Voltammetric Determination of Ascorbic Acid Content in Cabbage Using Anthraquinone Modified Carbon Paste Electrode

Alemu, T.; Zelalem, B; Amare, N

doi:https://doi.org/10.1155/2022/7154170

Journal of Chemistry

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Abstract Introduction Results and Discussion Conclusion Data Availability Conflicts of Interest References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 7154170 | https://doi.org/10.1155/2022/7154170

Voltammetric Determination of Ascorbic Acid Content in Cabbage Using Anthraquinone Modified Carbon Paste Electrode

T. Alemu,¹B Zelalem,¹and N Amare¹

Academic Editor: Bahaddurghatta Eshwaraswamy Kumara Kumara Swamy

Received25 Sept 2021

Revised02 Mar 2022

Accepted25 Mar 2022

Published27 Apr 2022

Abstract

In this study, a low-cost, sensitive, and efficient voltammetric method based on anthraquinone modified carbon paste electrode was developed for determination of ascorbic acid in cabbage samples. After cyclic voltammetry was used to investigate the electrochemical behavior of ascorbic acid and to study dependence of oxidative peak current on scan rate and pH, square wave voltammetric method was developed for direct determination of ascorbic acid in cabbage samples. In contrast to the unmodified carbon paste electrode, a remarkable enhancement in oxidative peak current at anthraquinone modified carbon paste electrode confirmed electrocatalytic property of the modifier towards oxidation of ascorbic acid. A better correlation coefficient for the dependence of peak current on the square root of scan rate () than on the scan rate () indicated that the oxidation of ascorbic acid at anthraquinone modified carbon paste electrode is predominantly governed by diffusion-controlled process. Square wave amplitude, square wave step potential, and square wave frequency are optimized for the investigation of AA in cabbage. The optimized values are 30 mV, 7 mV, and 35 Hz, respectively. Under the optimized method and solution parameters, an excellent linear response was observed between square wave voltammetric peak current of AQMCPE and concentration of ascorbic acid in the range to M with a better correlation coefficient () and detection limit ( M). The ascorbic acid content of the three cabbage samples from three different cabbage growing areas was found in the range – mg/g of powdered cabbage. Excellent recovery results between 95.042 and 96.139% for spiked ascorbic acid in cabbage samples confirmed the potential applicability of the developed method based on AQMCPE for the determination of ascorbic acid in real samples like cabbage.

1. Introduction

Ascorbic acid (AA), also known as vitamin C, is the most common electroactive molecule which exists widely in foods, beverages, animal feed, and pharmaceutical formulations and plays a paramount role as an antioxidant [1–3]. AA (Scheme 1) is water-soluble, slightly alcohol-soluble, and insoluble in chloroform, ether, and benzene [2]. AA plays a key role in body tissue growth and maintains connective tissues, which include bones, blood vessels, and skin meanwhile; AA is significant for biological metabolisms such as free radical scavenging and immunity development [3, 4].

AA is one of the food ingredients that plays a key role in supporting the antioxidant barrier of the body, which is best known under the common name vitamin C [5]. Though most animals can endogenously synthesize large quantities of AA, humans do not have the capability to synthesize AA [6], and so, it must be obtained entirely through one’s diet. Therefore, fruits, vegetables, and their products and other AA-rich sources like red and green peppers, broccoli, tomatoes, strawberries, brussels sprouts, etc. [2, 7] are needed to be consumed for the source of AA. The amount of AA may be reduced due to some processing and storage effects such as packaging material, prolonged storage, too high temperature, and light [8, 9]. Because of that, to improve the nutritive value and maintain natural properties, AA is usually added to dietary foods.

The amount of AA required in a healthy diet varies with age and gender. According to Health Canada dietary reference intakes, estimated average requirements and recommended dietary allowances, respectively, are for children (ages 1-3) 13 and 15 mg, for adult females 60 and 75 mg, and for adult males 75 and 90 mg/day [6]. However, AA amounts greater than 2,000 mg/day are not recommended because such high doses may lead to stomach upset, abdominal cramps, and diarrhea. In addition, excessive AA supplementation may have the potential to increase urinary oxalate and uric acid excretion, which could contribute to the formation of kidney stones, especially in individuals with renal disorders [10]. Monitoring of AA levels during production as well as quality control stages is important. AA has been linked to impressive health benefits, like for prevention and treatment of scurvy, common cold, anemia, hemorrhagic disorders, cancer, and wound healing as well as infertility [11–14]. Hence, the amount of AA in dietary foods must be known. Therefore, developing accurate, reliable, rapid, and user-friendly method for determining levels of AA is of great importance. The amount of AA in cabbage cultivated in the sampling areas has not been reported, and therefore, this work is also intended to determine the amount of AA in cabbage.

