Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume
46, Number 3, March 2008, pp. 261-268
Options for Rapid Analysis of Peptides and Proteins,
Using Wide-Pore, Superficially Porous, High-Performance Liquid
Chromatography Particles with Unique Bonded-Phase Ligands
Robert D. Ricker[1], Clifford B. Woodward III[1],
Kurt Forrer[2], Bernard J. Permar[1], and Wu Chen[1]
[1]Agilent Technologies, Life Science and Chemical Analysis,
2850 Centerville Rd., Wilmington, DE 19808 USA and
[2]Novartis
Pharma, Biotechnology WKL-681.546, 4002 Basel, Switzerland
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, a2-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.
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