APPLICATION OF A BETA-CYCLODEXTRIN SULFATE-IMMOBILIZED PRECOLUMN TO SELECTIVE ONLINE ENRICHMENT AND SEPARATION OF HEPARIN-BINDING PROTEINS BY COLUMN-SWITCHING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A column-switching high-performance liquid chromatography (HPLC) syste m which consisted of a beta-cyclodextrin (beta-CD) sulfate-immobilized hydrophilic vinyl-polymer gel precolumn and a reversed-phase analytic al column was developed for the selective on-line enrichment and separ ation of heparin-binding proteins. Of 15 proteins investigated, 10 pro teins having heparin-binding activity were retained on the beta-CD sul fate precolumn almost quantitatively, in contrast 5 proteins having no heparin-binding activity were not retained. Calibration graphs for ba sic fibroblast growth factor constructed at various sample volumes wer e nearly identical, indicating that the protein could be enriched by t his system. The system was successfully used for the selective separat ion of lysozyme in egg white. The beta-CD sulfate-immobilized precolum n showed no loss of analytical performance over 2 years during which a bout 400 samples were analysed.