Q: What is the relationship between pore size and particle size for an HPLC packing material?
A: The short answer is that these two properties of an HPLC packing are not related. Most reversed-phase HPLC columns are based on a stationary phase bonded to silica particles. These particles form the basis of the packing material. The most popular particle sizes are 5-, 3.5- and 3-μm particle diameters. Particles of <3-μm are becoming increasingly popular because of their increased column efficiency and relative independence of column efficiency on flow rate.
The pore size of HPLC particles can vary widely from product to product, but should be consistent within a particular product line of columns. There are two general categories of pore sizes. Small-pore particles have pores ranging from about 6-15 nm (60-150 Å), with the majority in the 8-12-nm range. Packings based on these particles generally are used for 'small molecule' separations, where the sample molecular weight is <≈1000 Da. Large-pore particles have pores of ≥ 30 nm (300 Å) and tend to be used more for large molecule separations, such as proteins.
Although it is convenient to draw HPLC packing materials to appear as tennis balls, with the bonded phase on the surface of a solid sphere, this is a poor representation. Silica is better represented as a rigid sponge, with >99% of the surface area inside the particle. The surface area of such particles is inversely proportional to the pore size, so large-pore columns have less surface area than small-pore ones.
As you can imagine, the chemistry and technology of silica particle synthesis are closely guarded secrets of the silica manufacturers. There are only a dozen or so manufacturers of silica, whereas there are many more companies that add the bonded phase to the silica particles and pack columns. There are several ways to make silica, but most start with a synthetic silica polymer formed from tetraethoxysilane or similar starting material. In one technique, the sol-gel procedure, a silica emulsion is converted into spherical beads that are dried into silica particles. Pore size and particle size are controlled by the pH, temperature and concentration of the silica sol used during particle preparation.
An alternate way to make silica particles is to make microparticles and aggregate them into larger particles, as illustrated in Figure 1. The pores result from the spaces between the microparticles, so larger microparticles make larger pores and vice versa.
As a rule of thumb, for easy access to the pores the pore size should be at least three times the hydrodynamic diameter of the molecule. The concept of hydrodynamic diameter is shown in Figure 2, where two molecules of equal molecular weight are shown; one is compact and one is rod-like. The hydrodynamic diameter is the diameter of the volume formed if the molecule is rotated in all directions, forming a spherical volume. For small molecules (<1000 Da), any of the small-pore columns will work. But for larger molecules, pore sizes of <15 nm may restrict entry, so larger-pore columns are selected.
For a given manufacturer’s product, the pore size will be fairly consistent. For example, manufacturer A may have particles of 8 nm pore size for one product line and 12 nm for another product line, whereas manufacturer B may use particles of 10 nm pore size for all its products. Rarely is the pore size a factor when choosing between different small-pore products.
The surface area of a particle is inversely proportional to the pore diameter, so a 5-μm particle size, 10-nm pore column will have approximately three times the surface area as a 5-μm, 30-nm pore column. Because retention is directly related to surface area, use of large-pore columns usually is not desirable when small-pore columns can be used. However, when an application results in retention times that are too large, even with high concentrations of organic solvent, a switch to a large-pore column can be one way to reduce retention.
In summary, the pore size of a column is selected so that the sample molecules have easy access to the pores. Smaller-pore columns are desired because of their higher surface area, as long as the analytes are sufficiently small to easily enter the pores. The particle size controls column efficiency or plate number. The pore size controls the surface area. Retention is controlled primarily by the surface area, bonded phase chemistry, mobile phase chemistry and column temperature.
This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his ongoing research with Lloyd Snyder, resulting in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com