In the Laboratory
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Analysis of Selenium in Brazil Nuts by Microwave Digestion and Fluorescence Detection Marie-Christine Sheffield Department of Geosciences, California State University, Chico, CA 95929 Tal M. Nahir* Department of Chemistry, California State University, Chico, CA 95929; *
[email protected] Selenium (Se) is a relatively rare element, but essential to life (1). The need for selenium is explained, in part, by its incorporation into selenoproteins (2), whose role is to break down potentially damaging peroxides in cell membranes. In addition, selenium is needed for the proper functioning of the thyroid gland. Brazil nuts, as well as kidney, liver, and wheat grown in North America, are good sources of selenium (3). Excessive anthropogenic emissions of seleno-compounds into the atmosphere and the aquatic ecosystem (4) have resulted in the classification of this element as an environmental pollutant. A main concern is the bioaccumulation of selenium as a result of overexposure to sources in the environment. Several instrumental methods have been considered for the determination of selenium in environmental samples (5). The following procedure for the analysis of selenium incorporates into a single experiment several important analytical techniques including microwave digestion (6, 7), fluorescence measurements of cation concentrations after treatment with complexing agents (8, 9), and a modified standard addition method (10). Readily available seleniumrich Brazil nuts are used here to generate a strong fluorometric response. Upper-level undergraduate students have been able to complete the procedure in a three-hour laboratory period. Experimental Procedure
Digestion and Derivatization A small quantity, 0.1 g of Brazil nuts, and 2.5 mL of concentrated nitric acid were placed in a Teflon cup, which was capped and placed inside the bomb assembly (Parr Microwave Digestion Bomb, Model 4781). Using a 700 W household microwave oven, the bomb was irradiated for 25 seconds at 70% power. Increasing the microwave settings beyond 30 seconds at 70% power made the bomb very difficult to open and increased the possibility of unexpected venting. Shorter runs and lower power levels often resulted in incomplete digestion. After allowing the bomb to cool for 30 min (an ice bath may be used to accelerate the cooling process), the whole solution was transferred from the Teflon cup into a 100-mL beaker. To reduce any Se(VI) in the sample to Se(IV), 4 mL of 6 M HCl was added, and the solution was brought to a gentle boil on a hot plate for five min After removing the solution from the hot plate, 10 mL of a 2.5% hydroxylammonium chloride/0.1% EDTA solution, and 20 mL of nanopure water were added to the beaker. The pH of the solution was adjusted to approximately two by adding concentrated aqueous ammonia solution (and 0.1 M HCl, if necessary). The derivatizing agent 2,3-diaminonaphthalene, 5 mL, was added to the selenite containing solution and the
resulting solution was placed in a water bath at 50 ⬚C for 30 min. The solution was then transferred to a 125-mL Erlenmeyer flask, nanopure water was added to a total volume of approximately 70 mL, and 10.0 mL of cyclohexane was pipetted to extract the piazselenol. After shaking the flask several times, 1.00 mL of the organic layer was diluted to 10.0 mL with cyclohexane. This solution, 2.00 mL, was placed in a 1-cm square cuvette for analysis by fluorescence spectrophotometry. All glassware was thoroughly rinsed with concentrated nitric acid to eliminate contamination. The net reaction is shown below: NH2
N Se
+ Se(IV) NH2
N
Reagents-Solutions The derivatization of selenite with 2,3diaminonaphthalene (DAN) (Spectrum, Gardena, CA) to form a piazselenol was carried out following a procedure suggested earlier (11). The derivatizing agent, 0.1% DAN was prepared by weighing approximately 25 mg DAN into a 125mL Erlenmeyer flask and adding 25 mL 0.1 M HCl. The flask was capped and shaken vigorously until a fairly homogeneous, cloudy, yellow-brown solution formed (~5 min). Cyclohexane, 10 mL, was added and the solution was again shaken for one min. The aqueous layer (bottom layer) was removed with a Pasteur pipet into a clean beaker, and the organic layer was poured into a designated waste bottle. The Erlenmeyer flask was then rinsed with water. The aqueous solution was transferred from the beaker back into the Erlenmeyer flask, another 10 mL cyclohexane was added, the flask was shaken, and again the aqueous layer was removed. The aqueous solution was transferred through a Whatman student-grade filter paper in a glass funnel into a 1-oz. amber Qorpak bottle. The collected solution needed to be clear and colorless. Fluorometric Analysis A three-dimensional (3D) scan was performed using a Hitachi F-4500. The scan speed was set at 240 nm/min, the excitation range between 350 and 400 nm, the emission range between 500 and 550 nm, and the sampling interval at 1 nm. Both the excitation and emission slits were set to 2.5 nm, the PMT voltage at 700 V, the response to auto, and the shutter control option was selected. A standard solution was prepared by treating 1.00 mL of stock 14.0 ppm Se(IV) (from dissolving SeO2 in water) with DAN using the same derivatization procedure as
