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Analytical assessment of the oscillating chemical reactions by use chemiluminescence detection
Talanta 44 (1997) 1463 – 1472
Analytical assessment of the oscillating chemical reactions by use chemiluminescence detection Rafael Jime´nez-Prieto *, Manuel Silva, Dolores Pe´rez-Bendito Department of Analytical Chemistry, Faculty of Sciences, Uni6ersity of Co´rdoba, E-14004 Co´rdoba, Spain Received 27 November 1996; received in revised form 7 February 1997; accepted 10 February 1997
Abstract This paper introduces the chemiluminescence (CL) detection in oscillating reaction-based determinations using the analyte pulse perturbation technique, a straightforward and expeditious approach to deriving quantitative analytical information from oscillating chemical reactions. The behavior of the H2O2 – KSCN – CuSO4-NaOH oscillating system in the presence of luminol was examined by using the proposed detection method and the classical potentiometric technique. Some analytical and practical aspects of both detection systems are discussed. A new analytical method for the determination of vitamin B6 based on the sequential perturbation produced by different amounts of this substance on the oscillating chemical system was also developed in order to assess the potential of CL detection for routine analyses. The calibration curve thus obtained was linear over the range 0.5 – 2.0 mmol of vitamin B6, and the precision and throughput were also quite good (3.04% as RSD and nine samples h − 1, respectively). The proposed method was validated by determining the vitamin in pharmaceutical preparations. © 1997 Elsevier Science B.V. Keywords: Oscillating reaction; Analyte pulse perturbation technique; Chemiluminescence detection; Vitamin B6; Pharmaceutical preparations
1. Introduction Some far-from-equilibrium chemical systems exhibit an oscillating behaviour as a result of their complex mechanisms including and autocatalytic step [1]; such systems are usually referred to as oscillating reactions. The use of these reactions has been the focus of much research in the area of theoretical and experimental chemical kinetics in recent years [2,3]. From the first paper about the quantitative use of oscillating reactions * Corresponding author.
published in 1978 by Tichonova et al. [4], some studies on the analytical applications of chemical oscillators have been developed [5–8]. The analyte pulse perturbation (APP) technique was developed fairly recently in order to facilitate the use of oscillating chemical reactions in routine quantitative analytical determinations [9]. It uses a continuous-flow stirred tank reactor (CSTR) to maintain oscillations for a long time (a few hours), thereby providing an inexhaustible indicator system for successively added analyte pulses; by contrast, classical determinations based on a closed system entail restarting the
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oscillating system for each new determination [4,6,7,10,11]. Research has shown that analytes exhibit various types of interactions with the H2O2 –KSCN– CuSO4 – NaOH oscillation chemical system; thus, sodium thiosulphate [9] and vanillin [12] diminish its oscillating amplitude and period, whereas gallic acid [13] and ascorbic acid [12] increase its amplitude, and reduced glutathione [14] and paracetamol [12] increase its period. These responses are linearly correlated with the analyte concentration in the injected sample—those for glutathione and paracetamol fit a second-order polynomial equation, however. The APP technique also allows the resolution of mixtures of species such as gallic acid and resorcinol [15]. It has become a solidly established tool for quantitative routine determinations including those of vanillin, paracetamol and ascorbic acid in foods and pharmaceutical samples [12]. Oscillations are usually monitored potentiometrically (with a platinum electrode). However, the H2O2 –KSCN – CuSO4 – NaOH system also exhibits oscillations in colour (between yellow and colourless), in the dissolved oxygen concentration [16] and, as shown more recently, in chemiluminescence in the presence of luminol [17,18]. The purpose of this work was to broaden the scope of application of the APP technique in oscillating reaction-based determinations by using a chemiluminescence (CL) detection system. To this end, we compared the performance of potentiometric and CL detection by use of the APP technique and then undertook the determination of vitamin B6 in pharmaceutical preparations by use of this novel detection methodology in farfrom-equilibrium dynamic systems.
2. Experimental
2.1. Reagents All reagents used were supplied by Merck in analytical reagent grade. Bidistilled water was used to prepare the solutions: (1) (1.5 M hydrogen peroxide), (2) (0.15 M sodium thiocyanate, 0.15 M sodium hydroxide and 1.95×10 − 3 M luminol)
and (3) [6.0× 10 − 4 M copper(II) sulphate]. The analyte solutions (sodium thiosulphate, gallic acid and paracetamol) were also prepared in bidistilled water, as were vitamin B6 solutions.
2.2. Apparatus The instrumental set-up used is depicted in Fig. 1. The oscillating assembly comprised a 10-ml glass reaction vessel (CSTR) fitted with a thermostated jacked connected to a Selecta 6000484 thermostat, an Eyela RC-2 magnetic stirrer for homogenization, a Gilson Minipuls-3 4-channel peristaltic pump (3 of the channels were used to deliver the reactants and the fourth to keep the volume of reaction mixture in the CSTR constant), a remote control for operating the pump that allowed instantaneous switching between several preset flow-rates, and a Metrohm Dossimat 665 autoburette for injection of analyte pulses. The CL detection system consisted of a Hitachi F-2000 fluorescence spectrophotometer the sample compartment of which was used to accommodate the CSTR and magnetic stirrer, and a PC-AT 12 MHz compatible computer equipped with a PC-Multilab PCL-812PG 12-bit analog-todigital converter (ADC) for data acquisition and processing. Poly(vinyl chloride) pumping tubes and PTFE tubing for the manifold were also used.
2.3. General procedure for CL-based determinations The CSTR, thermostated at 27.5°C, was loaded with the three reagent solutions (1–3), delivered by the peristaltic pump, to a final volume of 9.0 ml, and the mixture was homogenized by the magnetic stirrer. Then, the flow-rate was reset to the working conditions, viz. an overall reactant feed flow-rate of 0.9 ml min − 1 (i.e. 0.3 ml min − 1 through each reactant channel), the reaction products being removed at the same rate. After steady oscillating conditions were obtained, the system was perturbed by injecting micro-analyte volumes. Sodium thiosulphate, gallic acid and paracetamol were assayed in this way for comparison to potentiometric detection. Changes in the oscillation amplitude and period by effect of the perturbations
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Fig. 1. Experimental set-up for implementation of CL oscillating reaction-based determinations.
were used as measurements in order to construct pertinent calibration graphs. Data were acquired and processed by using software developed by the authors in Microsoft QuickBasic v. 4.0 language.
