Voltammetric Investigation of Ferulic Acid at Disposable Pencil Graphite Electrode

Ferulic acid (FA), a monohydroxycinnamic acid, is an antioxidant with multiple beneficial effects on human health, presenting also importance in the food and cosmetics industry. Its electrochemical behavior was investigated at the disposable and cost-effective pencil graphite electrode (PGE). Cyclic voltammetry emphasized its pH-dependent, diffusion-controlled oxidation. Using the optimized conditions (HB type PGE, Britton Robinson buffer pH 4.56) differential pulse and square-wave voltammetric techniques were applied for its quantitative determination in the range 4.00 × 10−7–1.00 × 10−3 mol/L FA. The developed methods were employed for the rapid and simple assessment of the FA content from a commercially available powder designed for cosmetic use.


Introduction
Ferulic acid (FA, 3-(4-hydroxy-3-methoxyphenyl)prop-2-enoic acid or 4-hydroxy-3methoxy cinnamic acid) was named after the plant (Ferula foetida) from which it was extracted for the first time in 1866 [1].FA is a phenolic compound, more specifically a derivative of the cinnamic acid, and contains in its structure a benzene ring with two functional groups (methoxy and hydroxyl), as well as a carboxylic moiety [2] (Figure S1).These structural characteristics are probably responsible for most of FA's properties.Thus, FA is a powerful natural antioxidant, having a strong scavenger effect for various free radicals (hydroxyl, superoxide, hydrogen peroxide and nitrogen dioxide) [3].
In the various studies published over the years, it has been proven that FA plays a crucial role in the human body and has countless beneficial effects: anti-inflammatory, antimicrobial, antiviral, antifungal, vasodilatory, antithrombotic, antiarrhythmic, hepatoprotective, anticarcinogenic, antiallergic, analgesic, neuroprotective, cholesterol-lowering, potential antidepressant, reducing the risk of chronic diseases as diabetes, and skin-protective (enhances wound healing) [3][4][5][6][7][8][9].FA helps increase sperm viability, is used as food additive, as a component in nutraceuticals and is a constituent in cosmetics and dermatological products [4,[9][10][11].On the other hand, it is worth mentioning that FA is a precursor for the production of vanillin, a flavor compound mainly used in food industry [10].This is particularly important considering that recently more and more emphasis is placed on the consumption of natural products.
FA is an ubiquitous compound in plant cell walls, constituent of lignocellulose, occurring as a result of the phenylalanine and tyrosine metabolism by the shikimic acid those reported for modified electrodes are obtained, but with a smaller number of steps and a lower consumption of reagents.
Powder for cosmetic preparations with a content of 98% FA extracted from the root of the Ferula assafoetida L. plant, supplied by Elemental SRL, Oradea, Romania, was purchased from a local drug store and used to test the analytical applicability of the developed voltammetric method.The FA content of the commercially available powder was assessed applying the standard addition method.The amount of powder necessary for the preparation of an FA solution with the theoretical concentration of 3.35 ×10 −3 mol/L was accurately weighed and dissolved with ethanol in a 10 mL volumetric flask.An aliquot of 0.06 mL from this solution was diluted with BRB pH 4.56 solution to obtain 10 mL of working solution which was analyzed by DPV at PGE. Differential pulse voltammograms were recorded for the 10 mL sample solution before and after each of the four additions of 0.05 mL of 4.00 × 10 −3 mol/L FA intermediary stock solution and the corresponding obtained peak currents were used to determine the FA concentration in the analyzed sample.
The 0.5 mm Rotring pencil graphite leads that acted as an active surface of the working electrode were purchased at once from a local bookstore.
The pH of the solutions was measured using a combined pH-sensitive glass electrode connected to a pH/mV-meter Consort P901 Scientific Instrument (Belgium).
The voltammetric measurements were made at an Autolab PGSTAT 12 with a GPES 4.9 software, as well as a Voltalab PST 050 Radiometer with a VoltaMaster 4.0 software.For the voltammetric recordings, the electrochemical cell contained 10.00 mL solution to be analyzed, in which the Ag/AgCl/KCl (3.00 mol/L) reference electrode, the platinum wire counter electrode and the PGE working electrode (if not stated otherwise) were immersed.The solid electrodes consisting of glassy carbon (GCE) and platinum (Pt) with a diameter of 0.30 cm and the corresponding geometrical surface area (A g ) of 0.0707 cm 2 were employed for comparison.The PGE (Figure S2) was prepared as described in a previous paper [19] and every time, 1.00 cm of the graphite lead was introduced in the solution so that the A g was always constant (0.1589 cm 2 ), thus ensuring the reproducibility of the measurements.
Cyclic voltammetry (CV) was used to investigate the voltammetric behavior of FA while, due to their higher sensitivity, differential pulse voltammetry (DPV) and squarewave voltammetry (SWV) were exploited for FA quantification.

