Thermoshaker

The antioxidant capacity (AC) of the films was tested using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and applying the QUENCHER method [39 (link)], which is based on the direct contact of a solid film with DPPH solution. The tested film was insoluble in the radical solution, and the reactions occurred at the sample’s surface. To determine the AC of the prepared films, 0.1 g of the film was ground in a mill and placed in a test tube to which 6 cm³ of 60.86 µmol/dm³ DPPH solution in methanol was added. Then, the test tube was put to a shaker (Thermoshaker, VWR International, LLC, Gdańsk, Poland) at 650 rpm for 15 min. Afterward, the test tube was kept in the dark place for 15 min, and then the UV-VIS spectrum of supernatant was registered at 517 nm using a UV-VIS spectrophotometer. The analysis was conducted in triplicate. The percentage of scavenging of the DPPH radical was calculated according to Formula (2): $$\%\mathrm{DPPH}=\frac{{\mathrm{A}}_0-{\mathrm{A}}_{\mathrm{s}}}{{\mathrm{A}}_0}100\%,$$
where A₀ is the absorbance of DPPH solution, and A_s the absorbance of DPPH solution after contacting the studied film.
A calibration curve based on Trolox solutions showing a linear dependence of the percentage of DPPH scavenging (% DPPH) on Trolox concentration was prepared. The AC of the tested film was expressed in μmol of Trolox equivalents per 100 g of the studied film [40 (link)].

For optimization and validation of the developed method, methanol stock solutions of commercially available dyeing mixtures (D1–D9, Table 6) at a concentration of 1 mg·mL⁻¹ were used as standards. In total, 10 μL of each of the stock solutions were placed in a PCR tube, then the solvent was evaporated under a nitrogen atmosphere at 40 °C using a sample concentrator (Liebisch, Germany). Subsequently, the residues were dissolved in 50 µL of BGE, centrifuged for 5 min at 12,000× g rpm (Microfuge 16 Centrifuge, Beckman Coulter, Krefeld, Germany), and analyzed.
Polyester threads of 1 or 4 cm length (consisting of approximately 50 single fibers) were used as a sample. Extraction was carried out in glass vials closed with caps with 50 μL of chlorobenzene at 100 °C in a thermoshaker (VWR, Radnor, PA, USA) operating at 1000 rpm for 0.5, 1, or 6 h, depending on the experiment. The preparation of five samples (P10, P13a, P13b, P17, P18) required some adjustments (see Table 8 for details).
The extracts were transferred to PCR tubes with the use of a micro syringe to avoid transferring microfibers. The extracts were then evaporated to dryness under gentle nitrogen flow (40 °C) and dissolved similarly to dye standards.