In the Laboratory
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Determination of Hypochlorite in Bleaching Products with Flower Extracts To Demonstrate the Principles of Flow Injection Analysis Luiz Antônio Ramos, Kátia Roberta Prieto, Éder Tadeu Gomes Cavalheiro,* and Carla Cristina Schmitt Cavalheiro Instituto de Química de São Carlos, Departamento de Química e Física Molecular Universidade de São Paulo, Av. Trabalhador São Carlense, 400 CEP 13566-950 São Carlos, SP Brazil; *
[email protected] Use of commonly available materials to demonstrate a concept is recognized as a method to gain students’ attention. An interesting material that can be used is the color of flowers. The crude extracts of flowers contain anthocyanins that were described by Timberlake and Bridle (1) as flavonoids derived from water-soluble flavilium salts. These anthocyanins are responsible for the red and blue colors of many vegetable tissues such as flower petals, fruit skins, plant stems, leaves, and so forth (2). The first application of the color changes of these extracts as a function of pH was presented by Geissman (3). Since then several articles in chemical education (4–11) have been based on crude extracts of vegetable tissues. We have used these color changes to demonstrate chemical equilibrium (12, 13), as indicators in acid–base titrations (14), in paper chromatography (15), and demonstrating the fundamentals of optical analytical methods (16, 17) by exploiting the low cost and easy preparation of the extracts. In this article we extend the use of these extracts to principles of analytical chemistry automation, with a flow injection analysis (FIA) procedure developed to determine hypochlorite in household bleaching products. The reaction is based on the discoloration of the crude flower extract in acidic medium. FIA comprises a group of techniques based on injection of a liquid sample into a moving, nonsegmented carrier stream of a suitable fluid. The injected sample forms a zone, which is then transported to a detector that records a desired physical parameter as it changes owing to the passage of the sample material through the flow cell (18). The FIA concept was originally developed by Ruzicka and Hansen (19) and received an important contribution of the Brazilian Group of Analytical Chemistry from CENA-USP (20, 21). FIA is a well-established, flow-based technology that has, during the last 25 years, brought speed, automation of solution handling, miniaturization, and low cost to the analytical laboratory. The literature on FIA, comprising close to 13,000 references, covers assays and applications from such diverse fields as pharmaceutical assays, environmental studies, oceanography, process control, agriculture, drug discovery, and clinical assays. FIA has also been used as a “front end”, or solution handling system, for an entire range of spectroscopic and electrochemical instruments since it offers sample pretreatment, matrix removal, and automated recalibration of detectors. Many articles have been presented concerning the use of FIA procedures in chemical education (22–39). Two interesting reviews about the development of the flow injection methods have been presented by Reis and co-workers (21, 40). www.JCE.DivCHED.org
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Theory The bleaching reaction involving the anthocyanins and the hypochlorite is not fully established. Several authors have studied the reaction of anthocyanins with oxidizing agents as presented by Iacobussi and Sweeny (41). In the case of a hydrogen peroxide oxidizing agent these authors agreed with Jurd (42) that the reaction below is the likely course of the oxidative process. + O
H 2 O2
OH colored
HO
OH H
OH
H
O
discolored
In this case the flavilium cation undergoes a cleavage in the center ring. For the oxidation by hypochlorite the reaction can be simply represented by − anthocyanin + ClO
discolored product
The flow injection determination of hypochlorite involves its mixture with the colored extract in acidic medium resulting in a diminution of the initial color, which is proportional to the ClO− concentration. Experimental
Preparation of the Flower Extract Depending on the time available, the students can prepare the crude extracts prior to the experiment or the instructor can prepare them. The extract can be prepared with fresh or frozen petals. Freezing the extract for an extended time is not recommended. To prepare the stock solution of the flower extract, immerse 25 g of petals (fresh or frozen) of Rhododendron simsii (azalea) in 100 mL of ethanol for 48 h at room temperature and then filter (3, 17). Alternatively any other species containing anthocyanins can be employed, in this work we also used Tibouchina granulosa (quaresmeira) as an alternative source of extract. After filtration the solvent is eliminated in a rotatory evaporator under vacuum at 40 ⬚C until no more solvent is distilled and a viscous liquid is obtained.
