Title: Separation of kafirins on surface porous RP-HPLC columns Authors
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 23, 2010
Publication Date: January 12, 2011
Citation: Bean, S., Ioerger, B.P., Blackwell, D.L. 2011. Separation of kafirins on surface porous RP-HPLC columns. Journal of Agricultural and Food Chemistry. 59:85-91. Interpretive Summary: Sorghum is known to have lowere protein degestibility than other cereal grains. The exact reason for this is not currently known. Sorghum proteins also cross-link extensively when heated and do not have the same functionality in food products as wheat proteins. To improve the utilization of sorghum, the reasons for the differences between sorghum proteins and other cereal proteins needs to be determined and sorghum germplasm with higher digestibility needs to be identified. Therefore, we investigated the use of new HPLC columns to provide faster, higher resolution methods for measuring the properties of sorghum flour. Improved methods for characterizing sorghum proteins will benefit scientists working to improve the quality of sorghum and as a tool to screen diverse germplasm to identify sorghum lines with improved properties. This method also reduces the consumption of the solvents used during analysis of the proteins by 53% thereby producing less waste and lowering the cost of analysis.
Technical Abstract: Surface porous HPLC columns were investigated for the separation of kafarins, 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 performed on a novel, alkaline stable surface porous C18 stationary phase. Resolution at alkaline pH was not as high; however, as with the traditional acidic acetonitrile mobile phases. When compared to fully porous stationary phases, the surface porous phases provided higher resolution with much lower separation times (17 min vs 40 min). Total peak areas were correlated to total protein content of sorghum (r squared = 0.96, n=10) and a method to measure in-vitro pepsin digestibility using RP-HPLC peak areas showed good correlation to the traditional nitrogen combustion method (r squared = 0.82, n=20). Thus, the surface porous stationary phases could be used only for more rapid separations, but also to provide simultaneous information on total protein content and digestibility.