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Determination of abscisic acid by cathodic stripping square wave voltammetry
Talanta 44 (1997) 1783 – 1792
Determination of abscisic acid by cathodic stripping square wave voltammetry P. Herna´ndez, M. Dabrio-Ramos, F. Pato´n, Y. Ballesteros, L. Herna´ndez * Departamento de Quı´mica Analı´tica, Uni6ersidad Auto´noma de Madrid, Madrid, Spain Received 10 October 1996; received in revised form 15 January 1997; accepted 12 February 1997
Abstract In this paper a study is accomplished on behavior in a mercury electrode, of the phytohormone abscisic acid and of the conditions of accumulation in a HMDE. A mechanism is proposed of reduction based on its electrochemical behavior and proving the product of the reduction through mass spectrometry of bulks. A method is proposed for the determination of Abscisic acid (ABA) with a quantification limit of 58 ng ml − 1. The procedure is applied wing determination of ABA in pears through the combination of high performance liquid chromatography (HPLC) with electrochemical quantification. © 1997 Elsevier Science B.V. Keywords: Abscisic acid; Phytohormone; Mercury electrode
1. Introduction Plant hormones or phytohormones are regulators that are produced by the plant itself. In low concentrations, they control the physiological processes. Phytohormones are classified into four groups: auxines, gibberellines, cytoquinines and inhibitors. Abscisic acid (ABA) belongs to the latter, which is quite different from the other groups of plant growth substances, as it inhibits or delays the physiological or biochemical process of plants [1]. Abscisic acid was isolated from young cotton fruit (Gossypium hirsutum) by a research group led initially by Corns and subsequently by Addi-
cot [2]. Its structure, shown in Fig. 1, was determined by Ohkuma and associates [2]. The levels of concentration of this inhibitor vary considerably from one plant species to another and depend on environmental conditions [1]. Philips and Waring [2] studied this variation in Acer pseudoplatanus buds and leaves, finding
* Corresponding author. 0039-9140/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 9 - 9 1 4 0 ( 9 7 ) 0 0 0 4 9 - 0
Fig. 1. Molecular structure of abscisic acid (ABA).
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minimum levels of ABA in June in the terminal buds and maxima in winter, precisely the opposite of the leaves. This suggests that the inhibitor formed in the leaves in summer shifts to the terminal buds at the start of autumn. Hormonal action depends on the hormone concentration, the presence and characteristics of the receptor and the elements involved in the signal transduction chain. The hormone-receiver complex, i.e., the activated receiver, is the first link in the signal transduction chain, which provokes a primary response. This in turn begins a series of changes which, as a whole, make up the complex physiological response. In the case of abscisic acid, there is no single biological activity, which instead is manifested in several types of physiological response. The ABA has a great inhibiting effect on the growth of many types of plants. It is not the only growth-inhibiting substance, but it is the only one that is non-toxic. A seed treated with ABA, for example, can germinate once it is placed in another medium. With another inhibitor, this seed does not germinate [2]. Seed germination is regulated by the level of ABA and gibberellins (phytohormones that stimulate cell division or prolongation, or both). Gibberellins induce the synthesis of a-amylase, while the ABA inhibits this action, stopping cell division and hence growth [3]. Abscisic acid accelerates the abscission process in flowers and fruit, as well as in the reproductive organs of a wide variety of plant species [4], and ABA is also responsible for the closure of the stomas in the leaves of some plants such as Triticum 6ulgare, thus producing a reduction in transpiration [5]. In addition to plant activity, it has been suggested that abscisic acid inhibits reproduction in insects and the growth of tumours in some types of mice [2]. Given its importance, abscisic acid has been studied widely from an analytic perspective ever since its discovery. The first technique used to identify this acid, at the start of the 1960s, was paper chromatography (PC), which was soon replaced by thin layer chromatography (TLC). Detection was by means of UV spectroscopy because
abscisic acid has an intense, well defined band [2]. As other techniques spread, however, TLC and PC was left as a mere technique for sample prepurification. Gas chromatography (GC) and high performance liquid chromatography (HPLC) began to be used in the 1970s to study abscisic acid, and coupled to mass spectroscopy (MS) are an excellent method of identification [6,7]. The problem in HPLC analysis is the low specificity of the UV detector. GC-MS is a powerful method for identification, but the samples must be cleaned up. The lower limit of detection of ABA is 50 ng in HPLC. Before the analysis, crude samples should be purified by solvent partitioning and preparative TLC or by a short column such as the C18 Sep-pak cartridge [2]. Bioassay and immunoassay has been also used for determination and quantification of abscisic acid, and does not require pre-purification, although the experimental period tends to be very long due to the preparation of the antigen and the antiserum [8]. In recent times, the same techniques used in previous years have continued to be used for the determination and quantification of ABA, although additional techniques such as ELISA have arisen. We have not, however, found any literature on the electrochemical study of this phytohormone. This analysis, discussed in the present paper, is approached using square wave voltammetry (SWV), given that the coupling of an accumulation step to an impulse technique would enable us to obtain detection thresholds in the order of ng ml − 1. The use of an electrochemical measurement provides an additional selectivity which allows one to avoid the needed pre-purification steps used on other methods, such as GC or HPLC-UV. Likewise, the limit of detection obtained in this proposed method for quantification of ABA is similar to those obtained by using other traditional methods.
