# Deposition Efficacy of Natural and Synthetic Antioxidants on Fabrics
## Materials and Methods
### Tannin Characterisation  
\({}^{31}\)_P NMR_.\({}^{31}\)P NMR analysis was performed after the derivatisation of accurately weighed tannin samples (10-15 mg) with the reagent 2-chloro-4,4',5,5'-tetramethyl-1,3,2-dioxapospholane, against cholesterol as internal standard according to methods previously published [35; 36; 37; 38], using an inverse-gated pulse-sequence on either a Bruker, Billerica, MA, USA, 300 MHz or a Bruker 700 MHz NMR spectrometer, controlled via TopSpin software packages.  
Gel permeation chromatography (_GPC_): Approximately 3.0 mg of tannin was suspended in 1.0 mL of glacial acetic acid/acetyl bromide (9:1 (v/v)) for 2 h in accordance with the method provided for lignins by [PERSON] et al. [39]. The solvent was then removed under reduced pressure, and the residue was dissolved in HPLC-grade THF and filtered over a 0.45 \(\upmu\)m syringe filter prior to injection. GPC analyses were performed as described before [40] using a Shimadzu, Kyoto, Japan, instrument, consisting of a controller unit (CBM-20A), a pumping unit (LC 20 AT) equipped with a 20 \(\upmu\)L sample loop, a degasser unit (DGU-20A3), a column oven (CTO-20 AC), a diode array detector (SPD-M20A), and a refractive index detector (RID-10A); the Shimadzu LabSolution software package (Version 5.42 SP3) was used for system control. Three analytical GPC columns (each 7.5 \(\times\) 30 mm) were connected in series for the analyses--an Agilent Technologies, Santa Clara, California, USA, PLgel 5 \(\upmu\)m 10 000 A, followed by an Agilent PLgel 5 \(\upmu\)m 1000 A and an Agilent PLgel 5 \(\upmu\)m 500 A. Calibration was performed with polystyrene standards (Sigma Aldrich/Merck KGaA, Darmstadt, Germany, MW range: 162-5 \(\times\) 10\({}^{6}\) g/mol). Final analyses of each sample were performed as described elsewhere [40].  
Folin-Ciocalteau test: The total phenolic content of the extracts was determined by the Folin-Ciocalteau method: 2.5 mL of Folin-reagent (diluted 10 times) was added to 0.5 mL of a solution of tannin in distilled water (0.10 mg/mL). After 2 min, 2.0 mL of sodium carbonate (7.5% (m/m)) was added. The mixture was heated at 50 \({}^{\circ}\)C for 5 min. Absorbance was read at \(\lambda\) = 765 nm in suitable cuvettes. A calibration curve was obtained following this approach using a solution of gallic acid in distilled water (0.5 mg/mL). Results are reported as mg of gallic acid equivalent (GAE) per mg of dry tannin (mg GAE/mg tannin) [41]. Measurements were done using a Shimadzu, Kyoto, Japan, 1800 UV spectrophotometer.  
Scalbert test: The proanthocyanidin content of pine tannins was determined by the Scalbert method [42]. A total of 5.0 mL of an acidic ferrous solution (77 mg FeSO\({}_{4}\cdot\)7 H\({}_{2}\)O in 500 mL HCl/BuOH (2/3)) was added to 0.5 mL of the aqueous tannin solution (1 mg/mL). Mixtures were heated at 95 \({}^{\circ}\)C for 15 min. Absorbance was read at \(\lambda\) = 530 nm. Results are expressed in cyanidin equivalents (CyaE) as mg per g of dry bark (mg CyaE/g bark). Measurements were done using a Shimadzu, Kyoto, Japan, 1800 UV spectrophotometer.