In the Laboratory
Spectrophotometric Determination of Total Sulfite in White Wine Samples Using Crude Extracts from Flowers Márlon Herbert Flora Barbosa Soares, Luiz Antonio Ramos, and Éder Tadeu Gomes Cavalheiro* Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905 São Carlos/SP, Brazil; *
[email protected] Geissman first described the color variation of flowers and its application in chemical education on the basis of color changes as a function of the pH of the medium (1). The red and blue colors of flowers are related to the presence of anthocyanins found in ethanol extracts of the flowers. Anthocyanins are defined by Timberlake and Bridle as flavonoids derived from water-soluble flavilium salts (2). Several teaching applications of crude extracts of flowers and other vegetal tissues have been presented using such materials in association with the definition, history, and separation of the pigments present in them (3–11). We have proposed the use of the color change in acidic and basic media to demonstrate the basics of chemical equilibria, their application as acid–base titration indicators, and the fundamental principles of optical methods of analysis and thin-layer chromatography (12–15), using crude flower extracts and the skin of black beans. The low cost and easy preparation of these or similar extracts should allow their utilization in any school. An important feature of such an experiment is its ability to catch the attention of the students, which facilitates the teaching process. In this report we describe the use of the crude extract of the Tibouchina granulosa flowers in the spectrophotometric determination of sulfite in white-wine samples. The analyses were performed by eight undergraduate students in a quantitative analysis course. The main didactic objectives are the demonstration of basic principles of optical methods of analysis, the use of alternative materials in chemical analysis, and the comparison of a new method with classical procedures. The demonstration of the possibility of using Rhododendron simsii (azalea) extract is also presented, suggesting that other flowers containing anthocyanins can be used. Students determined the sulfite content in white-wine samples during a quantitative spectrophotometric experiment using the discoloring reaction between sulfite and anthocyanin (eq 1), which gave results in good agreement with those from the classical iodometric procedure (16 ). The spectrophotometric procedure was optimized for pH, maximum wavelength, and extract concentration before its application in the classroom. Attracting the student attention by the use of a natural dye is a very important educational advantage.
OH
OH OH
OH HSO3−
+
HO
O
H
O OH
O
HO
+
O
GLU H OH
SO3H
GLU
(1)
Fundamentals Sulfite is frequently added to food and beverages, including wine, as an antioxidant to prevent enzymatic oxidation during the processing and storage of these products. However, the amount of sulfite should be controlled owing to the risk of allergic reactions in sensitive people (17 ). Many procedures have been proposed for the determination of sulfite in food and beverages, including use of SO2 gas separation, photometry, electrochemistry, chromatography, gravimetry, and chemiluminescence methods (18, 19). Sulfite also reacts with the flavilic group of the anthocyanins in a discoloring reaction (eq 1) (20). The addition of the sulfite anion in the C-4 position of the flavilic ring blocks electronic delocalization in the anthocyanin molecule and the color of a solution containing the pigment becomes less intense. Sulfite is usually present as an equilibrium of the free and ethanol-adduct forms in wine samples (21, 22). In the presence of naturally occurring ketones or aldehydes (R1 = H), only the bisulfite species reacts according the following reaction (16, 21, 22), and in the procedure described here the total SO32᎑ content is determined, since in basic media only sulfite is present (pKa = 7.2). OH
O R1
+
C
HSO3−
R2
R1
C
SO3−
(2)
R2
Experimental Procedures
Preparation of Crude Extract Three hundred grams of petals of T. granulosa, R. simsii, or any other appropriate flower, were immersed in 300 mL of ethanol for 48 h and then filtered through a qualitative paper. The solvent was eliminated on a rotary evaporator under vacuum (40 °C) until a viscous liquid was obtained. This product was dried to constant weight in a vacuum oven at 35 °C. Alternatively a hair dryer can be used to remove the solvent. Reagents and Solutions All the solutions were prepared using deionized water and analytical-grade reagents, which were used without further purification. A series of 100 mg L᎑1 (100 ppm = 7.93 × 10᎑4 mol L᎑1) sodium sulfite stock solutions were freshly prepared in 0.2 mol L᎑1 phosphate buffers pH 1–10. From these stock solutions, the reference solutions containing from 0 to 10 mg L᎑1 (ppm) sulfite were prepared just before use, by dilu-
JChemEd.chem.wisc.edu • Vol. 79 No. 9 September 2002 • Journal of Chemical Education
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In the Laboratory
0.40
pH 10 pH 9 pH 3 pH 7
Tibouchina granulosa
Absorbance
Absorbance
0.2
0.1
0.35
0.30
0.25
0.20
0.0
500
600
700
0
Wavelength / nm
Rhododendron simsii 2
4
6
8
Sulfite Concentration / (mg L᎑1)
10
Figure 1. Absorption spectra of the system Tibouchina granulosa crude extract–sulfite at different pH values, obtained by the teaching staff. Conditions: 4 g L-1 extract and 2 ppm sulfite in 0.2 mol L᎑1 phosphate buffer.