2. Experimental
2.1. Reagents and equipment
A 0.2 mg ml −1 solution of abscisic acid (ABA) in methanol prepared from the crystallized
P. Herna´ndez et al. / Talanta 44 (1997) 1783–1792
form supplied by SERVA (New York, USA). The solutions must by refrigerated and not exposed to light. Analysis quality reagents. Deionised water using Milliro-MilliQ (Water System) equipment. PAR polarographic analyser model 384B, equipped with a Ag/AgCl/KCl (3M) reference electrode and a platinum counter electrode. This polarograph was used for the square wave and cyclical voltammetry analyses using a hanging drop mercury electrode (HDME). Bioanalytical System Potentiostat BAS 100, equipped with a mercury pool electrode, a saturated calomel electrode as the reference electrode and a platinum electrode as the counter electrode. These were used for a coulometric analysis of the abscisic acid. HEWLETT PACKARD model 5890 Series II gas chromatograph with a HEWLETT PACKARD model 5971A spectrometry detector used to identify the coulometric reaction products. High performance liquid chromatography composed of a GILSON 302 pump connected to a GILSON 802C manometric module, with a GILSON 116 UV detector attached to the unit. The HPLC was used a the preparative separation method in a plant extract for subsequent electrochemical detection.
2.2. Procedure The polarographic cell contains a 10-ml buffer solution at a previously adjusted pH and a known concentration of abscisic acid. The O2 was eliminated by means of a stream of N2 for 5 min in the first measurement and 30 s in the subsequent potential scans. An accumulation time (tac) was applied at an accumulation potential (Eac) with a stirring speed of 400 r.p.m. The polarogram was recorded between −0.4 and − 1.1 V for a mercury drop electrode of 0.0278 cm2 average area, replaced for each measurement, against an Ag/ AgCl 3M electrode and a platinum counter electrode. The equilibrium time was 5 s. In order to establish the measurement conditions, the corresponding variable was modified in each analysis to find the optimum response value,
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establishing initial conditions of: square wave frequency ( f ) at 50 Hz with increments in the step wave potential (E) of 2 mV in each cycle, producing a scan rate of 100 mV s − 1. The pulse amplitude (a) was 20 mV.
2.2.1. Extraction procedure The procedure used was as follows: 51.35 g of pears was pulverised after prior freezing with liquid nitrogen. Methanol (200 ml) was added, ensuring contact by constant stirring at 4°C for 24 h. The extract was vacuum filtered on a G4 filter plate, and the resulting extract was evaporated at 40°C in a rotary evaporator to a minimum volume. The extract was centrifuged to remove pigments and lipid phases, dissolved in methanol and levelled to 10 ml. 2.2.2. Methylation of the ABA carboxyl group [14] 100 ml of a 50 mg ml − 1 ABA solution in methanol was taken and the solvent was evaporated by means of a N2 stream. The dry residue was dissolved in 100 ml of a mixture containing acetonitrile:water:methanol:pyridine (7:1:1:1), adding 5 ml of methylchloroformate (MCF), which acted as the methylating agent, 100 ml of chloroform and 100 ml of 1M sodium bicarbonate. The abscisic acid was methylated in this medium, and afterwards the methylated compound was extracted in the chloroform, which remained at the bottom. After separating the two phases, the chloroform was removed by evaporation using a N2 stream, leaving a dry residue that was dissolved in 100 ml of hexane.
3. Results and discussion
3.1. Influence of pH and accumulation potential on the abscisic acid reduction The purpose of this study was to ascertain the conditions that facilitate the greatest adsorption of abscisic acid on the surface of the mercury droplet. The measurements were made in different solutions containing 1.6 mg ml − 1 of ABA in HClO4, 1
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tion and the highest peak intensity corresponding to the electroreduction of the phytohormone appear. We chose an accumulation potential of Eac = − 0.4 V and pH 1 for the subsequent analyses. From the analysis of the variation in the pH, we obtain a shift in the peak potential towards more cathodic values, indicating an intervention of the protons in the reaction of the abscisic acid electroreduction. This shift shows two linear zones with differing slopes in the pH range under consideration. For pH levels between 0 and 2, the linear dependence fits the equation of the line: Ep = − 0.74− 0.05 pH;
r= 0.986
when the pH is between 2 and 4, the variation of the peak potential is linear, fitting the equation: Ep = − 0.64− 0.1 pH;
Fig. 2. Square wave voltammograms of abscisic acid reduction at different pH values: (a) pH 0; (b) pH 0.3; (c) pH 1; (d) pH 2 and (e) pH 3.
and 0.1 M, and H3PO4 0.04 M, obtaining the desired pH for the analysis in each case with KOH additions. An accumulation time of tac = 30 s was used, along with the rest of the conditions described in Section 2.2. The accumulation potential (Eac) was varied for each pH value. Fig. 2 shows the results for pH lower than 3. Beyond this pH, the width at half height grows considerably while the peak intensity diminishes, with a poor definition of the hormone reduction wave. In the concentrations at which it was found, the abscisic acid was not electroactive at basic medium. Our results suggest that the pH is achieved at pH =1, where the best wave defini-
r=0.993
in which peak potential is expressed in V. The point of intersection between the two straight lines produces the value of pK corresponding to the abscisic acid system, obtaining a value of pK= 1.9. A coulometric study was performed in a BAS100 electrochemical analyser using a cell with a mercury pool electrode as the working electrode. A perchloric acid-sodium perchlorate solution at pH 1 containing 200 g of abscisic acid was placed in the cell. Applying a potential of − 1.0 V, the ensuing number of electrons involved in the process was n= 4 [9]. To identify the product of the electrolysis, the solution was fed through a Sep-Pak C18 cartridge, after which the reduction product was eluted with methanol. The acid group of the molecule was methylated, and the product was injected into the GC-MS. The mass spectrum indicates the reduction of two double conjugated bonds (Fig. 3). The proposed reduction mechanism for the ABA is in Fig. 4.