Several methods have been developed for the determination of AA in different sources including pharmaceutical formulations, foods, fruits, and biological fluids. Chromatography [15], GC-MS [16], and spectrophotometry [8, 17] are among the commonly reported techniques for the determination of AA. However, these techniques have several disadvantages including long analysis time, expensive instrumentation, the requirement of special trained technicians, and environmental issues [18, 19]. In this regard, electrochemical methods are promising alternatives for the determination of electroactive species, because of their quick response times, low cost, simplicity of instrumentation, high sensitivity, and environmentally friendly [20–22]. Different attempts have been devoted to developing simple and rapid electrochemical methods for determination of AA in different samples [23–25]. Modifying the surface of the working electrode has of great importance since it improves electrochemical performance by improving sensitivity, electron conductivity, and surface area as well as mechanical properties [24, 25]. Cellulose actuate film modified glassy carbon electrode [1], Pt electrode [2], MWCNT/tetradecyltrimethylammonium bromide modified glassy carbon electrode [20], AgNPs/PVP modified glassy carbon electrode [24], and multiwall carbon nanotubes modified glassy carbon electrode [25] are among commonly reported electrode materials for the electrochemical determination of AA in pharmaceutical formulations and fruit juices. Recently, carbon paste electrodes (CPE) have gained a great deal of attention due to their wide electrochemical window, easy fabrication, low background current, low cost, chemical inertness, surface renewability, and eco-friendly. However, the kinetics, weak mechanical stability, and sensitivity issues while operating in analytical processes are some disadvantages of bare CPE which limit their analytical applications. To resolve these limitations, modification of CPE surface by modifiers has been developed [26, 27]. Modifiers, generally, can increase the electron transfer active sites at the modified electrode interface [27, 28] which in turn increases the sensitivity of the modified electrode. The present study is aimed at designing anthraquinone modified carbon paste electrode (AQMCPE) for determination of ascorbic acid in cabbage samples.

2. Experimental Part

2.1. Chemicals and Reagents

Ascorbic acid (≥99.0% Blulux, India), sodium monohydrogen phosphate (≥98%, Blulux Laboratories (p) Ltd., India), sodium dihydrogen phosphate (≥98%, Blulux Laboratories (p) Ltd., India), hydrochloric acid (Blulux, India), sodium hydroxide (Blulux, India) distilled water, graphite powder (Blulux, India), anthraquinone (CDH, India), and paraffin oil (CARELABMED, India) were among the chemicals used. All chemicals and reagents were of analytical grade and hence used without further purification.

2.2. Apparatus and Instruments

CHI760D electrochemical workstation (Austin, USA) connected to a personal computer with three electrode systems (unmodified carbon paste or anthraquinone modified carbon paste electrode as a working electrode, platinum coil as a counter electrode, and Ag/AgCl as a reference electrode) was used for voltammetric measurements. Electroanalytical digital balance (Nimbus, UK) and pH meter (Denver Instrument, Hungary) were used to measure mass and pH, respectively. An orbital shaker (Heidolph Unimax, England) and blender were also used.

2.3. Procedures

2.3.1. Preparation of Supporting Electrolyte

Phosphate buffer solutions (PBS) in the pH range 2.0–9.0 were prepared from a mixture of 0.1 M Na₂HPO₄ and 0.1 M NaH₂PO₄ in distilled water. 0.1 M NaOH and 0.1 M HCl solutions were used to adjust the pH of the buffer solutions.

2.3.2. Preparation of Standard Solutions of Ascorbic Acid

A stock solution of 10 mM AA was prepared by dissolving 0.1761 g of AA in 100 mL of pH 4 PBS. From the stock solution, while 2.0 mM AA solution was used for the cyclic voltammetric investigations, different concentrations (0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, and 4.0 mM) of AA were prepared in pH 4 PBS by serial dilution method.

