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Microchemical Journal
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Carbon-fibre microelectrodes coupled with square-wave voltammetry for the directanalysis of dimethomorph fungicide in natural waters
Thiago M.B.F. Oliveira a, Helena Becker a, Elisane Longhinotti a, Djenaine De Souza b,Pedro de Lima-Neto a, Adriana N. Correia a,⁎a Departamento de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Bloco 940 Campus do Pici, 60455-970, Fortaleza, CE, Brazilb Campus de Patos de Minas, Instituto de Química, Universidade Federal de Uberlândia, Av. Getúlio Vargas no 230 Centro, 38700-126 Patos de Minas, MG, Brazil
This paper describes the development and electrochemical behaviour of a quick, easy, cheap and eco-friendlyelectroanalytical procedure for the direct analysis of dimethomorph fungicide (DIM) in natural water samplesusing a carbon-fibre microelectrode (CFM) coupled with square-wave voltammetry (SWV). The optimizedexperimental and voltammetric parameters employed were a 0.04 mol L−1 Britton–Robinson buffer (pH3.0), a pulse potential frequency of 70 s−1, a pulse amplitude of 30 mV and a scan increment of 2 mV. It waspossible to observe a well-shaped oxidation peak at +1.25 V (vs. Ag/AgCl/saturated Cl–), which was relatedto a two-electron transfer in the quasi-reversible redox process affected by a strong adsorption of reactantsand products on the electrode surface. Analytical parameters such as linearity range, correlation coefficients,detection and quantification limits were evaluated and compared to similar results obtained using gas chro-matography coupled with mass spectrometry. However, by using SWV, no clean-up, extraction or pre-concentration procedures were necessary, making the electroanalytical procedure more feasible for analyticalroutine analysis.
In recent years, the requirement for the production of high-yieldand high-quality crops has increased the variety and quantity of pes-ticides employed in production, transport and particularly in the stor-age of agricultural products. Around 800 types of pesticides arewidely used today in agricultural practices, and their intensive andabusive use has led to an increase in the levels of residues presentin soils, waters and foods [1].
One particular kind of chemical pesticide associated with the pro-tection and conservation of agricultural compounds is the morpholineclass; these are used as systemic fungicides with protective and cura-tive properties to control powdery mildew and to prevent Peronos-porales germ disease in vegetables and fruits [2]. Additionally, somemorpholines have been listed as potential endocrine disrupters, dueto being potent inhibitors of the human sterol isomerase enzyme,which is part of the cholesterol biosynthesis pathway [3].
Among these compounds, dimethomorph {(E,Z)-4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morpholine} (DIM),is also suspected to be an endocrine disruptor, interfering in the func-tioning of the hormonal system in humans and animals [4]. Despite itsbeneficial effects for agricultural practices, DIM shows marked toxicity
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for both humans and other living organisms, including soil and watermicroflora, by modifying DNA profiles, causing undesirable geneticalchanges. After its application, DIM can accumulate in the environment,such as in surface and ground waters, waste water, sludge, soils, foodand other food products. Furthermore, it can concentrate in living or-ganisms, changing important biological functions, even at very low con-centrations [5].
As a consequence of the environmental and health problems sur-rounding the use of DIM, several precise and sensitive analytical pro-cedures have been developed all around the world for DIMquantification in a variety of food samples. All previously publishedstudies have described the simultaneous analysis of a large numberof agrochemical compounds, including DIM, employing liquid [6–9]or gas [10,11] chromatography in tandem with mass spectrometry.In these studies, the procedures required a previous step of extractionand clean-up of the samples.
In spite of the fact that DIM presents significant solubility in water,and thus can be released in the environment, no methodology for itsdetection in natural water samples can be found in the available liter-ature. Furthermore, to the best of our knowledge, no study using elec-troanalytical procedures to determine DIM, in any type of matrix, hasbeen previously reported. For this reason, the aim of this study wasthe development of a quick, easy, cheap and eco-friendly electroana-lytical procedure for the direct analysis of DIM in natural water sam-ples using a carbon-fibre microelectrode (CFM) in combination withsquare-wave voltammetry (SWV).
85T.M.B.F. Oliveira et al. / Microchemical Journal 109 (2013) 84–92
Several electroanalytical methods using microelectrodes, includingCFM, have been used in a variety of applications due to their advantagesover conventional electrodes [12]. The high current densities measured(due to spherical diffusion), the minimized ohmic drop and capacitiveeffects that make possible an increase in the signal-to-noise ratio, fastresponses and the effects of insensitivity in the convection process,allow the working of the microelectrode in organic solvents of low di-electric constant in the study of fast electron transfer reactions, coupledreactions, in flow systems and in vivo analysis [13]. In particular, CFMsare constituted by carbon fibres with small diameters that range fromseveral micrometres to several tens of micrometres, which offerssome advantageous properties including a wide-ranging positive po-tential window, simplicity of construction and low cost. As a conse-quence of these properties, CFMs can be used in the analyticaldetermination of various pesticides in different samples, without pre-treatment or clean-up steps, thus offering an excellent alternative tothe use of chromatographic techniques [14].
