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Copper determination in sugar cane spirits by fast sequential flame atomic absorption spectrometry using internal standardization
Microchemical Journal 96 (2010) 99–101
Contents lists available at ScienceDirect
Microchemical Journal 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 / m i c r o c
Copper determination in sugar cane spirits by fast sequential flame atomic absorption spectrometry using internal standardization Kelber Miranda, Amália G.G. Dionísio, Edenir R. Pereira-Filho ⁎ Universidade Federal de São Carlos, Centro de Ciências Exatas e de Tecnologia, Departamento de Química, Grupo de Análise Instrumental Aplicada, Rodovia Washington Luiz, km 235, Caixa Postal 676, CEP 13565-905, São Carlos — SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 November 2009 Received in revised form 18 February 2010 Accepted 18 February 2010 Available online 26 February 2010 Keywords: FS-FAAS Sugar cane spirit Cachaça Copper Internal standard
a b s t r a c t In this study, a fast and simple method is proposed for the determination of Cu in sugar cane spirits employing fast sequential flame atomic absorption spectrometry and the internal standard technique. First, Ag, Bi, Co and Ni were evaluated as internal standards to minimize transport interferences. The results demonstrated that Ag at a concentration of 2 mg L− 1 was effective. Under these conditions, Cu could be determined with a limit of detection of 15 µg L− 1. Then, Cu was determined in 5 sugar cane spirit samples using the proposed method and the results were compared with those obtained by inductively coupled plasma optical emission spectrometry after microwave oven acid digestion. The content of Cu varied from 0.66 to 6.64 mg L− 1. Accuracy and precision of the proposed method were evaluated by comparing the results obtained with both methods. A paired t-test at a 95% confidence level showed that the proposed method enabled the achievement of similar results as those obtained by ICP OES after acid digestion. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In Brazil nearly 30,000 industrial units are responsible to produce approximately 1.3 billion liters a year of a worldwide appreciated sugar cane spirit named “cachaça” [1]. This alcoholic beverage is produced by distillation after the fermentation of sugar cane and since Cu has been used to make the distillation container, this metal may contaminate the sugar cane spirit. During the distillation process a compound named verdigris [CuCO3Cu(OH)2] is formed on the container wall and it can be dissolved by acidic alcoholic vapors [2]. Some procedures like the use of active coal and ionic exchange resins have been used to remove cuprous ions from the distilled drink but unluckily they also remove other substances that contribute to a desirable flavor and fragrance. The alternative to use steel in the manufacture of the stills resulted in complaints of low sensory quality of the sugar cane spirit [3]. Trace levels of Cu are known to be essential in metabolism; however, higher doses may cause Wilson's disease and other deleterious health effects [4]. For this reason, the accurate determination of Cu is crucial to strengthen the agribusiness dedicated to the production and commercialization of Brazilian sugar cane spirits. The Brazilian Ministry of Agriculture, Livestock and Supply established quality standards for cachaça by fixing technical regulations [5]. Regarding inorganic contaminants, the maximum allowed level of Cu in Brazilian cachaça is 5 mg L− 1. ⁎ Corresponding author. E-mail address: [email protected] (E.R. Pereira-Filho). 0026-265X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2010.02.011
Flame atomic absorption spectrometry (FAAS) has been employed for rapid Cu determination in alcoholic beverages using matrixmatching calibration [6,7]. However, due to the fact that samples of Brazilian sugar cane spirits present alcohol contents that can vary in a great extent, as from 30 to 60% (v/v) [8], using this approach would be an exhausting task because the alcoholic grade needs to be determined for each sample and the standard solutions should be prepared in the same matrix. Therefore, the analyte addition technique is more appropriate than matrix-matching calibration to determine copper by FAAS in sugar cane spirits [9]. However, the execution of the analyte addition technique is laborious and slow. Internal standardization utilizing fast sequential FAAS (FS-FAAS) can also be adopted as a simple and efficient strategy to minimize transport interferences in FAAS [10,11]. When applying internal standardization a known amount of a selected element (the internal standard) is added to all blanks, reference solutions and samples. The signal of the internal standard (IS) is monitored together with the signal of the analytes of interest and a correction factor is calculated from the ratio of the initial and actual values of the IS. This correction factor is then applied to the signals of the analytes. The element to be used as an IS needs to present some characteristics: (1) shall not be present in the sample in a detectable concentration, (2) shall not interfere with the analytes, (3) shall itself be free from interferences and (4) shall be relatively neutral to changes in flame stoichiometry. The concentration of the IS shall result in a signal level typically from 0.1 to 0.4 absorbance. FS-FAAS is a sequential multi-element technique that keeps the advantages of conventional FAAS. When working in sequential mode,
