – – – – 202 – – – – – – –
*Direct introduction by CVG, (I) atomic emission and (II) ion emission.
2.3. Microwave-assisted acid digestion Samples (up to 500 mg) were transferred to the quartz vessels. Digestion efficiency using nitric acid solutions (6 mL) was evaluated in the following concentrations: 1, 2, 3, 7, and 14 mol L − 1 HNO3. After closing and capping the rotor, vessels were pressurized with 2.5, 5, 7.5, 10, 15 and 20 bar of oxygen. The gases were introduced into the vessels using the valve originally designed for pressure release after conventional acid sample digestion. Then, the rotor was placed inside the oven, and microwave-heating program was started by applying (i) 1000 W with a ramp of 5 min, (ii) 1000 W for 10 min, and (iii) 0 W for 20 min (cooling step) [30]. After digestion, the pressure of each vessel was carefully released. In this work, each run was performed using a minimum of four vessels. The resulting solutions were transferred to 30 mL polypropylene vials and diluted to the mark with water. Cleaning of digestion vessels was carried out with 6 mL of concentrated HNO3 in the microwave oven at 1000 W for 10 min and 0 W for 20 min for cooling. All statistical comparisons were performed using Student's t test, (GraphPad InStat Software Inc., Version 3.00, 1997). A significance level of P b 0.05 was chosen for all comparisons. 3. Results and discussion 3.1. Preliminary evaluation of digestion efficiency Preliminary tests were carried out with air at atmospheric pressure in order to evaluate the effect of oxygen pressure on whole milk powder digestion. Nitric acid concentration was varied in order to achieve a condition of efficient organic matter digestion using as low as possible concentrated acid solution, which was evaluated by the carbon content in digests and residual acidity determination. The same procedure was repeated using oxygen for pressurization of
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reaction vessels. In order to evaluate the effectiveness of digestion procedure, a selected tolerance level lower than 2000 mg L − 1 of carbon content in solution was chosen, which were considered appropriate for subsequent analysis by ICP OES and ICP-MS.
organic matter. This reaction could be effective with diluted nitric acid if digestion vessels were pressurized with oxygen.
3.1.1. Digestion procedure with air Preliminary experiments were performed using 500 mg of whole milk powder to evaluate the minimum nitric acid concentration that was sufficient to obtain suitable values of carbon content in digests. Thus, digestion using different nitric acid concentrations under atmospheric air pressure was tested. When 14 and 7 mol L − 1 HNO3 solutions were used, the carbon content in solution was lower than 532 ± 45 and 720 ± 114 mg L − 1, respectively. However, using 3 mol L − 1 HNO3, final digests presented a yellow color and carbon content values were higher, about 8000 mg L − 1. When 2 mol L − 1 HNO3 was used the digestion was not effective and solid residues remained as suspended particles with a deep brown color (carbon content values as high as 10,500 mg L − 1 were obtained). The residual acidity was also determined in final digests obtained from digestion procedure performed at atmospheric air. When using 7 and 14 mol L − 1 HNO3, which showed good digestion efficiency, the residual acidities were 44 and 64% (m/m), respectively. Although, digestions performed using 3 and 2 mol L − 1 did not present a good digestion efficiency of organic matter, lower residual acidities were obtained in final digests (13% m/m for both, 3 and 2 mol L − 1 HNO3). Final digests obtained using more concentrated nitric acid solutions (7 and 14 mol L − 1), presented a colorless aspect. Best results under these conditions, regarding carbon content in digests and residual acidity were obtained using 7 mol L − 1 HNO3.
