A general separation method of phenolic acids using pH-zone-refining counter-current chromatography and its application to oat bran.

pH-zone-refining counter-current chromatography technique for the separation of natural and synthetic mixtures has been widely used, especially for organic acids and alkaloids. Phenolic acids are very important compounds due to the potential treatment for a wide variety of diseases. However, there is not a general method for their separation. In this work, the conditions of pH-zone-refining counter-current chromatography, involving solvent systems, concentration of retainer and eluter, flow rate of mobile phase as well as sample pretreatment, were optimized to improve extraction efficiency and reduce separation time. Finally a general separation method for seven common phenolic acids has been established using pH-zone-refining counter-current chromatography. The separation of these phenolic acids was performed with a two-phase solvent system composed of methyl tert-butyl ether/acetonitrile/water at a volume ratio of 4.75: 0.25: 5, where 3mM trifluoroacetic acid was added to the organic stationary phase as a retainer and 3mM NH4OH was added to the aqueous mobile phase as an eluter. As a result, seven phenolic acids, including syringic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, p-hydroxybenzoic acid, ferulic acid and p-coumaric acid were successfully separated with the purities of 95.9%, 67.3%, 96.9%, 82.4%, 97.0%, 91.0%, and 97.2%, respectively. The established general method has been applied to the crude sample of oat bran pretreated with AB-8 resin. A total of 49.5mg of syringic acid, 109.2mg of p-coumaric acid and 184.5mg of ferulic acid were successfully purified in one run from 1.22g crude extract with the purities of 95.2%, 93.0%, and 91.8%, respectively.