Separation of kafirins on surface porous reversed-phase high-performance liquid chromatography columns

Surface porous high-performance liquid chromatography (HPLC) columns were investigated for the separation of kafirins, storage proteins of grain sorghum. Kafirins were successfully separated using C3, C8, and C18 surface porous stationary phases in less than 17 min. Separations using a monolithic C18 stationary phase were also developed and were slightly faster than those achieved on the surface porous C18 stationary phase. However, the resolution was higher on the latter column. Using an ammonium hydroxide/acetonitrile mobile phase, separations were performed on a novel, alkaline stable surface porous C18 stationary phase. The resolution at alkaline pH was not as high, however, as with the traditional acidic acetonitrile mobile phases. In comparison to fully porous stationary phases, the surface porous phases provided higher resolution with much lower separation times (17 versus 40 min). Total peak areas were correlated to total protein content of sorghum (r(2) = 0.96; n = 10), and a method to measure in vitro pepsin digestibility using reversed-phase (RP)-HPLC peak areas showed good correlation to the traditional nitrogen combustion method (r(2) = 0.82; n = 20). Thus, the surface porous stationary phases could be used not only for more rapid separations but also to provide simultaneous information on total protein content and digestibility.