2.4. CL determination of 6itamin B6 The CSTR, thermostated at 25°C, was filled, in this sequence, with 2.1 ml of 1.0 M hydrogen peroxide, 1.4 ml of 0.1 M sodium thiocynate, 1.05 ml of 0.1 M sodium hydroxide, 1.4 ml of 1.0× 10 − 3 M copper sulphate and 1.0 ml of 7.0×10 − 3 M luminol, the mixture being homogenized by magnetic stirring. Without delay, the peristaltic pump was actuated to deliver the three reactant streams — the overall feed stream was 0.6 M in H2O2, 4.0 ×10 − 2 M in NaSCN, 3.0× 10 − 2 M in NaOH, 2.0 ×10 − 4 M in CuSO4 and 1.0× 10 − 3 M in luminol — at a constant flow-rate of 1.8 ml min − 1. As soon as the signal and time increments for the oscillations levelled off, variable volumes (a few microlitres) of sample or standard containing also variable amounts of vitamin B6 in the rage 0.5 – 20 mmol were sequentially injected. Perturbations decreased the amplitude of the oscillat-
ing cycle by a factor proportional to the amount of vitamin B6 injected. After the system response was recorded, the flow-rate delivered by the pump was raised to 4.65 ml min − 1 for 150 s in order to expedite restoration of the steady state. Then, the initial flow-rate (1.8 ml min − 1) was reset—such an abrupt rise was facilitated by the fact that the oscillating state was not lost under the working conditions used. After the steady state was regained, the system was ready for a new determination.
3. Results and discussion The copper(II)-catalysed oscillating reaction between hydrogen peroxide and sodium thiocyanate has been widely studied by Orba´n in 1986 and 1990 and Luo et al. in 1988 [16,19,20]; who propose that the mechanism for this oscillating reaction consists of 30 kinetic steps involving 26 independent variables [21]. The centerpiece of the mechanism of this reaction, which contains the key of the oscillation, is the positive and negative feedback loops on which the autocatalytic process
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Table 1 Effect of experimental variables on the oscillating system, with potentiometric and CL detection Variable
Range
Measured parameter Amplitude
H2O2 NaSCN CuSO4 NaOH Temperature Luminol
0.4–0.8 M (3.0–9.0)×10−2 (1.0–3.0)×10−4 (1.0–7.0)×10−2 20–40°C (0.5–2.5)×10−3
M M M M
Selected value Period
Ea
CL
Ea
CL
¡ ¡ ¡ :cons. —
¡ ¡
¡ ¡ ¡ —
¡ ¡ ¡
0.5 M 5.0×10−2 2.0×10−4 5.0×10−2 27.5°C 6.5×10−4
M M M M
E, potentiometric detection. Arrows pointing upward and downward indicate increase or decrease, respectively, in the oscillating amplitude and period with increase in the variable concerned over the stated range. a Data from Jime´nez-Prieto et al. [9].
relies. Thus, the positive feedback loop produces the yellow superoxy copper(I) complex: OS(O)CN* +Cu + [SCN − ]n OS(O)CN − +Cu2 + +nSCN − H2O2 + Cu2 + +OH − HO2 −Cu(I)yellow + H2O which disappears in the negative feedback loop: HO2 −Cu(I)yellow +nSCN − Cu + [SCN − ]n +HO*2 the concentration of species Cu + [SCN − ]n being crucial for this feedback network. The light-producing reactions of luminol have been the subject of much research and are thus widely documented [22 – 25]. However, oscillating chemiluminescence (viz. that produced by addition of luminol to the H2O2 – KSCN–CuSO4 – NaOH oscillating system) is an intriguing phenomenon that was only recently described in the literature [17]. Although the information gathered from the few studies carried out so far on the interaction of luminol with this oscillating system necessitates some refining [18], it can be ascribed as: HO2 −Cu(I)yellow +LumH − Cu2 + +2OH − +Lum* −
Such a peculiar oscillating system has not yet been used for analytical determinations. In this work, we examined its analytical performance in combination with the analyte pulse perturbation (APP) technique and compared it with that of potentiometric detection. As a result, a new analytical method for the determination of vitamin B6 in pharmaceutical preparations was developed to validate the proposed use of CL oscillating-reaction based methods for routine analyses.
3.1. CL 6s potentiometric detection of oscillating chemical reactions The interaction of luminol with the H2O2 – KSCN–CuSO4 –NaOH oscillating system was examined with a view to its subsequent use for developing analytical determinations in combination with the APP technique. Thus, the effect of experimental variables potentially influencing the system performance was thoroughly assessed and the results compared with those obtained with potentiometric detection. The oscillating amplitude and period were the two measured parameters used in both cases. Table 1 shows the results of this comparative study. As can be seen, the oscillation period exhibited a similar behaviour with both potentiometric and CL detection; however, the oscillation
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Fig. 2. Time course of oscillations for the H2O2 – KSCN – CuSO4 – NaOH system in the absence and presence of perturbations with (A) and (B) sodium thiosulphate, (C) and (D) gallic acid, and (E) and (F) paracetamol, with potentiometric and CL detection in the presence of luminol.
