In the Laboratory
Detection of Catechol by Potentiometric-Flow Injection Analysis in the Presence of Interferents
W
Suzanne K. Lunsford* Department of Chemistry, Wright State University, Dayton, OH 45435; *
[email protected] Justyna Widera Department of Chemistry, Adelphi University, Garden City, NY 11530-0701 Hong Zhang Advanced Testing Laboratory, Cincinnati, OH 45242
The goal of this chemistry department has been to develop more advanced analytical labs for the third- and fourthyear chemistry majors that incorporate analytical techniques, biological molecules, and common interferents encountered in the detection of biological molecules. Catechol is a biomolecule of interest to neuroscientists as it is secreted in the brain and altered levels have been associated with mental and behavioral disorders such as schizophrenia, attention deficient disorder, Alzheimer’s disease, Parkinson’s disease, eating disorders, epilepsy, and amphetamine and cocaine addiction (1, 2). The presence of interferents commonly found in the human body such as ascorbic acid, uric acid, and acetaminophen may complicate catechol analysis. Amperometric and voltammetric techniques cannot be employed without prior removal of these interferents from catechol (3, 4). This experiment uses potentiometric-flow injection analysis (FIA) carried out on a crown ether-modified platinum electrode (dibenzo-18-crown-6; DB18C6) to detect catechol in the presence of common interferents. Potentiometric-FIA does not require prior separation and thus has an advantage over classical spectroscopic techniques. Potentiometric-FIA also does not require biological molecules to absorb light.1 Owing to their chemical stability many crown ethers have been developed as ion-selective electrodes (5–7). Berre et al. found it was possible to electrochemically polymerize various crown ethers on platinum electrodes (8). To propagate this type of electrochemical polymerization, the polymer solution should be free of moisture, require no synthesis, and include the supporting electrolyte, such as tetrabutylammonium tetrafluoroborate (TBATFB). The DB18C6 displays a black polymer film on the electrode surface after polymerization and can be reproduced easily. The goal of this experiment is to give students the electroanalytical lab skills needed for real-world applications, such as studying neurological disorders. This involves learning and using potentiometric-FIA instrumentation, carried out on a DB18C6-modified electrode for the detection of catechol in the presence and absence of common biological interferents. Experimental Procedure
Polymer Preparation Before electropolymerization, the dual working platinum electrode (MF-1021, BAS) was polished on nylon cloth using 2 µm diamond paste and then polished on microcloth using 0.05 µm alumina. The dual platinum electrode was www.JCE.DivCHED.org
•
rinsed with HPLC-grade acetonitrile and air dried for approximately 15 minutes. The electropolymerization of DB18C6 at a dual polished platinum electrode was carried out in a single-compartment cell. A platinum wire was the auxiliary electrode and the reference was an Ag兾AgCl兾3 M NaCl (MF-2074, BAS) electrode placed downstream in the single-compartment cell. The polymerization of DB18C6 was performed on a PAR 175 potentiostat–galvanostat at an applied potential of +3.2 V against Ag兾AgCl兾3 M NaCl for five minutes. The polymerized DB18C6-modified electrode was rinsed with acetonitrile and then immersed in a monomer-free TBATFB solution. A potential of +0.5 V was applied for 25 minutes. The flow injection system consisted of an Altex 100-A double reciprocating pump followed by an Altex injection valve (Cat. No. 905-42). Teflon tubing was used throughout the FIA studies. The potentiometric measurements were carried out with an Orion model 601A ionanalyzer and the FIA peaks were recorded by a Fisher Recordall 5000 series. The mobile phase was potassium phosphate buffer at a pH of 9.4 with a flow rate of 1.0 mL兾min. Hazards Sulfuric acid and TBATFB may cause eye, skin, and respiratory tract irritation. DB18C6 may cause eye and skin irriations. Catechol is corrosive to eyes and harmful if absorbed in skin. Catechol may cause respiratory and digestive tract irritation with possible burns. Additionally catechol may cause central nervous system depression and could target organs such as kidneys, central nervous system, liver, cardiovascular system, and red blood cells. Acetonitrile can cause skin irritation and target organs such as the kidneys, central nervous system, liver, and lungs. Results and Conclusions The students first analyzed the potentiometric-FIA response to catechol at various concentrations. The potentiometric-FIA response of the crown ether-modified electrode was linear over four orders of catechol concentration: 10᎑2– 10᎑5 M with a detection limit as low as 0.5 × 10᎑5 M (Figure 1). The students then analyzed the potentiometric-FIA response to catechol at 1 × 10᎑4 M in the presence of ascorbic acid at various concentrations. It was found that ascorbic acid did not significantly hinder the potentiometric-FIA response to catechol as long as the ascorbic acid:catechol concentra-
Vol. 84 No. 9 September 2007
•
Journal of Chemical Education
1471
In the Laboratory Table 1. Analysis of Catechol in the Presence of Ascorbic Acid, Uric Acid, and Acetaminophen (Aceta) Potential/mV
Figure 1. Flow injection potentiometric detection calibration curve of the dibenzo-18-crown-6 modified electrode for catechol: R2 = 0.9893.
