Spectrophotometric measurements and HPLC analysis
The antioxidant capacities, total phenolic and ascorbic acid contents of the samples evaluated by conventional spectrophotometric and HPLC techniques are shown in Table 1. The results showed good agreement between total phenolic and ascorbic acid contents and antioxidant capacity, except for the banana sample. Despite the lowest total phenolic and ascorbic acid contents, the banana sample has the highest antioxidant activity values. This finding corresponds well with that of a previous study. It is well known that phytochemicals such as phenolic substances and ascorbic acid as well as flavonoids, tocopherols, carotenoids, and even fibers may be responsible for scavenging of DPPH and ABTS radicals (Li et al. 2014; Tiveron et al. 2012). The results are also in accordance with the studies carried out with different fruits and vegetables (Li et al. 2014; Velioglu et al. 1998; Maduwanthi and Marapana 2021).
In order to get the highest analytical response in voltammetric studies, optimization parameters such as electrode modification, the film thickness in modification, and supporting electrolyte solution effect were investigated. First, thin films of the electro polymerized TSA/GCE modified sensor were prepared using the CV technique with one to ten cycles, and their DPV responses for 100 ppm Trolox were investigated (Fig S2). When the DPV responses were examined, the highest peak current was obtained at the TSA/GCE modified sensor electro polymerized with 5 cycles. Thus, the sensor with this film thickness was used in subsequent studies. Then, the supporting electrolyte solution environment was investigated in order to obtain a high voltammetric response. DPV responses at TSA/GCE modified sensor in 0.1 M KCl, LiClO4, NaClO4, NaCl, NaNO3, Na2SO4, and PBS (pH 7.4) electrolyte solutions of 100 ppm Trolox were investigated (Fig S3). The best DPV response was obtained in 0.1 M NaNO3 solution, and this solution medium was used in subsequent studies.
Surface analysis of the electro polymerized TSA/GCE modified sensor was carried out by SEM technique (Figure 1). A thin film structure was obtained with a film thickness of approximately 20.31 µm. Its surface was highly porous and many porous structures were seen. Such porous and rough structure increased the sensitivity of the modified sensor during detection.
Differential pulse voltammetric analyzes were performed in 0.1 M NaNO3 at a TSA/GCE modified sensor to determine the voltammetric oxidation behavior and detection limits of Trolox, gallic acid, and ascorbic acid. The concentrations of Trolox were increased as 0.083, 0.48, 2.05, 2.60, 3.15, 4.08, 4.41, 5.68, 6.82, 7.38, 8.68, 10.29, 10.94, 12.00 ppm from the Trolox standard solution to the electrolyte solution. A linear curve was obtained with the equation Ip (µA) = 0.8464 C (ppm) + 1.0954 in this concentration range (Figure 2). Concentrations of gallic acid were 0.30, 0.31, 0.45, 1.54, 2.08, 2.61, 3.13, 3.65, 5.17, 7.60, 8.54, 10.34, 12.96, 14.61, 15.41, 16.20, 19.20, 23.33, 26.46, 32.01, 34.48, 37.98, 41.45, 48.32, 55.10, 61.77, 68.36, 74.85, 82.25, 87.57, 93.79, 99.94 ppm in 0.1 M NaNO3. Two oxidation peaks were observed for gallic acid at approximately 350 mV (p1) and 750 mV (p2) at the TSA/GCE modified sensor (Figure 3). Linear curves in the concentration range of 10.34 – 99.94 ppm were obtained with the equations Ip1 (µA) = 0.1099 C (ppm) + 4.9467 for the first oxidation peak and Ip2 (µA) = 0.0254 C (ppm) + 0.4719 for the second peak (Figure 3). The concentrations of ascorbic acid were increased to 10.0, 13.6, 24.4, 31.4, 38.4, 45.2, 52.0, 58.6, 62.7, 71.7, 78.0, 84.3, 91.9, 96.7, 102.7 ppm from the ascorbic acid standard solution to 0.1 M NaNO3. In this concentration range, a linear curve was obtained with the equation Ip (µA) = 0.0391 C (ppm) + 0.048 (Figure 4). In addition, the LOD values obtained for Trolox, gallic acid, and ascorbic acid were calculated as 0.23, 0.40, 0.75 ppm, respectively, and the LOQ values were calculated as 0.77, 1.32, 2.51 ppm, respectively.
Simultaneous determinations of Trolox, gallic acid, and ascorbic acid were performed to test the selectivity of the method and the designed modified sensor. (Fig S4). Figure 5 shows the DPV responses at the poly (p-TSA) modified sensor as a result of increasing Trolox concentrations in the presence of constant concentrations of gallic acid and ascorbic acid. The anodic peak potentials for ascorbic acid, Trolox and gallic acid at the modified sensor were observed at +0.14, +0.25, and +0.41 V, respectively. Results of simultaneous voltammetric analysis for solutions with increased gallic acid and ascorbic acid concentrations and constant concentrations of other analytes were shown in Figure S5, Figure S6. These results showed that there was no interaction in the electrochemical determination of Trolox, gallic acid and ascorbic acid. Three analytes were detected sensitively, stable and with good anodic peak resolution at the poly (p-TSA) modified sensor. It can be said that simultaneous determination of three analytes in real sample extracts at the poly (p-TSA) modified sensor is possible.
Antioxidant capacity, total phenolic, and ascorbic acid contents in apricot, arugula, banana, cranberry, spinach and strawberry extracts used in spectrophotometric and chromatographic methods were determined electrochemically by DPV technique at a poly (p-TSA) modified sensor. Voltammetric calibration curves were generated for Trolox, gallic acid and ascorbic acid. Antioxidant capacity, total phenolic and ascorbic acid contents were calculated by finding the concentrations corresponding to the current values of the extracts from these calibration charts. The average of the data (µmol TE/100g DW, mg GAE/ 100g DW and µg/g DW respectively) calculated as a result of three voltammetric analyzes for each extract was shown in Table 2. Very low concentrations of Trolox, gallic acid, and ascorbic acid could be calculated in the extracts by electrochemical measurement.
Regression analysis was performed to correlate the results obtained by conventional methods with electrochemical measurements. The Pearson’s correlation coefficients between conventional techniques (spectrophotometric and HPLC) and voltammetric methods are shown in Table 3. High correlation was observed between voltammetric TEAC value and spectrophotometric DPPH (R2 = 0.985, p<0.01) and ABTS (R2 = 0.983, p<0.01) assays. The voltammetric total phenolic and ascorbic acid contents also signified strong correlations with those of spectrophotometric (R2 = 0.992, p<0.01) and HPLC (R2 = 0.995, p<0.01) results, respectively. Good compatibility between analytical techniques used in this study, voltammetric technique is promising as an alternative technique in terms of antioxidant capacity, total phenolic and ascorbic acid contents determination.