Isolation of bitter acids from hops ( H umulus lupulus L .) using countercurrent chromatography

Commercially available hops ( Humulus lupulus L.) bitter acid extracts contain a mixture of three major congeners (co‐, n‐, and ad‐) in addition to cis / trans diastereomers for each congener. Individual isomerized α‐acids were obtained by the consecutive application of two separate countercurrent chromatography methods. First, individual isomerized α‐acid congeners as a mixture of cis / trans diastereomers were obtained using a solvent system consisting of hexane and aqueous buffer. The second purification, capable of separating cis / trans diastereomers, was accomplished using a quaternary solvent system; an alternative procedure using β‐cyclodextrin followed by countercurrent chromatography was also investigated. The NaBH 4 reduction of the purified isomerized α‐acid compounds followed by countercurrent chromatography purification resulted in individual ρ iso α‐acids (>95%). Similarly, catalytic hydrogenation of the purified isomerized α‐acid compounds followed by countercurrent chromatography purification produced individual tetrahydro isomerized α‐acids (>95%). Reported herein is a widely applicable approach that focuses on three critical variables – solvent system composition, pH, and buffer‐to‐sample ratio – that enable the efficient purification of individual bitter acids (≥95%) from commercially available hops extracts.