For the evaluation of gas phase effect inside the digestion vessel, whole milk powder sample (500 mg) was digested using 2 mol L − 1 HNO3 with oxygen pressures ranging from 20 to 2.5 bar. As it can be seen in Fig. 1, the digestion procedure presented almost the same efficiency (carbon content in digests b2000 mg L − 1) for oxygen pressures from 20 to 7.5 bar. In these conditions, according to the ideal gas behavior the initial amounts of oxygen inside the reaction vessel were 1.94, 1.46, 0.97 and 0.73 g for 258 pressures of 20, 15, 10 and 7.5 bar, respectively. However, the efficiency of digestion decreased for lower oxygen pressure. Higher values of carbon content were observed for 5 and 2.5 bar of oxygen, i.e., 2217 ± 106 and 4528 ± 659 mg L − 1, respectively. In these conditions, the initial oxygen amounts inside reaction vessels were 0.49 and 0.24 g, respectively. The residual acidity was also determined. As expected, the residual acidity in final solutions presented similar values, ranging from 23 to 13% m/m (15 and 2.5 bar, respectively). As previously discussed [23,24], the HNO3 regeneration process was not dependent on the initial pressure. Nevertheless, results obtained in the present study indicate a closer relation between oxygen and the nitric acid amount inside the reaction vessels. When nitric acid concentration was lower than the minimum amount required to obtain an effective organic matter oxidation, the regeneration process could be considered decisive for digestion when oxygen was present inside the reaction vessel in a minimum initial amount of 0.73 g (7.5 bar). When lower amount of oxygen was used, the regeneration process was less effective and final digests showed carbon content in solution higher than 2000 mg L − 1. Therefore, in the range of oxygen pressure evaluated in this work, no significant changes were observed at pressures higher than 7.5 bar based on carbon content and residual acidity values and this pressure of oxygen was selected for further experiments. 3.3. Determination of Ca, Cd, Cu, Fe, Hg, K, Mg Mn, Mo, Na, Pb and Zn in CRM, whey, whole and non-fat milk powder samples
6000
100
5000
80
4000 60 3000 40 2000 20
1000 0
2.5
5
7.5
10
15
20
Residual acidity (%)
Accuracy was evaluated by using CRMs of non-fat and skim milk powders. Digestion of CRM, whole milk powder, skim milk powder and whey powder samples was performed using optimized conditions (500 mg of sample mass, 2 mol L − 1 HNO3 and 7.5 bar of O2). Results obtained for Ca, Cd, Cu, Fe, Hg, K, Mg Mn, Mo, Na, Pb and Zn in CRM presented a good agreement (better than 95%, Student's t test, P b 0.05) with certified values as it may be seen in Table 2. It is
Carbon content in digests (mg L-1)
3.1.2. Digestion procedure with oxygen pressure The effect of nitric acid concentration on digestion efficiency was also evaluated under 20 bar of oxygen pressure. Sample masses of 500 mg were used and carbon content was determined in final digests. Digestions were evaluated using nitric acid concentrations ranging from 14 to 1 mol L− 1. A similar effectiveness on organic matter oxidation was observed with solutions ranging from 14 to 2 mol L− 1 HNO3, where was possible to obtain final digests with carbon content values lower than 2000 mg L − 1 (1124 ± 235, 1345 ± 158, 1703 ± 68 and 1450 ± 50 mg L − 1 for solutions of 14, 7, 3 and 2 mol L − 1 HNO3, respectively). The digestion efficiency was low when using 1 mol L− 1 HNO3; final digests presented higher values of carbon content in solution (2345 ± 30 mg L − 1) and slightly brown color with solid residues remaining as suspended particles. The residual acidity in final digests was between 99% m/m (14 mol L − 1 HNO3) and 18% m/m (2 mol L − 1 HNO3). Based on these results the residual acidities obtained for digestions performed under atmospheric pressure were compared with those performed under oxygen pressure. When the same nitric acid concentration was evaluated, the residual acidity for digestion performed under oxygen pressure always presented higher values and the digested solutions were clear. A different behavior was observed in final digests obtained for experiments performed at atmospheric air. This result obtained for digestion performed under oxygen pressure may be due to the absence of NO2 in the gas phase, probably owing to the regeneration process of nitric acid promoted by oxygen, which leads to higher residual acidity values. In this sense, even using an acid solution as diluted as 2 mol L − 1 HNO3 to digest 500 mg of whole milk powder the carbon content values were always lower than 2000 mg L − 1 using 20 bar of oxygen. It results in a reduction in the nitric acid concentration around 8 times without decreasing the digestion efficiency when compared with the use of more concentrated nitric acid solution. As reported in previous studies [23,24], the oxidant action of nitric acid may be improved if a regenerating process occurs, which is mainly dependent on the amount of oxygen available in gas phase during the oxidation of the
3.2. Evaluation of different oxygen pressures
0
Oxygen pressure (bar) Fig. 1. Digestion efficiency of 500 mg of whole milk powder using 2 mol L− 1 HNO3; effect of oxygen pressure (gray bars) on digestion efficiency (horizontal line represents the arbitrarily chosen tolerance level of carbon content in solution = 2000 mg L− 1). Error bars represent the standard deviation (n = 3).