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Absorption Spectra To obtain the absorption spectra use 100 mg of the crude extract diluted with universal buffer pH 2.0 (43) to a final volume of 25.0 mL in a volumetric flask. From the spectra (absorbance versus wavelength), determine the wavelength of maximum absorption (λmax). Alternatively, the absorbance at λmax of solutions containing different quantities of crude extract can be measured and checked for adherence to the Beer– Lambert law. Preparation of the Carrier Solution The suggested carrier solution is the universal buffer prepared according to Perrin and Dempsey (43), containing 2.85 mM sodium tetraborate, 10 mM disodium hydrogen phosphate, and 6.7 mM trisodium citrate adjusted to pH 2.0 with hydrochloric acid. A pH of 2.0 was chosen after optimizing this parameter for the best sensitivity. Apparatus In this example, both static and flow measurements were carried out in a Genesys 20 spectrophotometer (Thermo Electron), using plastic cuvettes with optical pathlength of 1.0 cm. A homemade glass flow cell with 1.0-cm optical pathlength was used in the flow measurements. A 12-channel model 7618-50 Ismatec peristaltic pump was used for the introduction of the solutions. The FIA manifold was mounted with polyethylene tubing (0.8-mm i.d.). All the reference and sample solutions were injected manually into the carrier stream using a laboratory-constructed, three-piece injector-commutator made of Perspex (20, 44), with two fixed side bars and a sliding central bar that is moved for sampling and injection. The spectrophotometer used was interfaced with a microcomputer using software developed in the laboratory. Alternatively the data can be recorded with a two-channel, strip-chart recorder. The configuration of the merging zones manifold used is represented in Figure 1. In this system the carrier stream drives both sample and reactant to mix in the confluence, and the reaction occurs inside the coil. After mixing the reaction products are carried to the detector, and the absor-
inject position
sample position
bance is monitored at the λmax of the crude extract. The results are stored on the microcomputer or registered on the strip-chart recorder.
Comparative Method and Standardization of the Solutions The 5% sodium hypochlorite solution (Baker) was prepared and standardized iodometrically. Take 5.0 mL of this standard solution and dilute to 100 mL in a volumetric flask. To a 15.00-mL aliquot of the resulting solution add 5.0 mL of 5% KI, 5.0 mL of glacial acetic acid, and 50 mL of water (45). The iodine liberated is then titrated with previously standardized 0.100 mol L᎑1 sodium thiosulfate using starch as indicator (46). Commercial household products containing labeled hypochlorite concentrations of 2.5–3.0% can be purchased in local stores and their accurate content can be determined by a similar procedure by an initial dilution of 20.0 mL of the sample to 100 mL in a volumetric flask. Compare the results from the classical procedure with those from the flow injection procedure. The flow injection procedure was based on to the following parameters: 50-cm (250-µL) crude extract loop length, 25-cm (125-µL) sample loop length, 8 g L᎑1 crude extract solution, 400-cm tubular reaction coil, spectrophotometric detector wavelength at the λmax for the crude extracts (526 nm for azalea and 548 nm for quaresmeira). Hazards Ingestion of sodium hypochlorite may cause corrosion of mucous membranes, esophageal or gastric perforation and laryngeal edema. Inhalation may produce severe bronchial irritation and pulmonary edema. Iodine is toxic by either ingestion or inhalation. It is also a strong irritant to eyes and skin and could cause allergic reaction. Ingestion of ethanol can cause nausea, vomiting, flushing, mental excitement or depression, drowsiness, impaired perception, incoordination, stupor and coma. Ingestion of glacial acetic acid may cause severe corrosion of the mouth and gastrointestinal tract, vomiting, hematemesis, diarrhea, circulatory collapse and uremia. Hydrochloric acid is corrosive, burns may result from the inhalation of the fumes and from skin contact with or ingestion of strong acid. (Information derived from ref 47.) Preliminary Studies To Optimize the System
L1 W
CE R
C
B1
W C
D
S
W
L2
Figure 1. FIA diagram of the merging zones system: C the carrier, L1 the crude extract loop, L2 the sample loop, CE crude extract solution, S the hypochlorite solution samples, B1 the tubular reaction coil, D the spectrophotometric detector, R the recording device (microcomputer or analog recorder), W the waste.
