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Amperometric biosensing of cinnamic acid using a spinach-based biocatalytic electrode
441
Bioelectrochemistty
and Bioenergerics,
27 (1992) 441-447
A section of .I. Electroanal. Chem., and constituting Vol. 342 (1992) Elsevier Sequoia S.A., Lausanne
JEC BB 01498
Amperometric biosensing of cinnamic acid using a spinach-based biocatalytic electrode Joseph Wang * and Najih Naser Department
of Chemistry, New Mexico State University, Las Cruces, NM 88003 (USA)
(Received 6 November 1991)
Abstract A new amperometric bioelectrode for cinnamic acid, based on the cinnamic acid hydroxylase activity of spinach leaves, is described. Such a bioelectrode addresses the commercial unavailability and instability of the pure enzyme. The incorporation of spinach leaves within a carbon paste matrix thus results in a fast response (tg5%= 12 s) to dynamic changes in the substrate concentration. The effect of various experimental parameters, such as pH, applied potential, flow rate or paste composition, is explored. Flow injection measurements yield a detection limit of 2~ 10m4 M, with linearity up to 4X 10e3 M, and a relative standard deviation of 2.0% (n = 25). Analogous measurements of dopamine, based on the polyphenol oxidase activity of spinach leaves, are also reported.
INTRODUCTION
Cinnamic acid (3-phenyl-2-propenoic acid) is an important substance in the production of esters for the perfume industry. In addition, reliable quantitation of cinnamic acid is of great significance in certain food, photographic, polymer and pharmaceutical industries, as well as for clinical and toxicological studies [l]. Common analytical techniques for measuring cinnamic acid include gas and liquid chromatography [2,31, electrophoresis [41 or UV spectroscopy [51. Biosensing schemes, which may facilitate the real-time monitoring of cinnamic acid in various industrial processes, have not been reported. This paper describes a new amperometric tissue electrode for monitoring cinnamic acid. The use of tissue slices as biocomnonents of biocatalytic electrodes is receiving considerable attention [6-81. In particular, tissue bioelectrodes are
l
To whom correspondence
0302-4598/92/$05.00
should be addressed.
0 1992 - Elsevier Sequoia S.A. All rights reserved
442
attractive owing to the high activity and stability of their enzyme, which is confined in its own natural environment. The spinach leaves employed in the present work are rich with the enzyme cinnamic acid hydroxylase (CAI-I) which specifically catalyzes the conversion of cinnamic acid to p-coumaric acid [9]:
CH
CH HC/
cinnamic acid
H&
p-coumaric acid
Cinnamic acid can thus be monitored by amperometric (anodic) detection of the coumaric acid generated. In addition to its successful use for monitoring cinnamic acid concentrations, the spinach biosensor represents a unique example of employing tissues as a source of biocatalytic activity when the pure enzyme is not stable or commercially available. Earlier attempts to isolate the spinach CAH resulted in a rapid loss of the biocatalytic activity [9]. Another unique feature of the spinach electrode is its suitability for monitoring (at a different potential) substrates of another enzyme (polyphenol oxidase) present in the tissue. These and other performance characteristics are reported in the following sections. EXPERIMENTAL
Apparatus
Batch experiments were performed in a 10 ml electrochemical cell (model V-2, Bioanalytical Systems (BAS), W. Lafayette). The spinach/ carbon paste electrode, the reference electrode (Ag/AgCl, model RE-1, BAS) and the platinum wire auxiliary electrode were inserted into the cell through holes in its Teflon cover. A magnetic stirrer and a stirring bar provided the convective transport. The flow injection system used has been described previously [lo]. A thin-layer amperometric detector (model TL4, BAS) and a 20 ~1 sample loop were used. The reference electrode was held in a downstream compartment. All experiments were performed using an EG&G PAR model 364 polarographic analyzer, the output of which was recorded on a Houston Omniscribe strip-chart recorder. Electrode preparation
The mixed spinach/carbon paste electrode was prepared in the following manner. The spinach leaves (purchased from a local grocery store) were crushed
443
with a pestle and mortar. The desired amount of spinach (0.2 g) was thoroughly hand-mixed with 0.32 g mineral oil (Aldrich) and 0.48 g graphite powder (Acheson 38, Fisher). A portion of the spinach/carbon paste was packed firmly into the end of a glass tube (inside diameter, 3 mm; outside diameter, 5 mm) with a copper wire inserted from the other end for connection. Another portion of the paste was packed into the electrode cavity (diameter 3 mm) of a thin-layer flow detector. The surfaces were smoothed with a weighing paper. Reagents and procedures