3.2. Influence of the scan rate The scan rate was analysed in cyclic voltammetry over a 1.6 mg ml − 1 solution of ABA in HClO4 0.1 M at pH 1. In all the measurements, the accumulation time was tac = 30 s, and accumulation potential was Eac = − 0.4 V.
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Fig. 3. Mass spectrum for the methylated ABA (down) and for the methylated product of the ABA electrolysis (up).
The data obtained with an increase in the scan rate indicate a shift in the peak potential towards more cathodic values. This variation of the peak potential in the abscisic acid reduction behaves linearly with the logarithm of the scan rate, following the equation
Ep = − 0.7185− 0.0422 log 6;
r= 0.996
where Ep is the peak potential expressed in V, and log 6 is the decimal logarithm the scan rate. The intensity of the peak increases linearly with the scan rate, fitting the equation
P. Herna´ndez et al. / Talanta 44 (1997) 1783–1792
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Fig. 4. The proposed reduction mechanism for the ABA.
ip =0.0968+0.0021n;
r = 0.9994
where the peak intensity is expressed in mA and the scan rate in mV s − 1. This behaviour indicates that the abscisic acid reduction is produced by means of an adsorptive process controlled by diffusion [10] and is irreversible. By representing the logarithm of the peak intensity at different scan rates against the peak potential, we obtain a linear relation that fits the equation log ip = − 13.367 −19.785Ep;
r =0.995
Using the value of the slope and knowing the number of electrons consumed in the electroreduction of the ABA, we can calculate the charge transfer coefficient, obtaining a value a=0.31 [11]. The influence of the square wave frequency was calculated under the conditions described in the Section 2.2, with the exception of the frequency variation. The results indicate that there is a linear dependency of the peak intensity on the frequency in accordance with the ratio: ip =3.059× 10 − 2 +5.76 ×10 − 3f;
r =0.9998
expressing ip in mA and where f is the square wave frequency expressed in Hz. There is also a linear variation in the peak potential against the decimal logarithm of the frequency which fits the equation Ep = − 0.72− 0.044 log f;
r =0.997
where Ep is the peak potential expressed in V and log f is the logarithm of the square wave frequency.
This analysis of the square wave frequency enables us to ascertain kinetic parameters of the electroreduction reaction of the abscisic acid in the mercury electrode. The value of the slope can be used to calculate the charge transfer coefficient (a), which can be calculated via the expression DEp/D log f= RT/Fna obtaining a value of n= 1.58. By means of coulometry, we found that the number of electrons exchanged by the molecule was n= 4. Hence, the value of the load transfer is a= 0.33 [12]. Given that the peak intensity is linear with the square wave frequency, and that the peak potential is linear with the decimal logarithm of the square wave frequency, we can deduce that the electrochemical reduction process of the abscisic acid is of the adsorptive type controlled by diffusion. This concurs with the data obtained in the cyclic voltammetry analysis.
3.3. Influence of the square wa6e amplitude From the variation in the square wave amplitude, we can obtain the degree of absorption of the ABA over the measurement electrode, i.e. the coating coefficient (Г) of the mercury droplet used for measurement [12,13]. For this purpose we used an abscisic acid solution at 1.6 mg ml − 1 concentration in HClO4 at pH 1. We established an accumulation time of tac = 30 s and the rest of the conditions as described in Section 2.2. The results are shown in Fig. 5. In Fig. 5 we see an increase in the intensity of the peak with the amplitude of the square wave up to a value of a= 40 For higher values of the square wave amplitude, there is no significant increase in the peak intensity.
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For square wave amplitude values lower than 20 mV, there is a linear increase in the value of the peak intensity that fits the equation: ip =0.038+ 0.013a;
r =0.992
where the peak potential is expressed in mV and the peak intensity in mA. On the basis of the slope of the line obtained for small square wave amplitudes, and knowing that the electrode has an surface area of 0.0278 cm2, we can calculate a value for the coating coefficient of the mercury droplet of 1.990.7 ×10 − 11 mol cm − 2 [12,13].
3.4. Influence of the scan increment Variations in the value of the scan increment produce changes in the intensity of the peak, which affect the sensitivity of the method. At the same time, however, the width at half height also varies, which affects its selectivity. A compromise must therefore be sought between the two factors. In our measurements, we used a 1.6-mg ml − 1 solution of ABA at pH 1 in HClO4 0.1 M, which acted as the supporting electrolyte. An accumulation time of tac =30 s was set, using the rest of the initially established parameters. Applying selectivity and sensitivity criteria, we took E=5 mV as the optimum value for subsequent measurements as this combines a width at half height of w1/2 =95 mV with a peak intensity of ip = 0.709 mA.
Fig. 6. Variation in peak intensity with accumulation time for different concentrations of ABA: (a) 0.1 mg ml − 1; (b) 0.2 mg ml − 1; (c) 0.3 mg ml − 1; (d) 0.4 mg ml − 1; (e) 0.6 mg ml − 1; (f) 0.8 mg ml − 1; (g) 1.0 mg ml − 1; (h) 1.2 mg ml − 1; (i) 1.4 mg ml − 1 and (j) 1.6 mg ml − 1.
Fig. 6 graphs the results of varied accumulation times. In all cases, the adsorption of the hormone to the electrode reaches the highest peak intensity at an accumulation time of 10s. If this time is increased, the intensity of the peak diminishes. The optimum value is therefore tac = 10 s.
3.5. Influence of the buffer type
Fig. 5. Variation in peak intensity with square wave amplitude. Conditions in text.
