Using Very Small Particle Chromatographic Packings to Optimize Resolution and Speed in Peptide Separations

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Thomas E. Wheat, Beth Gillece-Castro, Ziling Lu, Uwe D. Neue, and Jeffrey R. Mazzeo [Waters]
WCBP, January 24-27, 2006
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The reversed phase LC peptide maps used for characterizing biopharmaceutical proteins must resolve all the peptides representing the entire sequence of the protein. This separation must be established in such a way that modifications of the protein can be recognized and measured. Analysis of such samples requires the best possible chromatographic resolution. To achieve these separations, maps are often developed with very long, shallow gradients on the order of one to three hours. Even with these methods, additional resolution is often required. The application of 1.7mm packing materials has been shown to improve resolution in peptide mapping by reducing dispersion. Investigation of the mechanisms underlying this improved resolution suggests that improved resolution can be obtained in shorter run times. The measured optimum flow rates are low, and such flow rates are consistent with the relatively slow diffusion of peptides. The relationship between linear velocity and particle size can be predicted from the van Deemter equation for molecules of this size. In these experiments, we describe the interactive effects of particle size, pore size, linear velocity, and gradient slope. The use of these smaller particles provides a mechanism for obtaining peptide maps that combine higher resolution with reduced analysis times.

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