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the instrument allows the measurement of a sequence of analytes in decrescent order of wavelengths in one monochromator scan. It is also possible to use the internal standard technique in a way that precision and accuracy of the measurement can be improved. When utilizing FSFAAS it is possible to apply internal standardization in an efficient and useful way that the application of the required correction factors is performed on-line by the instrument software automatically [12,13]. In the present paper, a fast and simple method to determine Cu in sugar cane spirits by FS-FAAS using internal standardization is presented and similar results as those obtained by inductively coupled plasma optical emission spectrometry (ICP OES) after acid digestion are achieved. 2. Materials and methods 2.1. Instrumentation FS-FAAS experiments were performed in a flame atomic absorption spectrometer (AA240FS; Varian, Mulgrave, Australia) fitted with a deuterium lamp for background correction. Silver, Bi, Co, Cu and Ni hollow cathode lamps were used as primary radiation sources and its electric currents were 4, 10, 7, 4 and 4 mA, respectively. In all experiments an oxidizing air/acetylene (13.5 L min− 1/2.0 L min− 1) flame was used. Copper was measured at 324.8 nm with a spectral bandwidth of 0.5 nm. 2.2. Reagents, analytical solutions and samples Deionized water (18 MΩ cm resistivity) generated from a Milli-Q® Plus Total Water System (Millipore Corp., Bedford, MA, USA) was used to prepare all solutions. Prior to use, all glassware and polypropylene flasks were washed with soap, soaked in 10% (v/v) HNO3 for 24 h, rinsed with deionized water and dried in such a manner to ensure that no contamination occurred. Reference solutions were prepared daily after appropriate dilution of the 1000 mg L− 1 Ag, Bi, Co, Cu and Ni stock standard solutions (Tec-Lab, Hexis, São Paulo, Brazil). Brazilian sugar cane spirit (cachaça) samples were purchased at the local market in São Carlos, São Paulo State, Brazil. 2.3. Determination of Cu in cachaça samples after total digestion A microwave oven system equipped with perfluoalcoxi (PFA) vessels and temperature sensor Multiwave, Anton-Paar (Graz, Austria) was employed for the digestion of the cachaça samples. A volume of 1 mL of cachaça was digested using 1 mL of HNO3 14 mol L− 1 and 6 mL of deionized water in a closed vessel. After decomposition, the digested samples were transferred to volumetric flasks and diluted to 10.0 mL with deionized water. The microwave oven heating program was performed in two steps: (1) 2 min at 300 W; and (2) 5 min at 600 W. All samples were digested in triplicate. Copper was determined in the diluted solutions by ICP OES (Vista AX, Varian, Mulgrave, Australia).
3. Results and discussion 3.1. Influence of alcohol content on Cu absorbance signals Brazilian sugar cane spirits may present alcohol contents that can vary from 30 to 60% (v/v). In the proposed method, the sugar cane spirit sample is diluted with 1.0 mol L-1 HNO3 1/1 (v/v) and, after dilution, the alcohol content can vary from 15 to 30%. Therefore, the influence of ethanol content on Cu absorbance signals was evaluated by comparing the slopes of analytical curves obtained from reference solutions containing 0.10–4.0 mg L− 1 Cu prepared in 0, 15, 20, 25 and 30% (v/v) ethanol. It can be seen in Fig. 1 that the slope of the analytical curve for Cu decreases when the alcoholic content increases. During the experiments we observed transport interferences: the nebulizer uptake rate decreased when the alcoholic content increased. These results confirm that if we wanted to determine Cu in sugar cane spirit samples with different alcoholic contents employing matrix-matching calibration we would have to access the alcoholic grade for each sample and prepare the standard solutions in the same matrix. This procedure would be hard-working.
3.2. Evaluation of Ag, Bi, Co and Ni as internal standards Silver, Bi, Co and Ni were tested as internal standards (IS). Analytical curves were established by spiking a cachaça sample with increasing concentrations of Cu (0.10–4.0 mg L− 1). The cachaça sample used for the evaluation did not contain Ag, Bi, Co, Cu and Ni in detectable concentrations. All the results can be seen in Table 1. The analytical signals obtained were corrected employing the internal standard technique for the four internal standards tested. More details about the internal standard technique can be found elsewhere [10]. The slope and its standard deviation of the analytical curve obtained by the standard calibration technique for copper (0.1460 ± 0.0011)CCu(mg L− 1) was compared with the slopes (±SD) of the analytical curves obtained with the internal standard technique using Ag, Bi, Co and Ni (0.1461 ± 0.0012, 0.1454 ± 0.0010, 0.1439 ± 0.0010 and 0.1449 ± 0.0013, respectively)CCu(mg L− 1) as internal standards. A paired t-test (n = 3) showed no differences between them at a 95% confidence level. The results demonstrate that Ag, Bi, Co and Ni are efficient as internal standards for correcting transport interferences observed during the determination of Cu in cachaça samples by FSFAAS.
