New agar microspheres for the separation and purification of natural products

A new type of agar chromatography media has been prepared with a yield over 80% using a water‐in‐oil emulsion technique. These microspheres have regular spherical shapes and particle diameters in the range 40–165 μm (average ∼90 μm). Cross‐linking of the resulting agar microspheres with epichlorohydrin and 1,4‐butanediol diglycidyl ether enhanced their mechanical and thermal stability. The alkaline conditions used during the cross‐linking reaction also decreased the content of ionized sulfate groups of the polysaccharide, thus reducing the nonspecific adsorption of positively charged molecules. The cross‐linked agar microspheres were functionalized with (i) branched poly(ethyleneimine) to obtain a stationary phase useful for the separation of proteins in an anion‐exchange mode and (ii) with poly‐β‐cyclodextrin enabling direct isolation and purification of puerarin from a crude extract of Radix puerariae. Using a 23.5 mL column loaded with 20 mg extract (0.85 mg/mL gel), puerarin with a purity of 96% was recovered with a yield of 86%.