2.8. Identification of chlorogenic acids by HPLC analysis
The identification of individual phenolics was carried
out using a Waters HPLC system, based on matching
retention times of standards. The HPLC system consisted
of, Waters 515 Binary HPLC pump, Waters automated
gradient controller, and Waters 2487 Dual Wavelength
Absorbance Detector. The column used was a hypersil
C18 column (25 cm 4.6 mm i.d. particle size 5 lm, pore
size 100A ˚ ). The compound were eluted with a gradient elution
of mobile phase A [5% acetonitrile in 0.035%–trifluoroacetic
acid (TFA)] and B (80% acetonitrile in 0.025%
TFA) where B increased from 10% to 20% in 10 min, to
50% by 20 min, was maintained at 50% for 5 min and
remained for 5 min before next injection