Among the electroanalytical techniques currently available for usewith CFM [15], SWV has proved to be extremely sensitive for the detec-tion of several pesticides [16–18]. Their use, by the appropriate applica-tion of a well-developed theoretical model considering current andpotential responses, allows valuable information concerning the kinet-ics andmechanisms of electron transfer to be obtained. This informationis important to identify the intermediate reactive species, to elucidatethe mechanism of action of pesticides and also to supply informationabout its redox properties in the environment. This is due to the factthat redox reactions that occur in the environment can be comparedwith redox reactions that occur at the electrified interface (electrode/solution) when electroanalytical techniques are employed [19].
2. Experimental section
2.1. Reagents and equipment
Electrochemical measurements were carried out using a potentio-stat (Autolab PGSTAT 30, Metrohm-Eco Chemie) equipped with alow-current module (low-current measurements with resolutionsdown to 0.3 fA), controlled by a personal computer, using GPES version4.9 software (General Purpose Electrochemical System, Metrohm-EcoChemie). An Ag/AgCl/ saturated Cl– electrodewas used as the referenceelectrode while the working electrode was a lab-made electrode con-structed from carbon-fibre micro-wire.
A Micronal B474 pH meter equipped with a 3.0 mol L−1 Ag/AgCl/KCl-glass combined electrode was used to adjust the pH values. Allthe solutions were prepared with water purified by a Milli-Q system(Millipore Corp.).
A stock solution of 1.0×10−3 mol L−1 of DIM (CAS: 110488-70-5),kindly supplied by Bayer-Brazil, was prepared daily by dissolving an ap-propriate quantity in acetonitrile and stored in a dark flask. A0.04 mol L−1 of Britton–Robinson (BR) buffer, prepared as describedin a previous paper [20], was used as the supporting electrolyte andthe pH was adjusted to the desired value by adding appropriateamounts of 1.0 mol L−1 NaOH stock solution.
A gas chromatography-mass spectrometer (GC–MS) system fromShimadzu, model GCMS-QP2010, equipped with a mass spectropho-tometer detector, in conjunction with a 30 m DB1-MS J&W Scientificcapillary column (100% methylpolysiloxane, 0.25 mm i.e., 1 mm filmthickness) was employed in chromatography data acquisition. Instru-ment operation and data processing was carried out with Labsolution(GC–MSsolution, release 2.30) software.
2.2. Construction and characterization of the microelectrode
The CFM was constructed from a 7 μm diameter carbon-fibre wireproduced by CTA-Brazil. The micro-wires were inserted into a 1.0 mLtip pipette and filled with epoxy resin. After this, the CFM was
polished using a mechanical polisher and glass paper of differentgranulations. Finally, the microelectrode was cleaned with waterand a microdisk surface was obtained.
The voltammetric characterization was carried out by studying theelectrochemical response of the potassium hexacyanoferrate (III) so-lutions in acid medium, due to its well-established behaviour, and thevoltammograms exhibited sigmoid profiles, characteristic of the utili-zation of microelectrodes [13].
2.3. Electrochemical working procedure
All measurements were performed under ambient conditions. Atwo-electrode configuration was used with the reference electrode(Ag/AgCl/saturated Cl–) also acting as counter electrode, and CFMwith 38.5 μm2 of electroative surface as working electrode. The ap-propriate solutions were transferred into the electrochemical celland the optimization of the analytical procedure for SWV was carriedout following a systematic study of the experimental parameters,such as pH of the medium, the pulse potential frequency (f), pulseamplitude (a) and the height of the potential step (ΔEs) or scan incre-ment, for a potential window ranging from 0 to +1.45 V.
All parameters were properly optimized since their values exertconsiderable influence on the sensitivity of voltammetric measure-ments [15,21]. The optimization was related to the maximum value ofpeak current and the maximum selectivity (half-peak width). Beforeeach experiment, a stream of N2 was passed through the solution for2 min to renew the electrode surface by removal of adsorbed products.
To accomplish that which has been mentioned above, the workingelectrode was placed in a measuring cell filled with 10 mL of a sup-porting electrolyte solution, and before initiating the measurements,some cyclic voltammetries at fast scan rates, in the potential intervalof the DIM oxidation process, were carried out for the best results ofthe CFM. A known concentration of DIM was added to the cell andthe experimental and voltammetric parameters were studied. In allmeasurements, the electrochemical cell was placed in a Faradaycage in order to minimize background noise.
