Autocatalytic Nature of Permanganate Oxidations ... - ACS Publications

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Anal. Chem. 2010, 82, 2580–2584

Autocatalytic Nature of Permanganate Oxidations Exploited for Highly Sensitive Chemiluminescence Detection Teo Slezak,† Jessica M. Terry,† Paul S. Francis,*,†,‡ Christopher M. Hindson,† Don C. Olson,§ Duane K. Wolcott,§ and Neil W. Barnett† School of Life and Environmental Sciences and Institute for Technology Research and Innovation, Deakin University, Geelong, Victoria 3217, Australia, and Global FIA, P.O. Box 480, Fox Island, Washington 98333 Manganese(II) salts catalyze the chemiluminescent oxidation of organic compounds with acidic potassium permanganate. The formation of insoluble manganese(IV) species from the reaction between manganese(II) and permanganate can be prevented with sodium polyphosphate, and therefore, relatively high concentrations of the catalyst can be added to the reagent before the lightproducing reaction is initiated. The rapid and intense emissions from these manganese(II) catalyzed chemiluminescence reactions provide highly sensitive detection and greater compatibility with liquid chromatography. The emission of light from chemical reactions can be utilized for sensitive detection with relatively simple instrumentation,1-7 but its application is limited by the number of reactions with sufficient chemiluminescence quantum yields under conditions suitable for routine analysis. Moreover, the emission intensity at any moment is determined not only by the number of reacting molecules and the overall quantum yield but also by the rate of the reaction.8 This aspect is crucial for chemiluminescence detection in flow-based analytical techniques such as liquid chromatography, where analytes are merged with the reagent to initiate the chemiluminescence reaction, and only a small portion of this moving solution is present in the flow cell (exposed to the photodetector) at any particular moment.9,10 Ideally, the reaction should be sufficiently fast so that very little tubing is required * Corresponding author. Phone: +61 3 5227 1294. Fax: +61 3 5227 1040. E-mail: [email protected]. † School of Life and Environmental Sciences, Deakin University. ‡ Institute for Technology Research and Innovation, Deakin University. § Global FIA. (1) Adcock, J. L.; Francis, P. S.; Barnett, N. W. Anal. Chim. Acta 2007, 601, 36–67. (2) Tsunoda, M.; Imai, K. Anal. Chim. Acta 2005, 541, 13–23. (3) Gorman, B. A.; Francis, P. S.; Barnett, N. W. Analyst 2006, 131, 616–639. (4) Francis, P. S.; Barnett, N. W.; Lewis, S. W.; Lim, K. F. Luminescence 2004, 19, 94–115. (5) Marquette, C. A.; Blum, L. J. Anal. Bioanal. Chem. 2006, 385, 546–554. (6) Brown, A. J.; Francis, P. S.; Adcock, J. L.; Lim, K. F.; Barnett, N. W. Anal. Chim. Acta 2008, 624, 175–183. (7) Gamiz-Gracia, L.; Garcia-Campana, A. M.; Huertas-Perez, J. F.; Lara, F. J. Anal. Chim. Acta 2009, 640, 7–28. (8) Pe´rez-Bendito, D.; Silva, M. In Chemiluminescence in Analytical Chemistry; Garcı´a-Campan ˜a, A. M., Baeyens, W. R. G., Eds.; Marcel Dekker: New York, 2001; pp 175-209. (9) Baeyens, W. R. G.; Schulman, S. G.; Calokerinos, A. C.; Zhao, Y.; Garcı´a Campan ˜a, A. M.; Nakashima, K.; De Keukeleire, D. J. Pharm. Biomed. Anal. 1998, 17, 941–953.

