In the Laboratory
Adsorptive Stripping Voltammetry of Environmental Indicators: Determination of Zinc in Algae
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C. Collado-Sánchez,* J. J. Hernández-Brito, J. Pérez-Peña, Mª. E. Torres-Padrón, and Mª. D. Gelado-Caballero Departmento de Química, Facultad de Ciencias del Mar, Universidad de Las Palmas de Gran Canaria, P.O. Box 550, Las Palmas, Spain; *
[email protected] The standard analytical methods used in monitoring heavy metals in the environment involve different techniques such as UV, AAS, et cetera. However, over the last decade new electrochemical methods have been developed (1). These methods provide advantages such as simple sample manipulation and low cost. In addition, they are easily carried out by inexperienced staff. The increasing interest in developing electroanalytical methods to monitor trace metals in the environment is well documented. In recent years (2, 3), authors have compared adsorptive stripping voltammetry (ASV) with AAS (4–6) and ICP (7), endorsing electroanalytical methods. Stripping methods are the most sensitive as metals are preconcentrated on the electrode before determination. Accumulation is carried out either by metal deposition (anodic stripping) or by adsorption of the metal chelated on the electrode (adsorptive stripping). This article describes a method for sample preparation and for the determination of average zinc content in algae (Cystoseira abis-marina C. Agardh, fucales, cystoseiracea) using adsorptive stripping voltammetry. These techniques are fast, inexpensive, sensitive, and simple when compared to standard procedures. Important pedagogic advantages can be found in the determination of pollutants such as zinc in algae. It allows students to use several analytical tools such as clean sampling and handling of algae, quality control of reagent impurities, wet digestion using a microwave oven, metal analysis using adsorptive stripping voltammetry, and so forth. Determination of metals in biological matrices gives rise to concepts such as bioindicator species and biomarkers. Discussion of uses of bioindicators, their advantages and limitations, is of considerable value in theoretical classes on environmental monitoring. The experiment requires a basic knowledge of electrochemistry and analytical chemistry and is meant for pregraduate or graduate students in chemistry or environmental courses. This experiment has been used for the laboratory component of a postgraduate electroanalytical course. Experimental Protocol
Hazards Hazardous substances such as mercury and acids should be handled according to the recommendations of the safety data sheet (International Chemical Safety Cards). Mercury is toxic when inhaled. Therefore, students should deal with mercury with care and supervision in a fume hood. Perchloric, hydrochloric, and nitric acids are strong oxidizing agents and could cause severe burns. Safety goggles, chemical-resistant gloves, and laboratory coats should be worn to prepare and carry out the experiment. Digestion of solutions must be performed in a fume hood. The mercury used in the exwww.JCE.DivCHED.org
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periment must be collected and disposed of in an appropriate waste container for subsequent distillation. The waste container should be isolated from any source of heat or ignition. The containers of this material may be hazardous when empty since they retain product residues (vapors, liquid).
Reagents and Good Practice Conditions Good laboratory practice conditions must be available to obtain a high degree of accuracy and low detection limits, especially in the determination of trace metals. The procedure requires careful sample handling and storage, proper container cleanliness, and the use of pure reagents. Handling during sampling and analysis is the main source of contamination. Thus, Teflon voltammetric cells are recommended rather than glass cells. Acid-cleaned plastic materials like polypropylene, polyethylene, and Teflon are used when they will be in contact with samples (beakers, digesters, or tweezers). Acid cleaning can be carried out by successive rinsing with diluted nitric (0.3 M) and hydrochloric acid (0.1 M) solutions and washing before their use with water of analytic quality (Millipore Milli-Q system) (8). The reagents and standards used for the sample analysis should be of the highest purity, including the electrode mercury. Most of the quality requirements are met by commercial reagents. The acids used to digest the sample and prepare the reagents are obtained commercially under several brand names such as Suprapure (Merck) and Aristar (BDH). Reagents of higher purity can be obtained by isothermal distillation (9). Perchloric acid and mercury, which are used in the experiment, are especially hazardous and must be handled with care. Sample calibration is carried out by using standard additions. Standard solutions are prepared by diluting atomic absorption standards in acid media. Hydrochloric acid (BDH, Sigma) is used at concentrations lower than 10᎑3 M. Standard zinc solutions (1 × 10᎑6 M) were prepared daily. Accuracy in preparing standards solutions and procedures used to control the quality of the determinations (replications) are important matters, which should be stressed to students. Sample handling is carried out on a laminar flow bench wearing plastic gloves during the whole process. Procedural Summary
Preparation of Samples It is important to clean the algae to avoid interference from organisms living in the algae (epyphytus). Cleaning is done by hand, wearing plastic gloves, and using plastic covered tweezers. After the samples are washed with seawater to remove salts, they are dried to constant weight for 48 h at 64 ⬚C. Then, they are washed with deionized water (Milli-Q
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In the Laboratory
Electrochemical Analysis Analysis of metal concentrations was carried out by ASV using a polarographic system (EG&G 384B), a digital plotter RE0093, and a mercury drop electrode (EG&G, 303A) with a Ag兾AgCl reference and platinum counter electrode. This method preconcentrates zinc on the mercury electrode as a complex with PDC (1 × 10᎑4M) (pyrrolidinedithiocarbamate) (12) and measures the reduction current of the adsorbed metal when a negative potential ramp is applied (Table 1). Oxygen is removed by purging the solution with nitrogen for 5 minutes. Metal analysis is performed in a diluted solution to avoid drop saturation and low pH conditions.
