In the Laboratory
Environmental Redox Potential and Redox Capacity Concepts Using a Simple Polarographic Experiment
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Alejandro Pidello Laboratory of Biological Chemistry, FCV, CIUNR, National University of Rosario, Bv. O. Lagos y ruta 33, 2170 Casilda, Argentina;
[email protected] The redox status of a system is the result of reactions that occur within the system and that involve the transfer of electrons. From a thermodynamic point of view, these reactions perform work. The work may be characterized by two components: the capacity component, dependent on the quantity of the matter and associated with mass, and the intensity (or potential) component, dependent on its position relative to a standard and not dependent on mass (1). The latter component determines the direction in which the capacity factor tends to evolve. In other words, the components that make possible the redox status characterization may belong to any of two types: dependent on, or independent of, the dimensions of the system. In fact, when two equal systems combine to form only one system, the amount of redox species (redox capacity component) is doubled while the redox potential (intensity component) remains constant. If the system is in equilibrium, both components are related by the Nernst expression (2). Consequently, either the redox potential (E ) or the redox capacity may be used without distinction to define the redox status of these systems (3–5). However, natural environments are typically in a state of redox disequilibria, since they generally have a considerable diversity of electroactive compounds undergoing redox reactions in several stages of nonequilibrium (6–9). The redox potential measured in these internal disequilibria conditions reflects mixed potentials (Emix) which results from the particular behaviors of the different redox pairs. In this situation, it becomes evident that the concentration of the oxidizing and reducing forms of each redox pair cannot be accurately defined by the measured potential (8, 10, 11). Under these conditions, a variation in oxidation or reduction capacity (12) would not be clearly detected by the redox potential measurements. As suggested by various authors, the complementary use of redox potential and redox capacity measurements improves the characterization of the redox status of these systems (6– 9). This situation makes it difficult to plan an undergraduate laboratory activity in applied biology (agronomy, veterinary, or environmental sciences) because students have little theoretical chemistry background and still need to understand the role of the redox status (redox potential and redox capacity) in biochemistry and microbial ecology of complex environments (such as soils, sediments, or ruminal fluids). A laboratory activity on this subject should establish a relationship between the two components that define the redox status. In this scenario, the students should understand that even when the intensity component (redox potential) makes it possible to predict the predominance of aerobic or anaerobic microbial processes, it is, in fact, the capacity component that provides information about the amount of microbial processes (microbial respiration or microbial 68
fermentation for example) that can be supported by the studied redox system (10). The aim of this laboratory experiment is to enable undergraduate students to distinguish clearly between redox potential and redox capacity concepts through concrete results obtained in a complex natural system such as soil, and to discuss the ecological significance of both concepts. The experiment consists in incorporating a small quantity of a reduced compound (hydroquinone; H2Q) into soil extracts, and measuring, simultaneously, the redox potential by means of a Pt electrode and the redox capacity by means of a polarographic technique. Experimental Procedure Instruments and Electrodes Differential pulse polarography was used to perform the polarographic analyses. A polarographic analyzer POL 150 and Tracemaster 5 software were used (Radiometer Analytical, France). The polarographic stand was formed by a polarographic cell (5–10 mL) equipped with a hanging mercury drop electrode (EK 290), a Pt electrode (XM 100), and a standard calomel electrode (SCE; XR 150, Radiometer Analytical, France). All potentials were measured versus a SCE. The operating polarographic conditions were: step duration, 0.2 s; step amplitude, 5 mV; pulse amplitude, 25 mV; pulse duration, 20 ns; and minimal and maximal current range, 10 nA and 1mA, respectively. The potential range was ᎑0.9 V to +0.3 V. The redox potential measurements were performed using a combined Pt electrode with a reference that matched the potential of a SCE (Model 97-98, Orion Research, USA). The electrode was calibrated between each measurement, using a solution containing 3 mmol/L potassium ferrocyanide, 3 mmol/L potassium ferricyanide, and 100 mmol/L KCl (13). The pH was measured with a combined ion-selective field effect transistor electrode (Durafet pH electrode series 079220, Leeds and Northrup, USA). In order to better define the redox conditions of the system, a correction to a pH = 7, E(w), was calculated as follows (14, 15) E(w) = E − 59(7 − pH)
Chemicals The disodium salt of ethylenediaminetetracetic acid (EDTA) solution (0.03 mol/L) was neutralized with 0.1 mol兾L NaOH and used as soil extractant. EDTA was chosen as extractant because it has good buffer and complexation capacities (16, 17) which ensure polarograms well adapted for teaching. A neutral pH was selected to reach a high buffering capacity of the EDTA and to ensure a minimal effect of protons on redox values. The reduction potential of this solution was ᎑0.005 V (vs SCE).