After the optimization of voltammetric parameters, analytical curveswere obtained in pure electrolyte by the standard additionmethod. Thestandard deviation of the mean current measured at the oxidation po-tential of DIM for 10 voltammograms of the blank solution in purifiedelectrolyte was used (Sb) [22,23] in the determination of the detectionand quantification limits (DL and QL, respectively) together with theslope of the straight line of the analytical curves (b) as follows:
DL ¼ 3Sbb
ð1Þ
QL ¼ 10Sbb
ð2Þ
The recovery experiments were carried out by adding a knownamount of DIM to the supporting electrolyte followed by standard ad-ditions from the DIM stock solution and plotting the resulting analyt-ical curves. All measurements were performed in triplicate. Therecovery efficiencies (%R) were calculated using Eq. (3). Here thevalue [DIM]found refers to the concentration obtained by extrapolatingthe analytical curves of the corresponding spiked samples:
%R ¼ DIM½ �foundDIM½ �added
100 ð3Þ
The precision and accuracy of the methodologies were tested withdifferent standard solutions of DIM and the relative standard devia-tions (RSD) were calculated as follows:
where S is the standard deviation of the mean current values obtainedat a know concentration and using the optimized parameters, and �x isthe mean peak current values.
2.4. Chromatography conditions
Chromatographic separations were made by the injection of 1 μL ofthe sample into the front inlet of the GC operating at 250 °C in thesplit ratio of 10.0. Helium (carrier gas) flowed at a rate of1.45 mLmin−1. The oven program started at 200 °C and was immedi-ately ramped at a rate of 40 °C min−1 to 250 °C and 15 °C min−1 witha hold at 290 °C for 3 min. The MS detector was interfaced with theGC at a temperature of 300 °C andwas auto-tuned using perfluorotribu-tylamine (PFTBA, tuning standard). Ionizationwas achieved by electronimpact using an emission current of 70 eV. The MSwas operated underSCAN mode, scanning from 30 to 500 m/z.
These experiments were performed in order to compare the resultsobtained by the use of CFM coupled with SWV. Additionally, in theGC–MS analysis, the QL and the DL were determined, similarly to thecalculations for the SWV experiments. However, in these experimentsthe Sb represents the standard deviation of the areas calculated valuesfrom the blank responses obtained using the chromatographic opti-mized parameter [22,23].
1.26
1.29
0.3
0.4
2.5. Application of methodology
After calculating the DL and QL for the determination of DIM in theBR buffer, the sensitivity, accuracy, reproducibility, precision of theprocedure, and the interference from natural water samples wereevaluated. This was done by means of analytical curves and recoverymeasurements with samples collected from three distinct dams locat-ed at three cities in the state of Ceará, Brazil.
The first sample was collected from the São Mateus dam, hereinaf-ter referred to as SMD, located in the city of Canindé, a semi-sterilenorth-eastern region. The second sample was collected from theAcarape do Meio dam, hereinafter referred to as AMD, located in thecity of Redenção, an urban area with a high degree of pollution thatis spreading to domestic sewerage and agricultural practices, suchas the cultivation of bananas, beans and maize. The third samplewas collected from the Gavião dam, hereinafter referred to as GD, lo-cated in the city of Pacatuba, which is responsible for the water sup-ply to the city of Fortaleza, the capital of the state of Ceará.
The samples were first strained in a 0.5 μm paper filter to removeall particulate components and maintained in a refrigerator even afteruse. Due to high DIM solubility, this study considered the totalamount of DIM to be dissolved in natural waters. These sampleswere used to prepare the adequate supporting electrolyte, whichhad been previously evaluated, by dissolving its components inthese samples. All voltammetric measurements were carried outwithout pre-treatment of the samples, and the recovery curveswere performed in triplicate.
2 4 6 8 10 121.17
1.20
1.23
Potential
pH
Ep
/ V
0.0
0.1
0.2
Current
Ip / nA
Fig. 1. Correlation between the peak currents (●) and peak potential (■) with pHvalues from SWV measurements of 4.60×10−5 mol L−1 DIM in 0.04 mol L−1 BR buff-er, on the CFM with f=100 s−1, a=50 mV and ΔEs=2mV.
3. Results and discussion
The SWV data acquired in this work were used to evaluate the ki-netics and mechanisms of DIM oxidation, and an eco-friendly meth-odology (using no toxic solvents in the extraction of the analyte,and with the working electrode having no toxic constituents in itscomposition) for the analysis of DIM in natural water samples. Tothis end, preliminary studies were performed for the optimizationof the values of experimental (pH of the medium) and voltammetricparameters (f, a and ΔEs) where it was possible to obtain the best an-alytical signal in terms of peak current (Ip) and peak potential (Ep).