List 1. Analysis of Zn in Algae 1. Collect plants 2. Dry the samples a. 48 h at 64 ⬚C b. 5–6 h at 105 ⬚C 3. Acid digestion: HNO3/HClO4 2:1 v/v 4. Irradiation with microwave oven, 90 min 5. Voltammetry analysis of Zn
Table 1. Electrochemical Parameters Parameters
Values
Purge time
5 min ᎑1.6 V
Deposition potential Deposition time
272
Initial potential
30 s ᎑1 V
Pulse height
25 x 10᎑6 V
Scan increment
5 x 10᎑6 V
Quiescence time
3s
Frequency
100 Hz
Scan speed
0.5 V/s
pH
7.4
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Peak Height Current / (10ⴚ8 A)
20
ⴚ8
10
Current / A
water) to remove any remaining salts. After they are dried at 105 ⬚C for 5–6 h, samples are ready for digestion. Several parts of the algae are selected to obtain a homogeneous mixture because metal concentrations may vary within the vegetal parts. Sample digestion is carried out in a microwave oven (CEM microwaves MDS-81D) using replicate aliquots of 0.25 g and a combination of nitric and perchloric acids with a 2:1 ratio (10–11). Digestion is performed using a nine-step program, with five steps of 5 min of irradiation at 100% power (630 W) and between them, four steps with 5 min of cooling. This sequence was repeated twice to ensure the complete destruction of the sample. The total time required to carry out the digestion is about 1.5 h. Finally, the residual acid solutions were diluted with 100 mL of deionized water in plastic volumetric flasks (List 1).
[Zn] = ⴚ (26.8 ⫾ 0.1) × 10ⴚ9 mol L 1
15
R 2 = 0.999
10
5
0 -1.4
-1.3
-1.2
-1.1
-1.0
Potential / V
0
10
20
30
40
50
60
[Zn] / (10ⴚ9 mol Lⴚ1)
70
Figure 1. Zinc determination: (left) sample zinc peak and (right) calibration by standard additions.
Thus, 400 µL of the acid sample solution is diluted to 10 mL (the capacity of the polarographic cell). The solution is buffered to 7.4 with a solution of 1 M BES [N,N-bis(2hydroxyethyl)-2-aminoethanesulfonic acid]兾0.5 M NH4OH and 40 µL of NH4OH (25%) to set pH quickly. Each sample is replicated and calibrated by standard additions (Figure 1) and one blank is analyzed by each student. Results are reported as µg of Zn per gram of dry weight algae (µg兾g). Data Analysis and Results Calibration is performed with standard additions. This procedure allows students to determine parameters, such as the relative standard deviation for each algae sample, based on analyses on the same sample used by different groups of students. Data quality depends on student performance in following the method, especially the spike accuracy for small volumes (less than 100 µL). Deviations among results from several students allow discussions about error sources and about whether the procedure has been followed correctly. Experimental study of annual variation of metal concentrations in the algae is suggested. Students should explain the measured metal concentrations taking into account the natural cycle of algae and metal pollution variability in the sampling area. They should also suggest possible experiments to confirm their hypothesis. The most important benefit is that students will appreciate the opportunity to understand reallife problems. They will find that the principles learned in the classroom are essential when undertaking environmental monitoring experiments.
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In the Laboratory
Conclusions
Literature Cited
A series of important didactic advantages is gained through metal determination in environmental matrices. Students learn to:
1. Electroanalytical Methods in Chemical and Environmental Analysis; Kalvoda, R., Ed.; Plenum Press: New York, 1987. 2. van den Berg, C. M. G. Anal. Chim. Acta 1991, 250, 265. 3. Wang, J. Fresenius J. Anal. Chem. 1990, 337, 506. 4. Golimowski, J.; Valenta, P.; Nürnberg H. W. Z. Lebensmittelunters. Frosch. 1979, 168, 353. 5. Sulek, A. M.; Elkins, E.; Zink, E. W. J. Ass. Offic. Anal. Chem. 1978, 61, 931. 6. Nürnberg, H. W. Sci. Total Envir. 1979, 12, 35. 7. Nimmo, M.; Fones, G. Anal. Chim. Acta 1994, 291, 321– 328. 8. Wang, J. Stripping Analysis: Principles, Instrumentation and Applications; VCH: Deerfield, FL, 1985; p 91. 9. Mass, R. P.; Dressing, S. A. Anal. Chem. 1983, 55, 808. 10. Navarro, M.; Lopez, M. C.; Lopez, H.; Sanchez, M. Anal. Chim. Acta, 1992, 257, 155–158. 11. Munda, I.; Hudnik, V. Marine Ecology 1988, 9, 213–225. 12. van den Berg, C. M. G. Talanta 1984, 31, 1069–1073.
• Carry out clean protocols for sampling and handling • Digest samples using advanced systems • Measure metals using electrochemical techniques of high accuracy and sensitivity • Explain environmental variability of annual distribution of metals in algae in the sampling area
Finally, discussion of the results could be used to emphasize the role of algae as coastal indicators of metal pollution and the advantage of this method when compared to the metal analysis in the seawater. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online.
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