2.4. Direct determination of Cu in cachaça samples using FS-FAAS and internal standardization In the proposed method, 5 mL of cachaça sample and 400 µL of a 50 mg L− 1 Ag, Co, Ni and 250 mg L− 1 Bi standard solution were pipetted in a 15 mL polypropylene flask. Then the volume was made up to 10 ml using 1.0 mol L− 1 HNO3 solution for Cu determination. After dilution, the final concentration was 10 mg L− 1 for Bi and 2 mg L− 1 for Ag, Co and Ni. Reference solutions containing 0.10–4.0 mg L− 1 Cu were used for calibration. The internal standards (Ag, Bi, Co and Ni) were added to all blanks, reference solutions and samples. Copper was determined on the most sensitive line (324.8 nm).
Fig. 1. Copper calibration curves without IS containing 0, 15, 20, 25 and 30% (v/v) ethanol by FS-FAAS.
K. Miranda et al. / Microchemical Journal 96 (2010) 99–101 Table 1 Wavelength, concentration used and linear regression equations obtained for the determination of Cu using four different internal standards (IS) and the standard calibration technique. IS
Wavelength (nm)
IS concentration (mg L− 1)
Silver 328.1 2.0 Bismuth 223.1 10.0 Cobalt 240.7 2.0 Nickel 232.0 2.0 Using the standard calibration technique
Linear regression equation (Cu) A = 0.1461CCu + 0.0041 A = 0.1454CCu + 0.0060 A = 0.1439CCu + 0.0051 A = 0.1449CCu + 0.0042 A = 0.1460CCu + 0.0053
(R = 0.9997) (R = 0.9996) (R = 0.9997) (R = 0.9997) (R = 0.9996)
R: correlation coefficient.
101
4. Conclusions Silver was effective as an internal standard, minimizing the transport interference observed, enhancing precision and accuracy on Cu determination in cachaça samples by FS-FAAS. The method proposed in this paper is fast, simple and enables the achievement of similar results as those obtained by ICP OES after microwave oven acid digestion. It can be considered an alternative to Cu determination in cachaça samples providing a substantial reduction in the total analytical time. Acknowledgements
3.3. Application of the proposed method Cu was determined in five cachaça samples purchased at the local market using Ag, Bi, Co and Ni as internal standards. Cu concentration varied from 0.66 to 6.64 mg L− 1. The analytical results using the proposed method were compared to the results obtained by ICP OES after microwave oven acid digestion. All the results are summarized in Table 2. The results obtained using Ag as internal standard are quite comparable with those obtained by ICP OES after digestion. A paired ttest demonstrated that there is no evidence for a systematic difference between the results obtained using Ag as internal standard and those obtained by ICP OES after digestion at a 95% confidence level. Considering the results obtained by ICP OES the most probable, the accuracy of the method was evaluated by determining the relative deviation (%) of the results found with and without the application of the IS technique. The deviations varied from − 13.4% to −20.9% using the standard calibration technique without IS, from − 1.9% to + 1.9% using Ag as IS, from −3.0% to + 3.7% using Bi as IS, from − 7.7% to +4.1% using Co as IS and from − 11.3% to + 2.6% using Ni as IS. It is important to point out that when using the standard calibration technique all results exhibited a negative bias, whereas the deviations were randomly distributed exhibiting positive and negative values in the presence of Ag as internal standard.
3.4. Analytical characteristics of the proposed method When using Ag as internal standard, the proposed method was able to determine Cu within a linear range from 0.1 to 4.0 mg L− 1. The analytical curve was defined by a typical linear equation, A = 0.1461CCu + 0.0041 (mg L− 1), with adequate linearity (r = 0.9997). The limit of detection (LOD) and the limit of quantification (LOQ), defined respectively as the concentration corresponding to 3 and 10-fold the standard deviation of the method blank divided by the slope of the analytical curve, was determined by analyzing ten replicates of the method blank. The LOD and the LOQ for the proposed method was 15 and 51 µg L− 1. Ferreira et al. [11] obtained a LOQ of 11 µg L− 1 for the determination of Cu in fruit juices using FS-FAAS and indium as internal standard.
