Iron 3 chloride hexahydrate

Acetic acid (99.5%), aluminum chloride hexahydrate, copper(II) chloride dihydrate, disodium hydrogen phosphate dihydrate, 96% ethanol, 36% hydrochloric acid, iron(III) chloride hexahydrate, methanol, phosphoric acid, potassium persulfate, potassium dihydrogen phosphate, potassium hexacyanoferrate(III), sodium acetate anhydrous, sodium carbonate anhydrous, sodium hydroxide, sodium nitrite, and trichloroAcetic acid were purchased from Chempur, Poland. Neocuproine was delivered by J&K Scientific, Germany. Folin–Ciocalteu reagent, acetonitrile, iron(II) sulfate heptahydrate, gallic acid were supplied by Merck, Germany, whereas rutin trihydrate by Roth, Germany. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), caffeine were purchased from Sigma-Aldrich, USA. All the chemicals were of analytical grade.

FeSO₄ × 7H₂O was used for a calibration curve. The compound was dissolved in methanol and diluted with water to obtain concentrations of 0.0125, 0.025, 0.05, 0.10, 0.15, 0.20, and 0.30 mg/mL. From the phenol samples, 1 mg/mL methanolic solutions were prepared and diluted with water to finally obtain seven concentrations: 0.1, 0.08, 0.06, 0.04, 0.03, 0.02, 0.01 mg/mL, respectively.
The assay was performed according to Benzie and Strain [29 (link)], with some modifications. The FRAP working solution was prepared prior to the start of the analysis: 0.3 mol acetate buffer (pH 3.6), 0.01 mol TPTZ (2,4,6-tripyridyl-s-triazine; Sigma-Aldrich, Poznań, Poland) in 0.04 mol HCl (POCH, Lublin, Poland) and 0.02 M FeCl₃ × 6H₂O in water (iron (III) chloride hexahydrate; Chempur, Poland) were mixed in a volumetric ratio of 10:1:1 and protected from light. Next, 75 µL of the phenol solutions or FeSO₄ × 7H₂O solutions were mixed with 2.25 mL of the FRAP working solution and 225 µL of water. The obtained mixtures were incubated at 37 °C for 30 min, and their absorbance was measured at 593 nm. Deionized water with a FRAP solution was used as a blank.

FeSO₄ × 7H₂O used for calibration curve construction was dissolved in methanol and further diluted with water to obtain concentrations of 0.0125, 0.025, 0.05, 0.10, 0.15, 0.20, and 0.30 mg/mL. From the phenol samples, 1 mg/mL methanol solutions were prepared and diluted with water to obtain seven concentrations: 0.1, 0.08, 0.06, 0.04, 0.03, 0.02, and 0.01 mg/mL.
The assay was performed according to Benzie and Strain [46 (link)] with some modifications. The FRAP working solution was prepared prior to the start of the analysis: 0.3 mol acetate buffer (pH 3.6), 0.01 mol TPTZ (2,4,6-tripyridyl-s-triazine; Sigma-Aldrich) in 0.04 mol HCl (POCh, Gliwice, Poland), and 0.02M FeCl₃×6H₂O in water (iron (III) chloride hexahydrate; Chempur, Poland) were mixed in a volumetric ratio of 10:1:1 and protected from light. Next, 20 µL of the phenol solutions or FeSO₄ × 7H₂O solutions were mixed with 2.25 mL of the FRAP working solution and 180 µL of water. The obtained mixtures were incubated at 37 °C for 30 min, and their absorbance was measured at 593 nm. Deionized water with FRAP solution was used as a blank.