3.6. Identification and Separation of Flavonoids and Phenolic Acids

The chromatographic separation of the flavonoids and phenolic acids was performed using ultra-high performance liquid chr.omatography (Model 1200, Agilent, Santa Clara, CA, USA). The flavonoids and phenolic acids were separated on an Agilent C18 (5 μm of pa.rticle size, 4.6 × 250 mm) reversed-phase column by gradient elution using 0.03 M orthophosphoric acid (A) and HPLC grade methanol (B). The gradient profile was: 0 min 40% B, 10 min 100% B, 15 min 100% B, and 20 min 40% B. The detector wavelengths were set at 280 and 360 nm. The flow rate and injection volume was 1 mL/min and 10 µL, respectively. Column temperature was set at 35 °C. Identification of the compounds was achieved by comparison of retention times with standards, UV spectra and UV absorbance ratios after co-injection of samples and standards. Standards including (−)-epicatechin (≥98%; CAS Registry number 490-46-0), (+)-catechin hydrate (≥98%; CAS Registry number 225937-10-0), kaempferol (≥97%; CAS Registry number 520-18-3), quercetin dihydrate (≥98%; CAS Registry number 6151-25-3), rutin hydrate (≥94%; CAS Registry number 207671-50-9), gallic acid monohydrate (>99%; CAS Registry number 5995-86-8), ferulic acid (>98%; CAS Registry number 1135-24-6), trans-cinnamic acid (≥98%; CAS Registry number 140-10-3), tannic acid (>99%; CAS Registry number 1401-55-4) and syringic acid (≥98.0%; CAS Registry number 530-57-4) were purchased from Sigma-Aldrich, city, Malaysia.