AI1 solutions were prepared with doubly-distilled water. Cinnamic acid (Fisher) and dopamine (Sigma) were used without further purification. The supporting electrolyte was 0.05 M phosphate buffer (pH 7.4). Amperometric detection was used in both the batch and flow injection systems. The desired working potential (usually + 0.9 V) was applied and transient currents were allowed to decay to a steady-state value. Solution stirring (400 rev/min) or flow (1.0 ml/min) were used in batch and flow injection experiments respectively. All measurements were performed at room temperature. RESULTS AND DISCUSSION
Figure 1 compares the amperometric response (at +0.90 V) to cinnamic acid obtained at unmodified and spinach-modified carbon paste electrodes. As expected, the unmodified surface does not respond to cinnamic acid. In contrast, a fast and sensitive anodic response (of the enzymatically produced coumaric acid) is observed at the tissue electrode. Hence convenient quantitation is possible with both batch (Fig. 1A) and flow injection (Fig. 1B) operations. The fast response 0 95%= 12 s) to the substrate is attributed to the close proximity of the biocatalytic and sensing sites, as is common .with mixed tissue/carbon paste electrodes [ll]. The rapid rise in the current is coupled to fast “wash-out” properties, thus resulting in flow injection rates of 135 samples h-l. The high reproducibility of the flow injection response should also be noted. The 15 peaks in Fig. 1B are a part of a series of 25 repetitive runs, which yielded a relative standard deviation of 2.0% (range, 60-63 nA; mean, 61 nA). Figure 2 shows the dependence of the cinnamic acid response on the operating potential and the solution pH. As expected for the anodic monitoring of coumaric acid, the oxidation current increases on changing the potential from 0.4 to 1.0 V, with the most rapid increase between 0.7 and 0.9 V. A peak-shaped pH profile with a gradual increase between 3.0 and 7.4 and a sharp decrease at higher values is observed. Since a peak-shaped pH profile, with an optimum pH of 4.6, was reported for the spinach CAH activity [9], the profile of Fig. 2(b) appears to reflect both the biocatalytic and detection requirements. The composition of the paste has a profound effect on the response of the tissue electrode. For example, pastes containing lo%, 20% and 30% (w/w) spinach yielded steady-state currents of 61
444
TIME Fig. 1. Current-time recordings at (a) the unmodified and (b) the spinach-modified (20% w/w) carbon paste electrodes. (A) Additions of 2x lo-’ M cinnamic acid (stirring rate, 400 rev/mitt); (B) repetitive flow injections of a 1 x 10d3 M cinnamic acid solution (flow rate, 1.0 ml/mitt). Sample volume, 20~1; operating potential, +0.9 V; electrolyte and carrier solutions, 0.05 M phosphate buffer (pH 7.4).
nA, 112 nA and 179 nA respectively for 2 X lop3 M cinnamic acid, reflecting the increase in the biocatalytic activity (conditions as in Fig. 1A). However, the best signal-to-noise characteristics were observed with the 20% (w/w> tissue electrode, which was thus employed in all subsequent work. The flow injection peak current for 1 X 10e3 M cinnamic acid gradually decreased (from 42 to 25 nA) upon increasing the flow rate from 0.5 to 2.4 ml/min. Such behavior is attributed primarily to the longer residence time of the sample plug in the working electrode compartment. The response time (t,,, ) decreased from 12 to 3 s upon increasing the flow rate. The concentration dependence of the tissue-modified electrode was also evaluated using a flow injection system. Figure 3 shows amperometric peaks for injections of cinnamic acid solutions of increasing concentration (0.5 X 10-3-10 X 10e3 M). The resulting calibration plot (also shown) exhibits linearity up to 4 X lop3 M, with curvature at higher concentrations. The slope of the initial linear portion is 26 nA/mM (correlation coefficient, 0.999). A detection limit of 2 x low4 M can be estimated from the signal-to-noise characteristics (S/N = 3) of the 5 X 10e4 M peak (a>. Hence 0.6 pg can be detected in the 20 ~1 solution. As expected for the surface immobilization of tissue materials, the spinach electrode exhibits long-term stability. No loss of biocatalytic activity was observed over a period of 13 days (with storage at 4°C). A slow decrease in the response
445
POTENTIAL/V 0.5
Fig. 2. Dependence of the response to 2 x lothe solution pH. Conditions as in Fig. lA(b).
