DOI:
10.1039/C4RA00286E
(Paper)
RSC Adv., 2014,
4, 14270-14280
Determination of trace amounts of thymol and caffeic acid in real samples using a graphene oxide nanosheet modified electrode: application of experimental design in voltammetric studies
Received
15th January 2014
, Accepted 11th March 2014
First published on 11th March 2014
Abstract
A graphene oxide nanosheet modified glassy carbon electrode (GO/GCE) was used for the determination of thymol (Thy) and caffeic acid (CA). The GO-based sensor was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. The oxidation peak current of the Thy and CA was determined in a 0.2 M Britton–Robinson (B–R) buffer solution and optimized by experimental design. A central composite rotatable design (CCRD) was used to evaluate the effects of the variables in the differential pulse voltammetry method (DPV). These variables include instrumental and experimental parameters. Under the optimized conditions the dynamic range for Thy and CA was from 2.0 to 200.0 μM and 1.5 to 270.0 μM and the detection limits were found to be 6.5 × 10−8 M and 2.5 × 10−9 M for Thy and CA, respectively. The electron transfer coefficient (α) as a kinetic parameter was also determined for the Thy and CA oxidation process. The results revealed that the modified electrode shows an improvement in anodic oxidation activity of Thy and CA due to a marked enhancement in the current response compared with the bare GCE. The modified electrode demonstrated good sensitivity, selectivity and stability. The prepared electrode was applied for the determination of Thy and CA in real samples such as a thyme plant and a eucalyptus inhaler drop.
1. Introduction
Thymol (2-isopropyl-5-methylphenol) is a naturally occurring compound found in plants such as Thymus and in the essential oil of various species of thyme (e.g. Thymus vulgaris L.). Thy has been used in inhibiting the peroxidation of liposome phospholipids in a concentration-dependent manner. The primary uses of Thy for medicinal purposes have been reported as a topical antifungal or antibacterial agent and also as a counter irritant in topical analgesic formulations. In addition, Thy has found applications in many different industrial fields such as production of perfumes, food flavoring, mouthwashes, cosmetics and also as a stabilizer to several therapeutic agents.1,2 The development of analytical techniques for the analysis of the Thy is important for increase quality and medical control applications. Many methods, including UV spectrophotometry,3 high performance liquid chromatography4 and liquid chromatography5 have been used for the determination of Thy.
Among cinnamic acid derivatives, caffeic acid (3,4-dihydroxycinnamic acid) is a kind of polyphenol that is widely distributed in higher plants as glycosides, esters and the free form. The caffeic acid (CA) is one of the most investigated derivatives not only because of its protective antioxidant behavior but also due to its effectiveness against immunoregulation diseases, asthma and allergic reactions.6 CA is one of the phenolic components that has been known as a part of the general plant with defense mechanism against infection predation.7 Additionally recent studies have shown that CA derivatives exhibited potential activity against HIV integrase and can inhibit HIV replication with moderate anti-HIV activity in cell culture.8
The electrochemical methods using chemically modified electrodes (CMEs) have been widely used as sensitive and selective analytical methods for detection of the trace amounts of biologically important compounds.9 In recent years, with the rapid development of nanostructured materials and nanotechnology in the fields of biotechnology and medicine have received considerable attention due to their novel properties.10
It was P. R. Wallace in 1946 that first wrote on the band structure of graphene and showed the unusual semimetallic behavior in this material. Since the discovery of graphene, more attention is focused to develop the application of the properties of graphene and its derivatives, because unique properties, namely ballistic conductivity, high elasticity, high mechanical strength, high surface area up to 2630 m2 g−1, and rapid heterogeneous electron transfer,11,12 make graphene a good candidate in many technological applications such as nanoelectronics, chemical/bio-sensors, nanocomposites, batteries, etc. The graphene is a two-dimensional sheet of sp2-bonded carbon atoms, which can be viewed as an extra-large polycyclic aromatic molecule.13 In addition, with the 2D structure, the monolayer graphene has its whole volume exposed to the environment, which can maximize the sensing effect. Graphene oxide (GO) is one of the most important derivatives of graphene which also has a large surface area, excellent conductivity and strong mechanical strength. In 1958, Hummers' reported14 the method that most commonly used today. In the GO, the contiguous aromatic lattice of graphene is interrupted by epoxide, alcohol, ketone carbonyl, and carboxylic groups. Recently, GO-based electrochemical sensors and biosensors have been utilized for the determination of dopamine,15 caffeine16 and glucose oxidase.17