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Before the optimization of the experimental parameters the absorption spectra were obtained for the crude extracts at pH 2.0. The resulting spectra are presented in Figure 2, with the maximum absorption at 526 nm for azalea and 548 nm for quaresmeira.
Flow Injection Parameters To obtain the best performance of the FIA system in relation to sensitivity, frequency of repeated analyses, economy of reagents, and so forth, some parameters should be optimized. In the present case a merging zones FIA configuration is used and involves a confluence and a reaction coil as presented in Figure 1. The sample (hypochlorite) and reagent (crude flower extract solution) loop sizes, which represent the
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In the Laboratory A 0.8
A
Absorbance
0.8
0.8
λmax ⴝ 526 nm
Absorbance
0.6
Absorbance
0.6
0.4
0.6 0.4 0.2
0.4
0
20
40
60
80
100
Loop / cm 0.2
0.2
0.0
0.0 400
450
500
550
600
650
700
0
20
Wavelength / nm
40
60
80
Time / s B 0.30
B
0.25
λmax ⴝ 548 nm
Absorbance
1.0
Absorbance
Absorbance
0.28
1.2
0.8 0.6
0.20
0.26 0.24 0.22 0.20 0.18 0.16 0.14
0.15
10
20
30
40
50
Loop / cm
0.10
0.4 0.05 0.2 0.00
0.0 400
450
500
550
600
650
700
0
20
Wavelength / nm
40
60
80
Time / s
Figure 2. Absorption spectra of the 3.0 mL of the 4.0 g L᎑1 crude extract solution of (A) azalea and of (B) quaresmeira in 10.0 mL of universal buffer pH 2.0.
Figure 3. (A) Effect of the crude extract loop length on the transient signal (conditions described in text) and (B) effect of the sample loop length on the transient signal resulting from the bleaching reaction between the crude anthocyanin extracts and the ClO− anion.
respective injected volumes, the length of the reaction coil, and the flow rate should be optimized before performing the analytical determinations. The best configuration is achieved when the maximum discoloring of the crude extract is observed. The use of pH 2.0 buffer was determined to give optimum results in a previous work (48).
the crude extract (8 g L᎑1) at a fixed flow rate of 2.9 mL min᎑1, with 50 µL of universal pH 2.0 buffer in the sample loop, and a 200-cm reaction coil. Increasing the loop length increases the absorbance, tending towards a constant value at 100-cm (500-µL) loop length. A 50-cm (250-µL) reagent loop was chosen to economize on reagent use and prevent peak spreading. The effect of the sample loop length is shown in Figure 3B with a fixed flow rate of a 2.9 mL min᎑1, 250 µL of crude extract, and a 200-cm reaction coil. Increasing the hypochlorite loop length decreased the absorbance until a constant value was reached at the 30-cm (150-µL) sample loop length. To minimize sample consumption, a 25-cm (125-µL) sample loop was chosen.
