Comparison of Heating Extraction Procedures for Al, Ca, Mg, and Mn in Tea Samples
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1 ANALYTICAL SCIENCES MARCH 2002, VOL The Japan Society for Analytical Chemistry 313 Comparison of Heating Extraction Procedures for Al, Ca, Mg, and Mn in Tea Samples Letícia M. COSTA,* Sandro T. GOUVEIA,** and Joaquim A. NÓBREGA* *Departamento de Química, Universidade Federal de São Carlos, P. O. Box 676, , São Carlos-SP, Brazil **Departamento de Físico-Química e Química Analítica, Universidade Federal do Ceará, Fortaleza-CE, Brazil A focused-microwave-assisted procedure was adopted for the extraction of Al, Ca, Mg, and Mn in tea leaves. The efficiency of extraction was evaluated using diluted acids and a water-soluble alkaline tertiary-amines solution. The extraction procedure was implemented in 5 min. A conventional hot-plate digestion procedure was employed for a comparison. A colorless digest was obtained for all tea samples and the total contents determined for each analyte were employed for estimating the efficiency of extraction. Tea infusions were also prepared. Subsequent determinations of Al, Ca, Mg, and Mn were carried out using flame atomic absorption spectrophotometry (FAAS) and inductively coupled plasma optical emission spectrometry (ICP-OES). In most cases, quantitative, or at least semi-quantitative, extractions were attained for Ca, Mg, and Mn. Lower recoveries were attained to Al, which seems to be related to compounds that bind this element in the sample matrix. Large variations in the trace-element composition of teas were observed; these results are discussed with reference to both extraction media and type of tea. (Received July 9, 2001; Accepted November 15, 2001) Introduction Tea is the second most consumed beverage in the world with an estimated billion cups consumed daily 1 and an estimated average consumption of 1 L of tea per person per day in the UK. 2 Stagg and Millin have emphasized the therapeutic action of teas owing to its anti-inflammatory action and antibacterial properties. 3 Teas are classified into three types, depending on the fermentation process. 4,5 One is green tea, produced by drying and roasting the leaves. Another is oolong, obtained by partial fermentation. The third one is black tea, which is fully fermented. The fermentation process involves an enzymatic oxidation of polyphenols, leading to the formation of chemical compounds that generate both the aroma and color of black tea. 6 Although tea is rich in minerals, such as Al, Ca, K, Mg, and Mn, the contribution of tea drinking as a mineral source is not clear because the bioavailability of many of these metals is uncertain, since the compounds in the matrix are generally not known. 7,8 A procedure was developed using tea-leaf slurries for determining Al, Ba, Mg, and Mn, which was compared to ash decomposition. The authors showed that similar amounts of these elements were leached after boiling in water for 5 min. 8 On the other hand, Zhou et al. showed that the Al concentration found in tea infusion depended on both the origin of the tea and the infusion time. For some tea types, Al extraction can be as low as 27% after four successive infusions in water. 9 Lamble and Hill proposed a focused-microwave-assisted To whom correspondence should be addressed. djan@terra.com.br digestion method for tea leaves from different sources. Digestions were completed in 35 min and quantitative recoveries were obtained for Al, Cu, K, Mg, Mn, and Zn. 2 For tea infusion, the efficiency of extraction is critically dependent on both agitation and the brewing time. Extraction with diluted acids is a simple and safe procedure that has been successfully applied to sample preparation. The use of diluted acids avoids the danger of a rapid pressure buildup in closed-vessels as well as the possibility of systematic errors caused by using strong oxidizing acids. 10 Microwave extraction using diluted acids is a simple alternative to sample preparation and its use is increasing. 11 Diluted acid solutions can more intensely absorb microwave energy, owing to their water content. 