In recent years, chemometric tools have been frequently applied to the optimization of analytical methods, because of their advantages such as a reduction in the number of experiments that lead to lower reagent consumption and considerably less laboratory work.18 Thus, these methods are faster to implement and more cost-effective than traditional univariate approaches. These enable the simultaneous study of several control factors and the development of mathematical models that permit estimate of the relevance and statistical significance of factors being studied. They also facilitate the evaluation of interaction effects between factors.19 The most appropriate multivariate techniques that are used in analytical optimization are response surface methodology (RSM). This methodology includes three steps: (1) central composite rotatable design (CCRD), (2) response surface modeling through regression analysis and (3) process factor optimization using the response surface models.20 The central composite design (CCD) was presented by Box and Wilson.21 A CCD combines a two-level full or fractional factorial design with additional points (star points) and at least one point at the center of the experimental region. This point is selected such that it has several properties, such as rotatability or orthogonality, in order to fit the quadratic polynomials. The CCD is a better alternative compared to the full factorial, three-level design as it demands a smaller number of experiments while providing comparable results. Therefore, this method has been the most accepted experimental design for second-order models.19,22
To the best of our knowledge, electrochemical behavior and voltammetric determination of Thy and CA using GO modified glassy carbon electrode has not been reported yet. In this paper, we describe the preparation and application of a grapheme oxide nanosheets modified glassy carbon electrode (GO/GCE) as a new sensor for the determination of Thy and CA in an aqueous buffer solution. In addition, we have evaluated the effect of various parameters on the oxidation of Thy and CA by experimental design method. We have examined this method for the voltammetric determination of Thy and CA in real samples such as eucalyptus inhaler drop, Thymex syrups and thyme plant.
2. Experimental
2.1 Instruments, chemicals and software
The cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronocoulometry measurements were carried out using a Sama 500 potentiostat (Isfahan, Iran). Electrochemical impedance spectroscopy (EIS) experiments were carried out using an Autolab potentiostat-galvanostat PGSTAT 35 (Eco Chemie Utrecht, Netherlands) equipped with NOVA 1.6 software. All the electrochemical studies were performed at 25 ± 1 °C with a three electrode assembly including a 50 mL glass cell, an Ag/AgCl electrode as the reference electrode and a platinum wire as the counter electrode. The working electrode was a modified glassy carbon electrode (2 mm diameter). A metrohm pH meter model 691 was also used for pH measurements. Fourier transform infrared (FTIR) spectra were recorded on a Nicolet magna FTIR-550 infrared spectrometer. Field emission scanning electron microscopy (FESEM) image was obtained using a Hitachi S4160 FESEM system. X-ray diffraction (XRD) patterns of the GO were carried out using X'Pert PRO, Philips system equipped with Cu Kα1 radiation (λ = 1.5406 Å). UV-vis absorption spectra was recorded with a UV-Perkin Elmer spectrophotometer (Lambda 2S). Demineral water was formed with an ultrapure water system (smart 2 pure, TKA, Germany).
All of the solutions were freshly prepared using demineral water. The pure form of thymol (Thy) and caffeic acid were purchased from Merck. A 0.2 M of B–R buffer solution was applied as supporting electrolyte and prepared from an acidic solution of CH3COOH, H3PO4 and H3BO3 by adjusting the pH with a saturated solution of NaOH. All other reagents were of analytical grade from Merck. The drug samples were purchased from local pharmaceutical company (Iran).
2.2 Synthesis of graphene oxide
GO was synthesized directly from graphite by a modified Hummers method.14 According to this method, at first 1 g graphite was stirred in 30.0 mL H2SO4 (98%) (in an ice bath) for 5 h. KMnO4 (6.0 g) and NaNO3 (1.0 g) were slowly added while keeping the temperature less than 20 °C. The mixture was then stirred at 50 °C for 45 min. Next, 100.0 mL demineral water was added and the mixture was heated at 70 °C for 30 min. The reaction was terminated by addition of demineral water (100.0 mL) and 30% H2O2 solution (5.0 mL). The resulting mixture was washed by repeated centrifugation and filtration, first with 10% HCl aqueous solution and then with demineral water. Finally, the GO product was obtained after drying process.
