Application of a beta-cyclodextrin sulfate-immobilized precolumn to selective on-line enrichment and separation of heparin-binding proteins by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatography (HPLC) system which consisted of a beta-cyclodextrin (beta-CD) sulfate-immobilized hydrophilic vinyl-polymer gel precolumn and a reversed-phase analytical column was developed for the selective on-line enrichment and separation of heparin-binding proteins. Of 15 proteins investigated, 10 proteins having heparin-binding activity were retained on the beta-CD sulfate precolumn almost quantitatively, in contrast 5 proteins having no heparin-binding activity were not retained. Calibration graphs for basic fibroblast growth factor constructed at various sample volumes were nearly identical, indicating that the protein could be enriched by this system. The system was successfully used for the selective separation of lysozyme in egg white. The beta-CD sulfate-immobilized precolumn showed no loss of analytical performance over 2 years during which about 400 samples were analysed.