Results and Discussion
Like any analytical study, the present one involved the optimization of various parameters for the FA determination and the investigation of its voltammetric behavior at the disposable PGE, having as the final goal the development of a sensitive method for its simple and rapid quantification in real samples.

Selection of the Working Electrode Material
The choice of PGE as a working electrode was based on the fact that graphite pencil leads have good electrochemical characteristics and by simply changing the pencil lead, a new electroactive surface can be ensured and thus the time-consuming cleaning step of the electrode surface is eliminated.The main components of graphite pencil leads are graphite and resin or high polymer.Depending on the ratio between these two components, there are several types of leads which present different hardness.The softer ones, labeled with B (from blackness) have a higher graphite content, while the harder ones, marked with H (from hardness) contain more polymer or resin [17].The FA voltammetric response on the classical solid working electrodes (GCE and Pt) as well as on various types of disposable PGEs (Figure 1) emphasized that the highest DPV signal corresponding to the FA oxidation was recorded at the HB type PGE (HB_PGE).The sensitivity (S, expressed in A × L/mol × cm 2 ) of the electrodes, an analyte-concentration-and A g -electrode-independent parameter, decreased in the order HB_PGE (0.3857) > H_PGE (0.3564) > 2H_PGE (0.3340) > GCE (0.2637) > B_PGE (0.2438) > Pt (0.1742) > 2B_PGE (0.1248).

Results and Discussion
Like any analytical study, the present one involved the optimization of various parameters for the FA determination and the investigation of its voltammetric behavior at the disposable PGE, having as the final goal the development of a sensitive method for its simple and rapid quantification in real samples.

