Options for Rapid Analysis of Peptides and Proteins, Using Wide-Pore, Superficially Porous, High-Performance Liquid Chromatography Particles with Unique Bonded-Phase Ligands

• Published in: Journal of chromatographic science 2008-03, Vol.46 (3), p.261-268
• Main Authors: Ricker, Robert D., Woodward, Clifford B., Forrer, Kurt, Permar, Bernard J., Chen, Wu
• Format: Article
• Language: English
• Subjects: alpha-Macroglobulins - analysis; Antibodies, Monoclonal - analysis; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; Glycophorin - analysis; Immunoglobulin M - analysis; Mass Spectrometry - instrumentation; Mass Spectrometry - methods; Particle Size; Peptides - analysis; Porosity; Proteins - analysis; Reproducibility of Results
• ISSN: 0021-9665; 1945-239X
• DOI: 10.1093/chromsci/46.3.261

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-µm cores and a 0.25-µm 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, α2-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.