Uhplc dad

Before the quantification of hydrogen peroxide and ferrous iron concentrations, the samples were filtered through 0.2-μm nylon filters (Merck Millipore®). The absorbance of filtered samples with H₂O₂ and dissolved ferrous iron were determined at 410 nm with titanium(IV) oxysulfate reagent and 1,10 phenanthroline solution at 510 nm using standard methods, DIN 38 402 H15 (LOQ was 2.9 · 10⁻² mM and SD was 4 · 10⁻³ mM) and ISO 6332 (LOQ was 4.5 · 10⁻³ mM and SD was 6.1 · 10⁻⁴ mM), respectively. The H₂O₂ and dissolved ferrous iron concentrations were obtained on the calibration curves. The concentration of sodium dodecyl sulphate was measured by colorimetric method adapted from (Koga et al. 1999 (link)) using methylene blue and chloroform. The detection limit of this method was 0.1 mg/L. The Fe³⁺-EDDS complex was determined by a 1200 Series system liquid chromatography with diode array detector (UHPLC-DAD) from Agilent Technologies (Waldbronn, Germany) according to Soriano-Molina et al. (2019 (link)). Dissolved organic carbon (DOC) and dissolved inorganic carbon (IC) were measured using filtered samples, with a Shimadzu-V CPH TOC analyser, which LOQ was 1 mg/L.

The hydroxycinnamic acids were identified and quantified using an Agilent Technologies UHPLC system with a diode array detector (UHPLC-DAD). The separation was carried out using a Zorbax Eclipse Plus C₁₈ rapid resolution column (50 mm × 2.1 mm i.d. 1.8 μm particle size). A binary phase solvent system was used: solvent A was 0.1% acetic acid dissolved in water, and solvent B was 0.1% acetic acid dissolved in methanol. The solvent gradient was as follows: 0–11 min, 9 to 14% B and 11–15 min, 15% B. The column temperature was set at 30°C, the flow rate was 0.7 mL/min, and the detector was set at 280 nm. The results were expressed as μg/g of dry weight (DW) as determined using methanolic solutions (1.5–50 μg/mL) of caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid as standards [11 (link)].