The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support (Grant 07/ 04515-4), to the fellowship to KM (Grant 09/01447-3) and to the Conselho Nacional de Desenvolvimento Tecnológico (CNPq) (Grant 308834/2006-2). References [1] Associação Brasileira de Bebidas. http://www.abrabe.org.br/cachaca.php (accessed 18.08.09). [2] B.S. Lima Neto, C.W.D. Bezerra, L.R. Polastro, P. Campos, R.F. Nascimento, S.M.B. Furuya, D.W. Franco, Copper in Brazilian sugar-cane spirits: quantification and control, Quím. Nova 17 (1994) 220. [3] A.D.J.B. Lima, M.D.G. Cardoso, L.G.D.L. Guimarães, J.M. De Lima, D.L. Nelson, Effect of copper removing substances on the amount of secondary compounds of sugar cane spirit, Quím. Nova 32 (2009) 845–848. [4] R.M. Cunha, E. Silva, E. Almeida, E.P.E. Valencia, V.F. Nascimento Filho, Determination of Fe, Cu and Zn in sugar-cane spirits commercialized in Southeastern Brazil by TXRF, J. Radioanal. Nucl. Ch. 260 (2004) 3–7. [5] Brasil, Ministério da Agricultura, Pecuária e Abastecimento, Instrução Normativa Nº 13 de 29/06/2005. Diário Oficial da União-Seção1, Nº 124 de 30 de Junho de 2005, pp. 3–4 (30.06.05). [6] E.A. Hernández-Caraballo, R.M. Avila-Gómez, T. Capote, F. Rivas, A.G. Pérez, Classification of Venezuelan spirituous beverages by means of discriminant analysis and artificial neural networks based on their Zn, Cu and Fe concentrations, Talanta 60 (2003) 1259–1267. [7] M. Navarro-Alarcon, C. Velasco, A. Jodral, C. Terrés, M. Olalla, H. Lopez, M.C. Lopez, Copper, zinc, calcium and magnesium content of alcoholic beverages and byproducts from Spain: nutritional supply, Food Addit. Contam. 24 (2007) 685–694. [8] R.A. Labanca, M.B.A. Glória, V.J.P. Gouveia, R.J.C.F. Afonso, Determination of copper and alcohol contents in sugar cane spirits produced in the state of Minas Gerais, Brazil, Quím. Nova 29 (2006) 1110–1113. [9] F.A. Honorato, R.S. Honorato, M.F. Pimentel, M.C.U. Araujo, Analytical curve or standard addition method: how to elect and design — a strategy applied to copper determination in sugarcane spirits using AAS, Analyst 127 (2002) 1520–1525. [10] S.L.C. Ferreira, A.S. Souza, G.C. Brandão, H.S. Ferreira, W.N.L. dos Santos, M.F. Pimentel, M.G.R. Vale, Direct determination of iron and manganese in wine using the reference element technique and fast sequential multi-element flame atomic absorption spectrometry, Talanta 74 (2008) 699–702. [11] S.L.C. Ferreira, E.G.P. Da Silva, L.A. Portugal, G.D. Matos, F.A. De Santana, M.G.A. Korn, A.C.S. Costa, Evaluation and application of the internal standard technique for the direct determination of copper in fruit juices employing fast sequential flame atomic absorption spectrometry, Anal. Lett. 41 (2008) 1571–1578. [12] H.D. Projahn, U. Steeg, J. Sanders, E. Vanciay, Application of the reference-element technique for fast sequential flame atomic-absorption spectrometry, Anal. Bioanal. Chem. 378 (2004) 1083–1087. [13] K. Miranda, E.R. Pereira-Filho, Potentialities of thermospray flame furnace atomic absorption spectrometry (TS-FF-AAS) in the fast sequential determination of Cd, Cu, Pb and Zn, Anal. Methods 1 (2009) 215–219.
Table 2 Determination of Cu in five cachaça samples using ICP OES after acid digestion and FS-FAAS without and with four different internal standards (n = 3). Sample
Cu ICP OES (mg L− 1)
Cu without IS (mg L− 1)
Cu with IS (Ag) (mg L− 1)
Cu with IS (Bi) (mg L− 1)
Cu with IS (Co) (mg L− 1)
Cu with IS (Ni) (mg L− 1)
Cachaça 1 Cachaça 2 Cachaça 3 Cachaça 4 Cachaça 5
0.67 ± 0.02 4.17 ± 0.08 1.24 ± 0.02 1.88 ± 0.07 6.70 ± 0.06
0.53 ± 0.01 3.61 ± 0.02 1.05 ± 0.02 1.56 ± 0.02 5.57 ± 0.04
0.66 ± 0.01 4.15 ± 0.06 1.25 ± 0.03 1.91 ± 0.01 6.64 ± 0.02
0.65 ± 0.01 4.12 ± 0.05 1.22 ± 0.03 1.95 ± 0.01 6.51 ± 0.04
0.62 ± 0.01 3.99 ± 0.06 1.16 ± 0.03 1.96 ± 0.01 6.36 ± 0.02
0.60 ± 0.01 4.10 ± 0.06 1.17 ± 0.02 1.93 ± 0.01 6.46 ± 0.02
IS: internal standard.