Journal of Chemical Education • Vol. 80 No. 1 January 2003 • JChemEd.chem.wisc.edu
In the Laboratory
A hydroquinone (98%, Fluka) solution (0.5 mol/L) was used to modify the redox conditions of the soil extract.
Soil Samples and Handling The loamy-clay soil used in this work is a vertic mollisol found in Casilda, in the humid pampa region of Argentina. The main soil characteristics were: organic carbon, 3.7%; total N, 2.3%; pH (H2O), 6.1; and cation exchange capacity, 21.7 cmolc kg᎑1. A soil water suspension (100 g of dry soil in 250 ml of 4 ⬚C distilled water) was successively passed through 2000 µm and 1000 µm mesh-size sieves by gentle washing with distilled water. Fractions of 2000–1000 µm mesh size (0.5 g) were treated with EDTA solution (2.5 mL). Three soil samples were examined: control soil–EDTA extract, and soil–EDTA extracts supplemented with 71 and 143 µmol of H2Q. Soil suspension tubes (11 mL) were purged with oxygen-free N2 (5 min) and placed in a horizontal shaker for 1 h. The tubes were centrifuged at 2000 rpm for 10 min and 250 µL of supernatants were diluted into 5 mL distilled deoxygenated water and introduced into the polarographic cell continuously flushed by oxygen-free N2 atmosphere. Hazards The chemicals used in this experiment must be handled with extreme caution. The CAS registry numbers are available in this issue of JCE Online.W Prevent skin and eye contact and avoid inhalation and heavy exposure. Gloves, eye protection, and a lab coat should be worn. Eye wash and safety equipment should be readily available. Results and Discussion Table 1 shows that the values of the redox potential in the control soil–EDTA extract were significantly higher ( p < .05) than those of soil–EDTA extracts where the redox capacity was modified by the incorporation of H2Q, and that
Table 1. Redox Potentials Values in the Nonsupplemented or H2Q-Supplemented Studied Soil Soil
E a /mV
E(w)b/mV
control soil extract
195(5)c
364(5)
control soil extract + H2Q (28.4 mmol/L)
75(5)
247(5)
control soil extract + H2Q (57.2 mmol/L)
64(3)
238(3)
a
E vs SCE.
Calculated values for E at pH = 7.0 and 25 ⬚C (14).
b c
SEM in parentheses.
no significant differences ( p < .05) were observed between the two supplemented treatments. These results were not modified when the values were corrected to pH = 7. In Figure 1, the average of polarograms of control soil– EDTA extracts was subtracted from the average curves of soil– EDTA extracts with H2Q additions. In the explored voltage range, concentration of electroactive compounds was higher in the extracts supplemented with 28.4 and 57.2 mmol/L of H2Q than in the control soil–EDTA extract. In both supplemented treatments, a peak centered at about ᎑0.020 V was observed. This peak, which explains why the intensity values were lower in supplemented treatments with respect to the control, corresponds to the typical peak observed in the polarogram of a H2Q–EDTA solution (Figure 2) and could therefore be clearly associated with the presence of H2Q. Figure 3 shows that the peak areas in the polarograms (which are not shown) were directly proportional to the added amount of H2Q. This result demonstrates that the redox capacity measurements are able to discriminate between the two supplemented treatments, in contrast with the results from the redox potential measurements.
400
300
Current / µA
Current / nA
1.5
200
1.4
1.3
100 1.2
0 -0.9
-0.6
-0.3
0.0
0.3
Potential / V Figure 1. Average of polarograms of the H2Q-supplemented soil– EDTA extracts after subtraction of the polarograms of the non-supplemented soil–EDTA extracts; [H2Q] = 28.4 µmol/L, solid line; [H2Q] = 57.2 µmol/L, dotted line.