3.1. Preliminary experiments
Preliminary SWV experiments were performed in 4.60×10−5 mol L−1 DIM solution with a scan potential from 0.0 to +1.5 Vvs. Ag/AgCl/saturated Cl– to evaluate the effects of the BR buffer, inthe pH interval of 2.0 to 12.0, on DIM oxidation. The decrease in protonconcentration as the pH increased, having a small effect on the SWV re-sponses, showed amaximumvalue at pH3.0, and above this value the Ipdecreased, as shown in Fig. 1. Moreover, as the slope ΔEp/ΔpH pre-sented values of –7.6 mV, which is a very small value compared to the–60 mV that corresponds to a 1 electron and 1 proton reaction, it canbe considered that the Ep value around+1.27 V does not seem to be af-fected by the concentration of H+, suggesting that DIM molecule pro-tonationwas not the determining step in the oxidationmechanism [24].
Additionally, some experiments using cyclic voltammetry were de-veloped in order to evaluate the reproducibility of the voltammetric re-sponses. For DIM, continuous cyclic voltammetries on CFM at100 mV s−1 showed that during the first anodic sweep, with a scan po-tential from+0.9 to +1.5 V, onewell-shaped oxidation peak appearedat around +1.25 V during the forward scan, and a poorly defined peakappeared in the reverse scan. It was also observed a no linear depen-dence between Ip and scan rate (v), and a displacement of the anodicprocess for more positive potentials, resulting in an increase of the sep-aration between the cathodic and anodic peak potentials (ΔEp=Epa−Epc). This data set featuring a quasi-reversible DIM oxidation processaccording diagnostic criteria developed for this technique. In subse-quent scans, the intensity of Ip is diminished by an order of aroundtwo, and Ep changed to more positive values, indicating the adsorptivenature of the redox process, in which the products of the oxidationremained adsorbed on the electrode surface. Additionally, these exper-iments show that stirring for only 2 min between each experiment wassufficient to renew the electrode surface, removing the adsorbed prod-ucts on the electrode surface to obtain suitable reproducibility in thevoltammetric responses.
3.2. Square-wave voltammetry parameters
When microelectrodes are employed, the variation of f has a com-plicated effect on Ip, because this variation corresponds to the simul-taneous variation of both parameters x and y, in which x=κf−1/2,y=rf–1/2, κ is a constant related to the standard heterogeneouscharge transfer rate constant (ks) by κ=ksD−1/2, r is the radius ofthe microelectrode, and in which D is the diffusion coefficient [25].
Based on this, the effect of f on Ip and Ep in the DIM oxidation pro-cess, using the CFM, was investigated from 10 to 100 s−1. The result-ing voltammograms showed that the increase in f promoted anincrease in Ip, and the reaction seemed to become less reversible
0.9 1.0 1.1 1.2 1.3 1.4 1.5-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
I / n
A
E / V
(A)
0.9 1.0 1.1 1.2 1.3 1.4 1.5-0.1
0.0
0.1
0.2
0.3
I / n
A
E / V
(B)
0.9 1.0 1.1 1.2 1.3 1.4 1.5-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
I / n
A
(C)
87T.M.B.F. Oliveira et al. / Microchemical Journal 109 (2013) 84–92
because both x and y decreased. As shown in Fig. 2, Ip increased until70 s−1 with the increase in f, and above this value the peak currentspresented lower values, characterizing the presence of the maximumat 70 s−1, a typical profile that occurs in a quasi-reversible redox pro-cess with adsorbed reactants and products.
According to SWV theory regarding spherical and disk microelec-trodes, Ep depends linearly on log (f), with the slope:
ΔEpΔ log fð Þ¼
�2:3RTαnF
ð5Þ
where R is the gas constant, T is the temperature, α is the electrontransfer coefficient, n is the number of electrons, and F is the Faradayconstant [25] Therefore, for DIM oxidation using CFM, Ep varies line-arly with the logarithmic value of f, and a plot of ΔEp vs. Δlog (f)gives a straight-line curve, as described by:
Ep ¼ 1:153 Vð Þ þ 0:0614log fð Þ ð6Þ
Using this slope, considering α=0.5 and T=298.15 K, andsubstituting the known values of R and F into the above relationshipsbetween Ep and f, the value of n was calculated as being equal to 2,considering the determining step of the redox reaction.
To confirm the DIM redox process type on CFM, SWV experimentswere performed in 4.60×10−5 mol L−1 DIM in 0.04 mol L−1 BR buffer(pH 3.0) realizing a scan potential from +0.9 to +1.5 V, with a=50mV and ΔEs=2mV. At different f values, one voltammetric peak to-wards the forward component and one peak towards the backwardcomponent of the currents were observed, as well as a well-shapedand symmetrical resultant curve, as shown in Fig. 3, indicating thatthe voltammetric responses for DIM oxidation do not achieve thesteady-state condition, as was expected, considering the use of micro-electrodes [26].
The separation between Ep in forward and backward componentswas practically constant, with Ep values closer to those observed inresultant responses. In addition, the width at half-height (ΔEp/2) ofthe resultant peak and also the ratio between the forward and back-ward peaks decreased when f values were increased. These responsesare in agreement with theoretical predictions for SWV voltammo-grams for quasi-reversible redox reactions affected by surface elec-trode reactions with adsorbed reactants and products [25].