Selection of the Working Electrode Material
The choice of PGE as a working electrode was based on the fact that graphite pencil leads have good electrochemical characteristics and by simply changing the pencil lead, a new electroactive surface can be ensured and thus the time-consuming cleaning step of the electrode surface is eliminated.The main components of graphite pencil leads are graphite and resin or high polymer.Depending on the ratio between these two components, there are several types of leads which present different hardness.The softer ones, labeled with B (from blackness) have a higher graphite content, while the harder ones, marked with H (from hardness) contain more polymer or resin [17].The FA voltammetric response on the classical solid working electrodes (GCE and Pt) as well as on various types of disposable PGEs (Figure 1) emphasized that the highest DPV signal corresponding to the FA oxidation was recorded at the HB type PGE (HB_PGE).The sensitivity (S, expressed in A × L/mol × cm 2 ) of the electrodes, an analyte-concentration-and Ag-electrodeindependent parameter, decreased in the order HB_PGE (0.3857) > H_PGE (0.3564) > 2H_PGE (0.3340) > GCE (0.2637) > B_PGE (0.2438) > Pt (0.1742) > 2B_PGE (0.1248).The selectivity and sensitivity of the voltammetric determinations are strongly influenced by the nature of the electroactive surface, which, most often, is modified in order to improve these characteristics.A simple procedure performed to change the electrode surface is the electrochemical pretreatment, which consists of applying either very negative or very positive potentials or scanning the potential between certain potential values.During this process, the surface is cleaned and oxygen-containing functional groups are created on it.However, the improvements brought by this treatment step to the voltammetric behavior of the analyte depend on both the analyzed species and the chemical and electrochemical applied conditions.FA oxidation signals recorded at HB_PGE pretreated electrochemically either potentiostatic (E constant) or potentiodynamic (CV) using three different supporting electrolytes were compared with that obtained at bare, untreated HB_PGE.As it can be observed from Table 1, the electrochemical pretreatment of the working electrode did not result in an improvement of FA anodic peak current.Therefore, for all following voltammetric investigations, an untreated HB_PGE was employed as the working electrode.The selectivity and sensitivity of the voltammetric determinations are strongly influenced by the nature of the electroactive surface, which, most often, is modified in order to improve these characteristics.A simple procedure performed to change the electrode surface is the electrochemical pretreatment, which consists of applying either very negative or very positive potentials or scanning the potential between certain potential values.During this process, the surface is cleaned and oxygen-containing functional groups are created on it.However, the improvements brought by this treatment step to the voltammetric behavior of the analyte depend on both the analyzed species and the chemical and electrochemical applied conditions.FA oxidation signals recorded at HB_PGE pretreated electrochemically either potentiostatic (E constant) or potentiodynamic (CV) using three different supporting electrolytes were compared with that obtained at bare, untreated HB_PGE.As it can be observed from Table 1, the electrochemical pretreatment of the working electrode did not result in an improvement of FA anodic peak current.Therefore, for all following voltammetric investigations, an untreated HB_PGE was employed as the working electrode.Since antioxidants are chemical species susceptible to being easily oxidized by atmospheric oxygen, both the stability of the FA stock and working solutions were studied.Thus, the variation of FA maximum anodic peak current was monitored during a week by recording the DPV curves for working solutions, freshly prepared at different time intervals, from the same stock solution kept in the refrigerator, when it was not in use.The results showed that the FA oxidation peak intensity slightly increased during the first three days being followed by a decrease (Figure S3).Although the peak current enhancement was only 2.57% one day after the preparation of the FA stock solution, for all subsequent measurements, a stock solution prepared on the day of the determinations was used.Voltammetry has the great advantage that it allows several measurements to be performed at the same solution without changing the concentration of the analyte, and this can only be achieved if the solution in the voltammetric cell is stable under the working conditions (usually ambient).In this context, the DPV anodic currents of FA recorded for 100 min on the same solution were almost constant (Figure S4), indicating that the working solution is stable enough to allow a large number of recordings (at least 50) to be made.

Influence of the Solution pH
Most often, the electrode processes of organic compounds involve, besides the electrons, protons.Therefore, the voltammetric behavior of the analyte depends on the solution pH and consequently the investigation of this chemical parameter is an important issue in the development of electroanalytical methods aimed for the quantitative determination of the corresponding species.The influence of the supporting electrolyte pH on FA voltammetric behavior at HB_PGE was investigated in the pH range 1.81 to 11.92 using the universal BRB and applying CV, DPV and SWV.
Cyclic voltammetric recordings emphasized that in the first direct scan, one anodic wave (a2) appeared (Figure 2a), while starting with the second forward scan, an additional smaller oxidation peak (a1) can be observed at less positive potentials (Figure 2b,c), up to a pH of about 8.00.Regardless of the scan number, a cathodic signal (c) appeared during the reversed potential sweep.This behavior was similar to that reported by Malagutti et al. [20] according to which the main oxidation signal (a2) corresponded to a two-step oxidation of the guaiacol moiety (similar to GCE, at HB_PGE, the two steps did not give distinct signals, only a small pre-wave can be observed at pH values below approximately 6).The cathodic signal was attributed to the reduction of the previously formed o-quinone, whose oxidation generated in the subsequent scans the anodic signal a1, corresponding to the quasi-reversible quinone-hydroquinone couple (the peaks a1 and c had close potentials, but the current of the cathodic peak was much higher than that of the oxidation signal).
the increase of the solution pH, indicating the participation of protons in the corresponding electrode reactions.The variations of the peak potential (Ep) of a2 signal vs. the solution pH were linear in the pH range 1.81-9.15,while for a1 and c, linear Ep = f(pH) dependencies were obtained in the range 1.81-7.96.The corresponding regression equations for each of the FA voltammetric signals showed slopes near to the theoretical value of 0.0592 V/pH from the Nernst equation, suggesting that in each considered electrode reaction, the number of exchanged electrons was equal to that of protons (Table 2).The highest current intensity of the FA main voltammetric signal (a2) was obtained in the pH range 3.29-4.56,while the more sensitive voltammetric techniques, DPV and The DPV and SWV recordings (Figure 2d) showed only one oxidation peak during the entire investigated pH range.However, all three applied electrochemical techniques demonstrated that the FA voltammetric peaks shifted towards less positive potentials with the increase of the solution pH, indicating the participation of protons in the corresponding electrode reactions.The variations of the peak potential (E p ) of a2 signal vs. the solution pH were linear in the pH range 1.81-9.15,while for a1 and c, linear E p = f(pH) dependencies were obtained in the range 1.81-7.96.The corresponding regression equations for each of the FA voltammetric signals showed slopes near to the theoretical value of 0.0592 V/pH from the Nernst equation, suggesting that in each considered electrode reaction, the number of exchanged electrons was equal to that of protons (Table 2).The highest current intensity of the FA main voltammetric signal (a2) was obtained in the pH range 3.29-4.56,while the more sensitive voltammetric techniques, DPV and SWV (Figure 2d), pointed out that pH 4.56 was the optimum value for FA quantitative determination at HB_PGE and therefore all subsequent measurements were performed in this medium.