2.3 Preparation of modified electrode
The GCE was carefully polished with 5.0 and 0.05 μm alumina slurry on a polishing cloth in sequence, then washed successively with HNO3–H2O (1
:
1), ethanol and demineral water in an ultrasonic bath for 15 min and dried in air. For fabrication of modified electrodes, the modifier suspension was prepared by dispersing 0.03 g GO in 200.0 mL H2O under ultrasonication for 30 min to obtain a homogeneous and stable suspension. The cleaned GCE was coated by casting proper volume of the brown suspension of GO and dried in the air. After the solvent was evaporated, the electrode surface was thoroughly rinsed with demineral water and dried in the air.
2.4 Design of experiment (DOE)
The optimization of effective chemical and instrumental parameters in the determination of Thy and CA were studied by response surface methodology (RSM). This method is a collection of statistical and mathematical techniques that is suitable for analysis and modeling of problems in which a response is influenced by several variables. It defines the effect of the independent variables, alone or in combination regarding the studied processes. The main advantage of RSM is to reduce the number of experimental trials needed to evaluate multiple variables and their interactions and provide the information necessary for design and process optimization. Additionally, this experimental response surface method generates a mathematical model which is presented in the graphical form.23
The CCRD is a useful method for optimization of the effective variables through analyzing the interaction between them in a minimum number of experiments. This design process consists of three parts: (1) a full factorial or fractional factorial design; (2) an additional design, often a star design in which experimental points are at a distance α from its center; and (3) a central point. Full uniformly rotatable central composite designs present the following characteristics: (1) they require an experiment number according to N = 2f + 2f + r, where f is the number of factors and r is the replicate number of the central point; (2) α-values depend on the number of variables and can be calculated by α = ±2f/4; (3) all factors are studied in five levels.24 The experiments are often carried out in randomized order in order to minimize the effects of unexplained variability in the actual responses due to extraneous factors.25 The response behavior could be related to the selected independent factors by a second-order polynomial. The generalized response surface model is shown as follows:
For instrumental parameter:
|
Yi = β0 + β1x1 + β2x2 + β3x3 + β11x12 + β22x22 + β33x32 + β12x1x2 + β13x1x3 + β23x2x3
| (1) |
For experimental parameter:
|
Yi = β0 + β1x1 + β2x2 + β11x12 + β22x22 + β12x1x2
| (2) |
where
Yi is the predicted response,
xi is the independent variable,
β0 is the intercept (constant),
βi,
βii and
βij are the linear, quadratic and interaction coefficients, respectively. In this work, we used the CCRD method for evaluate relevance of three instrumental factors (scan rate, step potential, modulation amplitude) and two experimental factors (pH and graphene oxide nanosheets content) effective on the DPV response MINITAB@ Release 16, a statistical software package, was used for data processing. The significance level of 95% was set for the mathematical model and surface response.
3. Results and discussion
3.1 Characterization of the prepared graphene oxide
The GO was characterized using FTIR, UV-Visible, SEM and XRD (Fig. 1). The spectrum of FT-IR of GO showed that the peak at 3420 cm−1 attributes to O–H stretching vibration, the peak at 1729 cm−1 attributes to C
O stretching vibration, the peak at 1626 cm−1 traits to deformation of O–H, the peak at 1215 cm−1 attributes to vibration of C–O (epoxy), and the peak at 1052 cm−1 characteristics to vibration of C–O (alkoxy). The experimental results demonstrated that some functional groups with negative charge were grafted in carbon ring of GO during its preparation. The SEM was used to investigate the surface morphological characters of the as-synthesized GO (Fig. 1B). It is observed that the GO has a typical flake-like shape with slight wrinkles on the surfaces.
 |
| Fig. 1 Characterizations of graphene oxide: (A) FT-IR spectrum of GO, (B) SEM image of GO, (C) XRD spectrum of GO, (D) UV-vis spectrum of GO. | |
The XRD pattern of the GO (Fig. 1C) displays a sharp diffraction peak at 9.91 degree attributed to the successful oxidation of the starting graphite. To further demonstrate the formation of GO, UV-vis spectroscopy was carried out (Fig. 1D). The GO has a characteristic band at 230 nm and a shoulder at 300 nm, corresponding to π–π* transitions of carbon–carbon bonds and n–π* transitions of carboxyl bonds, respectively.26 The analysis results are consistent with previous research27 which demonstrates that GO nanosheets were successfully synthesized.
