Electrochemical Behavior of Chloramphenicol on Carbon Electrodes in a Microelectrochemical Cell published by Ural Federal University eISSN 2411-1414; chimicatechnoacta.ru ARTICLE 2022, vol. 9(4), No. 20229409 DOI: 10.15826/chimtech.2022.9.4.09 1 of 5 Electrochemical behavior of chloramphenicol on carbon electrodes in a microelectrochemical cell Tatiana S. Svalova, Regina A. Zaidullina *, Margarita V. Medvedeva, Elizaveta D. Vedernikova, Alisa N. Kozitsina Institute of Chemical Engineering, Ural Federal University, Ekaterinburg 620009, Russia * Corresponding author: zaidullina.regina@urfu.ru This paper belongs to a Regular Issue. © 2022, the Authors. This article is published in open access under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Abstract Express determination of antibiotics is an extremely important task today. Portable electrochemical microdevices are a viable alternative to traditional methods of analysis. The development of such devices requires the study of redox processes in detail. This article is devoted to the comparative study of the electrochemical behavior of chloram- phenicol in water solvents in standard laboratory and portable micro- electrochemical cells. It was found that the electrochemical reduction of chloramphenicol proceeds via a 3-electron mechanism to the for- mation of a dimer. In the transition from the macrocell to the micro- cell, a decrease in the electrochemical reduction current and a shift of the peak potential to the cathode region are observed, which is appar- ently associated mainly with the type of the electrode material. The best characteristics of the direct electrochemical response were ob- tained in the differential pulse voltammetry mode. Under the selected operating parameters, the peak current of the electrochemical reduc- tion of chloramphenicol is linearly dependent on the concentration of the antibiotic in the range of 2∙10–3–1∙10–5 M with a detection limit of 3∙10–5 M. Obtained characteristics are sufficient for the quality control of pharmaceuticals and can be improved through the use of organic and hybrid modifiers of the working electrode surface. Keywords chloramphenicol express-determination electrochemical behavior voltammetry microcell screen-printed electrode Received: 14.07.22 Revised: 02.08.22 Accepted: 02.08.22 Available online: 16.08.22 1. Introduction Chloramphenicol (CAP) is a broad-spectrum antibiotic uti- lized in veterinary and medicine due to its high efficacy and low cost. However, CAP entering the aquatic environment does not decompose and accumulates therein. Thus, it con- taminats sediments and water systems and induces un- healthy effects, such as aplastic anemia (blood disorder), agranulocytosis, dosage independent suspected carcinogen- ity in humans, and also contributes to the development of bacteria resistance [1, 2]. Therefore, detection of chloram- phenicol in food and environmental objects is an important task [3]. To determine the trace amounts of chloramphenicol in laboratory practice, chromatographic [4], spectroscopic [5], enzyme immunoassay [6], and other methods of analy- sis are used [7]. However, all these approaches require ex- pensive equipment, reagents, and a rather lengthy analysis procedure. Of greatest interest is the electrochemical deter- mination of chloramphenicol [8], mainly due to the ultra- sensitivity of the determination, the possibility of miniatur- ization and the transition to portable test systems. Electro- chemical analytical microdevices have great prospects, as they are suitable for the mass production, representing in- expensive, disposable sensor systems. In the designs of such devices, as a rule, three-electrode systems made by screen printing are used [9]. For example, Pakapongpan et al. developed an electrochemical sensor using a magnetic screen-printed electrode (SPE), which demonstrated a de- tection limit of 10 nM for the detection of CAP [10]. Li et al. invented a laser-enabled flexible electrochemical sensor on finger, which can be used for rapid real-time in-site elec- trochemical identification of CAP in meat (LOD 10 μM) [11]. The advantages of such electrochemical systems with printed electrodes are low cost and the possibility of one- time use, which simplifies the analysis procedure and im- proves analytical performance by eliminating the stage of electrode surface regeneration. Transition from standard electrochemical cells to portable microdevices requires additional studies of the http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2022.9.4.09 mailto:zaidullina.regina@urfu.ru