In the analysis of the influence of pH, the optimum working value was set at pH 1. On the basis of this condition, we tested different types of electrolytes: hydrochloric acid–sodium chloride, sulphuric acid–sodium sulphate, and finally perchloric acid–sodium perchlorate. Solutions of all these electrolytes at different concentrations were prepared to fix the ionic strength of the medium. Sulphuric acid–sodium sulphate solutions produce the highest peak intensities, the higher, the greater the concentration of electrolyte. This solution was thus used as a buffer medium and a support electrolyte at a 1 M concentration.
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3.6. Influence of the ABA concentration In the study of the effect of abscisic acid concentration on the peak intensity, the optimum conditions of the medium were set at those discussed in the previous sections. The polarographic cell contained 10 ml of sulphuric acid-sodium sulphate solution at 1 M concentration, pH 1, which acted as a support electrolyte and different amounts of abscisic acid. An inert atmosphere was created by a stream of N2 prior to each measurement. An accumulation potential Eac = − 0.4 V was applied for 10 s, with an equilibrium time of 5 s. The initial scan potential coincided with Eac, and the final potential was set at − 1.2 V to permit the full development of the wave. Instrumental variables during the measurement stage were set at, pulse amplitude a =40 mV, scan increment E = 5 mV, and square wave frequency f=120 Hz, producing a scan rate of 600 mV s − 1. There is an increase in the peak intensity with the concentration, shown in the superimposed voltammograms in Fig. 7. This dependence of the peak intensity on concentration follows a linear relationship, which fits the expression ip = − 1.76×10 − 2 +7.89 ×10 − 4C;
HPLC was used as the preparatory technique in a 10-mm column of C18, using a 0.01M mixture of methanol:phosphoric acid at pH 3 (1:2 v/v) as the eluent at a flow of 1.8 ml min − 1, and a UV detector at a wavelength of 270 nm. The retention time of the abscisic acid was 19.51 min. The corresponding fraction was collected at the column outlet. For the purification process, we injected into the column a mixture containing 3 ml of extract and 6 ml of phosphoric acid with a 500 ml loop.
r= 0.999
The intensity of the peak is expressed in mA while the concentration is in ng ml − 1. The relative error of the method ranges between 0.3 and 0.8% in absolute values and the relative standard deviation varies between 2.1 and 7.8%, producing a detection threshold of 30 ng ml − 1 and a quantification threshold of 58 ng ml − 1.
4. Application After optimising the abscisic acid determination method, it was applied to an extract of pears taken straight from the tree at the initial growth stage. The extract obtained from little pears, like abscisic acid, contains many other compounds that interfere with voltammetric measurements, making purification necessary. For this purpose,
Fig. 7. Square wave voltammograms of abscisic acid reduction at different concentrations: r =residual current; (a) 75 ng ml − 1; (b) 125 ng ml − 1; (c) 150 ng ml − 1; (d) 250 ng ml − 1; (e) 300 ng ml − 1; (f) 350 ng ml − 1; (g) 400 ng ml − 1; (h) 450 ng ml − 1 and (i) 500 ng ml − 1.
P. Herna´ndez et al. / Talanta 44 (1997) 1783–1792
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This process was repeated four times, collecting a total volume of 20.3 ml at the outlet of the column. Square wave voltammetry was used for the measurement under the same conditions as the calibration of the abscisic acid. In order to find the ABA concentration contained in the extract, we performed standard additions. The intensity of the peak increases linearly with the concentration of the added ABA, fitting the equation: ip = 0.0529+ 2.108C;
r= 0.999
where the peak intensity is expressed in mA and the concentration in mg ml − 1 (Fig. 8). Extrapolating from the straight line, we find a 0.025 mg ml − 1 concentration of ABA in the measurement cell. Taking into account the successive dilutions of the sample, and the calculated extraction yield (78%), we can calculate that the amount of abscisic acid contained in the initial pear sample was 3.1 9 0.1 mg per gram of pear. This is the average value obtained in 5 samples for which the same extraction process was carried out. This method has been used since the signal obtained for the ABA through HPLC determination with UV detection remains under its quantification limit. In the same way, with a direct electrochemical measure, the nature of the sample produces adsorptions in the electrode that make the signal of the ABA to disappear. So, in this work, a separation through HPLC is combined with a very sensitive electrochemical detection. Hence, in whatever the vegetal sample it was necessary to determine ABA, this proposed method can be applied.
Acknowledgements The authors gratefully acknowledge financial support from the Direccio´n General de Investigacio´n of Spain under project PB92/132 and for a scholarship to F. Pato´n.
Fig. 8. Square wave voltammograms of real sample (a) and spiked samples with different concentrations: (b) 50 ng ml − 1; (c) 100 ng ml − 1; (d) 150 ng ml − 1; (e) 200 ng ml − 1; (f) 250 ng ml − 1; (g) 300 ng ml − 1; (h) 350 ng ml − 1 and (i) 400 ng ml − 1.
References [1] R.J. Weaver (Ed.), Reguladores del crecimiento de las plantas en la agricultura. Trillas, Mexico, 1985.