The structure of the DIM is very complex due to its large amountof conjugated double bonds in the structure, steric hindrance, donorand withdrawing groups of electrons, among others, whichmakes ox-idation mechanism difficult to understand. Evaluating the molecule
0 20 40 60 80 1000.0
0.1
0.2
0.3
0.4
0.50.9 1.2 1.5 1.8 2.1
1.22
1.23
1.24
1.25
1.26
1.27
1.28
Current
I p /
nA
f / s-1
Potential
log f
Ep / V
Fig. 2. Peak currents plotted as a function of frequency (●), and peak potential plottedas a function of the logarithm of frequency (■) for the SWV experiments, evaluatedwith 4.60×10−5 mol L−1 DIM in 0.04 mol L−1 in BR buffer (pH 3.0), on the CFMwith a=50 mV and ΔEs=2 mV.
E / V
Fig. 3. Square-wave voltammograms for oxidation of 4.60×10−5 mol L−1 DIM solutionon the CFM, with a=50 mV, ΔEs=2 mV and ƒ=10 s−1 (A), ƒ=50 s−1 (B) andƒ=100 s−1 (C), all showing the forward (- - -), backward (. . .) and resultant (—) com-ponents of the current, respectively.
as a whole, the double bond located in the vicinity of the aromaticsand morpholinic rings is more unprotected and susceptible to under-go reaction, indicating the most probable site of oxidation. These dataseem to have good correlation with reports of the literature [27,28],showing that oxidation of conjugated molecules require relativelyhigh oxidation potential, as that obtained for the DIM. Besides, fromthe analysis of SWV responses in conjunction with informationabout organic electrochemistry [27,28] it can be ascertained that themost feasible mechanism for DIM oxidation on CFM involves thetwo-electron oxidation with adsorption of reactants and productson the electrode surface.
The influence of a on Ip was also considered for the DIM oxidationprocess for values from 5 to 50 mV, and the voltammetric responsesindicated that an increase in Ip occurred as long as a values were
increased. However, only linear relationships occurred for a valueslower than 30 mV; above this value, the deviation from linearity oc-curred due to the fact that it is almost impossible to achieve steady-state conditions at high amplitude values. Moreover, all Ep valuesshifted to more positive values as long as a values increased, whichis typical for redox processes involving strong adsorption from prod-ucts and reactants on the electrode surface [15,29,30], as shown inpreliminary experiments using cyclic voltammetry. Thus, for analyti-cal applications, a value of 30 mV was chosen for a.
For ΔEs, the increase in this value will also increase the signal andsensitivity of the methodology. However, for larger values of this pa-rameter, a widening of the peaks may occur, thus diminishing the res-olution of the analysis. Therefore, an evaluation was made and theresponses showed that ΔEs promoted an increase in peak currentvalues until 6 mV. However, no linear dependence between Ip andΔEs was obtained, which is also typical of the voltammetric responsesin reactions involving reactants and products adsorbed on the elec-trode surface [31]. Consequently, the value for this parameter was2 mV in subsequent experiments.
3.3. Analytical curves
All the aforementioned parameters were employed not only todraw the analytical curves for the DIM oxidation process on CFM ina supporting electrolyte medium, but also to determine the sensitivityof the proposed methodology, and for applications in natural watersamples. For this reason, using the optimized parameters above, line-ar calibration curves were obtained from consecutive additions of thestock DIM solution into the electrochemical cell containing 10 mL ofthe BR buffer (pH 3.0). The SWV responses were recorded for a con-centration range from 3.15×10−6 to 1.44×10−4 mol L−1 of DIM,where the results obtained showed a proportional increase in Ipwith the increase of the concentration of DIM, with a small displace-ment in Ep, indicating strong adsorption of reagents and products ofthe redox reaction on the electrode surface, as shown in Fig. 4. Inthis step, five analytical curves were constructed, and the resultsshown represent a medium of the all obtained curves.
The analytical curves for DIM oxidation on CFM can therefore berepresented by:
Ip nAð Þ ¼ –2:65� 10–3 �0:21� 10–3� �
þ 1:34 �0:11� 10–2� �
DIM½ � μmol L–1� �
ð7Þ
with a negative interception and a correlation coefficient of 0.9996.Therefore, an evaluation of the presence of random errors was real-ized by a significance test in order to determine if the difference
0.6 0.8 1.0 1.2 1.40.00
0.04
0.08
0.12
0.16
0.20
0 40 80 120 1600.00
0.04
0.08
0.12
0.16
0.20
I / n
A
E / V
I p / n
A
[DIM] / µmol L-1
Fig. 4. Square-wave voltammograms for DIM in 0.040 mol L−1 BR at pH 3.0 on the CFM,with f=70 s−1, a=30 mV, ΔEs=2 mV, and concentrations in the interval from3.15×10−6 to 1.44×10−4 mol L−1 of DIM. Insert corresponds to the analytical curvesobtained from voltammograms.