3.2 Electrochemical characterizations of the modified electrode
The cyclic voltammograms of GCE and GO/GCE in 0.2 M B–R buffer solution (pH 4.7) containing 5.0 × 10−3 M [Fe(CN)6]3−/4− are shown in Fig. 2A. [Fe (CN)6]3−/4− produced a couple of well-defined redox waves at GC electrode with a peak-to-peak separation (ΔEp) of 200.0 mV at 100 mV s−1. When the electrode was coated with GO, the peak currents of redox peaks decreased obviously, which could be attributed to the negatively charged carboxyl group on GO surface,28 blocking the diffusion of [Fe(CN)6]3−/4− from solution to electrode surface. Moreover, the increase of ΔEp (250.0 mV) was observed at GO/GCE and it indicated that GO film could improve the irreversibility of the electrode reaction process.
 |
| Fig. 2 CVs (A) and EIS (B) of bare GCE (a) and GO/GEC (b) in 5 mM [Fe(CN)6]3−/4− solution containing. The scan rate of CV is 100 mV s−1; the frequency range of EIS was from 0.1 to 104 Hz at the formal potential of 0.2 V. | |
In order to get more information about the surface, electrochemical impedance spectroscopy (EIS) was employed. The Nyquist plot of the bare GCE and GO/GCE in 0.2 M B–R buffer solution (pH = 4.7) containing 5.0 × 10−3 M [Fe(CN)6]3−/4− and in the frequency range from 0.1 Hz to 104 Hz at the formal potential, 0.2 V, was shown in Fig. 2B. It can be seen that the impedance spectrum of the bare GCE electrode exhibits two distinct regions: (1) a semicircle, related to the charge-transfer process; (2) a 45° line defining a region of semi-infinite diffusion of species in the electrode. The value of the electron transfer resistance (Rct, semicircle diameter) depends on the dielectric and insulating features at the electrode–electrolyte interface.29 The electrode surface coverage (θ) can be calculated as:
|
θ (%) = [1 − (Rct bare/Rct modified)] × 100
| (3) |
The Rct for the GCE was 1.06 kΩ. The assembly of GO layer on the electrode surface lead to a lower rate of the electron transfer [(Rct) modified = 2.97 kΩ]. Using these values and eqn (3), the surface coverage value was estimated to be 65.0%.
It can be seen that a small well defined semicircle region/part at higher frequencies was obtained at the bare GCE, indicated small interface impedance. When GO was deposited on the surface of GCE, the impedance value was much larger than that at GCE. In this regard, the negative charges on GO film lead to appearance of the electrostatic repulsion into the electrode/solution system. Consequently, the rate of the electron transfer of Fe(CN)63−/4− probe on GO/GCE electrode reduces and impedance value is much larger than that at bare GCE. This is reflected by the appearance of the large semicircle part on the spectrum, corresponding to a charge-resistance, Rct = 2.97 kΩ. This phenomenon also demonstrates that GO was successfully immobilized on the GCE surface which is in good agreement with that found by cyclic voltammetry measurements.
After proving the presence of the modifier at the surface of the electrode by EIS, for electrocatalytic investigations of the modified electrode, cyclic voltammograms of this electrode were drown in the presence of probes solution of Fe2+/Fe3+ in 0.2 M B–R buffer solution (pH = 3.0) at various scan rates (not shown). An approximate estimate of the electrode surface coverage of the electrode was obtained using a 0.1 mM K3Fe(CN)6 solution as a probe at different scan rates. The electrode-surface coverage was calculated by the method used by Sharp et al.30 The peak current is related to the surface concentration of the electroactive species Γ by the following equation:
where
n represents the number of electrons involved in the reaction;
A is the surface area (0.0314 cm
2) of the GO/GCE;
Γ (mol cm
−2) is the surface coverage and the other symbols have their usual meanings. From the slope of the anodic peak current
vs. scan rate (not shown), the surface concentration of GNS was calculated 1.8 × 10
−6 mol cm
−2.
3.3 Response surface methodology
Effective instrumental parameters such as scan rate (0.02–0.18 V s−1, x1), step potential (0.001–0.004 V, x2) and modulation amplitude (0.01–0.09 V, x3) and experimental parameters such as pH (1.0–10.0, x1), graphene oxide nanosheets content (3.0–35.0 μL x2) were investigated by CCRD to obtain the optimum process. Therefore, for instrumental parameter f = 3, α = ±1.68, N (number of experiments) = 17 (with three repeats, r = 3) and levels (in coded values) were: −1.68, −1, 0, +1, +1.68 and for experimental parameters f = 2, α = ±1.41, N (number of experiments) = 11 (with three repeats, r = 3) and levels (in coded values) were: −1.41, −1, 0, +1, +1.41. The experiments were performed according to the design matrix with coded levels of parameters as shown in Tables 1 and 2. The DPV responses of a solution containing 20.0 μM of Thy or CA was obtained. Then, we calculated optimum value of each factor for Thy and CA with MINITAB® Release 16 software. These coded values were transformed to the actual values providing the optimum conditions of the factors. The results are shown in Table 3.