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between the confluence point and detection (to minimize band broadening) and a considerable proportion of the emitted light can be captured before the analyte zone is flushed out of the detector. One reagent that approaches these goals is acidic potassium permanganate, which over the last few decades has become widely used for detection in flow injection analysis and high-performance liquid chromatography.1 The intense red emission from this reagent upon reaction with various organic compounds has been identified as the 4T1 f 6A1 phosphorescence from manganese(II).11 A variety of potential enhancers of these chemiluminescence reactions have been examined;1 the two most commonly used, formaldehyde and sodium polyphosphate, both provide significant increases in emission intensity. However, formaldehyde also reacts with the reagent to produce an undesirable background response.12 The oxidation of various organic compounds with potassium permanganate is known to be autocatalytic13,14 (the rate of reaction is influenced by the concentration of reaction intermediates or products) which has been attributed to the formation of manganese(II) and colloidal manganese(IV).15-17 Several researchers have described a change in permanganate chemiluminescence intensity or reaction rate in the presence of manganese(II) salts.18-22 Agater and co-workers added 200 mM manganese(II) to accelerate the very slow chemiluminescent oxidation of (10) Kuroda, N.; Kai, M.; Nakashima, K. In Chemiluminescence in Analytical Chemistry; Garcı´a-Campan ˜a, A. M. , Baeyens, W. R. G. , Eds.; Marcel Dekker: New York, 2001; pp 393-425. (11) Adcock, J. L.; Francis, P. S.; Smith, T. A.; Barnett, N. W. Analyst 2008, 133, 49–51. (12) Townshend, A.; Murillo Pulgarı´n, J. A.; Alan ˜o´n Pardo, M. T. Anal. Chim. Acta 2003, 488, 81–88. (13) Launer, H. F. J. Am. Chem. Soc. 1932, 54, 2597–2610. (14) Launer, H. F.; Yost, D. M. J. Am. Chem. Soc. 1934, 56, 2571–2577. (15) Perez-Benito, J. F.; Arias, C.; Brillas, E. Int. J. Chem. Kinet. 1990, 22, 261– 287. (16) Farokhi, S. A.; Nandibewoor, S. T. Can. J. Chem. 2004, 82, 1372–1380. (17) Kova´cs, K. A.; Gro´f, P.; Burai, L.; Riedel, M. J. Phys. Chem. A 2004, 108, 11026–11031. (18) Agater, I. B.; Jewsbury, R. A.; Williams, K. Anal. Commun. 1996, 33, 367– 369. (19) Zhu, C.; Wang, L.; Li, Y.; Gao, F. Fenxi Huaxue 1997, 25, 387–390. (20) Thongpoon, C.; Liawruangrath, B.; Liawruangrath, S.; Wheatley, R. A.; Townshend, A. J. Pharm. Biomed. Anal. 2006, 42, 277–282. (21) Townshend, A.; Pulgarin, J. A. M.; Pardo, M. T. A. Anal. Bioanal. Chem. 2005, 381, 925–931. (22) Townshend, A.; Wheatley, R. A.; Chisvert, A.; Salvador, A. Anal. Chim. Acta 2002, 462, 209–215. 10.1021/ac9028399  2010 American Chemical Society Published on Web 02/17/2010

carbohydrates, but the lowest analyte concentration that they examined was 1 × 10-4 M.18 Zhu and co-workers determined manganese(II) based on its influence on the rate of the reaction between permanganate and 2,3-butanedione.19 Townshend and co-workers added 1 mM manganese(II) to the carrier stream of their flow injection analysis system, which they found increased signal intensity by 16% for one analyte,20 but decreased the signal by 51% and 80% for two other analytes.21,22 However, at that time, the emitting species in these reactions had not yet been confirmed11,23 (the luminescence was often erroneously attributed to the production of singlet oxygen1,20) and the full implications of these observations were not apparent. In this paper, we demonstrate that the presence of manganese(II), either formed by the degradation of permanganate or added to freshly prepared permanganate solutions, has a significant influence on the intensity and rate of chemiluminescence reactions with this reagent, which can be exploited for rapid and highly sensitive detection for flow analysis techniques such as high-performance liquid chromatography. EXPERIMENTAL SECTION Flow Injection Analysis. The manifold was constructed as previously described,24 except that the volume of the sample loop was 70 µL. The chemiluminescence detector either was a GloCel with single-inlet serpentine-channel reaction zone25 (Global FIA, Fox Island, WA) or was constructed in-house.24 Stopped Flow. Experiments were performed with a flow injection analysis manifold consisting of a programmable dual syringe pump (Model sp210iw, World Precision Instruments, Glen Waverly, Victoria, Australia), Valco six-port injection valve (SGE, Ringwood, Victoria, Australia), and GloCel chemiluminescence detector with dual-inlet serpentine-channel reaction zone25 (Global FIA). The dual-inlet design enabled the merging of solutions directly in front of the photomultiplier module (Electron Tubes model P30A-05; ETP, Ermington, NSW, Australia), and therefore, the entire chemiluminescence intensity versus time profile was captured. The serpentine reaction zone has previously been shown to enhance mixing efficiency compared to the conventional spiral configuration.25,26 The syringes were loaded with deionized water (carrier) and the permanganate reagent. After the injection loop was filled with the analyte solution, the pump was activated. Equivalent, precise volumes of the carrier and reagent solutions were dispensed, which propelled the analyte and reagent into the serpentine reaction channel, where it was held for a set period of time. The output signal from the photomultiplier module was recorded using “e-corder 410” data acquisition (eDAQ, Denistone East, NSW, Australia). Longitudinal dispersion of the analyte zone was minimized using the shortest possible length of tubing between the valve and detector. Inserting a small volume of analyte solution into a carrier stream in this manner, rather than dispensing an analyte stream from the syringe pump, enabled convenient and thorough flushing of the detector (by activating (23) Adcock, J. L.; Francis, P. S.; Barnett, N. W. J. Fluoresc. 2009, 19, 867–874. (24) Adcock, J. L.; Barnett, N. W.; Costin, J. W.; Francis, P. S.; Lewis, S. W. Talanta 2005, 67, 585–589. (25) Terry, J. M.; Adcock, J. L.; Olson, D. C.; Wolcott, D. K.; Schwanger, C.; Hill, L. A.; Barnett, N. W.; Francis, P. S. Anal. Chem. 2008, 80, 9817– 9821. (26) Mohr, S.; Terry, J. M.; Adcock, J. L.; Fielden, P. R.; Goddard, N. J.; Barnett, N. W.; Wolcott, D. K.; Francis, P. S. Analyst 2009, 134, 2233–2238.