Conc/M
Ascorbic Acid
Uric Acid
Aceta
Mixture of Three Interferents
0
6.8
6.8
6.8
6.8
1 X 10−5
6.4
5.6
3.6
5.6
−4
1 X 10
6.4
5.6
5.8
5.6
1 X 10−3
8.0
6.0
8.2
5.9
1 X 10−2
10.0
8.8
9.2
16.4
1 X 10−1
12.4
11.0
11.7
18.2
Note: The concentration for each of the three interferents is given in the first column. The catechol concentration is 1 X 10−4 M.
Figure 2. Structures of the target molecule and the interferents.
Figure 3. Detector response obtained with DB18C6 dual modified platinum electrode in flow injection analysis mode: (A) repeated injection responses of 10−4 M catechol and (B) repeated injection responses of a mixture (10−4 M catechol and 10−1 M ascorbic acid). The mobile phase was potassium phosphate buffer at a flow rate of 1.0 mL/min.
1472
Journal of Chemical Education
•
tion ratio was 10:1 or less (Table 1). Ascorbic acid and catechol both contain 1,2-dihydroxy moieties attached to a five or six-membered ring (Figure 2), and possibly ascorbic acid competes with catechol for the active sites at the crown ethermodified electrode surface. Thus there was an increase of FIA signal when the concentration of ascorbic acid (interferent) exceeded the concentration of catechol (targeted compound) by a ratio greater than 10:1. This is shown in Table 1 and in Figure 3. When tested individually, the uric acid and acetaminophen interferents act in the same manner as ascorbic acid, showing little interference at concentration ratios of 10:1 or less. Even at ratios higher than 10:1 they interfere less than ascorbic acid, presumably because they have structures that differ from catechol (Figure 2). Interesting observations were made when catechol detection was studied in the presence of the mixtures of the three interferents: ascorbic acid, uric acid, and acetaminophen (Table 1). At 10:1 or lower ratios of mixture of the three interferents to catechol, there is a decrease in potential. This behavior could be explained by the structures of the molecules. Structural similarities between catechol and ascorbic acid play an important role (as per the explanation above); however, hydrogen bond interactions between the studied molecules could also be significant. If some of the catechol molecules were involved in hydrogen bond formation with the interferents, they could be less susceptible to interaction with the poly(crown ether) moieties present at the electrode surface. This hypothesis provides a possible explanation of the FIA signal decrease at small concentrations of interferents, at ratios equal or smaller than 10:1. At the higher than 10:1 ratios of interferent mixture to catechol concentration, the FIA signal increases more significantly. At higher concentrations, interferent molecules more efficiently compete with catechol molecules for active poly(crown ether) sites at the modified electrode surface. This behavior is expressed by the increase of the FIA signal values with the increase of the interferent concentrations. This could explain also why a tenfold excess of ascorbic acid has more of an effect on the measured potential change than a tenfold excess of all three interferents together. Basically, the structural similarities between catechol and ascorbic acid and lack of the secondary interactions with other interferents molecules, allows ascor-
Vol. 84 No. 9 September 2007
•
www.JCE.DivCHED.org
In the Laboratory
bic acid to interact with poly(crown ether) more easily and efficiently. As the above discussion is based on the hypothesis of the possible hydrogen bonding between molecules, additional studies by NMR could provide further insight and present an opportunity for students to use to a new instrumental technique. Our future work will involve the NMR studies of this interesting phenomenon.