amplitude response was different for hydrogen peroxide and temperature. The optimum value for each variable in the CL detection study was selected with three criteria in mind, namely: (a) maximizing the stability of the oscillating system over time; (b) maximizing the oscillation amplitude; and (c) ensuring that the oscillation period would allow the effect of perturbations on it to be accurately determined. The high similarity in the behaviour of the oscillating period – the most important feature of an oscillating system whatever the detection system used—in relation to changes in the experimental variables suggests a coupled interaction between luminol and the oscillating system. In fact, considering that oscillations coincide in colour (between yellow and colourless) and in CL, then one can assume the yellow superoxy copper(I) complex to act as a catalyst in the oxidation of luminol by
hydrogen peroxide. On the other hand, the differences observed for the hydrogen peroxide concentration and temperature in comparing the oscillation amplitude responses can be ascribed to a substantial effect of these variables on the optimization of the CL luminol reaction—the stability of the oscillating system over time is compromised in no way, however. The similar effects of copper on the oscillation amplitude and period with both potentiometric and CL detection, and hence the seemingly null influence of this variable on the CL luminol reaction—despite its catalytic effect on it—can be ascribed to the high concentration of copper in the reaction medium: the reaction was thus poisoned, so increasing the copper concentration decreased the CL amplitude, similarly as in potentiometric detection. Under the selected experimental conditions, the oscillating curve was perturbed by using several
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Table 2 Comparison of the performance of CL and potentiometric detection in the oscillating reaction-base determination of thiosulphate, gallic acid and paracetamol Analyte
Determination method CL detection
Potentiometric detection Period
Amplitudea
Perioda
0.15 2.5e
1.0 – 18 0.28 0.71f
2.0 –18 0.73 1.33f
Period
Amplitudeb
0.20 2.1g
0.075 – 2.0 0.022 2.1h 14
Thiosulphate
Amplitude
Dynamic linear range (mmol) Detection limit (mmol) Precision (RSD)d (%) Throughput (samples h−1)
0.10 3.5e
Gallic acid
Amplitude
Dynamic linear range Detection limit (mmol) Precision (RSD)d (%) Throughput (samples h−1)
0.15 4.2g
Paracetamol
Amplitude
Periodc
Dynamic linear range Detection limit (mmol) Precision (RSD)d (%) Throughput (samples h−1)
0.5–7.0 0.13 2.27i 7
0.5 – 6.0 0.15 0.95i 8
0.4 – 3.0
10
10 – 12
0.6 – 1.9
11
a
Data from Jime´nez-Prieto et al. [9]. Data from Jime´nez-Prieto et al. [13]. c Data from Jime´nez-Prieto et al. [12]. d Relative standard deviation. From 11 determinations of e 1 and f 10 mmol of thiosulphate, b
amounts of various analytes, viz. sodium thiosulphate, gallic acid and paracetamol, which were chosen because each responded differently to perturbations of the oscillating system with potentiometric detection and thus afforded comparison with CL detection. Fig. 2 shows the typical responses thus obtained and their potentiometric counterparts. The responses were analysed in terms of the oscillation amplitude and period of the perturbed system for each analyte tested. Table 2 gives the analytical figures of merit for the proposed method and those obtained with potentiometric detection. Detection limits were taken to be the analyte concentrations giving analytical signals equal to three times the standard deviation (n=30) of the oscillation amplitude or period in the absence of perturbation (blank) [26]; also, the throughput was calculated from the time needed for the system to recover after each perturbation.
g
0.75 and
h
0.3 mmol of gallic acid, and i 2 mmol of paracetamol.
The results shown in Fig. 2 and Table 2 allow one to draw several interesting conclusions, namely: 1. As a rule, the detection limit and dynamic linear range was similar or better with CL detection except for gallic acid. This analyte exhibited a peculiar behaviour; the large increase in the amplitude with potentiometric detection (Fig. 2C) was more likely the result of the electroanalytical properties of this compound, which presumably forms a complex with copper in the medium [13], than of its interaction with the oscillating system since a perturbation by itself can never increase the oscillation amplitude. In this respect, CL detection is a more reliable means for assessing interactions between analytes and the oscillating system. On the other hand, the reactivity sequence obtained for the analytes (sodium
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thiosulphate \ gallic acid\ paracetamol) may be directly related to their reducing power. 2. CL detection provided poorer precision (as RSD) than potentiometric detection as the likely result of instrumental differences. Thus, the sharp change in CL signals (Fig. 2) results in some inaccuracy in locating its maximum. While the acquisition rate was more than adequate (the maximum A/D sampling rate was 30 kHz), the performance was constrained by the spectrofluorimeter’s time constant, which directly influenced the analog signal provided by the instrument.
3.2. New approach to analytical determinations: the CL quantitation of 6itamin B6 After both detection methods were assessed, the potential of CL oscillating reaction-based determinations for routine analyses was evaluated with the determination of vitamin B6 in pharmaceutical preparations. The addition of variable amounts of vitamin B6 to the CSTR where the H2O2 – KSCN–CuSO4 – NaOH-luminol oscillating reaction was developed decreased the amplitude of the cycles following the perturbation to an extent proportional to the analyte concentration in the sample. Fig. 3 shows a typical oscillation sequence obtained for the
Fig. 3. Typical profiles for the proposed CL oscillating reaction in the presence and absence of a vitamin B6 perturbation. Arrows indicate the times at which oscillations were perturbed. Zones A –C correspond to the oscillating steady state (A), the response of the oscillating system to the perturbation (B), and (C) recovery following the perturbation.
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proposed chemical system in the absence and presence of vitamin B6 perturbations. Fig. 3 shows the system oscillating in the steady state (zone A), its response to the perturbations (zone B) and the situation after the steady state conditions were restored (zone C).
3.2.1. Influence of experimental 6ariables The effects of the variables potentially influencing the behaviour of the oscillating system were thoroughly studied in the presence and absence of perturbations. The measured parameter used for this purpose was the ratio of the oscillating amplitude before and after each injection. The optimum value of each variable met the following two conditions: (a) it allowed the system to oscillate undisruptedly and to recover its steady state after each perturbation; and (b) the system response to vitamin B6 perturbations was maximal and so was the sensitivity of the method. Fig. 4 illustrates the influence of the different variables studied—their optimum values are given in Section 2. Some selected values did not correspond to the maximum value of the measured parameter (e.g. 2.0×10 − 2 and 1.5×10 − 2 M as the concentration of NaSCN and NaOH, respectively) because a compromise between sensitivity in the determination of vitamin B6 and stability of the oscillating system must be made. Thus, lower concentrations of both reactants caused the system to lose some stability on each injection. One other influential variable—not shown in Fig. 4—was the reactant feed rate, the effect of which was examined over the range 0.44–5.31 ml min − 1. The oscillation amplitude of both the unperturbed and the perturbed system were found to decrease with increase in this rate, with no net effect on the amplitude ratio. The oscillating period also decreased with increase in the feed rate. Because the system response was not affected, the optimum flow-rate value was taken to be that resulting in an appropriate oscillation period in the steady state, viz. 1.8 ml min − 1. On the other hand, the flow-rate used to restore the steadystate conditions after each perturbation was the highest possible in order to shorten the restoration time with no loss of the oscillating state –
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Fig. 4. Influence of the concentrations of hydrogen peroxide (A), copper sulphate (B), sodium thiocyanate (C), sodium hydroxide (D) and luminol (E), and of temperature (F), on the oscillating reaction in the presence of vitamin B6. Arrows indicate the selected values for the variables.
not even with the most concentrated sample. As shown in the Experimental section, such a rate was set at 4.65 ml min − 1 and applied for 150 s.
