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Guidance for Selecting the Correct Pore Size of Your Analytical Columns for Better RPLC Separations of Biomolecules (part 1)

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

  • J. Calvin Giddings first introduced the idea of pore confinement, where solutes lose freedom to diffuse and interact with the surface.  Both equilibrium   constants (entropy) and kinetics can be impacted, if pores are too small (1, 2).

  • Klaus Unger cited evidence that pore openings should be 10 times larger than analyte hydrodynamic diameter (in solution), in order to achieve unrestricted diffusion and optimum performance (defined as speed and resolution (3-5)). 

  • High speed separations of biomolecules using any type of particle morphology (totally porous or superficially porous) require that the particle pore diameter be “adequately sized” for the largest analytes in the sample.

  • Matching pore size to analytes may be the first and most important step in large molecule method development as convenient experiments become readily available.

Key Points to Remember

  • Chromatographers have typically used analyte molecular weight (MW) as a rough guideline for selecting the right pore size for analytical columns. 

  • MW is accurately known for small molecules and also known (usually) for proteins and other large molecules.

  • However, the physical size of a molecule in solution is much more important for pore access and may not be readily available or discernible. 

  • Molecular size generally increases with molecular weight (MW or Da), although it is not a linear relationship, especially for proteins. 

  • Particles for HPLC and UHPLC columns usually have a broad pore distribution range as shown in Figure 1 for HALO particles in four different nominal pore sizes.

  • Pore apertures for HALO 90 Å particles are large enough to allow typical pharmaceuticals and even small peptides (< 3-5 kDa) to travel virtually anywhere within the pore space to interact with stationary phase. 

  • Larger peptides may be excluded from many HALO 90 Å pores and usually benefit by moving to pores having somewhat larger diameter (e.g., HALO 160 Å). 

  • Both small and large proteins (up to 500 kDa) benefit from using particles whose pores are as large as 400 to 1000 Å (HALO 400 and 1000 Å).

  • HALO Fused-Core particles (superficially porous) offer unique advantages because very large pores can be made without sacrificing particle strength. 

  • High physical strength and the short diffusion path of the thinner superficially porous layer combine to allow high peak performance to be maintained at higher linear velocities, even for large molecules.

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.



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