Table 1 Levels of independent instrumental variables established based on five-level three-factor CCRD for Thy and CA
Symbols |
Variables |
Coded and actual levels |
−1.68 |
−1.0 |
0.0 |
1.0 |
1.68 |
X1 |
Scan rate (V s−1) |
0.02 |
0.06 |
0.1 |
0.14 |
0.18 |
X2 |
Step potential |
0.001 |
0.002 |
0.003 |
0.004 |
0.005 |
X3 |
Modulation amplitude (V) |
0.01 |
0.03 |
0.05 |
0.06 |
0.09 |
Table 2 Levels of independent experimental variables established based on five-level two-factor CCRD for Thy and CA
|
Symbols |
Variables |
Coded and actual levels |
−1.41 |
−1.0 |
0.0 |
1.0 |
1.41 |
For Thy |
X1 |
pH |
2.0 |
3.0 |
5.0 |
8.0 |
10.0 |
X2 |
GO contants (μL) |
3.0 |
8.0 |
15.0 |
20.0 |
25.0 |
For CA |
X1 |
pH |
1.0 |
3.0 |
5.0 |
6.0 |
7.0 |
X2 |
GO contants (μL) |
3.0 |
8.0 |
15.0 |
25.0 |
35.0 |
Table 3 Optimized parameters for the Thy and CA determination by DPV
Variables |
Coded optimized values for Thy |
Optimized values for Thy |
Coded optimized values for CA |
Optimized values for CA |
pH |
−0.3286 |
4.7 |
−1.3856 |
1.74 |
GO content (μL) |
0.7000 |
19.2 |
−0.8005 |
8.82 |
Scan rate (V s−1) |
0.6487 |
0.12 |
0.6625 |
0.13 |
Step potential (V) |
−0.3228 |
0.0026 |
−1.3818 |
0.0014 |
Modulation amplitude (V) |
0.2053 |
0.052 |
0.4587 |
0.057 |
The optimum response values can be obtained by the graphical analysis of the surface plots. Fig. 3 shows the surface plots obtained from DPV responses for instruments (A and B) and experimental (C and D) parameters for Thy and CA, respectively. The highlight points in this figure represent the optimum values. However, we used the optimum values for DPV determination of Thy and CA.
 |
| Fig. 3 (A) The contour plot of the combined effect of instrumental variables (A, B, C, correspond to scan rate, step potential and modulation amplitude) on DPV current of Thy, (B) the same plot for CA. (C) The contour plot of the combined effect of experimental variables (A, B correspond to pH and GO content) on DPV current of Thy, (D) the same plot for CA. | |
3.4 Electrochemical properties of Thy and CA at GO/GCE
In order to test the electrochemical activity of the GO/GCE, its CV responses were obtained from both Thy and CA solution at 100 mV s−1. The obtained results from Fig. 4A and B demonstrate that the oxidation peak currents are markedly higher than those at the bare GCE in the presence of 20 μM Thy or 20 μM CA. The cyclic voltammetric peak current of Thy and CA at the GO/GCE was about 1.6 and 2.2 times higher than that obtained at the bare GCE, respectively. Also for Thy a decrease (about 10 mV) was observed in peak potential at the GO/GCE. These results suggest that GO is an effective mediator in the oxidation process of Thy and CA. On the other hand, the obtained data clearly show that the addition of graphene oxide nanosheet over the surface of GCE definitely improves the characteristics of Thy and CA oxidation.
 |
| Fig. 4 CVs of GCE (a and c) and GO/GCE (b and d) in the absence (a and b) and presence (c and d) of 20.0 μM Thy in 0.2 M B–R buffer, pH = 4.7 (A) and in 20.0 μM CA in 0.2 M B–R buffer, pH = 1.7 (B), scan rate: 100 mV s−1. | |
The obvious enhancement of oxidation peak current indicates that GO has effective ability to oxidize Thy or CA which can be assigned to the attractive characteristics of GO, such as subtle electronic characteristics and strong adsorptive capability. The GO should have very strong adsorptive capability for Thy or CA, because there are several interactions between GO and them. First, the interaction, which leads to form hydrogen bonds and some hydrophobic force,31 since the OH and COOH groups on GO sheet can form strong hydrogen-bonding interaction with OH groups in Thy or CA. Second, Thy and CA have an aromatic structure, so maybe there is π–π stacking interactions between GO and them. These interactions cause the high loading efficiency of Thy or CA on GO.