1.1 -0.09
-0.06
-0.03
0.00
0.03
0.06
Potential / V Figure 2. Average of polarograms of H2Q–EDTA solution. The arrow shows the location in the potential range of the typical peak of this solution.
JChemEd.chem.wisc.edu • Vol. 80 No. 1 January 2003 • Journal of Chemical Education
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In the Laboratory
(producing 216 mol more of N2O per gram of soil) than the soil that received a lower dose.
Peak Area / µC
1.5
Acknowledgments 1.0
I wish to thank my colleagues at LQB-FCV for their help and encouragement in implementing this lab. The helpful comments of the reviewers are acknowledged. 0.5
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0.0 0
1
2
3
Supplemental Material
Detailed laboratory documentation for the students and notes for the instructor are available in this issue of JCE Online.
Concentration / (mmol/L)
Literature Cited Figure 3. Relation between the ᎑0.020 V-centered peak areas (µC) obtained in the polarograms of the supplemented soils and the amounts of H2Q added to soil–EDTA extracts.
The described experiment clearly illustrates the difficulty in defining the redox status of a natural system only through redox intensity (redox potential) measurements and shows that, as suggested by different authors (10), it is necessary to combine the measurements of the two redox components (potential and capacity) to better define the redox status of such complex systems. Moreover, this experiment also provides a platform for fruitful discussions that attempt to answer the following question: to what extent the concepts of redox potential and redox capacity may be useful to better explain the complex interactions occurring between biotic and abiotic processes in a given natural system? Taking into account the stoichiometric reactions that describe complete oxidation of H2Q (C6H4O2) using O2 or NO3᎑ as final electron acceptors are, C6H4O2 + 6O2 → 6CO2 + 2H2O C6H4O2 + 6NO3᎑ + 6H+ → 6CO2 + 3N2O + 5H2O it may be inferred that, even when redox potential values are not significantly different between two supplemented systems (Table 1), the observed variations in the reduction capacities between the two systems indicate the soil that received a higher amount of H2Q could support a greater microbial respiration activity (consuming 432 mol more of O2 per gram of soil), or a greater microbial denitrification activity
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1. Buffle, J.; Stumm, W. In Chemical and Biological Regulations of Aquatic Systems; Buffle, J.; DeVitre, R. R., Eds.; Lewis Publishers: Boca Raton, Florida, 1994; pp 1–42. 2. Segel, I. Biochemical Calculations, 2nd ed.; John Wiley and Sons: New York, 1976; p 174. 3. Thompson, M. L.; Kateley, J. L. J. Chem. Educ. 1999, 76, 95. 4. Anderson, R. H. J. Chem. Educ. 1993, 70, 940. 5. Guenther, W. B. J. Chem. Educ. 1967, 44, 46. 6. Nordstrom, D. K.; Munoz, J. L. Geochemical Thermodynamics, 2nd ed.; Blackwell Scientific Publications: Boston, 1994; p 302. 7. Grundl, T. Chemosphere 1994, 28, 613. 8. Lindberg, R. D.; Runnells, D. D. Science 1985, 225, 925. 9. Perdue, J. H.; Patrick, W. H. In Metal Contaminated Aquatic Sediments; Allen, H. E. Ed.; Ann Arbor Press: Chelsea, Michigan, 1995; pp 169–185. 10. Yao, H.; Conrad, R.; Wassmann, R.; Neue, H. U. Biogeochemistry 1999, 47, 269. 11. Whitfield, N. Limnol. Oceanogr. 1969, 14, 547. 12. Barcelona, M. J.; Holm, T. R. Environ. Sci. Technol. 1991, 25, 1565. 13. ZoBell, C. E. Bull. Am. Assoc. Pet. Geol. 1946, 30, 477. 14. Stumm, W.; Morgan, J. J. Aquatic Chemistry, 3rd ed.; John Wiley & Sons, Inc.: New York, 1996; p 464. 15. Glinski, J.; Stepniewski, W. Soil Aeration and Its Role for Plants; CRC Press Inc.: Boca Raton, Florida, 1985; p 109. 16. García-Armada, P.; Losada, J.; De Vicente-Perez, S. J. Chem. Educ. 1996, 73, 544. 17. Lanza, P. J. Chem. Educ. 1990, 67, 704.
Journal of Chemical Education • Vol. 80 No. 1 January 2003 • JChemEd.chem.wisc.edu
