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Molecules 2020, 25, 3191 7 of 24 HORAC (hydroxyl radical averting capacity) technique is based on the oxidation of fluorescein by hydroxyl radicals which are generated using Fenton reagent. Formed antioxidant blocks produced hydroxyl radicals [42,60]. Other method using the Fenton-like system to induce lipid peroxidation is lipid peroxidation inhibitory assay by measuring the thiobarbituric acid reactive substances (TBARS) [12,42,60]. This assay is based on the reaction of 2-thiobarbituric acid (TBA) with malondialdehyde (MDA) as one of the products of unsaturated lipids oxidation (Scheme 5) [61]. This method is limited by the possibility of MDA reaction with amino group to form Schiff base [62]. Scheme 5. Reaction of two molecules of 2-thiobarbituric acid with malondialdehyde [61]. Other radical probes, such as TRAP (total peroxyl radical trapping antioxidant parameter) monitoring the reaction between peroxyl radicals and the sample quenched the chemiluminescence, CUPRAC (cupric reducing antioxidant power) by which the reduction of Cu2+ is observed, Fremy’s salt (galvinoxyl radical), aroxyl radical (2,6-di-tert-butyl-4(4′-methoxyphenyl)phenoxyl radical) [42,60,63,64]. 3.2. Electrochemical Methods Electrochemical properties of various compounds can also be used for evaluation of their reducing and antioxidant properties. Electrochemical methods for the determination of total antioxidant capacity are widely used because of their sensitivity, speed, and low cost [65]. Essential electrochemical methods encompass cyclic voltammetry, amperometry, and biamperometry. Cyclic voltammetry (CV) is a method based on the measurement of oxidation potential (E1/2) intensity of the sample [43]. The oxidation potential is usually scanned linearly in time from an initial to a final value and back as a triangular waveform. Low values of oxidation potential reflect the tendency of molecule to donate an electron and indicate significant antioxidant capacity [42,43,45,65]. There are some limitations of this method because of the detection limit of 10−5 M and low resolution [65]. The amperometric technique involves the measurement of the flowing current intensity between a working and a reference electrode at a fixed value of potential generated by the oxidation-reduction reaction [42,65]. This method is based on the reduction of DPPH• radical at a glassy carbon electrode [65–67]. Biamperometry uses two identical polarized platinum electrodes with high sensitivity [42,65,68]. The potential difference (∆E) between the electrodes is controlled and the measurement depends on the reaction of the analyzed sample with redox couple [42,65]. 3.3. Chromatographic Methods Chromatography encompasses a group of broadly applicable methods in detection and separation of various compounds present in the analyte, but can also be used for antioxidant capacity measurement. The mostly preferred chromatographic methods are gas chromatography and high-performance liquid chromatography. High-performance liquid chromatography (HPLC) is used to separate, identify, and quantify the individual components present in the analyte. This method is based on the affinity of analyte to a stationary phase, which is usually non-polar (reverse phase), but also polar (normal-phase) and is placed inside the column. The chosen composition of the mobile phase (eluent) depends on the used stationary phase and interactions between analytes. The separation process depends on affinity of analyte toward the stationary and mobile phases. The analyte flows through column in a liquid mobilePDF Image | Antioxidant Activity Determination of Silver Nanoparticles
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