Optimization of high-performance liquid chromatographic peptide separations with alternative mobile and stationary phases.
Peptides are routinely separated with reversed-phase high-performance liquid chromatography using increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. While these separations may be improved by adjustments of gradient slope or substitutions of different solid-phase chemistries, many mixtures would benefit from systematic optimization of mobile phase components. Tryptic digests of cytochrome c from various species were separated on Waters Delta-Pak C18. The effects of varying pH as well as the concentration and type of ion-pair reagent were examined. In addition, low pH, ion-suppression/ion-pairing chromatography was inverted using a polymeric reversed-phase column at high pH with alkyl amine ion pairing. Finally, a tryptic digest of cytochrome c was resolved by ion-exchange chromatography with a strong cation-exchange high-performance liquid chromatography column. These data suggest a framework for dramatically changing the selectivity of peptide separations, leading to more satisfactory peptide mapping.
Young
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Waters Division of Millipore Corp., Milford, MA 01757.
Wheat
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D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000032:analysis; D006736:Horses; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000302:isolation & purification*; D013696:Temperature; D014357:Trypsin
D003574:Cytochrome c Group; D007202:Indicators and Reagents; D010455:Peptides; D014357:Trypsin