between the interception obtained in these analytical curves andthe standard values originated from random error [32]. The t-testwas used according to:
t ¼ �x−μð Þffiffiffin
ps
ð8Þ
where �x is the average from interception values obtained, μ is thestandard value expected in the case of the interception being zero, nis the number of determinations and s is the standard deviation ofthe current responses. The t value calculated was 2.65, which waslower than the critical value (tcritical=2.78) at an assurance level of95%, indicating that no considerable differences occurred betweenthe medium value calculated and the theoretical value, and the nega-tive interception was free from random errors.
As the analytical curves are free from random errors, the confi-dence interval (CI) for interception and slope was calculated accord-ing to:
CI¼ DIM½ � � tn−1ð Þs= ffiffiffin
p ð9Þ
where DIM½ � is themedium concentration of DIM, tn–1 is the calculatedt value, and the other symbols are similar to those used in the t-test.These analyses were also performed at an assurance level of 95%,and the values calculated can be represented by Eq. (7).
Additionally, the detection limit (DL) and quantification limit (QL)for DIM oxidation on CFM, according to previously optimized param-eters, were calculated employing the criteria presented in theExperimental section. Another important parameter analysed in thepresent study was the reproducibility in the voltammetric responses,which was evaluated in five different solutions containing7.86×10−6 mol L−1 of DIM, and the value of RSD obtained for n=5was lower than 1.0%. Additionally, the repeatability was also evaluatedin the same concentration as above and the RSD obtained for n=10wasaround 1.0%.
DIM was also evaluated using the chromatographic technique(GC–MS) in order to compare the results obtained with SWV on theCFM. The chromatographic determinations of DIM were performedusing the experimental parameters presented in the Experimentalsection, which are similar to the chromatographic conditions alreadyreported by Moreno et al. [33], and Hengel and Shibamoto [34].Fig. 5A shows a representative mass spectrum of DIM and Fig. 5Bshows the proposed fragmentation process of the mainly molecularion peaks that occur in the DIM molecule.
In addition, using helium gas as carrier gas at a flow rate of1.45 mL min−1, the analytical curve in pure Milli-Q was thenobtained by the use of the standard addition method, consideringthe range of concentration from 2.23×10−5 to 1.77×10−4 mol L−1.The analytical curves for GC–MS can therefore be represented by:
A ¼ –1:36� 10–5 þ 3:20 DIM½ � μmol L–1� �
ð10Þ
with a correlation coefficient of 0.9972. One of the obtained chro-matograms is presented in Fig. 6 showing the presence of the twochromatographic peaks, at retention times of 6.07 and 6.35 min,which correspond to Z-dimethomorph and E-dimethomorph, respec-tively. Additionally, a linear dependence of the peak areas can be ob-served with the increase in DIM concentration, which was used tocalculate the DL and QL values for GC–MS experiments. The data pre-sented here pertains only to the peak at 6.07 min.
The analytical parameters, or figures of merit, were evaluated forthe proposed procedure as well as for GC–MS experiments, accordingto the procedure described in the Experimental section. The correla-tion coefficient (r), which determines the degree of linearity betweenthe concentration of DIM and analytical responses, the standard devi-ation of the arithmetic mean of ten blank solutions for SWV or GC–MS
H3CO OCH3
Cl
O
N
OH
H3CO OCH3
Cl
C
O
H
N
O-
H3CO OCH3
Cl
OH
H3CO OCH3
Cl
OH
H3CO OCH3
Cl
OH
m\z 301
H3CO
H3CO
-
Cl
OH
Cl
OH
m\z 165
(A)
(B)
Fig. 5. (A) Representative mass spectrum of DIM for an isomer mixture. (B) Proposed fragmentation process for CG-MS data from DIM fungicide. Employed conditions: injection of1 μL of the sample; GC operating at 250 °C; Helium (carrier gas) flowed at a rate of 1.45 mL min−1; MS was auto-tuned using perfluorotributylamine (PFTBA, tuning standard); andoperated under SCAN mode, scanning from 30 to 500 m/z, where the ionization was achieved by electron impact using an emission current of 70 eV.
89T.M.B.F. Oliveira et al. / Microchemical Journal 109 (2013) 84–92
3 4 5 6 70
2000
4000
6000
8000
10000
(E)
H
OCH3
OCH3
N
O O
Cl
H
N
O O
OCH3
OCH3
Cl
time / min
(Z)
Fig. 6. Total ion chromatogram of analytical curve of DIM by GC–MS. Employed condi-tions: injection of 1 μL of the sample; GC operating at 250 °C; Helium (carrier gas) flo-wed at a rate of 1.45 mL min−1; MS was auto-tuned using perfluorotributylamine(PFTBA, tuning standard); and operated under SCAN mode, scanning from 30 to500 m/z, where the ionization was achieved by electron impact using an emission cur-rent of 70 eV.
experiments (Sb), the slope of the analytical curve (s), the detectionlimit (DL), and the quantification limit (QL), are shown in Table 1for SWV experiments on the CFM and for GC–MS experiments.