Note 1. Some biological compounds do not absorb light or they absorb light in the same region as another biological molecule of the study resulting in interference problems.
Literature Cited
Summary
1. Hassan, S. S.; Elnemma, E. M. Anal. Chem. 1989, 61, 2189.
The crown ether-modified electrodes as potentiometricFIA detectors for catechol in the presence of common interferents did not require prior separation. As illustrated in the literature (3, 4), simple amperometric and voltammetric techniques can not be employed without prior separation of the common interferents, which are oxidized at more negative potentials and can only be detected at concentrations of 10᎑1 to 10᎑3 M. The modified platinum DB18C6 potentiometric-FIA detector for catechol has achieved low detection limits (10᎑6 M), minimal interference, rapid analysis, and uses simple, low-cost instrumentation. The level of catechol and interferents concentrations depends on the nature and source of a sample. For example the content of human body fluids differs from animal fluids, and brain samples differ from blood or urine samples. In healthy patients the typical concentrations of interferents in human serum samples would be 10᎑4 M uric acid (9) and 10᎑5 M ascorbic acid (10) with catecholamine concentration ranging from 10᎑6 M (11) in blood to 0.6 M in mast cells (12). However in patients that are suffering from mental and behavioral disorders the concentrations of ascorbic acid and uric acid can radically decrease, while the concentration of catecholamine and other neurotransmitters significantly increases and acetaminophen concentration can reach even 10᎑4 M value (13).
2. Ma, Y. L.; Galal, A.; Zimmer, H.; Huang, Z. F.; Bishop, P. L.; Mark, H. B., Jr. Analytica Chemica Acta 1994, 21, 289.
W
4. Coury, L. A.; Huber, E. W.; Birch, E. M.; Heineman, W. R. Anal. Chem. 1988, 60, 553. 5. Kissinger, P. T.; Heineman, W. R. Laboratory Techniques in Electroanalytical Chemistry, 2nd ed.; Marcel Dekker: New York, 1976. 6. Mark, H. B., Jr.; Atta, N.; Ma, Y. L.; Petticrew, K. L.; Zimmer, H.; Shi, Y.; Lunsford, S. K.; Rubinson, J. F.; Galal, A. Bioelectrochem. Bioenergetics 1995, 38, 229. 7. Hubbard, A. T. J. Electroanal. Chem. 1969, 22, 165. 8. Berre, L. V.; Carlier, R.; Tallec, A.; Simonet, J. J. Electroanal. Chem. 1982, 143, 425. 9. Skinner, K. A.; White, C. R.; Patel, R.; Tan, S.; Barnes, S.; Kirk, M.; Darley-Usmar, V. J. Biol. Chem. 1988, 273, 24491– 24497. 10. Lykkesfeldt, J.; Prieme, H.; Loft, S.; Poulsen, H. E. British Medical Journal 1996, 313, 91. 11. Lagercrantz, H.; Bistoletti, P. Pediatric Research 1997, 11, 889– 893. 12. Albillos, A.; Dernick, G.; Horstmann, H.; Almers, W.; Alvarez, De Toledo G.; Lindau, M. Nature 1997, 389, 509.
Supplemental Material
A more detailed presentation of the experiment is available in this issue of JCE Online.
www.JCE.DivCHED.org
3. Adams, R. N.; Mccreery, R. L.; Tse, D. S. E. J. Med. Chem. 1976, 19, 37.
•
13. McKim, J. M., Jr.; Wilga, P. C.; Pregenzer, D. K.; Petrella, D. K. Pharmaceutical Discovery 2004, 1, 1.
Vol. 84 No. 9 September 2007
•
Journal of Chemical Education
1473