11 Concentrated acids can be less efficient microwave absorber and can generate high blank values. This work evaluated the extraction of Al, Ca, Mg, and Mn in tea leaves using diluted acid solutions heated by conductive or microwave-assisted ovens. These elements were chosen while considering their different behaviors in the extraction procedures. In spite of similar chemical characteristics, Odegård and Lund showed that Mg is less strongly bound to organic material than Ca in tea leaves. 7 On the other hand, previous work dealing with biological samples has showed that Mn is quantitatively extracted in most samples and Al is hardly extracted, even when using longer extraction times. 13 Experimental Samples The samples investigated were: erva cidreira (Cymbopogan citratus), erva-mate chimarrão (Ilex paraguariensis), both unfermented, matte (Ilex paraguariensis) partially fermented
2 314 ANALYTICAL SCIENCES MARCH 2002, VOL. 18 Table 1 Instrumental conditions for the determination of Al, Ca, Mg, and Mg by FAAS Element λ/nm Slit/nm Flame composition Al N 2O C 2H 2 Ca N 2O C 2H 2 Mg Air C 2H 2 Mn Air C 2H 2 tea, and black royal blend (Camellia sinensis), fully fermented tea. The first sample was collected in a home backyard. All others were bought in local markets. The Cymbopogan citratus leaves were rinsed with distilled and deionized water (Milli-Q, Millipore, 18 MΩ cm, Bedford, MA, USA) and dried for 72 h at 60 C. They were ground in a Whiley Mill (Microtec Model) with 1 mm sieves. Commercial tea samples were used without any treatment. Reagents and reference solutions All solutions were prepared from analytical-grade reagents and distilled-deionized Milli-Q water was used throughout. Acid diluted solutions (1% and 10% v/v) were prepared from the proper dilution of HNO 3 (Mallinckrodt, Darmsdat, Germany) and HCl (Carlo Erba, Italy) in water. A watersoluble tertiary-amines solution, named CFA-C (Coal-Fly Ash C, Spectrasol, Warwick, NY, USA), was diluted in Milli-Q water to 5% v/v. Additionally to these media, tea samples were also extracted using water. Aluminum (Al pellets, 3 8 mesh, %, Aldrich, Milwaukee, WI, USA) and Ca (CaCO 3, Merck, Poole, UK) stock solutions (1000 mg L 1 ) were prepared. Magnesium and Mn stock solutions, both containing 1000 mg L 1, were furnished by Merck. Reference multielementar solutions containing Al, Ca, Mg, and Mn were prepared by appropriate dilutions of these stocks. The acid concentrations of the reference solutions were matched with that used in each extraction procedure. Sample preparation Ash and wet digestion procedure. The total digestion was based on a procedure proposed by Manickum and Verbeek. 12 A mass of 1.0 g of tea samples was mineralized at 500 C during 1 h. The residue was treated with concentrated HNO 3 and evaporated to dryness before heating at 500 C for 15 min. The cooled residue was moistened with water and dissolved in concentrated HCl and water. After cooling, the colorless solution was transferred to a 50 ml volumetric flask. The sample mass was chosen according to the type of tea used and the sensitivity for each element. Samples were prepared in quintuplicate. Extraction with conventional heating A volume of 20 ml of HCl or HNO 3 (1 or 10% v/v) was added to tea sample masses of 0.2, 0.5 or 1.0 g. A 5% v/v CFA-C solution or water was also investigated as an extraction medium. The tea suspension was boiled during 5 min, and after cooling it was filtered (Framex 389) into a 50.0 ml volumetric flask and the volume was made up with water. All samples were prepared in triplicate. Blank solutions were prepared in each medium studied. Extraction with focused microwave oven Focused microwave-assisted extraction was conducted using the STAR 6 system (CEM, Matthews, NC, USA) with a nominal power of 950 ± 50 W. The extraction was carried out in borosilicate vessels, and then volumes of 20 ml of each acid solution (1 or 10% v/v) or water were added in approximately 0.1 or 0.5 g aliquots of four tea samples. Thereafter, the reaction vessels were heated for 3 