1.0
M cinnamic acid on (a) the operating potential and (b)
(5%/day) was observed after that period. The tissue/carbon paste configuration permits rapid renewal of its surface after such loss. Spinach leaves are also rich in the enzyme polyphenol oxidase [121 which catalyzes the conversion of catecholamines to the corresponding quinones. Hence spinach-modified electrodes can be used for monitoring important neurotransmitters in a manner analogous to bioelectrodes based on banana [ll] or eggplant [13]. For example, Fig. 4 illustrates the amperometric response to dopamine, under both flow injection and batch conditions, as recorded at unmodified and spinachcontaining carbon paste electrodes. Unlike the absence of response at the unmodified surface, the tissue electrode offers a rapid, sensitive and reproducible response to dopamine (through reductive monitoring of its quinone product). An injection rate of 90 samples h-’ can be realized from this dynamic behavior. The flow injection detection limit is around 5 X 10m5 M. Hence multianalyte sensing becomes possible by coupling the multienzyme activity of the spinach tissue with the use of different operating potentials. In conclusion, the cinnamic acid hydroxylating system of spinach has been exploited for the design of a new bioelectrode for cinnamic acid. The mixed spinach/carbon paste configuration thus responds rapidly to dynamic changes in
446
TIME
3805 ~li?O5
80I 2
I 4
I 8
I 8
I 10
CONCENTRATION/mM Fig. 3. Flow injection peaks for cinnamic acid solutions of increasing concentration: (a) 5 x 10e4 M, (b) 1 X 10V3 M, (c) 2 X 10e3 M, (d) 3 X lo-’ M, (e) 4 X 10e3 M, (0 5 x 10e3 M, (g) 6 X 10m3 M, (h) 8 x 10m3 M and (i) 1 X lo-* M. Also shown is the resulting calibration plot. Conditions as in Fig. lB(b).
=f 0
IPmin
I I I I I
i,
s
14 $ Pmin
I
TIME Fig. 4. Current-time recordings at (a) the unmodified and (b) the spinach-modified (20% w/w) carbon paste electrodes: (A) repetitive flow injections of a 1 X 10e3 M dopamine; (B) additions of 5 X 10m4 M dopamine. Operating potential, - 0.2 V, other conditions as in Fig. 1.
447
the cinnamic acid concentration. In addition to the quantitation of cinnamic acid, this study illustrates the sensing utility of tissue materials in cases where the pure enzyme is not available and the usefulness of the multienzyme activity of such materials for multianalyte detection. Hence this study further illustrates the versatility of using natural materials for biosensor work. Such behavior should benefit tissue-based assays of other analytes. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13
M. Grayson (Ed.), Encyclopedia of Chemical Technology, Vol. 6, Wiley, New York, 1981, p. 142. W. Greenaway, J. May and F. Whatley, J. Chromatogr., 472 (1989) 393. A. Janczyk and I. Wilczynska-Wojtulewicz, Chem. Anal. (Warsaw), 33, (1988) 535. S. Fujiwara and S. Honda, Anal. Chem., 58 (1985) 1811. M. Bounias, M.H. Daurade and Y. Lizzi, Analysis, 17 (1989) 201. G.a. Rechnitz, Anal. Chim. Acta, 180 (1986) 281. J. Wang, Electroanalysis, 3 (1991) 255. M.A. Arnold, Am. Lab. (Boston), 15(6) (1983) 34. P.M. Nair and L.C. Vining, Phytochemistry, 4 (1965) 161. J. Wang and L.D. Hutchins, Anal. Chem., 57 (1985) 1536. J. Wang and MS. Lin, Anal. Chem., 60 (1988) 1545. A. Sanchez-Ferrer, J. Villalba and F. Garcia-Carmora, Phytochemistry, 28 (1989) 1321. A. Navaratne, M.S. Lin and G.A. Rechnitz, Anal. Chim. Acta, 237 (1990) 107.