http://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0002-0263-2111 https://crossmark.crossref.org/dialog/?doi=https://doi.org/10.15826/chimtech.2022.9.4.09&domain=pdf&date_stamp=2022-8-16 Chimica Techno Acta 2022, vol. 9(4), No. 20229409 ARTICLE 2 of 5 electrode reactions because of the significant differences in ge- ometry of a working electrode and electrochemical cell, pa- rameters of diffusion and charge transfer, etc. [12, 13]. It can also influence the analytical signal parameters [14–17]. The aim of this article is the comparative study of the electrochemical behavior of CAP in standard laboratory and portable microelectrochemical cells, and the estimation of the possibility of quantitative electrochemical determina- tion of CAP using the direct electrochemical response. 2. Experimental 2.1. Reagents and Chemicals All solutions were prepared with ultrapure water from a Branstead Pacific TII Water Purification System. Britton– Robinson buffer solutions (BRB) (pH 2–10) were prepared from 0.04 M orthophosphoric acid, 0.04M acetic acid and 0.04 M boric acid; the pH values were adjusted with sodium hydroxide. The pH was measured with a FiveEasy pH Meter (Mettler Toledo). All chemicals were purchased from Sigma Aldrich: orthophosphoric acid, acetic acid, boric acid, so- dium hydroxide, lithium perchlorate, acetonitrile, potas- sium chloride, ferrocyanide (K4Fe[CN]6), potassium ferri- cyanide (K3Fe[CN]6). 2.2. Electrochemical research Cyclic voltammograms (CV), differential pulse voltammo- grams (DPV), square wave voltammograms (SWV) and chronoammograms (CA) were recorded on a potenti- ostat/galvanostat μAutolab Type III. The three-electrode standard cell (Figure 1a) consisted of a glassy carbon work- ing electrode (GCE), an Ag/AgCl/KCl reference electrode, and a carbon rod as an auxiliary electrode. A Micrux AIO- platform with a three-electrode system made by screen printing was used as a microelectrochemical cell (Figure 1b). 3. Results and Discussion The study of the electrochemical behavior of chloramphen- icol in water solutions was carried out both in the standard three-electrode cell and in the microcell. Cyclic voltammograms (Figure 2a), registered on the glassy carbon electrode, show a pronounced cathodic peak at a potential of –0.7 V and an anodic peak at a potential of 0.3 V, which increases with further cycling. Also, in the sec- ond cycle, a peak at a potential of –0.3 V appears, and the current of the cathodic peak at a potential of –0.7 V de- creases. It probably indicates a two-stage reduction process. On the CV registered in microcell, the electrochemical be- havior of CAP is similar (Figure 2b). The shift of the ca- thodic peak potential in the negative region can be associ- ated with the type of the electrode material and the differ- ences in effective surfaces (Figure 2c). Studies on the effect of pH on the electroreduction (Fig- ure 3) in the Britton–Robinson solution were carried out to estimate the proton influence. With an increase in pH, a shift of the peak to the cathodic region is observed. Thus, we can assume a proton-dependent reduction mechanism. In an aqueous medium ΔE/ΔpH = –43 mV, which indicates an equal number of protons and electrons involved in the reaction. The maximal peak current is observed at pH = 5 (Figure 3b). Figure 4 shows the dependences of the recovery peak current on the square root of the scan rate (ν1/2) in the po- tential scan rate range from 1 to 2000 mV/s. The peak cur- rent has a linear relationship with the square root of the scan rate over the pH range under study. Thus, the electro- chemical reduction of chloramphenicol is a diffusion-con- trolled process. The effective number of electrons, involved in CAP elec- troreduction, was estimated as 3, using the Randles-Sevcik theory (Equation 1) [18]: 𝐼p = (2.69 × 105) 𝑛 3 2⁄ 𝐴𝐷 1 2⁄ 𝑐 𝑣 1 2⁄ , (1) where Ip is the peak current, A; n is the number of trans- ferred electrons; A is the area of the electrode, cm2; D is the diffusion coefficient, cm2/s; c is the concentration, M; and ν is the potential scan rate, V/s. It is known that the diffusion coefficient for the nitroar- omatic compounds in aqueous media is ~10–5 cm2 [19], the diameter of GCE is 2 mm, the concentration of CAP is 2∙10–3 M (in BRB pH = 5), the scan rate is 2 V/s. Figure 1 The standard three-electrode cell (a); ‘Micrux’ AIO-platform for screen-printed electrodes (SPE) (b). Chimica Techno Acta 2022, vol. 9(4), No. 20229409 ARTICLE 3 of 5 Figure 2 CV registered in presence of 2∙10–3 M CAP in BRB pH = 7 on GCE (a), on GCE and SPE (b), CV registered in presence