2.3.3. Sampling and Real Sample Preparation

Three cabbage samples were collected from three different cabbage growing areas, namely, Guld, Karishewa, and Birbira. The three localities are in the Wag Hemra administrative zone of Amhara regional state, Ethiopia. In each sampling area, three farmlands were chosen randomly, and 2.0 kg cabbage sample was collected in each farmland. The samples were kept in plastic bags separately and then transported to the laboratory, Bahir Dar University, Ethiopia. Each cabbage sample was deeply washed with tap water to remove adsorbed soil, and other materials followed by distilled water, and then, after the outer part was removed, 1.0 kg cabbage sample was taken and chopped with a stainless steel knife. The chopped samples were spread onto drying trays at room temperature for three days. The dried cabbage samples then were grounded to a fine powder using a pestle and mortar. 100 g powdered cabbage sample from each farmland in each kebele was taken and mixed prior to extraction.

A mixture of 100 g powdered cabbage sample in each kebele was taken. The samples collected from each kebele were extracted in 665 ml pH 4 PBS at room temperature using an orbital shaker at 200 revolutions per minute for 48 hours. The extracts were filtrated using a Whatman No. 1 filter paper and were ready for analysis.

2.3.4. Preparation of Working Electrode

The unmodified carbon paste electrode (UCPE) was prepared by thoroughly mixing 70% () graphite powder and 30% () paraffin oil [29]. The mixture was homogeneously mixed by hand for 30 minutes using pestle and mortar. The homogenized paste was allowed to rest for a period of 24 hrs, and then, the paste was packed into the tip of the plastic tube. A copper wire was inserted from the backside of the plastic tube to provide electrical contact. The surface of the electrode was smoothed manually against a smooth white paper until a shiny surface is emerged.

Anthraquinone modified carbon past electrode (AQMCPE) was prepared by mixing 6.25 g of graphite powder with 1.5 g of anthraquinone in a small agate mortar for about five minutes. A 2.25 g (2.66 ml) of paraffin oil was added to the mixture followed by milling for 30 min to obtain a homogenous anthraquinone modified carbon paste electrode. The homogenized paste further was allowed to rest for 24 hrs. A desired amount of the prepared paste was packed into the end cavity of the plastic tube. The surface of the fabricated anthraquinone modified carbon paste electrode was then polished on a clean paper before being used.

2.3.5. Electrochemical Measurements

A three-electrode system with Ag/AgCl as a reference electrode, Pt coil as counter electrode, and bare CPE or AQMCPE was used for electrochemical measurements. Cyclic voltammetry was used to investigate the electrochemical behavior of AA at the surface of AQMCPE at various scan rates and pHs. Furthermore, square wave voltammetry was employed for the quantitative determination of AA in cabbage samples.

3. Results and Discussion

3.1. Cyclic Voltammetric Investigation of AA

3.1.1. Electrochemical Behavior of AA

The electrochemical behavior of ascorbic acid at UCPE and AQMCPE was studied using cyclic voltammetry. Figure 1 presents the cyclic voltammograms of (a) UCPE and (b) AQMCPE in the absence of AA and (c) UCPE and (d) AQMCPE containing 2.0 mM AA in pH 6.5 PBS at a scan rate of 100 mVs⁻¹. As can be seen from the figure, no peak was observed at UCPE and AQMCPE in the absence of AA (curves a and b, respectively). This indicated that the background current of the supporting electrolyte has no interfering signal during the electrochemical measurements on the UCPE (curve a) and AQMCPE (curve b) in the potential window between -300 and 1200 mV. On the other hand, an intensive oxidation peak was observed in the presence of AA at UCPE (curve c) and AQMCPE (curve d). A remarkable enhancement in the oxidation peak current and oxidation peak potential shifting to a less positive peak potential at AQMCPE indicated the catalytic role of the modifier towards the oxidation of AA.

