Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, December 20, 2014

HPLC Column PORE SIZE (or Pore Diameter) and Retention Time

Think of your typical porous bare silica support as a big sponge full of holes. All of those holes (pores) are where the sample will migrate through before emerging out the other side. With conventional chromatography supports, most of the interaction takes place inside the particle, not on the surface. The size and number of these openings relate to retention time. Besides particle size (particle diameter), pore size is one of the most important characteristics of silica based chromatography supports.

The pore size or pore diameter is often expressed in Angstroms (i.e. 80 A = 8 nm). The degree of porosity relates to the hydrodynamic volume of your sample and is inversely related to the surface area of the support. The larger the surface area of the support (smaller pore size), the longer the possible retention of the sample. For small drug molecule samples under 1,000 daltons (an estimate only) we often use high surface area supports with small pore sizes between 60 and 150 Angstroms (~ 200 to 500 square meters per gram). These provide high retention characteristics useful in separating apart many small compounds in one analysis run. For larger molecules (i.e. peptides and proteins), we employ supports with larger pore sizes (~300 Angstroms). Particles with small pores have larger surface areas which can provide more interaction with the sample. Note: Pore size is often determined using the BET Nitrogen adsorption/desorption equation. Due to endcapping of the support (e.g. C8 or C18), the actual value obtained is often 20-30% less than the original value.

When comparing bare silica columns or trying to identify similar conventional columns for use in a method, pore size must be considered. Manufacturer's publish the pore size in Angstroms (*sometimes in nm) for their different supports. Choosing columns with similar pore sizes is just one of many parameters needed to provide similar retention characteristics.