of redox indicator 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] on GCE and SPE, 0.1 KCl, scan rate 0.1 V/s (c). Figure 3 CVs registered in presence of 2∙10–3 M CAP on GCE, BRB pH = 2–10, scan rate 0.1 V/s (a); plot I = f(pH) (b) and plot E = f(pH) (c). Figure 4 CVs registered in presence of 2∙10–3 M CAP on GCE, BRB pH = 5 (a); plot peak recovery current vs scan rate (b). Based on the obtained results, we can assume that the electrochemical behavior of chloramphenicol in microcell accompanied with a pronounced cathodic peak corresponds to the 3-electron and 3-proton electroreduction of the nitro group with the formation of the corresponding dimer (Scheme 1). Chimica Techno Acta 2022, vol. 9(4), No. 20229409 ARTICLE 4 of 5 Scheme 1 Proposed mechanism of CAP electrochemical reduction in microcell. To achieve the best analytical characteristics of the CAP determination, we optimized the voltammetric mode both in standard and micro-electrochemical cells using CV, DPV and SWV. Figure 5 shows that during the changing registra- tion mode from the LV (–0.65 V) to the SWV (–0.54 V) and DPV (–0.56 V), the cathodic peak potential shifts to the an- odic region and the peak current increases significantly. The best characteristics were obtained using the DPV mode (Figure 5). Similar tendency was also observed in the Mi- crux cell. When registering the DPV of CAP in a microvolume of the sample (the volume of the sample was 50 µL), the cathodic peak current significantly decreased, which is probably asso- ciated with a complication of charge transfer and a less devel- oped surface of the screen-printed electrode (Figure 6). Thus, the process of electrochemical reduction of CAP, registered in DPV mode can be used as a direct analytical signal in the portable electrochemical cells for express de- termination of the antibiotic in microvolumes of samples. Under the chosen operating parameters, a calibration plot I = f(C) was obtained in model solutions of CAP (Fig- ure 7). The regression is linear (R2 = 0.9984) in the wide concentration range of the CAP. The detection limit, esti- mated according with the 3-sigma criterion, was 3∙10–5 M. The obtained characteristics are sufficient for the quality control of pharmaceuticals and can be improved using or- ganic and hybrid modifiers of the working electrode surface. 4. Conclusions In this work, the electrochemical behavior of chloramphen- icol in aqueous solvents was studied in the standard three- electrode cell and the portable electrochemical cell with the screen-printed three-electrode system. A similar RedOx be- havior of CAP was obtained, accompanied with the pro- nounced irreversible reduction peak corresponding to the 3-electron and 3-proton electrochemical conversion into the corresponding dimer. The best characteristics of the response were obtained in the differential pulse volt- ammetry mode. In the transition from the macrocell to the microcell, a decrease in the electroreduction current and a shift of the peak potential to the cathodic region are ob- served, which is apparently associated with the type of the working electrode. Under the experimentally optimized op- erating parameters, the current of the CAP electroreduction peak linearly depends on the antibiotic concentration in the range of 2∙10–3–1∙10–5 M with a detection limit of 3∙10–5 M and Figure 5 LV, scan rate 0.1 V/s (1); SWV, modulation amplitude 0.2 V, frequency 25 Hz (2); DPV, modulation amplitude 0.2 V, mod- ulation time 0.005 s (3). 2∙10–3 M chloramphenicol, glassy carbon electrode, BRB pH = 5. Figure 6 DPV, 2∙10–3 M chloramphenicol, SPE (1), GCE (2). BRB pH = 5, modulation amplitude 0.2 V, time modulation 0.005 s. Figure 7 Calibration plot I = f(CCAP) obtained in the Micrux cell on the SPE, DPV modulation amplitude 0.2 V, modulation time 0.005 s, BRB pH = 5. can be used as an analytical signal of an electrochemical sen- sor. An increase in the detection sensitivity can be achieved using organic and hybrid modifiers of the working electrode surface. Chimica Techno Acta 2022, vol. 9(4), No. 20229409 ARTICLE 5 of 5 Supplementary materials No supplementary materials are available. Funding This work was supported by the Presidential Grants Fund of the Russian Federation (grant no. MK-392.2022.1.3). Acknowledgments None. Author contributions Conceptualization: T.S.S., M.V.M. Data curation: M.V.M. Formal Analysis: M.V.M., E.D.V. Funding acquisition: T.S.S., M.V.M. Investigation: M.V.M., E.D.V. Methodology: T.S.S., M.V.M., E.D.V. Project administration: T.S.S. 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