min at 95 C, using a ramp time of 2 min necessary to reach the set temperature. The total heating time was 5 min. After cooling, the suspension was filtered and transferred to a 50.0 ml volumetric flask. Under each experimental condition, all samples were prepared in triplicate. Successive extractions of Al in black and green teas Based on a procedure proposed by Zhou et al., 9 an experiment was performed to evaluate the behavior of Al exposed to successive extractions. Samples of Ilex paraguariensis (green tea) and Camellia sinensis (black tea) were used in this experiment. A mass of 2 g of tea leaves was accurately weighed in a beaker and 50 ml of water was added. The water was boiled for 20 min. During infusion, the mixture was mechanically stirred. A volume of 40 ml of the infusion was removed and stored in a clean polyethylene bottle. After, another equivalent aliquot of water had been added, it was heated until boiling was again promoted. This procedure was repeated five times. Aluminum was determined in each infusion. This experiment showed the amount of Al successively extracted and the possibility to attain a quantitative recovery of this element. Determination of Al, Ca, Mg, and Mn by FAAS and ICP-OES Aluminum, Ca, Mg, and Mn in all tea extracts were determined by FAAS. The instrumental parameters are listed in Table 1. Sample dilution was made according to the element concentration and sensitivity. All extracts were previously diluted before Ca and Mg determinations. Before Ca determination, La(III) (10 mg ml 1 in 4% v/v HNO 3) was added to all solutions. A solution containing 1% v/v KCl was added as an ionization buffer before the Al determination. Some determinations were carried out using a Thermo Jarrel Ash (Franklin, MA, USA) Model Atom Scan 25 for sequential inductively coupled plasma optical emission spectrometry (ICP- OES). All of the experimental parameters were kept at standard conditions and the following emission lines were used for each element: Al I, ; Ca II, ; Mg I, ; and Mn I, nm. Results and Discussion The total contents of Al, Ca, Mg, and Mn in each sample were determined using a conventional acid-digestion procedure combining ash and wet digestion; the results are given in Table 2. These values were adopted as reference values to estimate the efficiency of extraction under each experimental condition. Extraction with conventional heating All determinations of elements in different extracts using FAAS and ICP-OES led to relative standard deviations lower than 5% for low concentrations, and usually lower than 2% for higher analyte concentrations (n = 3). The results obtained after 5 min of brewing of teas in boiling solutions of diluted acids, CFA-C, and water are shown in Figs. 1, 2, 3, and 4 for all elements. The extraction procedure with conventional hot-plate heating and diluted acids led to recoveries of around 60% for Ca, Mg, and Mn in all types of tea
3 ANALYTICAL SCIENCES MARCH 2002, VOL Table 2 Camellia sinensis (fully fermented) Ilex paraguariensis (partially fermented) Ilex paraguariensis (non fermented) Cympogan citratus (non fermented) Total content of Al, Ca, Mg, and Mn in teas Mean ± standard deviation, n = 5. Al/µg g 1 Ca/µg g 1 Mg/µg g 1 Mn/µg g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.5 Fig. 1 Aluminum extraction using conventional and microwave-assisted heating for different types of teas: (A) Ilex paraguariensis, (B) Ilex paraguariensis (green), and (C) Camellia sinensis. (Figs. 2, 3, and 4). Increasing the extraction time to 10 min did not improve the efficiency of extraction. Odegård and Lund concluded that the concentrations of Al, Ba, Ca, Mg, and Mn in tea infusion were constant after 5 min of extraction, and adopted this time for extractions. 