Investigation of FA Voltammetric Behavior at HB_PGE
Cyclic voltammetry is the frequently chosen technique for studying the voltammetric behavior of an analyte in certain conditions (in this case, at HB_PGE in BRB pH 4.56) because it provides information about the type and the reversibility of the redox process in which the species of interest participates, as well as on the number of involved electrons and the rate-limiting step.
In order to clarify the voltammetric behavior of FA at HB_PGE, the first two scans were recorded applying different scan rates (v).The cyclic voltammograms corresponding to the first potential scan (Figure 3a) presented two peaks, an oxidation one (a2) at potentials around 0.600-0.700V and a cathodic signal (c) at 0.350-0.400V.The positions of these peaks indicated that they did not belong to the same redox couple, so that FA was irreversibly oxidized in the process generating the peak a2.In the second potential scan (Figure 3b), a new anodic peak (a1) appeared at about 0.400-0.450V, which can be considered to be the counter-partner of the reduction signal (c).These correspond to a redox couple [20] that is quasi-reversible, due to the fact that the peak separation was more than 0.059/n V (n represents the number of electrons involved in the process) and the ratio of the peak currents (I pa1 /I pc ) was sub-unitary, the reduction reaction being faster.
Micromachines 2023, 14, x FOR PEER REVIEW 7 of 17 SWV (Figure 2d), pointed out that pH 4.56 was the optimum value for FA quantitative determination at HB_PGE and therefore all subsequent measurements were performed in this medium.