3.5 Effect of scan rate
Useful information involving electrochemical mechanism can usually be acquired from the relationship between peak current and scan rate. The effect of various scan rates on the oxidation of Thy and CA at the GO/GCE was obtained (not shown). According to the cyclic voltammetry data and eqn (5), for scan rates, the oxidation peak current values (Ipa) were linearly proportional to the square root of the scan rate when analyzing a solution of Thy (20 μM). This indicates that, at a sufficiently positive potential, the reaction is controlled by the diffusion of Thy. The linear regression equation is shown in eqn (5):32 |
Ipa = 3.01 × 105n[(1 − α)nα]1/2AD1/2Cυ1/2
| (5) |
where D is the diffusion coefficient, C is the bulk concentration of Thy, A is the electrode surface area, υ is the scan rate, α is the transfer coefficient that can range from zero to one, and n is the number of electrons transferred in the reaction. According to eqn (6)–(8):32 |
η = a + b log i
| (6) |
|
a = (2.3RT/nααF)log i
| (7) |
A Tafel plot is a useful parameter for evaluating the kinetic parameters. The Tafel plot (E vs. log
I), was drawn using the data derived from the rising part of the current–voltage curve at a scan rate of 50 mV s−1. The number of electrons involved in the rate-determining step (nα) and ionic exchanging current density (j0) can be estimated from the slope and the intercept of the Tafel plot, respectively. The obtained numerical value of 0.1409 V decade for Tafel slop indicates a one-electron process for a rate-limiting step, assuming a charge transfer coefficient of α = 0.58. Also, the value of j0 was found to be 3.8 × 10−3 μA cm−2 from the intercept of the Tafel plot.
The influence of scan rate (υ) on the electrochemical behavior of CA was obtained in the GO/GCE system with B–R buffer solution (pH = 1.7) containing CA (20.0 μM). For scan rates between 30.0 and 2100.0 mV s−1, the CA oxidation peak current also increases linearly with the square root of the scan rate suggesting that the reaction is diffusion-limited. The linear regression equation for CA was found to be:
|
Ip = 2.69 × 105n3/2AD1/2Cυ1/2
| (9) |
From the Tafel plot for CA, α and j0 was found to be 0.5 and 0.24 μA cm−2, respectively.
3.6 Chronocoulometry measurements
The oxidation of Thy and CA by GO/GCE was studied by chronocoulometry. Chronocoulograms obtained for Thy and CA solutions in concentrations ranging from 1.0 to 4.0 and 0.05 to 0.15 mM, respectively, using a potential step of 1000 mV (Fig. 5). For electroactive materials (Thy and CA in this case) with a diffusion coefficient of D the current for electrochemical reaction (at a mass transport limited rate) is described by the Cottrell equation:32 |
Q = 2nFAD1/2Cb1/2π−1/2t1/2
| (10) |
where D and Cb are the diffusion coefficient (cm2 s−1) and the bulk concentration (mol cm−3), respectively. Under the diffusion control condition, the plot of Q vs. t1/2 will be linear from which its slope, the value of D can be calculated. Inset (a) of Fig. 5 shows the experimental plots of Q vs. t1/2 with the best fits for different concentration of Thy. The resulting slopes of the straight lines were plotted vs. the Thy concentration (Fig. 5 inset b). The mean value of the D was found to be 6.0 × 10−7 cm2 s−1.
 |
| Fig. 5 Chronoamperograms of Thy in B–R buffer (pH 4.7) containing: 0.05 to 0.15 mM Thy, inset a: plot of i vs. t−1/2 for different concentrations of Thy, inset b: the plot of the slope of straight lines against the Thy concentration. | |
Also from the Cottrell equation, the diffusion coefficient of D for CA was estimated to be 1.0 × 10−5 cm2 s−1.
