title: Options for rapid analysis of peptides and proteins, using wide-pore, superficially porous, high-performance liquid chromatography particles with unique bonded-phase ligands.
abstract: The large size and complexity of many proteins constrains the reversed-phase high-performance liquid chromatography packings that are useful for their separation. Wide-pore, superficially porous, silica-based packings with solid 4.5-microm cores and a 0.25-microm porous outer layer (Poroshell) demonstrate a variety of characteristics that are beneficial for the separation of proteins. A shorter diffusion distance allows separations of large molecules at high linear velocities. This benefit over totally porous particles is clearly shown using separations of a peptide-protein standard. The structure and reduced surface area (4.5 m2/g) of these superficially porous particles simplifies interactions with its surface, resulting in improved peak shapes and resolution. Specialized bonding chemistries for low- and high-pH operation may be used to change band-spacing and achieve atypical separations. These rapid analysis options are demonstrated using protein standards and very high molecular weight glycosylated proteins including intact monoclonal antibodies, IgM, alpha2-macroglobulin, and glycophorin. In liquid chromatography-mass spectrometry analysis of a myoglobin peptide digest, bidentate-C18-bonded superficially porous packings achieve complete runs in 4 min and demonstrate an elution pattern that is unique from that of material bonded with sterically protected C18 ligands.
authors.affiliation: Agilent Technologies, Life Science and Chemical Analysis, 2850 Centerville Rd., Wilmington, DE 19808, USA. robert_ricker@agilent.com
authors.forename: Robert D
authors.initials: RD
authors.lastname: Ricker
authors.forename: Clifford B
authors.initials: CB
authors.lastname: Woodward
authors.forename: Kurt
authors.initials: K
authors.lastname: Forrer
authors.forename: Bernard J
authors.initials: BJ
authors.lastname: Permar
authors.forename: Wu
authors.initials: W
authors.lastname: Chen
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006021:Glycophorins / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D010316:Particle Size; D010455:Peptides / Q000032:analysis*; D016062:Porosity; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D000511:alpha-Macroglobulins / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D006021:Glycophorins; D007075:Immunoglobulin M; D010455:Peptides; D011506:Proteins; D000511:alpha-Macroglobulins