Investigation of FA Voltammetric Behavior at HB_PGE
Cyclic voltammetry is the frequently chosen technique for studying the voltammetric behavior of an analyte in certain conditions (in this case, at HB_PGE in BRB pH 4.56) because it provides information about the type and the reversibility of the redox process in which the species of interest participates, as well as on the number of involved electrons and the rate-limiting step.
In order to clarify the voltammetric behavior of FA at HB_PGE, the first two scans were recorded applying different scan rates (v).The cyclic voltammograms corresponding to the first potential scan (Figure 3a) presented two peaks, an oxidation one (a2) at potentials around 0.600-0.700V and a cathodic signal (c) at 0.350-0.400V.The positions of these peaks indicated that they did not belong to the same redox couple, so that FA was irreversibly oxidized in the process generating the peak a2.In the second potential scan (Figure 3b), a new anodic peak (a1) appeared at about 0.400-0.450V, which can be considered to be the counter-partner of the reduction signal (c).These correspond to a redox couple [20] that is quasi-reversible, due to the fact that the peak separation was more than 0.059/n V (n represents the number of electrons involved in the process) and the ratio of the peak currents (Ipa1/Ipc) was sub-unitary, the reduction reaction being faster.From Figure 3, it can be seen that the heights of FA voltammetric signals increased and their potentials shifted (towards more positive values for the anodic peaks and towards less positive potentials for the cathodic one), this last observation being characteristic for quasi-reversible and irreversible electrode processes.The nature and thus the limiting step of the electrode process corresponding to each peak were derived from the various dependencies of the peak height on the scan rate (Table 3).Hence, the FA main oxidation signal a2 was due to a diffusion-controlled process, while the electrode reactions corresponding to the redox couple generating the a1-c peaks pair were controlled by the analyte adsorption at the electrode surface.From Figure 3, it can be seen that the heights of FA voltammetric signals increased and their potentials shifted (towards more positive values for the anodic peaks and towards less positive potentials for the cathodic one), this last observation being characteristic for quasireversible and irreversible electrode processes.The nature and thus the limiting step of the electrode process corresponding to each peak were derived from the various dependencies of the peak height on the scan rate (Table 3).Hence, the FA main oxidation signal a2 was due to a diffusion-controlled process, while the electrode reactions corresponding to the redox couple generating the a1-c peaks pair were controlled by the analyte adsorption at the electrode surface.
At high scan rates, FA oxidation signal a2 seems to represent the sum of two unresolved signals (Figure 3).According to the literature data [20,21], this behavior would be due to a two-step process of one electron each, during which radical species are formed and can further be involved in electropolymerization reactions [22].
Repetitive voltammetric measurements performed at the same graphite pencil lead (Figure 4) showed that the anodic peak a2 was diminished with the increase of scan number (Figure 4a).For example, in the second and third SWV recordings, the peak decreased by 70% and 10%, respectively.Starting with the second scan, the anodic signal a1 appeared at less positive potentials and its intensity was enhanced by successive potential sweeps, this fact being further amplified in the square-wave voltammograms (Figure 4b), where the peak intensity was almost doubled in the second scan, while it increased with about 20% during the third scan.This observation, combined with the surface-confined character of the processes corresponding to the a1-c peaks pair, as well as the previously reported results [22], led to the conclusion that during the voltammetric measurements, FA electropolymerization took place.As a result, most probably a weak conductive polymeric film (due to the reduced increase of the a1 and c peaks intensities in the cyclic voltammograms) arose at the electrode surface, hindering the electron transfer between FA and the PGE surface, a fact reflected in the lowered intensity of peak a2.Thus, a new graphite lead must be used for each voltammetric measurement.Fortunately, due to the fact that PGE is disposable and cheap, this is not a disadvantage.