3.2.2. Determination of 6itamin B6 The oscillating system was perturbed with microvolumes of sample containing variable amounts of vitamin B6 (pyridoxine) between 0.5 and 20 mmol as described in Section 2. The measured parameter was the amplitude of the second response cycle (see zone C in Fig. 3). A plot of Table 3 Analytical figures of merit of the determination of vitamin B6 with the proposed CL method Dynamic linear range (mmol) Detection limit (mmol) Precision (RSD) (%)a Throughput (samples h−1) a
0.5 – 20 0.1 3.04 9
From 11 determinations of 5 mmol of vitamin B6.
oscillation amplitude against analyte concentration was linear throughout the range studied. Table 3 gives the analytical figures of merit for the determination. As can be seen, the proposed method is quite precise. Its throughput was calculated from the time needed for the system to regain its initial state after each perturbation; the result, 9 samples h − 1, confirms the goodness of the APP technique for the intended purpose. The potential interfering effects of structurally related species was also investigated. To this end, a fixed amount of pyridoxine (5 mmol) was injected jointly with variable amounts of pyridoxylamine, pyridoxal-5-phosphate, pyridoxal and 4-pyridoxic acid. All were found to interfere with the determination—particularly pyridoxamine, above an interferent-to-analyte ratio as low as 0.0015; this can be ascribed to its reducing power and the presence of primary amino groups in its structure, which probably had a marked dis-
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Table 4 Determination of vitamin B6 in pharmaceutical preparations Trade name
Nominal con- Founda (mg tent (mg g−1) g−1)
Error (%)
Paciumb Vertigumc Taurobetinad Trofimilinae
33.3 275.1 112.4 387.1
−1.1 −10.1 −7.4 −4.5
339 9 2479 13 10496 370930
a
Average of three individual determinations9 S.D. Other ingredients (in mg g−1) included: b diazepam (16.7); c dixirazine (41.3), inositol nicotinate (275.1), wheat starch (33) and lactose (317.7); d taurine (224.7), adenosine phosphate dipotassium salt (11.2), uridine phosphate dipotassium salt (22.5), vitamin B12 (0.22) and sucrose (343.8); e 129 mg of total homogenate of neurohomologous phospholipids of cortical grey matter equivalent to 2.55 mg of lipid phosphorus in terms of its components (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, diphosphoinositic acid and sphingomyelin).
turbing effect on the oscillating reaction [21]. These results are of a high interest with a view to developing sensitive determination methods for various pyridoxine derivatives, which could be used as a useful alternative to other liquid chromatographic detection systems.
3.2.3. Applications We assessed the potential of the proposed method for determining vitamin B6 in real samples of special interest. For this purpose, we selected several pharmaceutical preparations including pyridoxine as the active principle or a secondary ingredient. The nominal value in each preparation was used as reference —with provision for potential errors made in the manufacturing process — because the basic aim was simply to demonstrate the usefulness of the method for routine analyses of this type of sample. The sample preparation procedure was quite simple. Thus, solutions were all made in bidistilled water and the amount of sample used was dictated by its pyridoxine concentration. In those cases were dissolution was incomplete, the sole additional treatment required was filtration to a clear solution, an appropriate aliquot of which was subjected to the above-described procedure. The Trofimilina sample, a phospholipid ho-
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mogenate and hence of oily texture, was dissolved in chloroform and then extracted for vitamin B6 with bidistilled water. The results obtained are given in Table 4. As can be seen, they were quite consistent with the nominal values. No separation of the species accompanying vitamin B6 in the preparations studied was needed. The simplicity, expeditiousness and precision of the proposed method as regards sample pretreatment, and the good results it provides, make oscillating chemical systems and chemiluminescence determination useful couples for determining species in real samples of interest.
Acknowledgements The authors gratefully acknowledge financial support from the Spanish Direccio´n General Interministerial de Ciencia y Tecnologı´a (DGICyT) for the realization of this work as part of Project PB91-0840.
References [1] A.S. Tomlin, Anal. Proc. 30 (1993) 3072. [2] R.J. Field and M. Burger, Oscillation and Travelling Waves in Chemical Systems. Wiley, New York, 1985. [3] P. Gray and S.K. Scott, Chemical Oscillations and Instabilities. Clarendon Press, Oxford, 1990. [4] L.P. Tichonova, L.N. Zakrevskaya, K.B. Yatsimirskii, Talanta 33 (1978) 1991. [5] Z. Qinyuang, J. Chen, Fenxi Shiyanshi 7 (1988) 4. [6] M. Jiang, Y. Li, X. Zhuo, Z. Zhao, H. Wang, J. Mo, Anal. Chim. Acta 236 (1990) 411. [7] K.B. Yatsimirskii, P.E. Strizhak, T.S. Ivaschenko, Talanta 40 (1993) 1227. [8] R.T. Echols, M.K. Caroll, J.F. Tyson, Anal. Proc. 32 (1995) 3. [9] R. Jime´nez-Prieto, M. Silva, D. Pe´rez-Bendito, Anal. Chem. 67 (1995) 729. [10] Y. Liang, R. Yu, Gaodeng Xuexiao Huaxue Xuebao 9 (1988) 881. [11] Q. Zhang, J. Chen, Fenxi Shiyanshi 7 (1988) 4. [12] R. Jime´nez-Prieto, M. Silva, D. Pe´rez-Bendito, Analyst 122 (1997) 287. [13] R. Jime´nez-Prieto, M. Silva, D. Pe´rez-Bendito, Anal. Chim. Acta 321 (1996) 53. [14] R. Jime´nez-Prieto, M. Silva, D. Pe´rez-Bendito, Analyst 121 (1996) 563.