JChemEd.chem.wisc.edu • Vol. 79 No. 11 November 2002 • Journal of Chemical Education
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In the Laboratory
For safety reasons, gloves and safety goggles were worn at all times. All work was performed under a vacuum hood. Concentrated acids and bases should be handled with care. If excessive pressure builds up, the bomb can vent unexpectedly (12). Results and Discussion At low concentrations the fluorescence intensity increases linearly, but at high concentrations the fluorescence intensity decreases due to self-absorption (13). The nonlinear concentration versus fluorescence relationship for the fluorometric intensity of selenium in the Brazil nut was evident at selenium concentrations greater than 0.10 ppm (Figure 1). Diluting the sample allows for measurements in the linear range. A contour plot depicting the fluorometric response from a Brazil nut extract is shown in Figure 2. Isobars indicate an increase in fluorometric response with a Se signature peak at 378 nm (excitation) and 518 nm (emission). The concentration of the Se–DAN complex in the cyclohexane solution was determined by a modified standardaddition method (10), where the corrected fluorescence signal (Fluorescence intensity × V兾V0; where V0 is the initial volume of the unknown and V is the total volume) is plotted as the ordinate on the graph. A standard addition plot is shown in Figure 3. The selenium concentration in Brazil nuts varies greatly. The calculated value of 36.3 ± 0.6 ppm is within the reported range of 0.20 to 253 ppm (14). W
Supplemental Material
Fluorescence Intensity
Hazards
600 500 400 300 200 100 0 0
1
2
3
4
5
6
[Se(IV)] (ppm) Figure 1. Concentration vs fluorescence relationship for derivatized Se(IV) solutions. The fluorescence intensity decreases at higher concentrations due to self-absorption. The line is drawn through the data points to guide the eye.
540 535
Emission / nm
described above. Four sequential aliquots of 10.0 µL of the 1.40 ppm Se–DAN standard (tenfold dilution of initial 1.00 mL 14.0 ppm aqueous solution into 10.0 mL of cyclohexane) were added to the cuvette using a micropipet. A 3D scan was obtained and the maximum fluorescence response was recorded initially and after each of the standard additions.
530 525 520 515
40 35 30
20 15 10
510
25
5
505 350
355
360
365
370
375
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Excitation / nm Figure 2. Contour graph depicting the fluorometric response from a Brazil nut extract. The maximum intensity is shown at ex = 378 nm and em = 518 nm.
Acknowledgments Support for this work was provided through a CELT grant and from the Departments of Biology, Chemistry, and Geosciences at the California State University, Chico. The experiment was developed, in part, for incorporation into the integrated-laboratory sequence at the Department of Chemistry. We give a special thank you to Sean Breed and Michael Hurtado for their assistance in the laboratory. Literature Cited 1. 2. 3. 4. 5.
Sarquis, M.; Mickey, C. D. J. Chem. Educ. 1980, 57, 886–889. Schrauzer, G. N. J. Nutr. 2000, 130, 1653–1656. Rayman, M. Lancet 2000, 356, 233–241. Conde, J. E.; Alaejos, M. S. Chem. Rev. 1997, 97, 1979–2003. Haygarth, P. M.; Rowland, A. P.; Stürup, S.; Jones, K. C.
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Fluorescence Intensity × V/Vo
Instructions for the students and notes for the instructor are available in this issue of JCE Online. 80
y = 1084 (±15)x + 41.5(± 0.3)
70 60 50 40 30
[Se] = 0.0383 (±0.0006) ppm
20 10 0 -0.04
-0.03
-0.02
-0.01
0.00
0.01
0.02
0.03
Vs /Vo × [Se(IV)std] (ppm) Figure 3. Standard addition plot for a Brazil nut sample (0.1053 g). V0 is the volume of the unknown solution; Vs is the added volume of the standard solution; V = V0 + Vs and [Se(IV)std] is the concentration of the standard solution, 1.40 ppm here.
Journal of Chemical Education • Vol. 79 No. 11 November 2002 • JChemEd.chem.wisc.edu
In the Laboratory Analyst 1993, 118, 1303–1308. 6. Freeman, R. G.; McCurdy, D. L. J. Chem. Educ. 1998, 75, 1033–1034. 7. Goltz, D. M.; Hall, T.; Grant, A.; Segstro, E. J. Chem. Educ. 2000, 77, 1486–1488. 8. Haworth, D. T.; Starshak, R. J.; Surak, J. G. J. Chem. Educ. 1964, 41, 436–437. 9. White, C. E. J. Chem. Educ. 1951, 28, 369–372. 10. Harris, D. C. Exploring Chemical Analysis, 2nd ed.; W. H.
Freeman and Company: New York, 2001, p 354–355. 11. Whetter, P. A.; Ullrey, D. E. J. Assoc. Off. Anal. Chem. 1978, 61, 927–930. 12. Parr Instrument Company; Parr Operating Instructions for Parr Microwave Acid Digestion Bombs; No. 243M; Parr Instrument Company: Moline, Illinois, 1997, p 1–8. 13. Henderson, G. J. Chem. Educ. 1977, 54, 57–59. 14. Chang, J. C.; Gutenmann, W. H.; Reid, C. M.; Lisk, D. J. Chemosphere 1995, 30, 801–802.
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