Linear Range, Limits of Detection and Quantification
The influence of the FA concentration on the intensity of its main oxidation peak a2 was investigated in the range 1.00 × 10 −7 -2.00 × 10 −3 mol/L FA using the more sensitive voltammetric techniques, namely DPV and SWV (Figure 5).It can be observed that at higher FA concentrations, a third anodic signal appeared at more positive potentials (≥~1.00V), but its Ipa = f(c) was not investigated.The height of the anodic signal a2 increased with the enhance-  The influence of the FA concentration on the intensity of its main oxidation peak a2 was investigated in the range 1.00 × 10 −7 -2.00 × 10 −3 mol/L FA using the more sensitive voltammetric techniques, namely DPV and SWV (Figure 5).It can be observed that at higher FA concentrations, a third anodic signal appeared at more positive potentials (≥~1.00V), but its I pa = f(c) was not investigated.The height of the anodic signal a2 increased with the enhancement of the FA concentration from 4.00 ×10 −7 to ~1.00 × 10 −3 mol/L, but the linear dependence between the peak current (I pa2 ) and the analyte concentration was divided into two segments with different slopes.Thus, the calibration graph presented two linear ranges (Table 4) described by the following regression equations (Figure S5): I pa2 (A) = 0.0637 C (mol/L) + 2.00 × 10 −7 (R 2 = 0.9993) and I pa2 (A) = 0.0148 C (mol/L) + 5.00 × 10 −6 (R 2 = 0.9987) for DPV and I pa2 (A) = 0.1144 C (mol/L) + 2.00 × 10 −6 (R 2 = 0.9997) and I pa2 (A) = 0.0406 C (mol/L) + 1.00 × 10 −5 (R 2 = 0.9993) for SWV.The limits of detection (LOD) (Table 4) and quantification (LOQ) were calculated as 3.3 σ/b and 10 σ/b, respectively, where σ represents the standard deviation of the intercept of the regression equation of the lower linear range and b was the slope (0.0637 A×L/mol for DPV and 0.1144 A × L/mol for SWV) of the calibration curve corresponding to that concentration range.The obtained LOQs were 9.33 × 10 −7 and 5.52 × 10 −7 mol/L FA for DPV and SWV, respectively.According to the values of the slopes of the calibration graphs, SWV was more sensitive than DPV, but the performance characteristics of the two techniques are somewhat similar.Compared to other methods developed for FA voltammetric quantification, those presented in this paper, which use bare carbon-based electrodes, have linear ranges (of almost three orders of magnitude) and LODs (at 10 −7 mol/L level) similar or better than half of those reported in the literature till now (Table 4), all of them using modified working electrodes.The reproducibility of the HB_PGE voltammetric response expressed as a percentage relative standard deviation (RSD%) and calculated as a standard deviation × 100/average of ten measurements was estimated by both DPV and SWV at three concentration levels situated within the linear range of the methods, each recording being carried out on a new HB_PGE.The RSD% values (Table 5) obtained for each tested concentration fell within the corresponding accepted limits [55], thus demonstrating the good precision of the DPV and SWV methods developed for FA quantification at HB_PGE.RSD% values from other reported methods can be significantly compared with those obtained in our work only if they were given for the same concentration level.However, a search of the RSD% values reported in the papers published for FA voltammetric analysis revealed the fact that the RSD% values obtained in our study are similar to many of the previously published ones [26,29,31,37,41,43,46], while there are also some with lower RSD% values [20,34,35,44].

Investigation of Possible Interferents on FA Voltammetric Analysis at HB_PGE
The effect of some polyphenolic antioxidants on FA voltammetric analysis at HB_PGE was investigated in BRB pH 4.56 solution.From the huge number of phenolic phytochemicals, which can coexist with FA in various matrices, gallic acid, a hydroxybenzoic acid most commonly used as reference to express the total phenolic content of samples, the structurally related hydroxycinnamic acid, caffeic acid and the glycosylated bioflavonoid naringin (Figure S6) were tested as possible interferents in FA determination by DPV at HB_PGE at molar concentration ratios FA/interferent of 1:1 and 1:10.All examined polyphenols presented oxidation peaks at potentials quite different from that of FA (Figure S7), namely caffeic acid and gallic acid at about 0.300 V and naringin at ~0.890 V and ~1.150 V. Thus, in the given working conditions the investigated antioxidants did not significantly influence the FA oxidation peak current due to the fact that the separation of their peak potentials was about 0.300 V even if the FA anodic peak potential (~0.635 V) was shifted with about 0.03-0.04V in the negative direction in the presence of caffeic acid and gallic acid and with 0.02 V towards more positive values when it was mixed with naringin.Moreover, it must be specified that the FA signal did not vary more than ±5% in the presence of the tested interferents.

Figure 5 .
Figure 5. Differential pulse (a,b) and square-wave (c,d) voltammograms recorded at HB_PGE for different concentrations of FA in BRB pH 4.56 solutions.

Table 2 .
The regression equations and the determination coefficients (R 2 ) for the E p = f(pH) dependencies of each voltammetric signal of FA observed at HB_PGE.

Table 3 .
Various dependencies of the FA peak currents on the potential scan rate corresponding to the cyclic voltammograms from

Table 4 .
The performance characteristics of voltammetric methods reported in the literature for FA determination.

Table 5 .
Results of the reproducibility studies obtained for the voltammetric determination at HB_PGE in BRB pH 4.56 of FA at different concentration levels.