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[2] Nobutaka Takanashi, Chemistry of plant hormones. Department of Agricultural Chemistry. Faculty of Agriculture. The University of Tokio, Bunkio-Ku, Tokio, Japan. CRC Press, Boca Raton, Florida, pp. 201–248. [3] M.J. Chrispeels, J.E. Varner, Nature (London) 212 (1966) 1066. [4] R.M.F. Van Stevenich, Nature (London) 183 (1959) 1246. [5] C.J. Mittelheuser, R.F.M. Van Stevenich, Nature (London) 221 (1969) 281. [6] E.W. Weiler, Planta 144 (1979) 225–263. [7] A.G. Netting, B.V. Milborrow, A.M. Duffield, Phytochemistry 21 (1982) 385. [8] B. Anderson, N. Haggstrom, K. Anderson, J. Chro-
.
matogr. 157 (1978) 303. [9] M. Iwamoto, A. Webber, R.A. Osteryoung, Anal Chem. 56 (1984) 1202. [10] A.J. Bard and L.R. Falkner, Electrochemical Methods. Wiley, New York, 1980, p. 224. [11] R. Mallaby, G. Ryback, J. Chem. Soc. Perkin Trans. 2 (1972) 919. [12] M. Lovric, S. Komorsky-Lovric, R.W. Murray, Electrochem. Acta 33 (6) (1988) 739 – 744. [13] J.J. O’Dea, A. Ribes, J.G. Osteryoung, J. Electroanal. Chem. 345 (1993) 287 – 301. [14] P. Husek, LC-GC international, The Magazine Separation Sci. 59 (1992) 43 – 49.
.
Determination of abscisic acid by cathodic stripping square wave voltammetry P. Herna´ndez, M. Dabrio-Ramos, F. Pato´n, Y. Ballesteros, L. Herna´ndez * Departamento de Quı´mica Analı´tica, Uni6ersidad Auto´noma de Madrid, Madrid, Spain Received 10 October 1996; received in revised form 15 January 1997; accepted 12 February 1997
Abstract In this paper a study is accomplished on behavior in a mercury electrode, of the phytohormone abscisic acid and of the conditions of accumulation in a HMDE. A mechanism is proposed of reduction based on its electrochemical behavior and proving the product of the reduction through mass spectrometry of bulks. A method is proposed for the determination of Abscisic acid (ABA) with a quantification limit of 58 ng ml − 1. The procedure is applied wing determination of ABA in pears through the combination of high performance liquid chromatography (HPLC) with electrochemical quantification. © 1997 Elsevier Science B.V. Keywords: Abscisic acid; Phytohormone; Mercury electrode
1. Introduction Plant hormones or phytohormones are regulators that are produced by the plant itself. In low concentrations, they control the physiological processes. Phytohormones are classified into four groups: auxines, gibberellines, cytoquinines and inhibitors. Abscisic acid (ABA) belongs to the latter, which is quite different from the other groups of plant growth substances, as it inhibits or delays the physiological or biochemical process of plants [1]. Abscisic acid was isolated from young cotton fruit (Gossypium hirsutum) by a research group led initially by Corns and subsequently by Addi-
cot [2]. Its structure, shown in Fig. 1, was determined by Ohkuma and associates [2]. The levels of concentration of this inhibitor vary considerably from one plant species to another and depend on environmental conditions [1]. Philips and Waring [2] studied this variation in Acer pseudoplatanus buds and leaves, finding
* Corresponding author. 0039-9140/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 9 - 9 1 4 0 ( 9 7 ) 0 0 0 4 9 - 0
Fig. 1. Molecular structure of abscisic acid (ABA).
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P. Herna´ndez et al. / Talanta 44 (1997) 1783–1792
minimum levels of ABA in June in the terminal buds and maxima in winter, precisely the opposite of the leaves. This suggests that the inhibitor formed in the leaves in summer shifts to the terminal buds at the start of autumn. Hormonal action depends on the hormone concentration, the presence and characteristics of the receptor and the elements involved in the signal transduction chain. The hormone-receiver complex, i.e., the activated receiver, is the first link in the signal transduction chain, which provokes a primary response. This in turn begins a series of changes which, as a whole, make up the complex physiological response. In the case of abscisic acid, there is no single biological activity, which instead is manifested in several types of physiological response. The ABA has a great inhibiting effect on the growth of many types of plants. It is not the only growth-inhibiting substance, but it is the only one that is non-toxic. A seed treated with ABA, for example, can germinate once it is placed in another medium. With another inhibitor, this seed does not germinate [2]. Seed germination is regulated by the level of ABA and gibberellins (phytohormones that stimulate cell division or prolongation, or both). Gibberellins induce the synthesis of a-amylase, while the ABA inhibits this action, stopping cell division and hence growth [3]. Abscisic acid accelerates the abscission process in flowers and fruit, as well as in the reproductive organs of a wide variety of plant species [4], and ABA is also responsible for the closure of the stomas in the leaves of some plants such as Triticum 6ulgare, thus producing a reduction in transpiration [5]. In addition to plant activity, it has been suggested that abscisic acid inhibits reproduction in insects and the growth of tumours in some types of mice [2]. Given its importance, abscisic acid has been studied widely from an analytic perspective ever since its discovery. The first technique used to identify this acid, at the start of the 1960s, was paper chromatography (PC), which was soon replaced by thin layer chromatography (TLC). Detection was by means of UV spectroscopy because
abscisic acid has an intense, well defined band [2]. As other techniques spread, however, TLC and PC was left as a mere technique for sample prepurification. Gas chromatography (GC) and high performance liquid chromatography (HPLC) began to be used in the 1970s to study abscisic acid, and coupled to mass spectroscopy (MS) are an excellent method of identification [6,7]. The problem in HPLC analysis is the low specificity of the UV detector. GC-MS is a powerful method for identification, but the samples must be cleaned up. The lower limit of detection of ABA is 50 ng in HPLC. Before the analysis, crude samples should be purified by solvent partitioning and preparative TLC or by a short column such as the C18 Sep-pak cartridge [2]. Bioassay and immunoassay has been also used for determination and quantification of abscisic acid, and does not require pre-purification, although the experimental period tends to be very long due to the preparation of the antigen and the antiserum [8]. In recent times, the same techniques used in previous years have continued to be used for the determination and quantification of ABA, although additional techniques such as ELISA have arisen. We have not, however, found any literature on the electrochemical study of this phytohormone. This analysis, discussed in the present paper, is approached using square wave voltammetry (SWV), given that the coupling of an accumulation step to an impulse technique would enable us to obtain detection thresholds in the order of ng ml − 1. The use of an electrochemical measurement provides an additional selectivity which allows one to avoid the needed pre-purification steps used on other methods, such as GC or HPLC-UV. Likewise, the limit of detection obtained in this proposed method for quantification of ABA is similar to those obtained by using other traditional methods.