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[15] R. Jime´nez-Prieto, M. Silva, D. Pe´rez-Bendito, Anal. Chim. Acta 334 (1996) 323. [16] M. Orba´n, J. Am. Chem. Soc. 108 (1986) 6893. [17] J. Amrehn, P. Resch, F.W. Schneider, J. Phys. Chem. 92 (1988) 3318. [18] S. Sattar, I.R. Epstein, J. Phys. Chem. 94 (1990) 275. [19] M. Orba´n, React. Kinet. Catal. Lett. 42 (1990) 343. [20] Y. Luo, K. Kustin, I.R. Epstein, Inorg. Chem. 27 (1988) 2489. [21] Y. Luo, M. Orba´n, K. Kustin, I.R. Epstein, J. Am.
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Chem. Soc. 111 (1989) 4541. [22] K.D. Gundermann and F. McCapra, in Chemiluminescence in Organic Chemistry. Springer-Verlag, Berlin, 1987, Ch. VII. [23] A. Townshend, Anal. Proc. 22 (1985) 370. [24] J.S. Lancaster, P.J. Worsfold, Anal. Proc. 26 (1989) 362. [25] L.J. Kricka, P.E. Stanley, J. Biolumin. Chemilumin. 6 (1991) 203. [26] G.L. Long, J.D. Winefordner, Anal. Chem. 55 (1983) 712A.
Analytical assessment of the oscillating chemical reactions by use chemiluminescence detection Rafael Jime´nez-Prieto *, Manuel Silva, Dolores Pe´rez-Bendito Department of Analytical Chemistry, Faculty of Sciences, Uni6ersity of Co´rdoba, E-14004 Co´rdoba, Spain Received 27 November 1996; received in revised form 7 February 1997; accepted 10 February 1997
Abstract This paper introduces the chemiluminescence (CL) detection in oscillating reaction-based determinations using the analyte pulse perturbation technique, a straightforward and expeditious approach to deriving quantitative analytical information from oscillating chemical reactions. The behavior of the H2O2 – KSCN – CuSO4-NaOH oscillating system in the presence of luminol was examined by using the proposed detection method and the classical potentiometric technique. Some analytical and practical aspects of both detection systems are discussed. A new analytical method for the determination of vitamin B6 based on the sequential perturbation produced by different amounts of this substance on the oscillating chemical system was also developed in order to assess the potential of CL detection for routine analyses. The calibration curve thus obtained was linear over the range 0.5 – 2.0 mmol of vitamin B6, and the precision and throughput were also quite good (3.04% as RSD and nine samples h − 1, respectively). The proposed method was validated by determining the vitamin in pharmaceutical preparations. © 1997 Elsevier Science B.V. Keywords: Oscillating reaction; Analyte pulse perturbation technique; Chemiluminescence detection; Vitamin B6; Pharmaceutical preparations
1. Introduction Some far-from-equilibrium chemical systems exhibit an oscillating behaviour as a result of their complex mechanisms including and autocatalytic step [1]; such systems are usually referred to as oscillating reactions. The use of these reactions has been the focus of much research in the area of theoretical and experimental chemical kinetics in recent years [2,3]. From the first paper about the quantitative use of oscillating reactions * Corresponding author.
published in 1978 by Tichonova et al. [4], some studies on the analytical applications of chemical oscillators have been developed [5–8]. The analyte pulse perturbation (APP) technique was developed fairly recently in order to facilitate the use of oscillating chemical reactions in routine quantitative analytical determinations [9]. It uses a continuous-flow stirred tank reactor (CSTR) to maintain oscillations for a long time (a few hours), thereby providing an inexhaustible indicator system for successively added analyte pulses; by contrast, classical determinations based on a closed system entail restarting the
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oscillating system for each new determination [4,6,7,10,11]. Research has shown that analytes exhibit various types of interactions with the H2O2 –KSCN– CuSO4 – NaOH oscillation chemical system; thus, sodium thiosulphate [9] and vanillin [12] diminish its oscillating amplitude and period, whereas gallic acid [13] and ascorbic acid [12] increase its amplitude, and reduced glutathione [14] and paracetamol [12] increase its period. These responses are linearly correlated with the analyte concentration in the injected sample—those for glutathione and paracetamol fit a second-order polynomial equation, however. The APP technique also allows the resolution of mixtures of species such as gallic acid and resorcinol [15]. It has become a solidly established tool for quantitative routine determinations including those of vanillin, paracetamol and ascorbic acid in foods and pharmaceutical samples [12]. Oscillations are usually monitored potentiometrically (with a platinum electrode). However, the H2O2 –KSCN – CuSO4 – NaOH system also exhibits oscillations in colour (between yellow and colourless), in the dissolved oxygen concentration [16] and, as shown more recently, in chemiluminescence in the presence of luminol [17,18]. The purpose of this work was to broaden the scope of application of the APP technique in oscillating reaction-based determinations by using a chemiluminescence (CL) detection system. To this end, we compared the performance of potentiometric and CL detection by use of the APP technique and then undertook the determination of vitamin B6 in pharmaceutical preparations by use of this novel detection methodology in farfrom-equilibrium dynamic systems.
2. Experimental
2.1. Reagents All reagents used were supplied by Merck in analytical reagent grade. Bidistilled water was used to prepare the solutions: (1) (1.5 M hydrogen peroxide), (2) (0.15 M sodium thiocyanate, 0.15 M sodium hydroxide and 1.95×10 − 3 M luminol)
and (3) [6.0× 10 − 4 M copper(II) sulphate]. The analyte solutions (sodium thiosulphate, gallic acid and paracetamol) were also prepared in bidistilled water, as were vitamin B6 solutions.