Figure 2. Typical analytical curve for sulfite in the presence of the crude extract of (upper curve) Tibouchina granulosa, detection at λ = 583 nm and (lower curve) Rhododendron simsii, detection at λ = 626 nm. Conditions: 0–10 ppm sulfite and 4 g L᎑1 extract in 0.2 mol L᎑1 phosphate buffer pH 9.0.
tion with 0.2 mol L᎑1 phosphate buffers pH 1–10. The sulfite determinations were performed at pH 9, which gave the best results (see below). The use of ppm units was decided on the basis of the Brazilian law exigencies for food additives. The crude-extract stock solutions were prepared with 0.4 g of the dried extract in 0.1 L of distilled water, resulting in a final concentration of 4 g L᎑1. If another vegetal species is used the extract amount should be controlled by adding enough extract to place the initial absorbance in the range of 0.2 to 0.4. Samples were 0.25 mL aliquots of white wine diluted to 10 mL with phosphate buffer pH 9.
Hazards
Apparatus During the parameter-optimization steps the UV–vis absorption spectra were recorded in a diode array spectrophotometer (Hewlett-Packard, model 8452A, USA) using a quartz cuvette with an optical path of 1.0 cm. All classroom measurements were carried out using an analog spectrophotometer (Micronal B-380, Brazil) and a quartz cuvette with optical path of 1.0 cm, in order to demonstrate to the students how to measure an absorption spectrum and to determine the optimum wavelength for the analytical procedure. Procedures Calibration curves were obtained using solutions containing 2.5 mL of the crude extract and appropriate aliquots of the standard sulfite solution to produce final concentrations in the 2–10 ppm range. All solutions were adjusted with 0.2 M phosphate buffer (pH 9) to give a final volume of 10 mL in volumetric flasks. The samples were as above except for using 0.25 mL of the diluted white wine instead of the standard sulfite solution. The absorbance was monitored at 583 nm (T. granulosa) or 626 nm (R. simsii). Iodometry is recommended as one standard method for determining sulfite in Brazilian food and beverages. Typically an excess of KIO3 is added to the sample and acidified with H2SO4. The excess I2 is back-titrated with standard Na2S2O3 solution in the presence of starch as indicator (23).
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Sulfite is tolerated at relatively high doses, since it is easily oxidized to sulfate. However, it should be noted that according to Taylor et al. (24 ) the ingestion of more than 4 g of sodium sulfite by humans can cause toxic symptoms such as intestinal irritation, vomiting, and diarrhea. The white wine should not be ingested. Results and Discussion The students were asked to measure the absorbance of the crude extract solutions in both acidic (pH 3.0) and basic (pH 9.0) medium, at 5 nm intervals from 400 to 700 nm, in order to determine the wavelength of maximum absorption (λmax). For T. granulosa the spectra revealed the λmax at 530 nm at pH 3.0 and at 583 nm at pH 9.0. However these values can change a little if flowers from other species are used. As the reaction of sulfite and anthocyanin is not favored in acidic conditions, the best sensitivity was investigated using solutions containing 4 g of the crude extract in 1000 mL of 0.2 mol L᎑1 phosphate buffer at the desired pH and 2 ppm sulfite. Best results were observed at pH 9.0, as shown by the data in Figure 1. This experiment was performed by the teaching staff prior to the laboratory class. At pH > 11.0 the anthocyanins are unstable owing to the formation of chalcones (25). The differences in the structure of anthocyanins associated with the color changes at different pH values are described by Brouillard et al. (25–27 ). Then the students were instructed to prepare solutions containing the crude extract, reference solutions with increasing concentrations of sulfite, and the buffer, as described above. These data were used to obtain the analytical curve, which was linear at least from 0 to 10 ppm sulfite. Figure 2 presents a typical discoloring analytical curve obtained by the students for T. granulosa, described by y = 0.423 – 0.008x (r = .997, n = 5), and the analytical curve obtained by the teaching staff for R. simsii, a popular ornamental species found worldwide. Again, a linear dependence in relation to the
Journal of Chemical Education • Vol. 79 No. 9 September 2002 • JChemEd.chem.wisc.edu
In the Laboratory
Acknowledgments
Table 1. Sulfite Determination in White Wine Using Crude Anthocyanin Extracts from Two Species Total SO32᎑/(mg L᎑1)
Extract
a
|%E|
Proposed Method
Iodometric Method
Tibouchina granulosa
248.8
257.6
3.4
Rhododendron simsii
267.2
257.6
3.7
a|%E|
= |[iodometric – proposed)/iodometric] × 100|.