3.1.2. Effect of pH

To investigate whether a proton has participated during the oxidation of AA, the effect of pH on the oxidation peak current response of AQMCPE was investigated using cyclic voltammetric measurements. Cyclic voltammograms of AQMCPE in various pH values (in the range 3.0–7.0) of PBS containing 2.0 mM AA are shown in Figure 2(a). The oxidation peak current at the surface of AQMCPE is observed to increase with pH value from 3.0 to 4.0 and then decreased when further increasing solution pH up to a value of 7 (curve A of Figure 2(b)). Accordingly, pH 4 was selected as an optimum pH of buffer solution for the subsequent experiments.

(a)

(b)

The dependence of the peak potential on the pH of the buffer solution was also investigated. Figure 2(b), B presents the oxidative peak potential versus the pH. As can be seen from the plot, the oxidation peak potential shifted in the negative potential direction with increasing pH. The observed peak potential shift indicated the participation of proton during the oxidation of AA at the surface of the modified electrode.

3.1.3. Effect of Scan Rate

To investigate the reversibility and the reaction kinetics of AA at the surface at AQMCPE, the effect of scan rate on the oxidation peak current and peak potential of AA was studied at various scan rates in the range 25–225 mVs⁻¹. Figure 3 depicts cyclic voltammograms of AQMCPE in pH 4.0 PBS containing 2.0 mM AA at different scan rates. As can be seen from the figure, the observed peak potential shift in the positive direction with increasing scan rate confirms the irreversibility of the oxidation reaction of AA at the AQMCPE.

To investigate whether the oxidation process of AA at AQMCPE is predominantly diffusion controlled or surface confined process, the correlation coefficients for the linear plots of the oxidative peak current vs. the scan rate (inset of Figure 3(a)) and oxidative peak current vs. the square root of scan rate (inset of Figure 3(b)) were compared. As can be seen from the plots, a better correlation coefficient for the dependence of peak current on the square root of scan rate () than on the scan rate () indicated that the oxidation of AA at AQMCPE is predominantly governed by diffusion controlled process [24]. This result is also supported by Figure 4 which shows the plot of log Iap vs. log . The anodic peak current is linearly related to the scan rate and described as log log , and . Moreover, a slope of 0.49, which is very close to the theoretical slope of 0.5 for diffusion limited electrode process confirmed the electrode process for the oxidation of AA at the surface of AQMCPE, is predominantly diffusion controlled process [30].

3.2. Square Wave Voltammetric Investigation of AA

Square-wave voltammetry (SWV), which is the most advanced and the most sophisticated technique in the family of pulse voltammetric techniques, is effective and rapid electroanalytical method due to its ability to discriminate against background currents, good sensitivity, and low detection limits [14]. Hence, it was selected for the quantitative determination of AA in cabbage samples.

3.2.1. Optimization of Square Wave Parameters for AA Determination

For further analysis, selected square wave voltammetric parameters such as square wave frequency, square wave amplitude, and square wave step potential were optimized to investigate the effect of each parameter on the oxidative peak current of AA at AQMCPE.

(1) Square Wave Amplitude. In order to investigate the effect of square wave amplitude on the oxidation peak current of AA, square wave voltammetric measurements of 2.0 mM AA in pH 4.0 PBS were recorded in the range of 10 to 50 mV at constant step potential and wave frequency (4 mV and 15 Hz, respectively). Figure 5 presents the effect of the amplitude on the oxidative peak current of AA at AQMCPE. As shown in the inset of Figure 5, upon increasing amplitude, a linear increase in the peak current was observed accompanied by peak broadening when the amplitude was greater than 30 mV. Thus, 30 mV was chosen as the optimum square wave amplitude which is a compromise between the peak height and the peak shape.

(2) Square Wave Step Potential. The effect of square wave step potential on the oxidation peak current response of AA at the surface of AQMCPE was studied at various step potentials (3.0–10 mV). As depicted in Figure 6, the anodic peak current increased with the increasing square wave step potential at constant amplitude (30 mV) and frequency (15 Hz). However, the increment of oxidation peak current is accompanied by peak broadening when the step potential was greater than 7.0 mV (inset of Figure 6). Hence, as a compromise between the peak current enhancement and peak broadening with increasing step potential, a step potential of 7.0 mV was chosen as the optimum square wave step potential for the subsequent experiment.