Contents lists available at ScienceDirect
Microchemical Journal 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 / m i c r o c
Copper determination in sugar cane spirits by fast sequential flame atomic absorption spectrometry using internal standardization Kelber Miranda, Amália G.G. Dionísio, Edenir R. Pereira-Filho ⁎ Universidade Federal de São Carlos, Centro de Ciências Exatas e de Tecnologia, Departamento de Química, Grupo de Análise Instrumental Aplicada, Rodovia Washington Luiz, km 235, Caixa Postal 676, CEP 13565-905, São Carlos — SP, Brazil
a r t i c l e
i n f o
Article history: Received 17 November 2009 Received in revised form 18 February 2010 Accepted 18 February 2010 Available online 26 February 2010 Keywords: FS-FAAS Sugar cane spirit Cachaça Copper Internal standard
a b s t r a c t In this study, a fast and simple method is proposed for the determination of Cu in sugar cane spirits employing fast sequential flame atomic absorption spectrometry and the internal standard technique. First, Ag, Bi, Co and Ni were evaluated as internal standards to minimize transport interferences. The results demonstrated that Ag at a concentration of 2 mg L− 1 was effective. Under these conditions, Cu could be determined with a limit of detection of 15 µg L− 1. Then, Cu was determined in 5 sugar cane spirit samples using the proposed method and the results were compared with those obtained by inductively coupled plasma optical emission spectrometry after microwave oven acid digestion. The content of Cu varied from 0.66 to 6.64 mg L− 1. Accuracy and precision of the proposed method were evaluated by comparing the results obtained with both methods. A paired t-test at a 95% confidence level showed that the proposed method enabled the achievement of similar results as those obtained by ICP OES after acid digestion. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In Brazil nearly 30,000 industrial units are responsible to produce approximately 1.3 billion liters a year of a worldwide appreciated sugar cane spirit named “cachaça” [1]. This alcoholic beverage is produced by distillation after the fermentation of sugar cane and since Cu has been used to make the distillation container, this metal may contaminate the sugar cane spirit. During the distillation process a compound named verdigris [CuCO3Cu(OH)2] is formed on the container wall and it can be dissolved by acidic alcoholic vapors [2]. Some procedures like the use of active coal and ionic exchange resins have been used to remove cuprous ions from the distilled drink but unluckily they also remove other substances that contribute to a desirable flavor and fragrance. The alternative to use steel in the manufacture of the stills resulted in complaints of low sensory quality of the sugar cane spirit [3]. Trace levels of Cu are known to be essential in metabolism; however, higher doses may cause Wilson's disease and other deleterious health effects [4]. For this reason, the accurate determination of Cu is crucial to strengthen the agribusiness dedicated to the production and commercialization of Brazilian sugar cane spirits. The Brazilian Ministry of Agriculture, Livestock and Supply established quality standards for cachaça by fixing technical regulations [5]. Regarding inorganic contaminants, the maximum allowed level of Cu in Brazilian cachaça is 5 mg L− 1. ⁎ Corresponding author. E-mail address: [email protected] (E.R. Pereira-Filho). 0026-265X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2010.02.011
Flame atomic absorption spectrometry (FAAS) has been employed for rapid Cu determination in alcoholic beverages using matrixmatching calibration [6,7]. However, due to the fact that samples of Brazilian sugar cane spirits present alcohol contents that can vary in a great extent, as from 30 to 60% (v/v) [8], using this approach would be an exhausting task because the alcoholic grade needs to be determined for each sample and the standard solutions should be prepared in the same matrix. Therefore, the analyte addition technique is more appropriate than matrix-matching calibration to determine copper by FAAS in sugar cane spirits [9]. However, the execution of the analyte addition technique is laborious and slow. Internal standardization utilizing fast sequential FAAS (FS-FAAS) can also be adopted as a simple and efficient strategy to minimize transport interferences in FAAS [10,11]. When applying internal standardization a known amount of a selected element (the internal standard) is added to all blanks, reference solutions and samples. The signal of the internal standard (IS) is monitored together with the signal of the analytes of interest and a correction factor is calculated from the ratio of the initial and actual values of the IS. This correction factor is then applied to the signals of the analytes. The element to be used as an IS needs to present some characteristics: (1) shall not be present in the sample in a detectable concentration, (2) shall not interfere with the analytes, (3) shall itself be free from interferences and (4) shall be relatively neutral to changes in flame stoichiometry. The concentration of the IS shall result in a signal level typically from 0.1 to 0.4 absorbance. FS-FAAS is a sequential multi-element technique that keeps the advantages of conventional FAAS. When working in sequential mode,