From a close analysis of the results obtained for DL and QL by theuse of SWV combined with CFM, and by the use of GC–MS proce-dures, it can be observed that the use of CFM combined with theSWV technique provided an alternative method for the analytical de-termination of DIM due to the possibility of its use in the simple andreliable determination in concentrations comparable those obtainedby gas chromatography combined with mass selective detection,which is a of the standard procedure used in analysis of this com-pound [34].
The proposed methodology presented in this study is eco-friendlyand suitable for DIM detection, and it can be an alternative procedureto the traditional gas chromatography methodologies, which involvethe inconvenience of being expensive, require time-consumingmulti-residue methods, involve difficulties in the preparation andclean-up steps of the samples, and, most significantly, employ organicsolvents, which can damage the environment [6–11].
Additionally, the repeatability and reproducibility values calculat-ed in the analytical measurements obtained in the present study indi-cated good precision and accuracy of the electroanalytical procedureand the possibility of its application in complex samples.
Despite DIM showing marked toxicity for both humans and otherliving organisms, Brazilian governmental agencies have not limitedthe use of DIM in agricultural practices and have not established, by
Table 1Parameters of the analytical curves obtained in DIM detection using CFM combinedwith SWV and GC–MS experiments (linearity range; slope of the analytical curves; r:correlation coefficient; Sb: standard deviation from the arithmetic mean of ten blanksolutions; DL: detection limits; and QL: quantification limits). The voltammetric datawere evaluated using medium values from five analytical curves and the chromato-gram data were evaluated using three analytical curves.
Parameter SWV GC–MS
Linearity range 3.15×10−6 to1.44×10−4 mol L−1
2.23×10−5 to1.77×10−4 mol L−1
Intercept –2.65×10−12 A –1.36×10−5 (a.u.)Slope 1.34×10−6 A mol−1 L 3.20×1010 (a.u.) L mol−1
specify laws, the permitted maximum limits in natural waters orfoods. Moreover, the United States Environmental Protection Agency(US-EPA) has also not established the maximum contaminant levelfor DIM in natural and drinking water. However, the EPA's level forchronic exposure to residues of DIM has been established as960 ppb for children of 1–6 years of age, and up to 3400 ppb for indi-viduals who are 13 years and older [35]. Thus, the proposed electro-analytical procedure could represent an interesting alternative toquantify DIM residues in natural waters.
3.4. Application of electroanalytical methodology in natural watersamples
The analytical procedure shown above was applied for naturalwater samples collected in three different dams in the state ofCeará, as described in the Experimental section. The collecting pointswere selected based on the different levels of organic matter, includ-ing industrial and domestic pollution. The water samples were used,as received, with only one filtration step having been previously car-ried out to prepare the supporting electrolyte (0.04 mol L−1 BR bufferwith pH adjusted to 3.0 by the addition of adequate quantities of1.0 mol L−1 NaOH solution), and the analytical curves were againobtained by SWV on CFM experiments to evaluate the influence ofsample components in the voltammetric responses.
The analytical curves performed in the natural water samples canbe represented by Eqs. (10), (11) and (12) for SMD, AMD and GD, re-spectively:
Ip Að Þ ¼ −4:36� 10−12 �1:11� 10−12� �
þ 1:50
� 10−6 �4:50� 10−8� �
DIM½ � mol L−1� �
ð11Þ
Ip Að Þ ¼ −4:05� 10−12 �7:76� 10−13� �
þ 1:46
� 10−6 �3:66� 10−8� �
DIM½ � mol L−1� �
ð12Þ
Ip Að Þ ¼ −4:01�10−12 �3:71�10−13� �
þ 1:45�10−6 �1:30�10−7� �
DIM½ � mol L−1� �
ð13Þ
Similar to that observed in the supporting electrolyte, the analyticalcurves evaluated in natural water samples also presented negative inter-ceptions. For this, the t-test was applied for three curves, similar to thateffectuated in the BR buffer, and all t values were calculated consideringthe use of triplicate experiments. Thus, 3.02, 2.25 and 1.55 were the tvalues obtained for SMD, AMD and GD, respectively. These values werelower than the scheduled critical value (tcritical=4.30) at an assurancelevel of 95%, indicating that no considerable differences occurred be-tween the calculated medium values and the theoretical values, andthe negative interceptions were free from random errors.