3.7 Calibration curve and the detection limit
Initial studies of the voltammetric behavior of the drugs were performed using DPV. The DPV technique was used because of several advantages compared to cyclic voltammetry, such as better resolution and negligible charging current contribution to the background current. The DPV is suitable for the analysis of drug mixtures containing electrochemically active substances. The DPV responses of Thy and CA solution in different concentration were investigated under optimized experimental conditions using a GO/GCE. Fig. 6 shows the DPVs obtained for the oxidation of different concentrations of Thy and CA at the GO/GCE in 0.2 M B–R buffer solution. From these voltammograms it can be seen that the peak currents of both Thy and CA increase linearly with concentration.
 |
| Fig. 6 DPVs of GO/GCE in 0.2 M B–R buffer solution (pH 4.7) containing different concentrations of Thy (A) and in 0.2 M B–R buffer solution (pH 1.7) containing different concentrations of CA (B). Inset show the calibration curve for Thy or CA. | |
The oxidation peak current of Thy was measured in B–R buffer solutions at pH 4.7 and plotted against the bulk concentration of Thy. The dependence of peak current on the Thy concentration (see inset (a) of Fig. 6A) was found to be linear for concentration ranges of 1.5–200.0 μM. The respective calibration equation for these concentration ranges is as followed:
|
y = 0.027x + 0.314, R2 = 0.994 (for 1.5 × 10−6 to 200.0 × 10−6 M)
| (11) |
Linear regression equations for peak current vs. CA concentration were also determined for CA concentration ranges 1.5 × 10−6 to 3.5 × 10−5 M and 3.5 × 10−5 to 2.7 × 10−4 M (see Fig. 6B and inset a). The respective calibration equations are:
|
y = 0.726x − 0.203, R2 = 0.999 (for 1.5 × 10−6 to 3.5 × 10−5 M)
| (12) |
|
y = 0.158x + 18.047, R2 = 0.994 (for 3.5 × 10−5 to 2.7 × 10−4 M)
| (13) |
The decrease of sensitivity (slope) in the second linear range is likely due to kinetic limitation. From the analysis of these data, we estimate that the lowest limit of detection of Thy and CA is of the order of 6.5 × 10−8 M (n = 17) and 2.5 × 10−9 M (n = 20), respectively. The relative standard deviation (R.S.D) of 2.0 × 10−5 M Thy (n = 9) was 1.23% which showed excellent reproducibility. The stability of the GO/GCE was investigated by measuring the oxidation response of CA (2.0 × 10−5 M) over a period of 20 days. The RSD of 3.8% demonstrates that the GO/GCE exhibits excellent stability over time.
3.8 Simultaneous determination of Thy and CA
One of the main purposes of this study was to assess the applicability of modified electrode for the determination of Thy and CA in a mixture solution of them separately based of the detection of their electrochemical responses. Differential pulse voltammetry was used to estimate the simultaneous determination of Thy and CA. In the first stage, according to the curve of I vs. pH for Thy and CA, the optimum pH for maximum response of two compounds was found to be 3.0. So in the first test, in mixtures of Thy and CA, the concentration of one species was changed, while the concentration of the other species remained constant and the results were shown in Fig. 7A. From the curves, it can be seen that the peak current of CA is proportional to the CA concentration in the range of 1.2 to 120.0 μM, when the concentration of Thy was kept at 20.0 μM. As seen in Fig. 7A, higher concentrations of CA do not strongly interfere with the detection of Thy. In the second test, in mixtures of Thy and CA, the concentration of CA was kept at 10.0 μM, while the concentration of Thy was changed in the range of 10.0 to 50.0 μM. The peak current of Thy increases proportionally, without any interference of CA concentration (Fig. 7B).
 |
| Fig. 7 DPV of GO/GCE in 0.2 M B–R buffer solution (pH = 3.0), (A) containing 20.0 μM Thy and different concentration of CA from inner to outer, 1.8–120.0 μM. Inset (a): plot of peak current as a function of CA concentration for the linear concentration range 1.8–120.0 μM. (B) containing 10.0 μM CA and different concentration of Thy from inner to outer, 10.0–50.0 Thy. Inset (a): plot of peak current as a function of Thy concentration for the linear concentration ranges of 10.0–50.0 μM. | |
Calibration parameters for the simultaneous determination of Thy and CA are shown in insets (a) of Fig. 7A and B. The slope of the linear regression line for the calibration curves of each species is nearly equal to slope of the linear regression when each one is analyzed independently. This indicates that CA and Thy do not interfere with each other when analyzed simultaneously.