Table 2. Determination of Sulfite Content of White Wine by Students and Teaching Staff Results from
Total SO32᎑/(mg L᎑1) Proposed Method Iodometric Method
|%E|a
Student group 1
278.0
280.7
1.0
Student group 2
295.8
301.5
1.9
Student group 3
260.8
272.6
4.3
Student group 4
295.2
340.2
13.2
273.2 ± 2.1 b
277.7 ± 2.2 b
Teaching staff
1.6
= |[iodometric – proposed)/iodometric] × 100|. bMean of 3 determinations ± SD. a|%E|
sulfite concentration described by the equation y = 0.286 – 0.009x (r = .998, n = 5) was observed. Table 1 presents the results of the determination of sulfite in the same white wine sample, using the crude extracts from T. granulosa and R. simsii under the same conditions. The results are within ±3–4% of results from the iodometric procedure. This suggests that any flower extract containing anthocyanins can be used in such determinations. The presence of anthocyanins in the vegetal tissue is indicated by a red, blue, or violet color (1–4 ). In this work we used only flower extracts, but other vegetal tissues (fruit skins, red cabbage leaves, etc.) also contain such substances (7). The results obtained by the students working in groups of two are presented in Table 2. One commercial wine was analyzed for sulfite content using T. granulosa crude extract. Calculations were performed considering the dilutions of each sample. The results were also compared with those from the iodometric titrations and parallel determinations performed by the teaching staff, but these were not revealed to the students until the final discussion. The results suggest a good agreement between the iodometric method and the proposed method for the same wine sample. The latter is easier to perform and does not involve a visual detection of the endpoint as in iodometry. An abnormal error was observed for group 4 and was included in the final evaluation of the results as an interesting point for discussion. As the Brazilian law allows up to 300 ppm of total sulfite in beverages the sample was still within regulatory limits. This is approximately the accepted value in many countries (28).
We are indebted to Edward R. Dockal for the final review of the manuscripts and Brazilian agency FAPESP for financial support (98/13873-0) and MHFBS fellowship (99/03726-3). Literature Cited 1. Geissman, T. A. J. Chem. Educ. 1941, 18, 108. 2. Timberlake; C. F.; Bridle, P. In The Flavonoids—Part I; Harborne, J. B.; Mabry, T. J.; Mabry, H., Eds.; Academic: New York, 1975; pp 215–224. 3. Alkema, J. Seager, S. L. J. Chem. Educ. 1982, 59, 183. 4. Geissman, T. A.; Crout, D. H. G. Organic Chemistry of Secondary Plant Metabolism; Freeman: San Francisco, 1969; pp 182–209. 5. Forster, M. J. Chem. Educ. 1978, 55, 107. 6. Séquin-Frey, M. J. Chem. Educ. 1981, 58, 301. 7. Mebane, R. C.; Rybolt, T. R. J. Chem. Educ. 1985, 62, 285. 8. Curtright, R. D.; Rynearson, J. A.; Markwell, J. J. Chem. Educ. 1994, 71, 682. 9. Fossen, T.; Cabrita, L.; Andersen, O. Food Chem. 1998, 63, 435. 10. Gibson, J. F. Educ. Chem. 1997, 9, 123. 11. Larson, R. A. Phytochemistry 1988, 27, 969. 12. Couto, A. B.; Ramos, L. A.; Cavalheiro, E. T. G. Quim. Nova 1998, 21, 221. 13. Soares, M. H. F. B.; Antunes, P. A.; Cavalheiro, E. T. G. Quim. Nova 2001, 24, 408–411. 14. Soares, M. H. F .B.; Boldrin-Silva, M. V.; Cavalheiro, E. T. G. Eclet. Quim. 2001, 26, 225–234. 15. Soares, M. H. F. B.; Couto, A. B.; Ramos, L. A.; Cavalheiro, E. T. G. Book of Abstracts, European Conference on Analytical Chemistry, Euroanalysis XI, Sept 3–9, 2000; Portuguese Chemical Society: Lisbon, Portugal, 2001; p 284. 16. Harris, D. C. Quantitative Chemical Analysis, 4th ed.; Freeman: New York, 1996; p 442. 17. Harris, D. C.; Quantitative Chemical Analysis, 4th ed.; Freeman: New York, 1996; pp 236, 473. 18. Decnop-Weever, L. G.; Kraak, J. C. Anal. Chim. Acta 1997, 337, 125–131. 19. Sarudi, J.; Kelemen, J. Talanta 1998, 45, 1281–1284. 20. Berké, B.; Chèze, C.; Vercauteren, J.; Deffieux, G. Tetrahedron Lett. 1998, 39, 5771. 21. Szekeres, L. Talanta 1974, 21, 1. 22. Joslyn, M. A.; Braverman, J. B. S. Adv. Food Res. 1954, 5, pp 97–160. 23. Normas Analíticas do Instituto Adolfo Lutz, 3rd ed.; Governo do Estado de São Paulo: São Paulo, 1985; p 107. 24. Taylor, S. L.; Higley, N. A.; Bush, R. K. Adv. Food Res. 1986, 30, pp 1–76. 25. Brouillard, R.; Dubois, J. E. J. Am. Chem. Soc. 1977, 99, 1359. 26. Brouillard, R; Delaporte, B. J. Am. Chem. Soc. 1977, 99, 8461. 27. Brouillard, R.; Iacobucci, G. A.; Sweeny, J. G. J. Am. Chem. Soc. 1982, 104, 7585. 28. Selinger, B. Chemistry in the Marketplace, 5th ed.; Harcourt Brace: Sydney, Australia, 1997.
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