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the instrument allows the measurement of a sequence of analytes in decrescent order of wavelengths in one monochromator scan. It is also possible to use the internal standard technique in a way that precision and accuracy of the measurement can be improved. When utilizing FSFAAS it is possible to apply internal standardization in an efficient and useful way that the application of the required correction factors is performed on-line by the instrument software automatically [12,13]. In the present paper, a fast and simple method to determine Cu in sugar cane spirits by FS-FAAS using internal standardization is presented and similar results as those obtained by inductively coupled plasma optical emission spectrometry (ICP OES) after acid digestion are achieved. 2. Materials and methods 2.1. Instrumentation FS-FAAS experiments were performed in a flame atomic absorption spectrometer (AA240FS; Varian, Mulgrave, Australia) fitted with a deuterium lamp for background correction. Silver, Bi, Co, Cu and Ni hollow cathode lamps were used as primary radiation sources and its electric currents were 4, 10, 7, 4 and 4 mA, respectively. In all experiments an oxidizing air/acetylene (13.5 L min− 1/2.0 L min− 1) flame was used. Copper was measured at 324.8 nm with a spectral bandwidth of 0.5 nm. 2.2. Reagents, analytical solutions and samples Deionized water (18 MΩ cm resistivity) generated from a Milli-Q® Plus Total Water System (Millipore Corp., Bedford, MA, USA) was used to prepare all solutions. Prior to use, all glassware and polypropylene flasks were washed with soap, soaked in 10% (v/v) HNO3 for 24 h, rinsed with deionized water and dried in such a manner to ensure that no contamination occurred. Reference solutions were prepared daily after appropriate dilution of the 1000 mg L− 1 Ag, Bi, Co, Cu and Ni stock standard solutions (Tec-Lab, Hexis, São Paulo, Brazil). Brazilian sugar cane spirit (cachaça) samples were purchased at the local market in São Carlos, São Paulo State, Brazil. 2.3. Determination of Cu in cachaça samples after total digestion A microwave oven system equipped with perfluoalcoxi (PFA) vessels and temperature sensor Multiwave, Anton-Paar (Graz, Austria) was employed for the digestion of the cachaça samples. A volume of 1 mL of cachaça was digested using 1 mL of HNO3 14 mol L− 1 and 6 mL of deionized water in a closed vessel. After decomposition, the digested samples were transferred to volumetric flasks and diluted to 10.0 mL with deionized water. The microwave oven heating program was performed in two steps: (1) 2 min at 300 W; and (2) 5 min at 600 W. All samples were digested in triplicate. Copper was determined in the diluted solutions by ICP OES (Vista AX, Varian, Mulgrave, Australia).
3. Results and discussion 3.1. Influence of alcohol content on Cu absorbance signals Brazilian sugar cane spirits may present alcohol contents that can vary from 30 to 60% (v/v). In the proposed method, the sugar cane spirit sample is diluted with 1.0 mol L-1 HNO3 1/1 (v/v) and, after dilution, the alcohol content can vary from 15 to 30%. Therefore, the influence of ethanol content on Cu absorbance signals was evaluated by comparing the slopes of analytical curves obtained from reference solutions containing 0.10–4.0 mg L− 1 Cu prepared in 0, 15, 20, 25 and 30% (v/v) ethanol. It can be seen in Fig. 1 that the slope of the analytical curve for Cu decreases when the alcoholic content increases. During the experiments we observed transport interferences: the nebulizer uptake rate decreased when the alcoholic content increased. These results confirm that if we wanted to determine Cu in sugar cane spirit samples with different alcoholic contents employing matrix-matching calibration we would have to access the alcoholic grade for each sample and prepare the standard solutions in the same matrix. This procedure would be hard-working.
3.2. Evaluation of Ag, Bi, Co and Ni as internal standards Silver, Bi, Co and Ni were tested as internal standards (IS). Analytical curves were established by spiking a cachaça sample with increasing concentrations of Cu (0.10–4.0 mg L− 1). The cachaça sample used for the evaluation did not contain Ag, Bi, Co, Cu and Ni in detectable concentrations. All the results can be seen in Table 1. The analytical signals obtained were corrected employing the internal standard technique for the four internal standards tested. More details about the internal standard technique can be found elsewhere [10]. The slope and its standard deviation of the analytical curve obtained by the standard calibration technique for copper (0.1460 ± 0.0011)CCu(mg L− 1) was compared with the slopes (±SD) of the analytical curves obtained with the internal standard technique using Ag, Bi, Co and Ni (0.1461 ± 0.0012, 0.1454 ± 0.0010, 0.1439 ± 0.0010 and 0.1449 ± 0.0013, respectively)CCu(mg L− 1) as internal standards. A paired t-test (n = 3) showed no differences between them at a 95% confidence level. The results demonstrate that Ag, Bi, Co and Ni are efficient as internal standards for correcting transport interferences observed during the determination of Cu in cachaça samples by FSFAAS.