2.2. Apparatus The instrumental set-up used is depicted in Fig. 1. The oscillating assembly comprised a 10-ml glass reaction vessel (CSTR) fitted with a thermostated jacked connected to a Selecta 6000484 thermostat, an Eyela RC-2 magnetic stirrer for homogenization, a Gilson Minipuls-3 4-channel peristaltic pump (3 of the channels were used to deliver the reactants and the fourth to keep the volume of reaction mixture in the CSTR constant), a remote control for operating the pump that allowed instantaneous switching between several preset flow-rates, and a Metrohm Dossimat 665 autoburette for injection of analyte pulses. The CL detection system consisted of a Hitachi F-2000 fluorescence spectrophotometer the sample compartment of which was used to accommodate the CSTR and magnetic stirrer, and a PC-AT 12 MHz compatible computer equipped with a PC-Multilab PCL-812PG 12-bit analog-todigital converter (ADC) for data acquisition and processing. Poly(vinyl chloride) pumping tubes and PTFE tubing for the manifold were also used.
2.3. General procedure for CL-based determinations The CSTR, thermostated at 27.5°C, was loaded with the three reagent solutions (1–3), delivered by the peristaltic pump, to a final volume of 9.0 ml, and the mixture was homogenized by the magnetic stirrer. Then, the flow-rate was reset to the working conditions, viz. an overall reactant feed flow-rate of 0.9 ml min − 1 (i.e. 0.3 ml min − 1 through each reactant channel), the reaction products being removed at the same rate. After steady oscillating conditions were obtained, the system was perturbed by injecting micro-analyte volumes. Sodium thiosulphate, gallic acid and paracetamol were assayed in this way for comparison to potentiometric detection. Changes in the oscillation amplitude and period by effect of the perturbations
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Fig. 1. Experimental set-up for implementation of CL oscillating reaction-based determinations.
were used as measurements in order to construct pertinent calibration graphs. Data were acquired and processed by using software developed by the authors in Microsoft QuickBasic v. 4.0 language.
2.4. CL determination of 6itamin B6 The CSTR, thermostated at 25°C, was filled, in this sequence, with 2.1 ml of 1.0 M hydrogen peroxide, 1.4 ml of 0.1 M sodium thiocynate, 1.05 ml of 0.1 M sodium hydroxide, 1.4 ml of 1.0× 10 − 3 M copper sulphate and 1.0 ml of 7.0×10 − 3 M luminol, the mixture being homogenized by magnetic stirring. Without delay, the peristaltic pump was actuated to deliver the three reactant streams — the overall feed stream was 0.6 M in H2O2, 4.0 ×10 − 2 M in NaSCN, 3.0× 10 − 2 M in NaOH, 2.0 ×10 − 4 M in CuSO4 and 1.0× 10 − 3 M in luminol — at a constant flow-rate of 1.8 ml min − 1. As soon as the signal and time increments for the oscillations levelled off, variable volumes (a few microlitres) of sample or standard containing also variable amounts of vitamin B6 in the rage 0.5 – 20 mmol were sequentially injected. Perturbations decreased the amplitude of the oscillat-
ing cycle by a factor proportional to the amount of vitamin B6 injected. After the system response was recorded, the flow-rate delivered by the pump was raised to 4.65 ml min − 1 for 150 s in order to expedite restoration of the steady state. Then, the initial flow-rate (1.8 ml min − 1) was reset—such an abrupt rise was facilitated by the fact that the oscillating state was not lost under the working conditions used. After the steady state was regained, the system was ready for a new determination.
3. Results and discussion The copper(II)-catalysed oscillating reaction between hydrogen peroxide and sodium thiocyanate has been widely studied by Orba´n in 1986 and 1990 and Luo et al. in 1988 [16,19,20]; who propose that the mechanism for this oscillating reaction consists of 30 kinetic steps involving 26 independent variables [21]. The centerpiece of the mechanism of this reaction, which contains the key of the oscillation, is the positive and negative feedback loops on which the autocatalytic process
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Table 1 Effect of experimental variables on the oscillating system, with potentiometric and CL detection Variable
Range
Measured parameter Amplitude
H2O2 NaSCN CuSO4 NaOH Temperature Luminol
0.4–0.8 M (3.0–9.0)×10−2 (1.0–3.0)×10−4 (1.0–7.0)×10−2 20–40°C (0.5–2.5)×10−3
M M M M
Selected value Period
Ea
CL
Ea
CL
¡ ¡ ¡ :cons. —
¡ ¡
¡ ¡ ¡ —
¡ ¡ ¡
0.5 M 5.0×10−2 2.0×10−4 5.0×10−2 27.5°C 6.5×10−4
M M M M
E, potentiometric detection. Arrows pointing upward and downward indicate increase or decrease, respectively, in the oscillating amplitude and period with increase in the variable concerned over the stated range. a Data from Jime´nez-Prieto et al. [9].
relies. Thus, the positive feedback loop produces the yellow superoxy copper(I) complex: OS(O)CN* +Cu + [SCN − ]n OS(O)CN − +Cu2 + +nSCN − H2O2 + Cu2 + +OH − HO2 −Cu(I)yellow + H2O which disappears in the negative feedback loop: HO2 −Cu(I)yellow +nSCN − Cu + [SCN − ]n +HO*2 the concentration of species Cu + [SCN − ]n being crucial for this feedback network. The light-producing reactions of luminol have been the subject of much research and are thus widely documented [22 – 25]. However, oscillating chemiluminescence (viz. that produced by addition of luminol to the H2O2 – KSCN–CuSO4 – NaOH oscillating system) is an intriguing phenomenon that was only recently described in the literature [17]. Although the information gathered from the few studies carried out so far on the interaction of luminol with this oscillating system necessitates some refining [18], it can be ascribed as: HO2 −Cu(I)yellow +LumH − Cu2 + +2OH − +Lum* −
Such a peculiar oscillating system has not yet been used for analytical determinations. In this work, we examined its analytical performance in combination with the analyte pulse perturbation (APP) technique and compared it with that of potentiometric detection. As a result, a new analytical method for the determination of vitamin B6 in pharmaceutical preparations was developed to validate the proposed use of CL oscillating-reaction based methods for routine analyses.