Sample and Reagent Loop Sizes The sample and reagent loops define their injected volumes according to the cylinder volume, V = π r 2l where V is the sample or reagent volume, r is the radius of the tube used to prepare the loop, and l is the loop length. If r and l are measured in centimeters the volume will be determined in cm3 and can be converted to mL or other convenient units. The effect of loop length on the flow injection absorbance plot, for loop sizes ranging from 50 to 500 µL, is shown in Figure 3. Figure 3A represents the effect of loop length of www.JCE.DivCHED.org
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Effect of Flow Rate and Length of the Reaction Coil The effect of the flow rate in the peak height was studied in the range of 1.5–6.9 mL min᎑1. The peak height reached a maximum value at 4.2 mL min᎑1 and then decreased with further increase of the flow rate. The decrease is caused by the short reaction time caused by the rapid flow rate. Tak-
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ing into consideration the stability of the pump, peak shape, and sampling time, the flow rate of the reagent carrier solution was adjusted to 4.2 mL min᎑1. A sample throughput of 80 sample h᎑1 was achieved by using that flow rate. Once the parameters described above were fixed, optimization of reaction coil length was studied using lengths between 100 and 400 cm. The longer coil lengths increase the degree of dispersion but shorter lengths do not allow for completion of the reaction. The optimum length with both acceptable dispersion and sufficient reaction resident time was found in the 400-cm reaction cell.
A 0.36 0.32
Absorbance
0.28 0.24 0.20 0.16 0.12 0.08 0.04 0.00 0
500
1000
1500
2000
2500
3000
3500
Time / s
Analytical curves were obtained for the determination of hypochlorite using both azalea and quaresmeira crude extracts. The FIA system represented in Figure 1 was used under the optimized conditions previously described: 8 g L᎑1 crude extract concentration, 50-cm (250-µL) crude extract loop, 25-cm (125-µL) hypochlorite loop in the concentration range 1.45–8.60 × 10᎑3 mol L᎑1, 400-cm reaction coil, and a flow rate of 4.2 mL min᎑1. The flow injection responses obtained under such conditions with azalea and quaresmeira are shown in Figures 4A and 4B, respectively. The resulting calibration curves followed the equations:
B 0.24
Absorbance
0.20 0.16 0.12 0.08 0.04 0.00 0
500
1000
1500
2000
2500
3000
3500
Time / s
Absorbance
Figure 4. Transient signals, obtained in triplet, when different ClO− concentrations are injected in the FIA system under the optimzed conditions described in the text using (A) azalea and (B) quaresmeira crude extracts. From left to right the hypochlorite concentrations are 8.56, 7.14, 5.71, 4.28, 2.85, 1.43, 2.85, 4.28, 5.71, 7.14, and 8.56 x 10᎑3 mol L᎑1.
0.5 crude extract
Absorbance
0.4
P1
0.42 0.36 0.30 0.24 0.18 0.12 0.06 0.0
4.0
8.0
ⴚ3
[Hypochlorite] / (10
P2
12
mol / L)
0.3 P3 S2
S1 P4
0.2
Results and Discussion
S3 P5
A = 0.3770 − 23.79 [ ClO− ] R = 0.9996, n = 6
(azalea)
(4)
A = 0.2264 − 14.30 [ClO−] R = 0.9995, n = 6
(quaresmeira)
(5)
The results suggest that any anthocyanin-containing flowers can be used in such determinations. The negative sign in the slope of the analytical curves represents the decrease in the absorbance caused by the discoloring reaction. The correlation coefficients show that the Beer–Lambert law is obeyed in this concentration range and that the parameters of the flow system are well adjusted. A limit of detection of 1.5 × 10᎑4 and 1.4 × 10᎑4 mol L᎑1 (3 × SDblank兾slope) (49) was calculated for the azalea and quaresmeira curves, respectively. Three commercial household bleaching samples containing hypochlorite were analyzed in relation to the ClO− content, using the more sensitive azalea crude extract. The flow injection diagrams for both the standard set and the samples is presented in Figure 5. The data for the analytical curve are detailed in the figure caption. The results obtained in the
0.1
Table 1. Results for the Determination of ClO− in Commercial Household Bleaching Products
0.0 0
500
1000
1500
2000
2500
3000
Time / s Figure 5. Transient signals observed for the standards in the analytical curve and the three samples, injected in triplicate into the FIA system. Standards were: 0 (crude extract), 1.52 (P1), 3.04 (P2), 6.07 (P3), 9.11 (P4) and 12.1 (P5) x 10᎑3 mol L᎑1 hypochlorite solutions and S1, S2, and S3 were the commercial household product samples. Inset shows a plot of the hypochlorite standards.