Bioelectrochemistty
and Bioenergerics,
27 (1992) 441-447
A section of .I. Electroanal. Chem., and constituting Vol. 342 (1992) Elsevier Sequoia S.A., Lausanne
JEC BB 01498
Amperometric biosensing of cinnamic acid using a spinach-based biocatalytic electrode Joseph Wang * and Najih Naser Department
of Chemistry, New Mexico State University, Las Cruces, NM 88003 (USA)
(Received 6 November 1991)
Abstract A new amperometric bioelectrode for cinnamic acid, based on the cinnamic acid hydroxylase activity of spinach leaves, is described. Such a bioelectrode addresses the commercial unavailability and instability of the pure enzyme. The incorporation of spinach leaves within a carbon paste matrix thus results in a fast response (tg5%= 12 s) to dynamic changes in the substrate concentration. The effect of various experimental parameters, such as pH, applied potential, flow rate or paste composition, is explored. Flow injection measurements yield a detection limit of 2~ 10m4 M, with linearity up to 4X 10e3 M, and a relative standard deviation of 2.0% (n = 25). Analogous measurements of dopamine, based on the polyphenol oxidase activity of spinach leaves, are also reported.
INTRODUCTION
Cinnamic acid (3-phenyl-2-propenoic acid) is an important substance in the production of esters for the perfume industry. In addition, reliable quantitation of cinnamic acid is of great significance in certain food, photographic, polymer and pharmaceutical industries, as well as for clinical and toxicological studies [l]. Common analytical techniques for measuring cinnamic acid include gas and liquid chromatography [2,31, electrophoresis [41 or UV spectroscopy [51. Biosensing schemes, which may facilitate the real-time monitoring of cinnamic acid in various industrial processes, have not been reported. This paper describes a new amperometric tissue electrode for monitoring cinnamic acid. The use of tissue slices as biocomnonents of biocatalytic electrodes is receiving considerable attention [6-81. In particular, tissue bioelectrodes are
l
To whom correspondence
0302-4598/92/$05.00
should be addressed.
0 1992 - Elsevier Sequoia S.A. All rights reserved
442
attractive owing to the high activity and stability of their enzyme, which is confined in its own natural environment. The spinach leaves employed in the present work are rich with the enzyme cinnamic acid hydroxylase (CAI-I) which specifically catalyzes the conversion of cinnamic acid to p-coumaric acid [9]:
CH
CH HC/
cinnamic acid
H&
p-coumaric acid
Cinnamic acid can thus be monitored by amperometric (anodic) detection of the coumaric acid generated. In addition to its successful use for monitoring cinnamic acid concentrations, the spinach biosensor represents a unique example of employing tissues as a source of biocatalytic activity when the pure enzyme is not stable or commercially available. Earlier attempts to isolate the spinach CAH resulted in a rapid loss of the biocatalytic activity [9]. Another unique feature of the spinach electrode is its suitability for monitoring (at a different potential) substrates of another enzyme (polyphenol oxidase) present in the tissue. These and other performance characteristics are reported in the following sections. EXPERIMENTAL
Apparatus
Batch experiments were performed in a 10 ml electrochemical cell (model V-2, Bioanalytical Systems (BAS), W. Lafayette). The spinach/ carbon paste electrode, the reference electrode (Ag/AgCl, model RE-1, BAS) and the platinum wire auxiliary electrode were inserted into the cell through holes in its Teflon cover. A magnetic stirrer and a stirring bar provided the convective transport. The flow injection system used has been described previously [lo]. A thin-layer amperometric detector (model TL4, BAS) and a 20 ~1 sample loop were used. The reference electrode was held in a downstream compartment. All experiments were performed using an EG&G PAR model 364 polarographic analyzer, the output of which was recorded on a Houston Omniscribe strip-chart recorder. Electrode preparation
The mixed spinach/carbon paste electrode was prepared in the following manner. The spinach leaves (purchased from a local grocery store) were crushed
443
with a pestle and mortar. The desired amount of spinach (0.2 g) was thoroughly hand-mixed with 0.32 g mineral oil (Aldrich) and 0.48 g graphite powder (Acheson 38, Fisher). A portion of the spinach/carbon paste was packed firmly into the end of a glass tube (inside diameter, 3 mm; outside diameter, 5 mm) with a copper wire inserted from the other end for connection. Another portion of the paste was packed into the electrode cavity (diameter 3 mm) of a thin-layer flow detector. The surfaces were smoothed with a weighing paper. Reagents and procedures