2. Experimental
2.1. Reagents and equipment
A 0.2 mg ml −1 solution of abscisic acid (ABA) in methanol prepared from the crystallized
P. Herna´ndez et al. / Talanta 44 (1997) 1783–1792
form supplied by SERVA (New York, USA). The solutions must by refrigerated and not exposed to light. Analysis quality reagents. Deionised water using Milliro-MilliQ (Water System) equipment. PAR polarographic analyser model 384B, equipped with a Ag/AgCl/KCl (3M) reference electrode and a platinum counter electrode. This polarograph was used for the square wave and cyclical voltammetry analyses using a hanging drop mercury electrode (HDME). Bioanalytical System Potentiostat BAS 100, equipped with a mercury pool electrode, a saturated calomel electrode as the reference electrode and a platinum electrode as the counter electrode. These were used for a coulometric analysis of the abscisic acid. HEWLETT PACKARD model 5890 Series II gas chromatograph with a HEWLETT PACKARD model 5971A spectrometry detector used to identify the coulometric reaction products. High performance liquid chromatography composed of a GILSON 302 pump connected to a GILSON 802C manometric module, with a GILSON 116 UV detector attached to the unit. The HPLC was used a the preparative separation method in a plant extract for subsequent electrochemical detection.
2.2. Procedure The polarographic cell contains a 10-ml buffer solution at a previously adjusted pH and a known concentration of abscisic acid. The O2 was eliminated by means of a stream of N2 for 5 min in the first measurement and 30 s in the subsequent potential scans. An accumulation time (tac) was applied at an accumulation potential (Eac) with a stirring speed of 400 r.p.m. The polarogram was recorded between −0.4 and − 1.1 V for a mercury drop electrode of 0.0278 cm2 average area, replaced for each measurement, against an Ag/ AgCl 3M electrode and a platinum counter electrode. The equilibrium time was 5 s. In order to establish the measurement conditions, the corresponding variable was modified in each analysis to find the optimum response value,
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establishing initial conditions of: square wave frequency ( f ) at 50 Hz with increments in the step wave potential (E) of 2 mV in each cycle, producing a scan rate of 100 mV s − 1. The pulse amplitude (a) was 20 mV.
2.2.1. Extraction procedure The procedure used was as follows: 51.35 g of pears was pulverised after prior freezing with liquid nitrogen. Methanol (200 ml) was added, ensuring contact by constant stirring at 4°C for 24 h. The extract was vacuum filtered on a G4 filter plate, and the resulting extract was evaporated at 40°C in a rotary evaporator to a minimum volume. The extract was centrifuged to remove pigments and lipid phases, dissolved in methanol and levelled to 10 ml. 2.2.2. Methylation of the ABA carboxyl group [14] 100 ml of a 50 mg ml − 1 ABA solution in methanol was taken and the solvent was evaporated by means of a N2 stream. The dry residue was dissolved in 100 ml of a mixture containing acetonitrile:water:methanol:pyridine (7:1:1:1), adding 5 ml of methylchloroformate (MCF), which acted as the methylating agent, 100 ml of chloroform and 100 ml of 1M sodium bicarbonate. The abscisic acid was methylated in this medium, and afterwards the methylated compound was extracted in the chloroform, which remained at the bottom. After separating the two phases, the chloroform was removed by evaporation using a N2 stream, leaving a dry residue that was dissolved in 100 ml of hexane.
3. Results and discussion
3.1. Influence of pH and accumulation potential on the abscisic acid reduction The purpose of this study was to ascertain the conditions that facilitate the greatest adsorption of abscisic acid on the surface of the mercury droplet. The measurements were made in different solutions containing 1.6 mg ml − 1 of ABA in HClO4, 1
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tion and the highest peak intensity corresponding to the electroreduction of the phytohormone appear. We chose an accumulation potential of Eac = − 0.4 V and pH 1 for the subsequent analyses. From the analysis of the variation in the pH, we obtain a shift in the peak potential towards more cathodic values, indicating an intervention of the protons in the reaction of the abscisic acid electroreduction. This shift shows two linear zones with differing slopes in the pH range under consideration. For pH levels between 0 and 2, the linear dependence fits the equation of the line: Ep = − 0.74− 0.05 pH;
r= 0.986
when the pH is between 2 and 4, the variation of the peak potential is linear, fitting the equation: Ep = − 0.64− 0.1 pH;
Fig. 2. Square wave voltammograms of abscisic acid reduction at different pH values: (a) pH 0; (b) pH 0.3; (c) pH 1; (d) pH 2 and (e) pH 3.