3.1. CL 6s potentiometric detection of oscillating chemical reactions The interaction of luminol with the H2O2 – KSCN–CuSO4 –NaOH oscillating system was examined with a view to its subsequent use for developing analytical determinations in combination with the APP technique. Thus, the effect of experimental variables potentially influencing the system performance was thoroughly assessed and the results compared with those obtained with potentiometric detection. The oscillating amplitude and period were the two measured parameters used in both cases. Table 1 shows the results of this comparative study. As can be seen, the oscillation period exhibited a similar behaviour with both potentiometric and CL detection; however, the oscillation
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Fig. 2. Time course of oscillations for the H2O2 – KSCN – CuSO4 – NaOH system in the absence and presence of perturbations with (A) and (B) sodium thiosulphate, (C) and (D) gallic acid, and (E) and (F) paracetamol, with potentiometric and CL detection in the presence of luminol.
amplitude response was different for hydrogen peroxide and temperature. The optimum value for each variable in the CL detection study was selected with three criteria in mind, namely: (a) maximizing the stability of the oscillating system over time; (b) maximizing the oscillation amplitude; and (c) ensuring that the oscillation period would allow the effect of perturbations on it to be accurately determined. The high similarity in the behaviour of the oscillating period – the most important feature of an oscillating system whatever the detection system used—in relation to changes in the experimental variables suggests a coupled interaction between luminol and the oscillating system. In fact, considering that oscillations coincide in colour (between yellow and colourless) and in CL, then one can assume the yellow superoxy copper(I) complex to act as a catalyst in the oxidation of luminol by
hydrogen peroxide. On the other hand, the differences observed for the hydrogen peroxide concentration and temperature in comparing the oscillation amplitude responses can be ascribed to a substantial effect of these variables on the optimization of the CL luminol reaction—the stability of the oscillating system over time is compromised in no way, however. The similar effects of copper on the oscillation amplitude and period with both potentiometric and CL detection, and hence the seemingly null influence of this variable on the CL luminol reaction—despite its catalytic effect on it—can be ascribed to the high concentration of copper in the reaction medium: the reaction was thus poisoned, so increasing the copper concentration decreased the CL amplitude, similarly as in potentiometric detection. Under the selected experimental conditions, the oscillating curve was perturbed by using several
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Table 2 Comparison of the performance of CL and potentiometric detection in the oscillating reaction-base determination of thiosulphate, gallic acid and paracetamol Analyte
Determination method CL detection
Potentiometric detection Period
Amplitudea
Perioda
0.15 2.5e
1.0 – 18 0.28 0.71f
2.0 –18 0.73 1.33f
Period
Amplitudeb
0.20 2.1g
0.075 – 2.0 0.022 2.1h 14
Thiosulphate
Amplitude
Dynamic linear range (mmol) Detection limit (mmol) Precision (RSD)d (%) Throughput (samples h−1)
0.10 3.5e
Gallic acid
Amplitude
Dynamic linear range Detection limit (mmol) Precision (RSD)d (%) Throughput (samples h−1)
0.15 4.2g
Paracetamol
Amplitude
Periodc
Dynamic linear range Detection limit (mmol) Precision (RSD)d (%) Throughput (samples h−1)
0.5–7.0 0.13 2.27i 7
0.5 – 6.0 0.15 0.95i 8
0.4 – 3.0
10
10 – 12
0.6 – 1.9
11
a
Data from Jime´nez-Prieto et al. [9]. Data from Jime´nez-Prieto et al. [13]. c Data from Jime´nez-Prieto et al. [12]. d Relative standard deviation. From 11 determinations of e 1 and f 10 mmol of thiosulphate, b
amounts of various analytes, viz. sodium thiosulphate, gallic acid and paracetamol, which were chosen because each responded differently to perturbations of the oscillating system with potentiometric detection and thus afforded comparison with CL detection. Fig. 2 shows the typical responses thus obtained and their potentiometric counterparts. The responses were analysed in terms of the oscillation amplitude and period of the perturbed system for each analyte tested. Table 2 gives the analytical figures of merit for the proposed method and those obtained with potentiometric detection. Detection limits were taken to be the analyte concentrations giving analytical signals equal to three times the standard deviation (n=30) of the oscillation amplitude or period in the absence of perturbation (blank) [26]; also, the throughput was calculated from the time needed for the system to recover after each perturbation.
g
0.75 and
h
0.3 mmol of gallic acid, and i 2 mmol of paracetamol.
The results shown in Fig. 2 and Table 2 allow one to draw several interesting conclusions, namely: 1. As a rule, the detection limit and dynamic linear range was similar or better with CL detection except for gallic acid. This analyte exhibited a peculiar behaviour; the large increase in the amplitude with potentiometric detection (Fig. 2C) was more likely the result of the electroanalytical properties of this compound, which presumably forms a complex with copper in the medium [13], than of its interaction with the oscillating system since a perturbation by itself can never increase the oscillation amplitude. In this respect, CL detection is a more reliable means for assessing interactions between analytes and the oscillating system. On the other hand, the reactivity sequence obtained for the analytes (sodium
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thiosulphate \ gallic acid\ paracetamol) may be directly related to their reducing power. 2. CL detection provided poorer precision (as RSD) than potentiometric detection as the likely result of instrumental differences. Thus, the sharp change in CL signals (Fig. 2) results in some inaccuracy in locating its maximum. While the acquisition rate was more than adequate (the maximum A/D sampling rate was 30 kHz), the performance was constrained by the spectrofluorimeter’s time constant, which directly influenced the analog signal provided by the instrument.
3.2. New approach to analytical determinations: the CL quantitation of 6itamin B6 After both detection methods were assessed, the potential of CL oscillating reaction-based determinations for routine analyses was evaluated with the determination of vitamin B6 in pharmaceutical preparations. The addition of variable amounts of vitamin B6 to the CSTR where the H2O2 – KSCN–CuSO4 – NaOH-luminol oscillating reaction was developed decreased the amplitude of the cycles following the perturbation to an extent proportional to the analyte concentration in the sample. Fig. 3 shows a typical oscillation sequence obtained for the
Fig. 3. Typical profiles for the proposed CL oscillating reaction in the presence and absence of a vitamin B6 perturbation. Arrows indicate the times at which oscillations were perturbed. Zones A –C correspond to the oscillating steady state (A), the response of the oscillating system to the perturbation (B), and (C) recovery following the perturbation.