2.4. Direct determination of Cu in cachaça samples using FS-FAAS and internal standardization In the proposed method, 5 mL of cachaça sample and 400 µL of a 50 mg L− 1 Ag, Co, Ni and 250 mg L− 1 Bi standard solution were pipetted in a 15 mL polypropylene flask. Then the volume was made up to 10 ml using 1.0 mol L− 1 HNO3 solution for Cu determination. After dilution, the final concentration was 10 mg L− 1 for Bi and 2 mg L− 1 for Ag, Co and Ni. Reference solutions containing 0.10–4.0 mg L− 1 Cu were used for calibration. The internal standards (Ag, Bi, Co and Ni) were added to all blanks, reference solutions and samples. Copper was determined on the most sensitive line (324.8 nm).
Fig. 1. Copper calibration curves without IS containing 0, 15, 20, 25 and 30% (v/v) ethanol by FS-FAAS.
K. Miranda et al. / Microchemical Journal 96 (2010) 99–101 Table 1 Wavelength, concentration used and linear regression equations obtained for the determination of Cu using four different internal standards (IS) and the standard calibration technique. IS
Wavelength (nm)
IS concentration (mg L− 1)
Silver 328.1 2.0 Bismuth 223.1 10.0 Cobalt 240.7 2.0 Nickel 232.0 2.0 Using the standard calibration technique
Linear regression equation (Cu) A = 0.1461CCu + 0.0041 A = 0.1454CCu + 0.0060 A = 0.1439CCu + 0.0051 A = 0.1449CCu + 0.0042 A = 0.1460CCu + 0.0053
(R = 0.9997) (R = 0.9996) (R = 0.9997) (R = 0.9997) (R = 0.9996)
R: correlation coefficient.
101
4. Conclusions Silver was effective as an internal standard, minimizing the transport interference observed, enhancing precision and accuracy on Cu determination in cachaça samples by FS-FAAS. The method proposed in this paper is fast, simple and enables the achievement of similar results as those obtained by ICP OES after microwave oven acid digestion. It can be considered an alternative to Cu determination in cachaça samples providing a substantial reduction in the total analytical time. Acknowledgements
3.3. Application of the proposed method Cu was determined in five cachaça samples purchased at the local market using Ag, Bi, Co and Ni as internal standards. Cu concentration varied from 0.66 to 6.64 mg L− 1. The analytical results using the proposed method were compared to the results obtained by ICP OES after microwave oven acid digestion. All the results are summarized in Table 2. The results obtained using Ag as internal standard are quite comparable with those obtained by ICP OES after digestion. A paired ttest demonstrated that there is no evidence for a systematic difference between the results obtained using Ag as internal standard and those obtained by ICP OES after digestion at a 95% confidence level. Considering the results obtained by ICP OES the most probable, the accuracy of the method was evaluated by determining the relative deviation (%) of the results found with and without the application of the IS technique. The deviations varied from − 13.4% to −20.9% using the standard calibration technique without IS, from − 1.9% to + 1.9% using Ag as IS, from −3.0% to + 3.7% using Bi as IS, from − 7.7% to +4.1% using Co as IS and from − 11.3% to + 2.6% using Ni as IS. It is important to point out that when using the standard calibration technique all results exhibited a negative bias, whereas the deviations were randomly distributed exhibiting positive and negative values in the presence of Ag as internal standard.
3.4. Analytical characteristics of the proposed method When using Ag as internal standard, the proposed method was able to determine Cu within a linear range from 0.1 to 4.0 mg L− 1. The analytical curve was defined by a typical linear equation, A = 0.1461CCu + 0.0041 (mg L− 1), with adequate linearity (r = 0.9997). The limit of detection (LOD) and the limit of quantification (LOQ), defined respectively as the concentration corresponding to 3 and 10-fold the standard deviation of the method blank divided by the slope of the analytical curve, was determined by analyzing ten replicates of the method blank. The LOD and the LOQ for the proposed method was 15 and 51 µg L− 1. Ferreira et al. [11] obtained a LOQ of 11 µg L− 1 for the determination of Cu in fruit juices using FS-FAAS and indium as internal standard.