the pump for an extended period of time without filling the injection loop) after each profile was collected, which ensured that the manganese(II) product of the reaction did not affect subsequent tests. Reagent Stability Studies. Standard solutions were combined with the permanganate reagent by sequential aspiration of solutions through a multiposition valve (model C25Z, Valco) and the GloCel detector with single-inlet serpentine-channel reactor, using a milliGAT pump (Global FIA).25 The pump and valve were controlled via a LabJack U12 data acquisition board, using software written in LabVIEW 8, which was also used to record the output from the photomultiplier module. This automated system was programmed to repeatedly aspirate 150 µL of reagent (valve position 1; 167 µL/s), 1500 µL of standard solution (position 2; 167 µL/s), and 1000 µL of deionized water (position 3; 100 µL/s), with a 60 min pause after every fifth cycle. High Performance Liquid Chromatography (HPLC). Separation was performed using a HP1100 liquid chromatography system (Agilent Technologies, Forest Hill, Victoria, Australia) with a Chromolith SpeedROD column (RP-18 end-capped, 50 mm length × 4.6 mm i.d.) and 5 mm monolithic guard column (Merck, Kilsyth, Victoria, Australia). The system was configured as previously described,27,28 except that a GloCel detector with singleinlet serpentine-channel reactor25 was used. The column eluate merged with the permanganate reagent at a T-fitting immediately prior to entering the detector. The injection volume was 20 µL. UV-Visible Absorption. Spectra were obtained using a Cary 300 Bio UV-visible spectrophotometer (Varian, Mulgrave, Australia) with 10 mm path length, sealable quartz cuvettes (Starna, Baulkham Hills BC, NSW, Australia). Reagents. Unless otherwise stated, the acidic potassium permanganate reagent (1 × 10-3 M) was prepared by dissolving potassium permanganate (Chem-Supply, Gillman, SA, Australia) in a 1% (m/v) sodium polyphosphate (+80 mesh; SigmaAldrich, Castle Hill, NSW, Australia) solution and adjusted to pH 2.5 with sulfuric acid. When required, manganese(II) sulfate (Ajax, Sydney, NSW, Australia) was added to the permanganate reagent. RESULTS AND DISCUSSION Acidic potassium permanganate solutions containing sodium polyphosphate have limited stability due to both the degradation of the oxidant29 and a pH dependent hydrolysis of the polyphosphate.30 Solutions that provide the optimum conditions for chemiluminescence detection (e.g., 1 mM KMnO4, 1% m/v sodium polyphosphate, adjusted to pH 2.5 with H2SO4)1 are sufficiently stable for most applications but show some variation over 48 h.31 Comparison of a freshly prepared reagent solution with others that had been left standing for 6 months (one protected from light and one exposed to natural light) revealed large differences in (27) Slezak, T.; Francis, P. S.; Anastos, N.; Barnett, N. W. Anal. Chim. Acta 2007, 593, 98–102. (28) Costin, J. W.; Lewis, S. W.; Purcell, S. D.; Waddell, L. R.; Francis, P. S.; Barnett, N. W. Anal. Chim. Acta 2007, 597, 19–23. (29) Dash, S.; Patel, S.; Mishra, B. K. Tetrahedron 2009, 65, 707–739. (30) Omelon, S. J.; Grynpas, M. D. Chem. Rev. 2008, 108, 4694–4715. (31) Spectrophotometric monitoring revealed a ∼1% decrease in absorption at 525 nm (λmax of permanganate) over 48 h. The chemiluminescence signal with a morphine standard increased by approximately 25% over the same period of time.