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proposed chemical system in the absence and presence of vitamin B6 perturbations. Fig. 3 shows the system oscillating in the steady state (zone A), its response to the perturbations (zone B) and the situation after the steady state conditions were restored (zone C).
3.2.1. Influence of experimental 6ariables The effects of the variables potentially influencing the behaviour of the oscillating system were thoroughly studied in the presence and absence of perturbations. The measured parameter used for this purpose was the ratio of the oscillating amplitude before and after each injection. The optimum value of each variable met the following two conditions: (a) it allowed the system to oscillate undisruptedly and to recover its steady state after each perturbation; and (b) the system response to vitamin B6 perturbations was maximal and so was the sensitivity of the method. Fig. 4 illustrates the influence of the different variables studied—their optimum values are given in Section 2. Some selected values did not correspond to the maximum value of the measured parameter (e.g. 2.0×10 − 2 and 1.5×10 − 2 M as the concentration of NaSCN and NaOH, respectively) because a compromise between sensitivity in the determination of vitamin B6 and stability of the oscillating system must be made. Thus, lower concentrations of both reactants caused the system to lose some stability on each injection. One other influential variable—not shown in Fig. 4—was the reactant feed rate, the effect of which was examined over the range 0.44–5.31 ml min − 1. The oscillation amplitude of both the unperturbed and the perturbed system were found to decrease with increase in this rate, with no net effect on the amplitude ratio. The oscillating period also decreased with increase in the feed rate. Because the system response was not affected, the optimum flow-rate value was taken to be that resulting in an appropriate oscillation period in the steady state, viz. 1.8 ml min − 1. On the other hand, the flow-rate used to restore the steadystate conditions after each perturbation was the highest possible in order to shorten the restoration time with no loss of the oscillating state –
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Fig. 4. Influence of the concentrations of hydrogen peroxide (A), copper sulphate (B), sodium thiocyanate (C), sodium hydroxide (D) and luminol (E), and of temperature (F), on the oscillating reaction in the presence of vitamin B6. Arrows indicate the selected values for the variables.
not even with the most concentrated sample. As shown in the Experimental section, such a rate was set at 4.65 ml min − 1 and applied for 150 s.
3.2.2. Determination of 6itamin B6 The oscillating system was perturbed with microvolumes of sample containing variable amounts of vitamin B6 (pyridoxine) between 0.5 and 20 mmol as described in Section 2. The measured parameter was the amplitude of the second response cycle (see zone C in Fig. 3). A plot of Table 3 Analytical figures of merit of the determination of vitamin B6 with the proposed CL method Dynamic linear range (mmol) Detection limit (mmol) Precision (RSD) (%)a Throughput (samples h−1) a
0.5 – 20 0.1 3.04 9
From 11 determinations of 5 mmol of vitamin B6.
oscillation amplitude against analyte concentration was linear throughout the range studied. Table 3 gives the analytical figures of merit for the determination. As can be seen, the proposed method is quite precise. Its throughput was calculated from the time needed for the system to regain its initial state after each perturbation; the result, 9 samples h − 1, confirms the goodness of the APP technique for the intended purpose. The potential interfering effects of structurally related species was also investigated. To this end, a fixed amount of pyridoxine (5 mmol) was injected jointly with variable amounts of pyridoxylamine, pyridoxal-5-phosphate, pyridoxal and 4-pyridoxic acid. All were found to interfere with the determination—particularly pyridoxamine, above an interferent-to-analyte ratio as low as 0.0015; this can be ascribed to its reducing power and the presence of primary amino groups in its structure, which probably had a marked dis-
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Table 4 Determination of vitamin B6 in pharmaceutical preparations Trade name
Nominal con- Founda (mg tent (mg g−1) g−1)
Error (%)
Paciumb Vertigumc Taurobetinad Trofimilinae
33.3 275.1 112.4 387.1
−1.1 −10.1 −7.4 −4.5
339 9 2479 13 10496 370930
a
Average of three individual determinations9 S.D. Other ingredients (in mg g−1) included: b diazepam (16.7); c dixirazine (41.3), inositol nicotinate (275.1), wheat starch (33) and lactose (317.7); d taurine (224.7), adenosine phosphate dipotassium salt (11.2), uridine phosphate dipotassium salt (22.5), vitamin B12 (0.22) and sucrose (343.8); e 129 mg of total homogenate of neurohomologous phospholipids of cortical grey matter equivalent to 2.55 mg of lipid phosphorus in terms of its components (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, diphosphoinositic acid and sphingomyelin).
turbing effect on the oscillating reaction [21]. These results are of a high interest with a view to developing sensitive determination methods for various pyridoxine derivatives, which could be used as a useful alternative to other liquid chromatographic detection systems.
3.2.3. Applications We assessed the potential of the proposed method for determining vitamin B6 in real samples of special interest. For this purpose, we selected several pharmaceutical preparations including pyridoxine as the active principle or a secondary ingredient. The nominal value in each preparation was used as reference —with provision for potential errors made in the manufacturing process — because the basic aim was simply to demonstrate the usefulness of the method for routine analyses of this type of sample. The sample preparation procedure was quite simple. Thus, solutions were all made in bidistilled water and the amount of sample used was dictated by its pyridoxine concentration. In those cases were dissolution was incomplete, the sole additional treatment required was filtration to a clear solution, an appropriate aliquot of which was subjected to the above-described procedure. The Trofimilina sample, a phospholipid ho-
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mogenate and hence of oily texture, was dissolved in chloroform and then extracted for vitamin B6 with bidistilled water. The results obtained are given in Table 4. As can be seen, they were quite consistent with the nominal values. No separation of the species accompanying vitamin B6 in the preparations studied was needed. The simplicity, expeditiousness and precision of the proposed method as regards sample pretreatment, and the good results it provides, make oscillating chemical systems and chemiluminescence determination useful couples for determining species in real samples of interest.
Acknowledgements The authors gratefully acknowledge financial support from the Spanish Direccio´n General Interministerial de Ciencia y Tecnologı´a (DGICyT) for the realization of this work as part of Project PB91-0840.
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