and 0.1 M, and H3PO4 0.04 M, obtaining the desired pH for the analysis in each case with KOH additions. An accumulation time of tac = 30 s was used, along with the rest of the conditions described in Section 2.2. The accumulation potential (Eac) was varied for each pH value. Fig. 2 shows the results for pH lower than 3. Beyond this pH, the width at half height grows considerably while the peak intensity diminishes, with a poor definition of the hormone reduction wave. In the concentrations at which it was found, the abscisic acid was not electroactive at basic medium. Our results suggest that the pH is achieved at pH =1, where the best wave defini-
r=0.993
in which peak potential is expressed in V. The point of intersection between the two straight lines produces the value of pK corresponding to the abscisic acid system, obtaining a value of pK= 1.9. A coulometric study was performed in a BAS100 electrochemical analyser using a cell with a mercury pool electrode as the working electrode. A perchloric acid-sodium perchlorate solution at pH 1 containing 200 g of abscisic acid was placed in the cell. Applying a potential of − 1.0 V, the ensuing number of electrons involved in the process was n= 4 [9]. To identify the product of the electrolysis, the solution was fed through a Sep-Pak C18 cartridge, after which the reduction product was eluted with methanol. The acid group of the molecule was methylated, and the product was injected into the GC-MS. The mass spectrum indicates the reduction of two double conjugated bonds (Fig. 3). The proposed reduction mechanism for the ABA is in Fig. 4.
3.2. Influence of the scan rate The scan rate was analysed in cyclic voltammetry over a 1.6 mg ml − 1 solution of ABA in HClO4 0.1 M at pH 1. In all the measurements, the accumulation time was tac = 30 s, and accumulation potential was Eac = − 0.4 V.
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Fig. 3. Mass spectrum for the methylated ABA (down) and for the methylated product of the ABA electrolysis (up).
The data obtained with an increase in the scan rate indicate a shift in the peak potential towards more cathodic values. This variation of the peak potential in the abscisic acid reduction behaves linearly with the logarithm of the scan rate, following the equation
Ep = − 0.7185− 0.0422 log 6;
r= 0.996
where Ep is the peak potential expressed in V, and log 6 is the decimal logarithm the scan rate. The intensity of the peak increases linearly with the scan rate, fitting the equation
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Fig. 4. The proposed reduction mechanism for the ABA.
ip =0.0968+0.0021n;
r = 0.9994
where the peak intensity is expressed in mA and the scan rate in mV s − 1. This behaviour indicates that the abscisic acid reduction is produced by means of an adsorptive process controlled by diffusion [10] and is irreversible. By representing the logarithm of the peak intensity at different scan rates against the peak potential, we obtain a linear relation that fits the equation log ip = − 13.367 −19.785Ep;
r =0.995
Using the value of the slope and knowing the number of electrons consumed in the electroreduction of the ABA, we can calculate the charge transfer coefficient, obtaining a value a=0.31 [11]. The influence of the square wave frequency was calculated under the conditions described in the Section 2.2, with the exception of the frequency variation. The results indicate that there is a linear dependency of the peak intensity on the frequency in accordance with the ratio: ip =3.059× 10 − 2 +5.76 ×10 − 3f;
r =0.9998
expressing ip in mA and where f is the square wave frequency expressed in Hz. There is also a linear variation in the peak potential against the decimal logarithm of the frequency which fits the equation Ep = − 0.72− 0.044 log f;
r =0.997
where Ep is the peak potential expressed in V and log f is the logarithm of the square wave frequency.
This analysis of the square wave frequency enables us to ascertain kinetic parameters of the electroreduction reaction of the abscisic acid in the mercury electrode. The value of the slope can be used to calculate the charge transfer coefficient (a), which can be calculated via the expression DEp/D log f= RT/Fna obtaining a value of n= 1.58. By means of coulometry, we found that the number of electrons exchanged by the molecule was n= 4. Hence, the value of the load transfer is a= 0.33 [12]. Given that the peak intensity is linear with the square wave frequency, and that the peak potential is linear with the decimal logarithm of the square wave frequency, we can deduce that the electrochemical reduction process of the abscisic acid is of the adsorptive type controlled by diffusion. This concurs with the data obtained in the cyclic voltammetry analysis.
3.3. Influence of the square wa6e amplitude From the variation in the square wave amplitude, we can obtain the degree of absorption of the ABA over the measurement electrode, i.e. the coating coefficient (Г) of the mercury droplet used for measurement [12,13]. For this purpose we used an abscisic acid solution at 1.6 mg ml − 1 concentration in HClO4 at pH 1. We established an accumulation time of tac = 30 s and the rest of the conditions as described in Section 2.2. The results are shown in Fig. 5. In Fig. 5 we see an increase in the intensity of the peak with the amplitude of the square wave up to a value of a= 40 For higher values of the square wave amplitude, there is no significant increase in the peak intensity.
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For square wave amplitude values lower than 20 mV, there is a linear increase in the value of the peak intensity that fits the equation: ip =0.038+ 0.013a;
r =0.992
where the peak potential is expressed in mV and the peak intensity in mA. On the basis of the slope of the line obtained for small square wave amplitudes, and knowing that the electrode has an surface area of 0.0278 cm2, we can calculate a value for the coating coefficient of the mercury droplet of 1.990.7 ×10 − 11 mol cm − 2 [12,13].
3.4. Influence of the scan increment Variations in the value of the scan increment produce changes in the intensity of the peak, which affect the sensitivity of the method. At the same time, however, the width at half height also varies, which affects its selectivity. A compromise must therefore be sought between the two factors. In our measurements, we used a 1.6-mg ml − 1 solution of ABA at pH 1 in HClO4 0.1 M, which acted as the supporting electrolyte. An accumulation time of tac =30 s was set, using the rest of the initially established parameters. Applying selectivity and sensitivity criteria, we took E=5 mV as the optimum value for subsequent measurements as this combines a width at half height of w1/2 =95 mV with a peak intensity of ip = 0.709 mA.
