2.3. Purification of chlorogenic acid
The procedure of Ky, Noirot, and Hamon (1997) was
slightly modified and employed for purification of CGA.
Coffee conserve was dispersed in water and ammonium
sulfate was added to a final concentration of 20 g/l in
order to precipitate proteins by an increase in ionic
strength followed by addition of 4% phosphoric acid to
render the CGA more soluble in ethyl acetate. Extracts
were treated with petroleum ether (40–60 C) to remove
lipids and pigments. De-pigmented extracts were further
treated with chloroform to remove caffeine and wax.
The residue was washed with ethyl acetate, distilled and
air dried to obtain a light creamy colored powder. The
CGA in the powder was estimated by UV-Spectrophotometry
(AOAC, 2005) and characterized by NMR (Bruker
Avance 500 MHz).