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Table 1. Chemiluminescence Signal (Peak Height) for Various Analytes (5 × 10-6 M) with Freshly Prepared and Stored Acidic Potassium Permanganate, Using Flow Injection Analysis Methodology chemiluminescence signal (mV) analyte

freshly prepared

6 months old (dark)

6 months old (natural light)

adrenalone tyramine 3-aminophenol 4-aminophenol 4-aminoresorcinol

50 88 35 300 1220

8 15 5 10 80

9 175 64 32 200

chemiluminescence intensity with various analytes (Table 1). As expected, the solution stored in the dark produced much lower chemiluminescence signals than the fresh solution, due to the slow degradation of both oxidant and polyphosphate. However, the decrease in chemiluminescence intensity was less evident for the reagent exposed to light, and in some cases, it produced an emission greater than that obtained with the freshly prepared reagent. The degradation of permanganate occurs at a faster rate when exposed to light.32 Spectrophotometric measurements showed that the concentration of the reagent stored under natural light had decreased by 29.5%, compared to 5.7% for the permanganate protected from light. The increase in chemiluminescence signal from the more degraded reagent was, therefore, attributed to manganese(II) catalysis of the light-producing reaction pathway, which counteracted the deleterious effects of lower oxidant and enhancer concentrations on emission intensity. This enhancement was further explored by adding manganese(II) sulfate (0.3 mM) to freshly prepared permanganate solutions (1 mM, adjusted to pH 2.5). Without sodium polyphosphate, the manganese(II) salt produced a rapid color change from the characteristic purple of permanganate to a murky maroon red, indicating the formation of manganese dioxide (Guyard reaction33). However, when sodium polyphosphate (1% m/v) was present, this change was not observed. The addition of manganese(II) produced a substantial increase in chemiluminescence intensity, which became even greater over the first 24 h after preparation (Figure 1). Repeated reactions with the analyte standard during the subsequent 24 h showed very little variation (relative standard deviation was 0.8%), and therefore, all subsequent experiments were performed using reagents left to stand for one day. As shown in Figure 1, the change in chemiluminescence intensity mirrored the slow oxidation of manganese(II) by permanganate (measured as a decrease in absorbance at 525 nm). A corresponding rise in absorbance below 325 nm indicated the formation of intermediate manganese species.6,34 The relatively small enhancement (or even inhibition) of the chemiluminescence response reported by Townshend and co-workers20–22 may, therefore, have been due to the merging of the manganese(II), (32) Rees, T. J. Chem. Educ. 1987, 64, 1058. (33) Orba´n, M.; Epstein, I. R. J. Am. Chem. Soc. 1989, 111, 8543–8544. (34) Spectrophotometric measurement of manganese(III) (λmax ) 516 nm; Milovanovic, G. A.; Pastor, F. T.; Petkovic, G. M.; Todorovic, M. Microchim. Acta 2004, 144, 51-56) was impeded by the high molar absorptivity of the permanganate ion.

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Figure 1. Increase in chemiluminescence intensity (with tyramine) for a reagent containing 1 × 10-3 M potassium permanganate and 3 × 10-4 M manganese(II) sulfate in 1% m/v sodium polyphosphate, adjusted to pH 2.5 with sulfuric acid, and the decrease in reagent absorbance at 525 nm.