Fig. 6. Variation in peak intensity with accumulation time for different concentrations of ABA: (a) 0.1 mg ml − 1; (b) 0.2 mg ml − 1; (c) 0.3 mg ml − 1; (d) 0.4 mg ml − 1; (e) 0.6 mg ml − 1; (f) 0.8 mg ml − 1; (g) 1.0 mg ml − 1; (h) 1.2 mg ml − 1; (i) 1.4 mg ml − 1 and (j) 1.6 mg ml − 1.
Fig. 6 graphs the results of varied accumulation times. In all cases, the adsorption of the hormone to the electrode reaches the highest peak intensity at an accumulation time of 10s. If this time is increased, the intensity of the peak diminishes. The optimum value is therefore tac = 10 s.
3.5. Influence of the buffer type
Fig. 5. Variation in peak intensity with square wave amplitude. Conditions in text.
In the analysis of the influence of pH, the optimum working value was set at pH 1. On the basis of this condition, we tested different types of electrolytes: hydrochloric acid–sodium chloride, sulphuric acid–sodium sulphate, and finally perchloric acid–sodium perchlorate. Solutions of all these electrolytes at different concentrations were prepared to fix the ionic strength of the medium. Sulphuric acid–sodium sulphate solutions produce the highest peak intensities, the higher, the greater the concentration of electrolyte. This solution was thus used as a buffer medium and a support electrolyte at a 1 M concentration.
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3.6. Influence of the ABA concentration In the study of the effect of abscisic acid concentration on the peak intensity, the optimum conditions of the medium were set at those discussed in the previous sections. The polarographic cell contained 10 ml of sulphuric acid-sodium sulphate solution at 1 M concentration, pH 1, which acted as a support electrolyte and different amounts of abscisic acid. An inert atmosphere was created by a stream of N2 prior to each measurement. An accumulation potential Eac = − 0.4 V was applied for 10 s, with an equilibrium time of 5 s. The initial scan potential coincided with Eac, and the final potential was set at − 1.2 V to permit the full development of the wave. Instrumental variables during the measurement stage were set at, pulse amplitude a =40 mV, scan increment E = 5 mV, and square wave frequency f=120 Hz, producing a scan rate of 600 mV s − 1. There is an increase in the peak intensity with the concentration, shown in the superimposed voltammograms in Fig. 7. This dependence of the peak intensity on concentration follows a linear relationship, which fits the expression ip = − 1.76×10 − 2 +7.89 ×10 − 4C;
HPLC was used as the preparatory technique in a 10-mm column of C18, using a 0.01M mixture of methanol:phosphoric acid at pH 3 (1:2 v/v) as the eluent at a flow of 1.8 ml min − 1, and a UV detector at a wavelength of 270 nm. The retention time of the abscisic acid was 19.51 min. The corresponding fraction was collected at the column outlet. For the purification process, we injected into the column a mixture containing 3 ml of extract and 6 ml of phosphoric acid with a 500 ml loop.
r= 0.999
The intensity of the peak is expressed in mA while the concentration is in ng ml − 1. The relative error of the method ranges between 0.3 and 0.8% in absolute values and the relative standard deviation varies between 2.1 and 7.8%, producing a detection threshold of 30 ng ml − 1 and a quantification threshold of 58 ng ml − 1.
4. Application After optimising the abscisic acid determination method, it was applied to an extract of pears taken straight from the tree at the initial growth stage. The extract obtained from little pears, like abscisic acid, contains many other compounds that interfere with voltammetric measurements, making purification necessary. For this purpose,
Fig. 7. Square wave voltammograms of abscisic acid reduction at different concentrations: r =residual current; (a) 75 ng ml − 1; (b) 125 ng ml − 1; (c) 150 ng ml − 1; (d) 250 ng ml − 1; (e) 300 ng ml − 1; (f) 350 ng ml − 1; (g) 400 ng ml − 1; (h) 450 ng ml − 1 and (i) 500 ng ml − 1.
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This process was repeated four times, collecting a total volume of 20.3 ml at the outlet of the column. Square wave voltammetry was used for the measurement under the same conditions as the calibration of the abscisic acid. In order to find the ABA concentration contained in the extract, we performed standard additions. The intensity of the peak increases linearly with the concentration of the added ABA, fitting the equation: ip = 0.0529+ 2.108C;
r= 0.999
where the peak intensity is expressed in mA and the concentration in mg ml − 1 (Fig. 8). Extrapolating from the straight line, we find a 0.025 mg ml − 1 concentration of ABA in the measurement cell. Taking into account the successive dilutions of the sample, and the calculated extraction yield (78%), we can calculate that the amount of abscisic acid contained in the initial pear sample was 3.1 9 0.1 mg per gram of pear. This is the average value obtained in 5 samples for which the same extraction process was carried out. This method has been used since the signal obtained for the ABA through HPLC determination with UV detection remains under its quantification limit. In the same way, with a direct electrochemical measure, the nature of the sample produces adsorptions in the electrode that make the signal of the ABA to disappear. So, in this work, a separation through HPLC is combined with a very sensitive electrochemical detection. Hence, in whatever the vegetal sample it was necessary to determine ABA, this proposed method can be applied.
Acknowledgements The authors gratefully acknowledge financial support from the Direccio´n General de Investigacio´n of Spain under project PB92/132 and for a scholarship to F. Pato´n.
Fig. 8. Square wave voltammograms of real sample (a) and spiked samples with different concentrations: (b) 50 ng ml − 1; (c) 100 ng ml − 1; (d) 150 ng ml − 1; (e) 200 ng ml − 1; (f) 250 ng ml − 1; (g) 300 ng ml − 1; (h) 350 ng ml − 1 and (i) 400 ng ml − 1.
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