Significant improvements in reversed-phase column performance have been made during the last 10-15 years; the resulting new column technology has enabled faster and more effective separations for small molecules such as pharmaceuticals. However, with pharmaceutical and biopharmaceutical companies racing to develop and commercialize new drugs such as monoclonal antibodies, antibody-drug conjugates, mAb fragments, etc., the needs for fast, efficient separations of proteins and peptides are increasing rapidly.

For such higher molecular weight and complex analytes, it is necessary to use larger-pore size columns to achieve narrow, symmetrical peaks and high peak capacity. Improved performance for bioseparations can be achieved by providing these larger biomolecules unrestricted access to the stationary phase, which resides predominantly in the particles’ pores. In order to avoid a trial-and-error approach for selecting the right column and pore size, some practical guidance is provided below.

Historical Perspective and References

Key Points to Remember

Figure 1

Size Matters (for Biomolecules)

Size differences are unimportant for HPLC and UHPLC of small molecules, which are typically orders of magnitude smaller than average pore openings in the particles, and such analytes enjoy the same freedom of diffusion as they do in the mobile phase.  As molecules become larger, the size-exclusion chromatography (SEC) mode begins to overlap with retention modes such as the reversed-phase chromatography (RPC) mode.  

Consensus has not yet been reached on how large pores should be to avoid unnecessary loss of HPLC performance. There is empirical evidence that a certain amount of size exclusion can be tolerated by the stationary phase retention mechanism. Solutes should be about 1/10 the size of the average pore in order to avoid serious performance loss due to restricted diffusion, slower mass transfer and insufficient stationary phase access.

Two RPLC separations of peptides and smaller proteins on columns with two different pore sizes are shown in Figure 2 below.  Note that the larger proteins (cytochrome c, lysozyme) show broader peaks on the smaller pore size column.  The larger pore size column (160 Ångstrom) produces sharper, more symmetrical peaks, and improved retention (better access to stationary phase).

Figure 2  Improved Peak Width and Symmetry with Larger Pore Size

Figure 2

List of References

1.  J. C. Giddings, Unified Separation Science, Chapter 2, Entropy Effects in Porous Media, Wiley (1991). 
2.  J. C. Giddings, Dynamics of Chromatography: Principles and Theory, CRC Press, Boca Raton, FL (2002).
3.  K. Unger, Porous Silica, Chapter 9 Size-Exclusion, Elsevier (1979).
4.  F. Eisenbeis and S. Ehlerding, Kontakte 1/78 (1978) 22-29.
5.  G. Barka and P. Hoffman, J. Chromatogr., 389 (1987) 273-278.

In part 2 of this LabNote, we will explore further the importance of pore size selection for columns when developing methods and carrying out separations of proteins, monoclonal antibodies and their fragments.

For more information about HALO columns for separations of peptides, proteins, monoclonal antibodies, their fragments, and glycans, please visit our web site at  Detailed information on these products is available in the HALO catalog.