Pore Size Selection of HPLC Columns and Its Influence on Analytical Results
Introduction
In HPLC, the pore size of a column refers to the average size of the pores or cavities in the packing particles, ranging from 100 to 300 Å. In porous packing materials, more than 95% of interactions between the stationary phase and target analytes occur inside these particles (that is, within the pores). Therefore, pore size is one of the most important parameters of a chromatographic packing material.
Both the pore size value and its distribution of a packing material play a critical role in chromatographic separation and purification:
- Large-pore packing materials extend the residence time of macromolecular solutes on the surface of the stationary phase, achieving adequate separation and improving peak shape. Therefore, large-pore packing materials are suitable for separating macromolecular compounds or molecules with large hydrodynamic volumes.
- Small-pore packing materials provide higher specific surface area, supporting larger sample loading capacity and retaining small organic compounds. Therefore, small-pore packing materials are suitable across small organic molecules to polymers.
Selecting Pore Sizes Based on Molecular Weight
The choice of column pore size is determined by molecular weight.
An analyte molecule can only enter the interior of a porous packing particle only when its molecular diameter is smaller than the pore diameter; moreover, to minimize the impact on analytical performance, the pore diameter should be at least three times the molecule diameter. In general, the selected column pore size is often more than four times the hydrodynamic diameter of the sample.
Therefore, in theory, compounds with molecular weights below 10,000 should be analyzed using a 120 Å column; compounds with molecular weights greater than 10,000 and less than 200,000 should be analyzed using a 300 Å column; and compounds with molecular weights greater than 200,000 require a gel column.
In practice, the selection is slightly different. If the molecular weight of a compound is below 2,000, a column with pore size between 80 Å and 180 Å are selected; If above, a 300 Å column is selected. Fully porous 300 Å columns are particularly suitable for the separation of proteins and peptides.
What Happens When the Pore Size Is Too Small?
When the molecular size of an analyte is too large for the pores of the packing material, separation performance can deteriorate significantly.
For example, when a 120 Å packing is used to separate a large molecule, the resolution can be poor, since good resolution on a reversed-phase column requires the analyte being able to enter the pores of the packing particles and interact with the bonded chains on the internal pore surfaces. When the analyte is too large to enter the pores efficiently, the effective interaction between the analyte and the stationary phase is reduced, resulting in bad retention, poor resolution and a negative impact on peak shape.
Application Example: Lactoferrin
A customer of Welch Materials uses an Xtimate C4, 120 Å column to analyze lactoferrin, obtaining the following chromatogram. Severe peak splitting and unsatisfactory peak shape can be easily observed.
Since lactoferrin has a molecular weight ranging from 75,000 to 80,000, analysts have switched to an Ultisil XB-C4, 300 Å column, obtaining the following chromatogram. The peak shape is considerably improved.
To help with future customers, Xtimate C4, 300 Å columns were later developed in early 2024 following this application case. Compared to Ultisil XB-C4, 300 Å, Xtimate C4, 300 Å has a pH range of 1.0-12.5, offering excellent stability for high pH applications. Xtimate C4 is also better suited for MS and ELSD detectors.
Conclusion
Pore size is an important parameter that can significantly affect HPLC analytical results. Selecting an appropriate pore size according to the molecular weight of the analyte is essential for achieving optimal chromatographic resolution and peak symmetry.