C.A. Bizzi et al. / Spectrochimica Acta Part B 66 (2011) 394–398 Table 2 Elements determined in milk CRMs by ICP OES (mean and confidence interval in μg g− 1, n = 5). Digestion conditions: 2 mol L− 1 HNO3 and 7.5 bar O2. Analyte
Ca Cd Cu Fe Hga K Mg Mn Mo Na Pb Zn a b
397
Table 4 Figures of merit for the proposed digestion procedure based on diluted nitric acid and oxygen pressure.
NIST 1549 (non-fat milk powder)
BCR 151 (skim milk powder)
Parameters
Proposed digestion procedure
Certified
Found
Certified
Found
13 000 ± 500 0.0005 ± 0.0002 0.7 ± 0.1 1.78 ± 0.10 0.0003 ± 0.0002 16 900 ± 300 1200 ± 300 0.26 ± 0.6 0.34b 4970 ± 100 0.019 ± 0.003 46.1 ± 2.2
13 218 ± 1293 b 0.05 0.63 ± 0.11 1.82 ± 0.11 b 0.004 17 101 ± 579 1195 ± 61 0.235 ± 0.051 0.310 ± 0.062 5183 ± 1053 b 1.15 46.9 ± 0.5
– 0.101 ± 0.008 5.23 ± 0.08 50.1 ± 1.3 0.101 ± 0.01 – – – – – 2.002 ± 0.026 –
11 844 ± 127 0.100 ± 0.001 5.24 ± 0.002 48.7 ± 1.9 0.102 ± 0.002 16 390 ± 455 1229 ± 14 0.269 ± 0.015 0.314 ± 0.022 4202 ± 85 1.92 ± 0.11 49.7 ± 3.1
Sample mass, mg Acid concentration, mol L− 1 Oxygen pressure, bar Carbon content in digests, mg L− 1 Residual acidity, % m/m
500 2 7.5 b1700 b18
Determination by CVG-ICP-MS. Non-certified value.
blank mean) obtained by ICP OES determination for Ca, Cd, Cu, Fe, K, Mg, Mn, Mo, Na, Pb and Zn was (0.81, 0.05, 0.18, 0.11, 0.59, 0.01, 0.02, 0.44, 0.11, 1.15, 0.07) μg g − 1, respectively. For Hg, a LOQ of 0.004 μg g − 1 was obtained by CVG-ICP-MS. The main analytical figures of merit and optimized conditions of the proposed digestion procedure under oxygen pressure in relation to the conventional wet digestion are shown in Table 4. 4. Conclusions
important to mention that milk powder is a complex matrix to bring into solution by conventional digestion procedures. It is a mixture of proteins, fats and sugars, with different amounts and compositions depending on the milk characteristics. As an example, whole milk powder is composed by 26.3, 26.7 and 38.4% (m/m) of protein, fat and sugar, respectively, while skim milk powder is 36.2, 0.8 and 52.0% (m/ m) of protein, fat and sugar, respectively and, whey powder is 12.9, 1.1 and 74.5% (m/m) of protein, fat and sugar, respectively [2]. However, even with different matrix compositions for different types of milk, the proposed procedure presented quantitative recoveries for Ca, Cd, Cu, Fe, K, Mg Mn, Mo, Na, Pb and Zn by ICP OES determination and for Hg by CVG-ICP-MS determination. In addition, the results obtained for whole milk powder digested by conventional wet digestion (6 mL of 14 mol L − 1 nitric acid, 300 mg of sample mass, 200 °C, 50 bar, polytetrafluorethylene vessels, Model Ethos 1, Milestone system) [31] presented a good agreement (better than 95%, Student's t test, P b 0.05) when compared with results obtained by the proposed procedure. This fact confirms that the proposed procedure, using diluted nitric acid under oxygen pressure was suitable for the determination of these analytes in milk powder samples. The same digestion procedure was applied for Ca, Cd, Cu, Fe, Hg, K, Mg Mn, Mo, Na, Pb and Zn determination in whole milk powder, skim milk powder and whey powder and results are shown in Table 3. The limit of quantification (LOQ, analyte concentration corresponding to the sample blank value plus 10 standard deviations of the
Table 3 Elements determined in commercial milk samples by ICP OES (mean and standard deviation in μg g− 1, n = 5). Digestion conditions for proposed procedure: 6 mL 2 mol L− 1 HNO3 and 7.5 bar O2. Analyte