7 The results obtained here are in agreement with this previous comment. The recoveries of Al were lower than 50% for oolong and partially fermented teas (Figs. 1(A) and 1(B)) and around 90% for black tea (Fig. 1(C)) in diluted acids. Aluminum was not extracted in Cymbopogan citratus tea (non-fermented). This is an indication that Al is present as different compounds in different types of teas. We could suppose that the low Al extraction observed in Cymbopogan citratus tea (nonfermented) could be due to an elevated concentration of Si in this sample. The bioavailability of the elements in tea leaves is still unknown. Thus, extraction results can be seen as preliminary data to estimate ingested concentrations. The aluminum concentration could be critical for patients with renal failure; however, for most samples Al was poorly extracted in most media, reaching values as low as 5% in Ilex paraguariensis (non-fermented tea, Fig. 1(B)). On the other hand, the efficiency of extraction of Ca, Mg, and Mn was generally higher than that. Depending on the tea type, up to 55 and 86% of Ca and Mg, respectively (Figs. 2(B) and 3(B)), were extracted. In all tea samples Mg was more easily extracted than Ca, showing that the former is less strongly bound to the matrix. Odegård and Lund 7 previously showed this behavior. A 5% v/v CFA-C solution was efficient for the extraction of Ca, Mg and Mn in all teas, except for Cymbopogan citratus (non-fermented), in which the extraction was lower than 40% for Mn (Fig. 4(D)). The Al extraction was lower than 10% in the CFA-C medium for all tea samples (Fig. 1). Focused microwave-assisted extraction The tea suspension stayed for 5 min under boiling when using conventional hot-plate heating. Extraction procedures with a microwave oven were carried out in a 5 min total time program. According to previous results using conventional heating, extending the extraction time to 10 min did not improve the
4 316 ANALYTICAL SCIENCES MARCH 2002, VOL. 18 Fig. 2 Calcium extraction using conventional and microwave-assisted heating for different types of teas: (A) Ilex paraguariensis, (B) Ilex paraguariensis (green), (C) Camellia sinensis, and (D) Cymbopogan citratus. Fig. 3 Magnesium extraction using conventional and microwave-assisted heating for different types of teas: (A) Ilex paraguariensis, (B) Ilex paraguariensis (green), (C) Camellia sinensis, and (D) Cymbopogan citratus.
5 ANALYTICAL SCIENCES MARCH 2002, VOL Fig. 4 Manganese extraction using conventional and microwave-assisted heating for different types of teas: (A) Ilex paraguariensis, (B) Ilex paraguariensis (green), (C) Camellia sinensis, and (D) Cymbopogan citratus. efficiency of extraction. Aluminum was not quantitatively extracted in all teas using acid solutions; the highest extraction values were attained with black tea samples in which more than 80% of Al was extracted under the most favorable conditions (Fig. 1(C)). Water was extracted lower than 10% in non-fermented and semi-fermented teas, and values of around 40% were obtained for black tea (Fig. 1). Wieteska et al. studied the extraction procedures for preparing of vegetable samples, and demonstrated that the addition of HF is essential for the quantitative dissolution of Al. 14 Calcium extraction was higher than 80% with diluted acids in all types of tea, except for semi-fermented tea, in which lower values were extracted (Fig. 2). Magnesium was extracted above 60% in an acid medium in all teas (Fig. 3). Once again, the extraction of Mg was higher than Ca in a water medium (Figs. 2 and 3). Manganese was more easily extracted in water and an acid medium, with values higher than 30% and 60%, respectively (Fig. 4). For both heating procedures, Mn extraction was more difficult in non-fermented tea. Hill and Lamble 2 showed the variability of trace-metal levels in tea leaves from different origins, and also that Al and Mn were poorly extracted in Camomile tea. We found a similar behavior for Cymbopogan citratus tea (non-fermented). Successive extraction of Al in black and green teas We evaluated the behavior of Al extracted in two types of tea: non-fermented (Ilex paraguariensis) and fully fermented (Camellia sinensis). The results are presented in Fig.5. There are considerable variations in the Al levels in different tea leaves. Camellia sinensis (black tea, fully fermented) contains nearly six-fold more Al than Ilex paraguariensis (green tea, non-fermented). It is possible that this variation may be due to different soil conditions as well as different harvesting Fig. 5 Successive extractions of Al in green and black teas. periods. The Camellia sinensis tea is grown in acid soil and tolerates high levels of Al, thus accumulating elevated quantities in its leaves. 