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