Multimodal liquid chromatography columns for the separation of proteins in either the anion-exchange or hydrophobic-interaction mode.
Several high-performance stationary phases suitable for protein chromatography were synthesized. Columns packed with these materials could be operated independently in either the anion-exchange or hydrophobic-interaction mode. Two approaches were used to prepare these materials. In the first method, a polyamine was adsorbed on the surface of macroporous silica and then crosslinked with a multifunctional oxirane. The hydrophobicity of the crosslinking agent and the extent of interconnection were used to modulate the electrostatic and solvophobic interactions. The second approach also utilized a crosslinked polyamine stationary phase; however, the forces of interaction were attenuated through controlled acylation of surface amines with a small anhydride molecule. The resolving ability of these columns, functioning in either mode, was comparable to commercial high-performance liquid chromatographic columns, designed to operate by a single retention mechanism. Column selectivity for proteins was completely different in each mode. Protein fractions collected from a multimodal column, operated in the anion-exchange mode, could be further purified by rechromatographing them on the same column in the hydrophobic-interaction mode. Utility of the multimodal column was demonstrated with the fractionation of several cytochromes and ferredoxins from the cyanobacterium Microcystis aeruginosa.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D000458:Cyanobacteria / Q000032:analysis; D003574:Cytochrome c Group / Q000302:isolation & purification; D009113:Muramidase / Q000302:isolation & purification; D010047:Ovalbumin / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D012260:Ribonucleases / Q000302:isolation & purification
D003574:Cytochrome c Group; D011506:Proteins; D010047:Ovalbumin; D012260:Ribonucleases; D009113:Muramidase