Ca Cd Cu Fe Hgb K Mg Mn Mo Na Pb Zn
Considering many strategies to organic matter digestion for metal determination, the use of oxygen as auxiliary reagent to decrease the nitric acid amount could be considered an efficient alternative to perform sample digestion. Experimental data suggesting the regeneration of HNO3 could be explained by the oxygen atmosphere that improves the effectiveness of digestion using diluted nitric acid solutions. In addition, it was possible to observe the need of a minimum oxygen amount before digestion procedure in order to promote the regeneration process of nitric acid. Consequently, the use of diluted nitric acid associated with pressurized oxygen atmosphere was proven to be feasible and could be recommended for milk powder digestion, reducing the volume of reagents and the amount of digestion residues. Using digestion vessels under oxygen pressures ranging from 7.5 to 20 bar, it was possible to digest sample masses of up to 500 mg with an amount equivalent to only 0.86 mL of concentrated nitric acid. For digestion performed in the same conditions, but without oxygen pressure, a volume of 7 mL of concentrated nitric acid was necessary to assure the effectiveness of organic matter digestion. These results demonstrate a decrease of more than 8 times of the required volume of concentrated nitric acid. Using optimized conditions the carbon content in digests value was lower than 1700 mg L − 1, which was considered suitable for further analysis. Agreement better than 95% (Student's t test, P b 0.05) was obtained for different CRMs and it was possible to determine Ca, Cd, Cu, Fe, K, Mg Mn, Mo, Na, Pb and Zn by ICP OES and Hg by CVG-ICP-MS in whole milk powder, skim milk powder and whey powder. Another advantage is related to the use of only diluted nitric acid without the need of other auxiliary solutions. It is also important to mention that these developments are in agreement with the recommendations of green chemistry [32,33].
Whey powder
Non-fat milk powder
Whole milk powder
Proposed procedure
Proposed procedure
Proposed procedure
Wet digestiona
Acknowledgements
8720 ± 197 b 0.05 0.276 ± 0.089 4.77 ± 0.17 0.006 ± 0.001 11 484 ± 53 931 ± 13 0.481 ± 0.010 0.357 ± 0.014 3990 ± 25 b 1.15 23.6 ± 0.1
12 763 ± 717 b 0.05 1.07 ± 0.12 3.57 ± 0.02 0.004 ± 0.0002 15 392 ± 389 1186 ± 95 0.326 ± 0.001 0.191 ± 0.013 5844 ± 136 b 1.15 41.9 ± 0.5
8773 ± 88 b 0.05 0.314 ± 0.003 3.01 ± 0.07 b 0.004 11 257 ± 108 825 ± 15 0.226 ± 0.006 0.167 ± 0.009 3869 ± 132 b 1.15 32.5 ± 0.2
8598 ± 344 b0.05 0.323 ± 0.010 3.10 ± 0.03 b0.004 11 032 ± 221 817 ± 33 0.219 ± 0.011 0.174 ± 0.003 3946 ± 158 b1.15 31.9 ± 1.0
The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Proc. Nr. 573672/2008-3 and Instituto Nacional de Ciências e Tecnologias Analíticas Avançadas Proc. Nr. 573894/2008-6), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Process 2006/59083-9) for grants and fellowships. Appendix A. Supplementary data
a
Conventional wet digestion in closed system using concentrated HNO3 (details in Ref. [31]). b Determination by CVG-ICP-MS.
Supplementary data to this article can be found online at doi:10.1016/j.sab.2011.04.013.
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C.A. Bizzi et al. / Spectrochimica Acta Part B 66 (2011) 394–398
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