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. All of those holes are where the sample will migrate through before emerging out the other side. The size and number of those holes relate to retention time. Besides particle size (diameter), pore size is one of the most important characteristics of silica based chromatography supports.

The pore size or pore diameter (often expressed in Angstroms and which relates to the hydrodynamic volume of your sample) is inversely related to the surface area of the support. The larger the surface area of the support (smaller pore size), the longer the retention of the sample. For small drug molecule samples under 1,000 daltons (an estimate only) we often use high surface area supports (small particle sizes) with pore sizes between 60 and 120 Angstroms (~ 500 to 300 square meters per gram). These provide high retention characteristics useful in separating apart many compounds in one run. For larger molecules we employ supports with larger pore sizes (~300 Angstroms). Described in a different way, smaller particle sizes have larger surface areas with longer retention times. 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 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 will often provide similar retention characteristics. Accurate column void volumes are needed to calculate many standard chromatography performance calculations, but they are also critical for determining if you have any retention at all. T zero and the (k') K prime, capacity or retention factors, all rely on accurate column void volume values. It is one of the first calculations you must make BEFORE developing or running any HPLC method [link to more info:]. Once determined, you must follow up by injecting an unretained sample into the column to determine the actual value.