3.9 Analytical application
3.9.1 Determination of Thy in Thymex syrups. This medication is prepared from the standardized extract of thyme and used in the treatment of upper respiratory tract inflammation. Electrochemical methods can be used to evaluate low concentrations of Thy present in the Thymex syrups. A glassy carbon electrode modified with graphene oxide nanosheets was applied for the determination of Thy in Thymex syrups (0.17% w/v, 0.0011 M). The concentration of CA in Thymex syrups was lower than the determination limit of modified electrode therefore we determined the recovery percentage for CA. Determination of Thy and CA was achieved by the standard addition method. The average recovery for Thy and CA were 100.1% and 98.9%; respectively. The analytical results are shown in Table 4.
Table 4 Determination of Thy and CA in real samples
Sample |
Added (μM) (Thy) |
Added (μM) (CA) |
Found (μM) (Thy) |
Found (μM) (CA) |
Recovery (%) (Thy) |
Recovery (%) (CA) |
Thymex syrups |
0.00 |
0.00 |
2.80 |
Not detected |
— |
— |
2.00 |
3.00 |
4.60 |
3.20 |
95.90 |
106.60 |
4.00 |
4.50 |
7.10 |
4.10 |
104.40 |
91.20 |
Eucalyptus inhaler drop |
0.00 |
0.00 |
1.09 |
0.98 |
— |
— |
4.00 |
2.00 |
5.20 |
3.09 |
102.00 |
103.60 |
6.50 |
3.00 |
7.20 |
3.70 |
94.90 |
93.00 |
Thyme plant |
0.00 |
0.00 |
2.30 |
0.83 |
— |
— |
3.00 |
4.00 |
4.90 |
5.10 |
92.50 |
105.50 |
6.00 |
8.00 |
8.70 |
8.41 |
104.50 |
95.30 |
3.9.2 Simultaneous determination of Thy and CA in eucalyptus inhaler drop. Eucalyptus inhaler drop is applied in the treatment of symptomatic relief of coughs and blocked noses in cold, flu and bronchitis. In order to test the capability of our Thy detection method in a pharmaceutical product, the GO/GCE was used to detect the Thy and CA concentration in eucalyptus inhaler drop (3% w/v, 0.0199 M). 1.00 mL of eucalyptus inhaler drop sample was diluted 100 times with deionized water. Then, an appropriate amount of the solution was diluted by B–R buffer (pH = 3.0) and was transferred into the electrochemical cell for DPV analysis. Each measurement was carried out thrice. The analytical results are shown in Table 4. The recovery is between 94.9% and 102.0% for Thy and 93.0% and 103.6% for CA. The results are acceptable and show that the proposed method could be effectively used for the determination of Thy and CA in commercial samples.
3.9.3 Determination of Thy and CA in thyme plant. The Thy and CA have been reported in thyme plant. To our knowledge, the determination of Thy and CA in thyme extract by DPV has not been reported yet. In this paper, we report the oxidation of Thy and CA by a GO/GCE electrode. Determination of Thy and CA in thyme extract was achieved by the standard addition method, yielding a plot of peak height vs. peak potential. Under these conditions, the average recovery for Thy and CA were 100.4% and 98.5%, respectively.Recovery rates of samples ranged from 91.2% to 106.6% (Table 4), indicating that the procedure is free from matrix interference. The results are acceptable and the proposed method could be used for the determination of Thy and CA in extract.
4. Conclusion
A novel GO-based electrochemical sensor for Thy and CA was successfully fabricated and the performance of this sensor was dramatically improved due to the excellent electrical conductivity, strong adsorptive ability and large effective surface area of GO. We used CCRD method to optimize voltammetric parameters for the Thy and CA determination. This method permitted analysis of the interaction between the variables with a minimum number of experiments. The results show that the oxidation peak current of Thy and CA increased obviously on GO/GCE. The detection limit for Thy and CA, the electron transfer coefficient (α), and diffusion coefficient (D) were calculated from differential pulse voltammetry, cyclic voltammetry and chronocoulometry responses. In addition, high sensitivity and selectivity, reproducibility of the voltammetric responses, low detection limit, ease of preparation and surface regeneration, makes the proposed modified electrode very useful for accurate determination of Thy and CA. The fabricated sensor was applied to detect Thy and CA in real samples with satisfying recoveries. These results indicated that GO is a good candidate of advanced electrode materials and could be combined with other functional materials to fabricate the sensing interface for more applications in the fields of electroanalysis.
Acknowledgements
The authors are grateful to University of Kashan for supporting this work by Grant no. 256450-2.
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