AI1 solutions were prepared with doubly-distilled water. Cinnamic acid (Fisher) and dopamine (Sigma) were used without further purification. The supporting electrolyte was 0.05 M phosphate buffer (pH 7.4). Amperometric detection was used in both the batch and flow injection systems. The desired working potential (usually + 0.9 V) was applied and transient currents were allowed to decay to a steady-state value. Solution stirring (400 rev/min) or flow (1.0 ml/min) were used in batch and flow injection experiments respectively. All measurements were performed at room temperature. RESULTS AND DISCUSSION
Figure 1 compares the amperometric response (at +0.90 V) to cinnamic acid obtained at unmodified and spinach-modified carbon paste electrodes. As expected, the unmodified surface does not respond to cinnamic acid. In contrast, a fast and sensitive anodic response (of the enzymatically produced coumaric acid) is observed at the tissue electrode. Hence convenient quantitation is possible with both batch (Fig. 1A) and flow injection (Fig. 1B) operations. The fast response 0 95%= 12 s) to the substrate is attributed to the close proximity of the biocatalytic and sensing sites, as is common .with mixed tissue/carbon paste electrodes [ll]. The rapid rise in the current is coupled to fast “wash-out” properties, thus resulting in flow injection rates of 135 samples h-l. The high reproducibility of the flow injection response should also be noted. The 15 peaks in Fig. 1B are a part of a series of 25 repetitive runs, which yielded a relative standard deviation of 2.0% (range, 60-63 nA; mean, 61 nA). Figure 2 shows the dependence of the cinnamic acid response on the operating potential and the solution pH. As expected for the anodic monitoring of coumaric acid, the oxidation current increases on changing the potential from 0.4 to 1.0 V, with the most rapid increase between 0.7 and 0.9 V. A peak-shaped pH profile with a gradual increase between 3.0 and 7.4 and a sharp decrease at higher values is observed. Since a peak-shaped pH profile, with an optimum pH of 4.6, was reported for the spinach CAH activity [9], the profile of Fig. 2(b) appears to reflect both the biocatalytic and detection requirements. The composition of the paste has a profound effect on the response of the tissue electrode. For example, pastes containing lo%, 20% and 30% (w/w) spinach yielded steady-state currents of 61
444
TIME Fig. 1. Current-time recordings at (a) the unmodified and (b) the spinach-modified (20% w/w) carbon paste electrodes. (A) Additions of 2x lo-’ M cinnamic acid (stirring rate, 400 rev/mitt); (B) repetitive flow injections of a 1 x 10d3 M cinnamic acid solution (flow rate, 1.0 ml/mitt). Sample volume, 20~1; operating potential, +0.9 V; electrolyte and carrier solutions, 0.05 M phosphate buffer (pH 7.4).
nA, 112 nA and 179 nA respectively for 2 X lop3 M cinnamic acid, reflecting the increase in the biocatalytic activity (conditions as in Fig. 1A). However, the best signal-to-noise characteristics were observed with the 20% (w/w> tissue electrode, which was thus employed in all subsequent work. The flow injection peak current for 1 X 10e3 M cinnamic acid gradually decreased (from 42 to 25 nA) upon increasing the flow rate from 0.5 to 2.4 ml/min. Such behavior is attributed primarily to the longer residence time of the sample plug in the working electrode compartment. The response time (t,,, ) decreased from 12 to 3 s upon increasing the flow rate. The concentration dependence of the tissue-modified electrode was also evaluated using a flow injection system. Figure 3 shows amperometric peaks for injections of cinnamic acid solutions of increasing concentration (0.5 X 10-3-10 X 10e3 M). The resulting calibration plot (also shown) exhibits linearity up to 4 X lop3 M, with curvature at higher concentrations. The slope of the initial linear portion is 26 nA/mM (correlation coefficient, 0.999). A detection limit of 2 x low4 M can be estimated from the signal-to-noise characteristics (S/N = 3) of the 5 X 10e4 M peak (a>. Hence 0.6 pg can be detected in the 20 ~1 solution. As expected for the surface immobilization of tissue materials, the spinach electrode exhibits long-term stability. No loss of biocatalytic activity was observed over a period of 13 days (with storage at 4°C). A slow decrease in the response
445
POTENTIAL/V 0.5
Fig. 2. Dependence of the response to 2 x lothe solution pH. Conditions as in Fig. lA(b).