permanganate and analyte online (which allowed very little time for the equilibration of manganese species) and the absence of polyphosphates in the reagent solution. The results of a 72 h stability study, using a permanganate reagent containing 6 × 10-4 M manganese(II) sulfate, is included in the Supporting Information (Figure S1). The concentration of manganese(II) sulfate that produced the greatest enhancement in chemiluminescence response was found to be dependent on both the instrumental approach and the particular analyte. Nevertheless, the optimum concentration was generally 0.3-0.6 mM. Typical data from these experiments is shown in the Supporting Information (Figure S2). The influence of manganese(II) sulfate on the chemiluminescence intensity from reactions between acidic potassium permanganate and analytes from four important classes (neurotransmitters,24,35 adrenergic amines,27,36 opiate alkaloids,28,37 and antioxidants38,39) that have been previously detected with this reagent is shown in Table 2; up to a 73-fold increase in chemiluminescence response was observed under these conditions. The chemiluminescence intensity versus time profiles for various analytes were examined using the stopped-flow approach. In general, the greatest increases in emission intensity were obtained from reactions that were relatively slow in the absence of manganese(II). Most notably, although permanganate chemiluminescence has previously been used to detect adrenergic phenolic amines after chromatographic separation,27,36 the rates of these reactions without added manganese(II) are not optimal for flow-through detection (see, for example, the intensity versus time profiles for synephrine in Figure 2); maximum emission (35) Lee, Y.-T.; Whang, C.-W. J. Chromatogr., A 1997, 771, 379–384. (36) Percy, D. W.; Adcock, J. L.; Conlan, X. A.; Barnett, N. W.; Gange, M. E.; Noonan, L. K.; Henderson, L. C.; Francis, P. S. Talanta 2010, 80, 2191– 2195. (37) Hill, L. A.; Lenehan, C. E.; Francis, P. S.; Adcock, J. L.; Gange, M. E.; Pfeffer, F. M.; Barnett, N. W. Talanta 2008, 76, 674–679. (38) Conlan, X. A.; Stupka, N.; McDermott, G. P.; Barnett, N. W.; Francis, P. S. Anal. Methods 2010, 2, 171–173. (39) Francis, P. S.; Costin, J. W.; Conlan, X. A.; Bellomarino, S. A.; Barnett, J. A.; Barnett, N. W. Food Chem., Submitted for publication (March 2009).

Table 2. Chemiluminescence Signal (Peak Height) for Reactions between Various Analytes (1 × 10-5 M) and Acidic Potassium Permanganate (1 × 10-3 M), with and without Additional Manganese(II) Sulfate (6 × 10-4 M), Using Flow Injection Analysis Methodology intensity (mV) analyte neurotransmitters and metabolites dopamine vanilmandelic acid homovanillic acid serotonin adrenergic amines octopamine synephrine tyramine hordenine opiate alkaloids morphine codeine oripavine pseudomorphine antioxidants resveratrol quercetin gallic acid caffeic acid

KMnO4

KMnO4 + Mn(II)

enhancement factor

63 38 71 915

174 363 333 2408

2.8 9.7 4.7 2.6

21 9 49 34

888 643 1017 777

41.5 72.8 20.8 23.1

1992 5 1158 2575

11050 41 7117 3700

5.5 8.0 6.1 1.4

15 306 8 47

93 478 47 113

6.4 1.6 5.7 2.4

intensity for these analytes occurred between 10 and 35 s, and the signal did not decay to half its maximum intensity until 40-80 s. However, adding 0.3 mM manganese(II) to the permanganate solution decreased the time to reach maximum intensity to 1.3-2.0 s (Figure 2). Increasing the manganese(II) concentration to 0.6 and 1.0 mM resulted in small additional increases in reaction rate, which were accompanied by improvements in maximum intensity (peak height) but decreases in the total chemiluminescence emission (peak area) (see Figure S3 in Supporting Information). The distribution of light within the flow injection analysis detection cells was examined by continuously merging the reactant solutions at the T-piece and capturing the emitted light with a digital SLR

Figure 2. Chemiluminescence intensity versus time profiles (using the stopped-flow technique) for the reaction of synephrine with acidic potassium permanganate with (A) 0, (B) 0.1, (C) 0.3, and (D) 0.6 mM manganese(II) sulfate added to the reagent. Each reagent contained 1% m/v sodium polyphosphate and was adjusted to pH 2.5 prior to adding manganese(II).