15 The water quality can also influence the Al levels. 16 To estimate body absorption, brewing in water is an important factor to evaluate the bioavailability of Al species. This aspect is important for patients with renal failures because Al can be accumulated by the body, resulting in neurological diseases. It is necessary as an intake control of foods with high amounts of this metal. The composition of Al species could vary depending on the method of tea production. For non-fermented teas, most of the leached Al is bound to large and small organic compounds. In semi-fermented and fully fermented teas, Al is mainly present
6 318 ANALYTICAL SCIENCES MARCH 2002, VOL. 18 as both free and bound forms to small stable organic compounds. 17 This aspect was proved by comparing the results obtained for green and black teas. The extraction of Al in black teas was higher than that observed in green teas (Fig. 5) owing to the more stable high molecular masses of compounds present in this latter sample. On the other hand, in black teas Al is free or associated to compounds with lower molecular masses. Generally, Al is poorly absorbed by the body. However, in organic complexes with small molecular masses, such as citrates, the Al-complexes are more bioavailable than inorganic complexes, such as hydroxide. 15 Conclusion The extraction of Al, Ca, Mg, and Mn in teas is dependent on both the type of compound of each element bound in the sample matrix and the extraction solution employed. Conductive and microwave-assisted heating are both effective, with the main advantage coming from the fact that the microwave-assisted procedure allows a better control of the total energy transferred to each sample medium. In most studied media the extraction efficiency decreases according to the following order: Mn > Ca > Mg > Al; the higher efficiency of Mg extraction compared to Ca is better observed in a water medium. The efficiency of extraction using only water can be considered to be useful preliminary data in nutritional studies. From an analytical point of view, extraction in diluted acid media can be considered as a simple and fast procedure for obtaining semi-quantitative data. Acknowledgements Letícia Malta Costa and Sandro Thomaz Gouveia would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for fellowships. Joaquim de Araújo Nóbrega are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação de Amparo á Pesquisa do Estado de São Paulo for research grants. References 1. A. Marcos, A. Fisher, G. Rea, and S. J. Hill, J. Anal. At. Spectrom., 1998, 13, K. Lamble and S. J. Hill, Analyst, 1995, 120, G. V. Stagg and D. J. Millin, J. Sci. Food Agric., 1975, 26, M. Serafini, A. Ghiselli, and A. Ferro-Luzzi, Eur. J. Clin. Nutr., 1996, 50, C. Wang, C. Ke, and J. Yang, J. Radioanal. Nuclear Chem., 1993, 173, P. Valera, F. Pablos, and A. G. González, Talanta, 1996, 43, K. E. Odegård and W. Lund, J. Anal. At. Spectrom., 1997, 12, J. J. Powell, T. J. Burden, and R. P. H. Thompson, Analyst, 1998, 123, C. Y. Zhou, J. Wu, H. Chi, M. K. Wong, L. L. Koh, and Y. C. Wee, Sci. Total Environ., 1996, 177, C. Y. Zhou, M. K. Wong, and L. L. Koh, J. Anal. At. Spectrom., 1996, 11, M. K. Wong, W. Gu, and T. L. Ng, Anal. Sci., 1997, 13, C. K. Manickum and A. A. Verbeek, J. Anal. At. Spectrom., 1994, 9, J. A. Nóbrega, Y. Gélinas, A. Krushevska, and R. M. Barnes, J. Anal. At. Spectrom., 1997, 12, E. Wieteska, A. Zióek, and A. Drzewińska, Anal. Chim. Acta, 1996, 330, K. R. Koch, B. Pougnet, and S. Villiers, Analyst, 1989, 114, J. J. Powell, S. M. Greenfield, H. G. Parkes, J. K. Nicholson, and R. P. H. Thompson, Food Chem. Toxicol., 1993, 31, S. B. Erdemoğlu, K. Pyrzyniska, and S. Güçer, Anal. Chim. Acta, 2000, 411, 81.
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