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Rel Errorb (%)
Sample
Label (%)
Iodometrica
FIAa
1
2.5–3.0
2.65 ± 0.01
2.66 ± 0.02
᎑0.38
2
2.5–3.0
2.43 ± 0.00
2.53 ± 0.04
᎑4.1
3
2.5–3.0
2.92 ± 0.04
3.01 ± 0.02
᎑3.0
a
n = 3.
b
[(iodometric − FIA)/iodometric] x 100.
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In the Laboratory
sample determination in comparison with the classical iodometric determination of the hypochlorite (46) are summarized in Table 1. The t-test applied to the two sets of results showed that the iodometric and flow injection procedures agree at the 95% confidence level. Acknowledgment The authors acknowledge financial support from FAPESP (Process 00/14486-2 and 04/00407-4). W
Supplemental Material
Notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Timberlake, C. F.; Bridle, P. The Flavonoids–Part I; Harborne, J. B., Mabry, T. J., Mabry, H., Eds.; Academic Press: New York, 1975; pp 215–224. 2. Larson, R. A. Phytochemistry 1988, 27, 969–978. 3. Geissman, T. A. J. Chem. Educ. 1941, 18, 108–110. 4. Geissman, T. A.; Crout, D. H. G. Organic Chemistry of Secondary Plant Metabolism; Freeman, Cooper & Cia: San Francisco, 1969; pp 182–209. 5. Forster, M. J. Chem. Educ. 1978, 55, 107–108. 6. Sequinfrey, M. J. Chem. Educ. 1981, 58, 301–305. 7. Mebane, R. C.; Rybolt, T. R. J. Chem. Educ. 1985, 62, 285– 285. 8. Curtright, R. D.; Rynearson, J. A.; Markwell, J. J. Chem. Educ. 1994, 71, 682–684. 9. Fossen, T.; Cabrita, L.; Andersen, O. Food Chem. 1998, 63, 435–440. 10. Gibson, J. F. Educ. Chem. 1997, 9, 123–125. 11. Terci, D. B. L.; Rossi, A. V. Quim. Nova. 2002, 25, 684–688. 12. Couto, A. B.; Ramos, L. A.; Cavalheiro, E. T. G. Quim. Nova. 1998, 21, 221–227. 13. Soares, M. H. F. B.; Boldrin-Silva, M. V.; Cavalheiro, E. T. G. Eclet. Quim. 2001, 26, 225–234. 14. Soares, M. H. F. B.; Antunes, P. A.; Cavalheiro, E. T. G. Quim. Nova. 2001, 24, 408–411. 15. Soares, M. H. F. B.; Couto, A. B.; Ramos, L. A.; Cavalheiro, E. T. G. Conference on Analytical Chemistry. Book of Abstracts, XI Euroanalysis; Lisbon, 2000; P-284. 16. Okumura, F.; Soares, M. H. F. B.; Cavalheiro, E. T. G. Quim. Nova. 2002, 25, 680–683. 17. Soares, M. H. F. B.; Ramos, L. A.; Cavalheiro, E. T. G. J. Chem. Educ. 2002, 79, 1111–1113. 18. Ruzicka, J. Flow Injection, 2nd ed.; FIAlab Instruments, Inc. Leaders in Flow Injection Technology. 2002. CD-ROM. 19. Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta. 1975, 78, 145– 157. 20. Ruzicka, J.; Stewart, J. W. B.; Zagatto, E. A. G. Anal. Chim. Acta. 1976, 81, 387–396.