1.0
M cinnamic acid on (a) the operating potential and (b)
(5%/day) was observed after that period. The tissue/carbon paste configuration permits rapid renewal of its surface after such loss. Spinach leaves are also rich in the enzyme polyphenol oxidase [121 which catalyzes the conversion of catecholamines to the corresponding quinones. Hence spinach-modified electrodes can be used for monitoring important neurotransmitters in a manner analogous to bioelectrodes based on banana [ll] or eggplant [13]. For example, Fig. 4 illustrates the amperometric response to dopamine, under both flow injection and batch conditions, as recorded at unmodified and spinachcontaining carbon paste electrodes. Unlike the absence of response at the unmodified surface, the tissue electrode offers a rapid, sensitive and reproducible response to dopamine (through reductive monitoring of its quinone product). An injection rate of 90 samples h-’ can be realized from this dynamic behavior. The flow injection detection limit is around 5 X 10m5 M. Hence multianalyte sensing becomes possible by coupling the multienzyme activity of the spinach tissue with the use of different operating potentials. In conclusion, the cinnamic acid hydroxylating system of spinach has been exploited for the design of a new bioelectrode for cinnamic acid. The mixed spinach/carbon paste configuration thus responds rapidly to dynamic changes in
446
TIME
3805 ~li?O5
80I 2
I 4
I 8
I 8
I 10
CONCENTRATION/mM Fig. 3. Flow injection peaks for cinnamic acid solutions of increasing concentration: (a) 5 x 10e4 M, (b) 1 X 10V3 M, (c) 2 X 10e3 M, (d) 3 X lo-’ M, (e) 4 X 10e3 M, (0 5 x 10e3 M, (g) 6 X 10m3 M, (h) 8 x 10m3 M and (i) 1 X lo-* M. Also shown is the resulting calibration plot. Conditions as in Fig. lB(b).
=f 0
IPmin
I I I I I
i,
s
14 $ Pmin
I
TIME Fig. 4. Current-time recordings at (a) the unmodified and (b) the spinach-modified (20% w/w) carbon paste electrodes: (A) repetitive flow injections of a 1 X 10e3 M dopamine; (B) additions of 5 X 10m4 M dopamine. Operating potential, - 0.2 V, other conditions as in Fig. 1.
447
the cinnamic acid concentration. In addition to the quantitation of cinnamic acid, this study illustrates the sensing utility of tissue materials in cases where the pure enzyme is not available and the usefulness of the multienzyme activity of such materials for multianalyte detection. Hence this study further illustrates the versatility of using natural materials for biosensor work. Such behavior should benefit tissue-based assays of other analytes. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13
M. Grayson (Ed.), Encyclopedia of Chemical Technology, Vol. 6, Wiley, New York, 1981, p. 142. W. Greenaway, J. May and F. Whatley, J. Chromatogr., 472 (1989) 393. A. Janczyk and I. Wilczynska-Wojtulewicz, Chem. Anal. (Warsaw), 33, (1988) 535. S. Fujiwara and S. Honda, Anal. Chem., 58 (1985) 1811. M. Bounias, M.H. Daurade and Y. Lizzi, Analysis, 17 (1989) 201. G.a. Rechnitz, Anal. Chim. Acta, 180 (1986) 281. J. Wang, Electroanalysis, 3 (1991) 255. M.A. Arnold, Am. Lab. (Boston), 15(6) (1983) 34. P.M. Nair and L.C. Vining, Phytochemistry, 4 (1965) 161. J. Wang and L.D. Hutchins, Anal. Chem., 57 (1985) 1536. J. Wang and MS. Lin, Anal. Chem., 60 (1988) 1545. A. Sanchez-Ferrer, J. Villalba and F. Garcia-Carmora, Phytochemistry, 28 (1989) 1321. A. Navaratne, M.S. Lin and G.A. Rechnitz, Anal. Chim. Acta, 237 (1990) 107.