(3) Square Wave Frequency. To further investigate the effect of square wave frequency on the oxidation peak current response of AQMCPE for AA determination at optimized amplitude and step potential, SWV results were taken. As can be seen from Figure 7, the height of the oxidation peak current increased with increasing the square wave frequency. However, the peak current increment was accompanied by peak broadening after 35 Hz wave frequency (inset of Figure 7). Hence, as a compromise between the increased peak current and peak broadening with increasing square wave frequency, a frequency of 35 Hz was chosen as the optimum value in the subsequent experiments.

3.2.2. Calibration Curve and Method Detection Limit

Under the optimum experimental conditions and method parameters, the dependence of square wave voltammetric oxidative peak current on the concentration of AA and inherited sensitivity of the method was investigated in the range of 0.05 to 4.0 mM. Figure 8 shows background corrected square wave voltammograms of various concentrations of AA in pH 4.0 PBS at AQMCPE. As can be seen from the inset of Figure 8, the oxidation peak current response of AQMCPE was linearly varied with the concentration of AA. The regression equation of anodic peak current vs. AA concentration was obtained as (inset of Figure 8) with a correlation coefficient of 0.9993, and limit of detection (3 s/m) and limit of quantification ( s/m) for were calculated to be and , respectively.

3.2.3. Determination of AA in Cabbage Samples

The applicability of AQMCPE for the determination of AA was evaluated by applying it to determine the AA content in cabbage samples. In this study, three cabbage samples from three different cabbage growing areas (Guld, Karishewa, and Birbira) were prepared to determine AA content in the cabbage samples. The developed method was used for the determination of AA content in the cabbage samples prepared as described under the experimental part. Figure 9 presents the square wave voltammograms for cabbage extracts under the optimized experimental conditions. Analyses were done using the mean value of triplicate measurements of each sample. As depicted from the inset of Figure 9 and Table 1, the AA content of cabbage samples in increasing order is Birbira, Karshewa, and Guld. The determination of AA in these samples was carried out according to the linear regression equation formulated for the calibration curve. The observed difference in AA content could be ascribed to the difference in the agronomical differences between the localities where they were cultivated. The detected AA content of the three cabbage samples calculated using the regression equation is summarized in Table 1.

3.2.4. Recovery Study of the Developed Method

The applicability of the developed method for the determination of AA in cabbage samples was validated using its recovery results for spiked standard AA from Karishewa cabbage sample. The experiment was carried out by spiking the analyzed cabbage samples with standard 2 mM AA solutions. Figure 10 presents the square wave voltammograms for (a) unspiked 10 ml of Karishewa cabbage extract and (b) first spiked and (c) second spiked of the extract each with 5.0 ml of 2.0 mM standard AA. The recovery result summarized in Table 2 showed excellent recovery of the method developed in the range 95.042–96.139% confirming the applicability of the developed method for determination of AA in cabbage samples.

3.2.5. Interference Study

To further evaluate the selectivity of the method to AA determination, the influence of possible interference was investigated by adding different amounts of uric acid (UA) to 1.6 mM of AA (Figure 11). As can be seen in Table 3, the addition of various concentrations of UA was shown no interference on the response current of AA with an associated error of less than 2.15%, indicating the accuracy and selectivity of the method towards AA.

3.3. Comparison of the Present Method with Other Reported Methods

The performance of the developed method was compared with selected previously reported voltammetric methods on AA determination in terms of linear dynamic range limit of detection and nature of the electrode substrate. The developed method using AQMCPE, which is the cheapest carbon-based electrode material, showed comparable performance with the other previously reported methods (Table 4).

4. Conclusion

In this study, anthraquinone modified carbon paste electrode was prepared by simple mixing procedure, and the preparation of the electrode does not require any pretreatment. Cyclic voltammetry and square wave voltammetry were used for the characterization and quantification of AA at AQMCPE, respectively. Compared to unmodified CPE, the AQMCPE enhanced the oxidation peak towards AA and showed pronounced catalytic property towards the oxidation of AA. Moreover, a negative shift of the oxidation peak potential at AQMCPE indicated the catalytic role of the modifier towards the oxidation of AA. The results showed a wide linear concentration range and comparable LOD with previously reported works. The developed sensor was successfully employed to detect AA in cabbage extracts with reasonable results, revealing its promising practical applicability.

Data Availability

Data availability is as open as possible and as closed as necessary.

Conflicts of Interest

The authors declare no conflict of interest.

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