The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support (Grant 07/ 04515-4), to the fellowship to KM (Grant 09/01447-3) and to the Conselho Nacional de Desenvolvimento Tecnológico (CNPq) (Grant 308834/2006-2). References [1] Associação Brasileira de Bebidas. http://www.abrabe.org.br/cachaca.php (accessed 18.08.09). [2] B.S. Lima Neto, C.W.D. Bezerra, L.R. Polastro, P. Campos, R.F. Nascimento, S.M.B. Furuya, D.W. Franco, Copper in Brazilian sugar-cane spirits: quantification and control, Quím. Nova 17 (1994) 220. [3] A.D.J.B. Lima, M.D.G. Cardoso, L.G.D.L. Guimarães, J.M. De Lima, D.L. Nelson, Effect of copper removing substances on the amount of secondary compounds of sugar cane spirit, Quím. Nova 32 (2009) 845–848. [4] R.M. Cunha, E. Silva, E. Almeida, E.P.E. Valencia, V.F. Nascimento Filho, Determination of Fe, Cu and Zn in sugar-cane spirits commercialized in Southeastern Brazil by TXRF, J. Radioanal. Nucl. Ch. 260 (2004) 3–7. [5] Brasil, Ministério da Agricultura, Pecuária e Abastecimento, Instrução Normativa Nº 13 de 29/06/2005. Diário Oficial da União-Seção1, Nº 124 de 30 de Junho de 2005, pp. 3–4 (30.06.05). [6] E.A. Hernández-Caraballo, R.M. Avila-Gómez, T. Capote, F. Rivas, A.G. Pérez, Classification of Venezuelan spirituous beverages by means of discriminant analysis and artificial neural networks based on their Zn, Cu and Fe concentrations, Talanta 60 (2003) 1259–1267. [7] M. Navarro-Alarcon, C. Velasco, A. Jodral, C. Terrés, M. Olalla, H. Lopez, M.C. Lopez, Copper, zinc, calcium and magnesium content of alcoholic beverages and byproducts from Spain: nutritional supply, Food Addit. Contam. 24 (2007) 685–694. [8] R.A. Labanca, M.B.A. Glória, V.J.P. Gouveia, R.J.C.F. Afonso, Determination of copper and alcohol contents in sugar cane spirits produced in the state of Minas Gerais, Brazil, Quím. Nova 29 (2006) 1110–1113. [9] F.A. Honorato, R.S. Honorato, M.F. Pimentel, M.C.U. Araujo, Analytical curve or standard addition method: how to elect and design — a strategy applied to copper determination in sugarcane spirits using AAS, Analyst 127 (2002) 1520–1525. [10] S.L.C. Ferreira, A.S. Souza, G.C. Brandão, H.S. Ferreira, W.N.L. dos Santos, M.F. Pimentel, M.G.R. Vale, Direct determination of iron and manganese in wine using the reference element technique and fast sequential multi-element flame atomic absorption spectrometry, Talanta 74 (2008) 699–702. [11] S.L.C. Ferreira, E.G.P. Da Silva, L.A. Portugal, G.D. Matos, F.A. De Santana, M.G.A. Korn, A.C.S. Costa, Evaluation and application of the internal standard technique for the direct determination of copper in fruit juices employing fast sequential flame atomic absorption spectrometry, Anal. Lett. 41 (2008) 1571–1578. [12] H.D. Projahn, U. Steeg, J. Sanders, E. Vanciay, Application of the reference-element technique for fast sequential flame atomic-absorption spectrometry, Anal. Bioanal. Chem. 378 (2004) 1083–1087. [13] K. Miranda, E.R. Pereira-Filho, Potentialities of thermospray flame furnace atomic absorption spectrometry (TS-FF-AAS) in the fast sequential determination of Cd, Cu, Pb and Zn, Anal. Methods 1 (2009) 215–219.
Table 2 Determination of Cu in five cachaça samples using ICP OES after acid digestion and FS-FAAS without and with four different internal standards (n = 3). Sample
Cu ICP OES (mg L− 1)
Cu without IS (mg L− 1)
Cu with IS (Ag) (mg L− 1)
Cu with IS (Bi) (mg L− 1)
Cu with IS (Co) (mg L− 1)
Cu with IS (Ni) (mg L− 1)
Cachaça 1 Cachaça 2 Cachaça 3 Cachaça 4 Cachaça 5
0.67 ± 0.02 4.17 ± 0.08 1.24 ± 0.02 1.88 ± 0.07 6.70 ± 0.06
0.53 ± 0.01 3.61 ± 0.02 1.05 ± 0.02 1.56 ± 0.02 5.57 ± 0.04
0.66 ± 0.01 4.15 ± 0.06 1.25 ± 0.03 1.91 ± 0.01 6.64 ± 0.02
0.65 ± 0.01 4.12 ± 0.05 1.22 ± 0.03 1.95 ± 0.01 6.51 ± 0.04
0.62 ± 0.01 3.99 ± 0.06 1.16 ± 0.03 1.96 ± 0.01 6.36 ± 0.02
0.60 ± 0.01 4.10 ± 0.06 1.17 ± 0.02 1.93 ± 0.01 6.46 ± 0.02
IS: internal standard.