Figure 3. Photographs of chemiluminescence from the single-inlet serpentine flow cell of the GloCel detector. Parts (a), (b), and (c) show the reaction between synephrine and acidic potassium permanganate, with 0, 0.3, and 0.6 mM manganese(II) sulfate, respectively, using an exposure time of 5 min. Parts (d), (e), and (f) show the reaction of morphine with the same three reagent conditions and an exposure time of 2.5 s. Each reagent contained 1% m/v sodium polyphosphate and was adjusted to pH 2.5 prior to adding manganese(II). The reagent and analyte (1 × 10-3 M) were continuously merged at a T-piece immediately prior to entering the detector.

camera (Figure 3). Without manganese(II) sulfate, the chemiluminescence from the reaction of permanganate and synephrine was spread throughout the flow cell, with a slightly more intense emission emanating from the outer “coils” of the serpentine channel (Figure 3a). In agreement with intensity versus time profile “A” (Figure 2), it appears that the reaction mixture would continue to emit light for a significant amount of time after exiting the detection zone. In contrast, the reactions in the presence of manganese(II) sulfate produced an intense emission in the center of the detection zone (Figure 3b,c), which decreased considerably before the solution exited the cell. Although the uncatalyzed reaction with morphine is already fast (Figure 3d), the increase in reaction rate, producing more intense chemiluminescence in the first coil of the serpentine channel, is evident in Figure 3e,f. The acidic potassium permanganate reagents were applied to postcolumn chemiluminescence detection for the reversed-phase HPLC separation of adrenergic amines. As shown in Figure 4, the faster, more intense emissions using the reagent containing additional manganese(II) translate to more sensitive postcolumn detection for HPLC, enabling the full potential of the acidic potassium permanganate reagent to be realized. Limits of detection for octopamine, synephrine, tyramine, N-methyltyramine, and hordenine were improved by over an order of magnitude (5 × 10-8, 4 × 10-8, 7 × 10-8, 8 × 10-8, and 1 × 10-7 M, respectively). Furthermore, the limit of detection for synephrine with the manganese(II) catalyzed reaction using flow injection analysis methodology was 1.2 × 10-9 M, which also represents an order of magnitude improvement compared to that obtained under previously optimized conditions.27,40 A relatively small enhancement was obtained for the reaction of permanganate with morphine (Table 2), which was found (using stopped flow) to reach maximum intensity within 2 s even without added manganese(II). Nevertheless, using flow injection analysis (40) Francis, P. S.; Brown, A. J.; Bellomarino, S. A.; Taylor, A. M.; Slezak, T.; Barnett, N. W. Luminescence 2009, 24, 90–95.

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CONCLUSIONS Permanganate chemiluminescence has been used to detect a wide variety of compounds, including biomolecules, antioxidants, pharmaceuticals, pesticides, and pollutants.1 The greater control of reaction rates and enhanced emission intensities obtained using manganese(II) catalysis will improve sensitivity and compatibility with liquid chromatography (and related flow-analysis techniques), providing superior figures of merit for existing procedures and the ability to extend this attractive mode of detection to new analytical applications.

Figure 4. Chromatograms for the separation of five adrenergic amines (each at 1 × 10-5 M) using acidic potassium permanganate chemiluminescence detection, (A) with and (B) without 0.3 mM manganese(II) added to the reagent. Peaks: 1 ) octopamine; 2 ) synephrine; 3 ) tyramine; 4 ) N-methyltyramine; 5 ) hordenine.

methodology, the limit of detection for morphine using the permanganate reagent with manganese(II) was 4.6 × 10-11 M, which was an order of magnitude better than that obtained under the same experimental conditions except without manganese(II) added to the permanganate reagent (5.4 × 10-10 M) and was superior to all previous limits of detection established for this reagent.1 Another interesting consequence of the differences in enhancement is a change in reagent selectivity. For example, without added manganese(II), dopamine produced a greater response than vanilmandelic acid (Table 2), but in the presence of manganese(II), vanilmandelic acid produced approximately twice the signal obtained for dopamine.

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ACKNOWLEDGMENT The authors thank the Institute for Technology, Research and Innovation (Deakin University) for financial support, Dr. Jacqui Adcock (School of Life and Environmental Sciences, Deakin University) for developing the software used for the stability studies, Elizabeth Zammit (School of Life and Environmental Sciences, Deakin University) for assistance during flow injection analysis experiments, and Donna Edwards (Knowledge Media Division, Deakin University) for the photography. Three authors (T.S., J.M.T. and C.M.H.) acknowledge receipt of an Australian Postgraduate Award. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review December 14, 2009. Accepted February 7, 2010. AC9028399