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21. Reis, B. F.; Gine, M. F.; Kronka, A. M. Quim. Nova. 1989, 12, 82–91. 22. Hansen, E. H.; Ruzicka, J. J. Chem. Educ. 1979, 56, 677– 680. 23. Ruzicka, J.; Hansen, E. H.; Hansing. A. U. Anal. Chim. Acta. 1982, 134, 55–71. 24. Betteridge, D. Freseniu’s Z. Anal. Chem. 1982, 312, 441–442. 25. Meyerhoff, M. E.; Kovach, P. M. J. Chem. Educ. 1983, 60, 766–768. 26. Rios, A.; Decastro, M. D. L.; Valcarcél, M. J. Chem. Educ. 1986, 63, 552–553. 27. Keller, J. W.; Gould, T. F.; Aubert, K. T. J. Chem. Educ. 1986, 63, 709–711. 28. Stults, C. L. M.; Wade, A. P.; Crouch, S. R. J. Chem. Educ. 1988, 65, 645–647. 29. McKelie, I. D.; Cardwell, J. D.; Cattrall, R. W. J. Chem. Educ. 1990, 67, 262–263. 30. Nóbrega, J. A.; Mozeto, A. A.; Alberici, R. M. J. Chem. Educ. 1991, 68, 966–968. 31. Davis, L. C. J. Chem. Educ. 1993, 70, 511–513. 32. Carroll, M. K.; Tyson, J. F. J. Chem. Educ. 1993, 70, A210– A210. 33. Echols, R. T.; Tyson, J. F. Analyst. 1995, 120, 1175–1179. 34. Hauser, P. C.; Cates. N. E. J. Chem. Educ. 1996, 73, 191– 192. 35. Nóbrega, J. A.; Rocha, F. R. P. J. Chem. Educ. 1997, 74, 560– 562. 36. Wolfe, C. A. C.; Oates, M. R.; Hage. D. S. J. Chem. Educ. 1998, 75, 1025–1028. 37. Elsholz, O. Anal. Chim. Acta. 1998, 366, 217–221. 38. Sartini, R. P; Zagatto, E. A. G.; Oliveira, C. C. J. Chem. Educ. 2000, 77, 735–737. 39. Rocha, F. R. P.; Martelli, P. B.; dos Reis, B. F. Quim. Nova. 2000, 23, 119–125. 40. Reis, B. F. Quim. Nova. 1996, 19, 51–58. 41. Iacobucci, G. A.; Sweeny, J. G. Tetrahedron 1983, 39, 3005– 3038. 42. Jurd, L. Tetrahedron 1968, 24, 4449–4449. 43. Perrin D. D.; Dempsey, B. Buffers for pH and Metal Ion Control; Chapman and Hall: London, 1974. 44. Bergamin, H.; Medeiros, J. X.; Reis, B. F.; Zagatto, E. A. G. Anal. Chim. Acta, 1978, 101, 9–16. 45. Determinaçao de Hipoclorito em Agua Sanitaria. http:// www.chemkeys.com/bra/md/eddns_2/detiod_1/ddheas_2/ ddheas_2.htm (accessed Sep 2005). 46. Christian, G. D. Analytical Chemistry, 5th ed.; Wiley & Sons: New York, 1994. 47. Budavari, S.; O’Neil, M. J.; Smith, A.; Heckelman, P. E.; Kinneary, J. F. The Merck Index, 12th ed.; Merck & Company Incorporated: Whitehouse Station, NJ, 1996. 48. Schmitt, C. C.; Ramos, L. A.; Moraes, F. C.; Cavalheiro, E. T. G. Conference on Analytical Chemistry. Book of Abstracts, XII Euroanalysis, Germany, 2002; p 210. 49. Miller, J. C.; Miller, J. N. Estadistica para Química Analítica; Addison-Wesley: Wilmington, DE, 1993.
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