Additionally, the analysis of the equations for three samples ofwater indicated that the analytical sensitivities, defined by the slopeof the analytical curves, are practically constant even when comparedwith those obtained from the BR buffer, independent of the origin ofthe sample. As such, the paired t-test was applied to observe ifthere were variations between the measured inclinations in the sup-porting electrolyte and in the natural water samples due to randommeasurement errors, and if the differences between the samplescould also contribute to the variation between the measurements[36]. Therefore, the paired t-test was used according to:
t ¼ dffiffiffin
psd
ð14Þ
where d and sd are, respectively, the mean and standard deviations of d,the difference between the values obtained in the supporting electrolyte
Table 2Analytical parameters obtained in natural water samples for DIM detection using CFM combined with SWV. The same parameter evaluated in supporting electrolyte. All data wereevaluated using medium values from three analytical and recovery curves.
SMD AMD GD
LR (mol L−1) 3.15×10−6 to 1.44×10−4
r 0.9998 0.9998 0.9997CIi (A) ±1.11×10−12 ±7.76×10−13 ±3.71×10−13
91T.M.B.F. Oliveira et al. / Microchemical Journal 109 (2013) 84–92
and in natural water samples, and n is the number of determinations (inthis case, n=3). In this way, the t values calculated were 2.81, 3.00 and2.67 for SMD, AMD and GD, respectively, which produced lower valuesthan the scheduled critical value (tcritical=4.30) at an assurance level of95%, confirming that the inclinations obtained in all natural water sam-ples were similar to the inclinations obtained in the BR buffer.
Consequently, it was possible to conclude that the analytical sen-sitivities in these complex samples and in the supporting electrolytewere practically equal, and that these experiments were free fromrandom errors. Therefore, Ip was practically unaffected by the pres-ence of components in the natural water samples. The small differ-ences that were present in the curves can be related to theelectrochemical responses that can occur with other organic and inor-ganic molecules present in natural water samples.
To confirm the uniformity in the responses in the electrolyte andin natural water, the DL and QL were calculated and are shown inTable 2. Here it can be seen that the DL and QL values calculated ineach sample are statistically identical, indicating that CFM can beused with success for direct electrochemical determination of DIMin natural water samples.
In order to quantify the small interference effects in the electro-chemical responses, recovery curves were constructed for these natu-ral water samples. To achieve this, the samples were artificially spikedwith 3.15×10−6 mol L−1 of stock standard solution, since DIM wasnot detected in these samples as collected. Recovery curves were ac-quired by the standard addition method and the recovery percentagewas graphically identified, with the abscissa axis referring to the con-centration of DIM in the electrochemical cell. Extrapolating the curvealong this axis yields the sample concentration, allowing the calcula-tion of the recovery values. All curves were constructed in triplicate.The results obtained for recovery percentages are shown in Table 2.
Additionally, the recovery data obtained were used to calculatethe accuracy of the procedure using SWV on CFM in natural watersamples. The accuracy represents, statistically, the concordance be-tween the added concentration and the recovered concentration.Therefore, the accuracy was calculated from the BIAS parameteraccording to:
%BIAS ¼ x−x0x0
� �100 ð15Þ
where x is the average from recovered concentrations and x0 is theadded concentration [36].
Therefore, the calculated BIAS values were 0.20%, –0.64% and–0.25% for SMD, AMD and GD, respectively. Based on the Internation-al Conference on Harmonization (ICH) [23], the recovery, the BIASand the RSD values from recovery curves presented in Table 2 arevery satisfactory for analytical procedures, and for this reason themethodology employing CFM combined with SWV is suitable forthe direct analysis of DIM in natural water samples.
4. Conclusions
The electroanalytical detection of DIM has been reported here forthe first time, and the results indicate that the use of SWV allows theevaluation of the kinetics and electrochemical mechanisms for theDIM oxidation process on CFM, which is of considerable importancesince it allows for a better understanding of DIM behaviour in the en-vironment and of the possible interferences in the application of thisprocedure in complex samples, such as natural waters.
All experimental data presented here show that the use of CFM forDIM determination was possible with a detection limit of3.90×10−7 mol L−1 (151.1 μg L−1), along with a high level of re-peatability and reproducibility. The comparison between the analyti-cal data obtained for the determination of DIM using SWV on CFMand GC–MS led to very coherent results, indicating that the proposedelectroanalytical procedure is an important tool to detect DIM resi-dues in small concentrations, without the need for pre-treatment orclean-up of the samples. As such, the proposed methodology is aneco-friendly and important tool for the direct detection of DIM in nat-ural waters, and an alternative procedure to the traditional gas chro-matography methodologies.
Acknowledgements
The authors wish to thank the Brazilian research funding institu-tions CNPq, CAPES and FINEP for their financial support. D. De Souzaalso wishes to thank the CNPq (process 35.0014/2010.8) and FUNCAP(process DCR-0039-1.06/09) for the PhD grants, and T. M. B. F. Oli-veira would like to thank the UERN for the award of a fellowship.
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