Effect of bed compression on high-performance liquid chromatography columns with gigaporous polymeric packings

The behavior of chromatographic columns packed with gigaporous, highly cross-linked styrenic particles was investigated for use in protein separation by reversed-phase chromatography at high flow velocities. Stainless-steel columns which were 3.5 or 7.5 cm long and had an inner diameter of 0.46 mm were slurry packed with 8 or 20 mm diameter spherical particles of 4000 A mean pore size by using methanol as the packing fluid. It was found that the conditions employed during the packing process have a dramatic effect on the properties of such columns and that this can be attributed in part to the deformability of the particles. An increase in the packing pressure to approximately 6000 p.s.i. (41 MPa) resulted in a higher mass-transfer efficiency for the column with a concomitant decrease in permeability. This is ascribed to a decrease in the interstitial porosity with increasing packing pressure since the experimentally measured plate heights for these columns were found to agree quantitatively with theoretical predictions that relate changes in the interstitial porosity to intraparticle mass transfer. However, the theoretically derived relationship between porosity, permeability, and efficiency does not hold for columns packed at pressures higher than 6000 p.s.i., in which case the total column porosity was found to be high while the permeability and column efficiency were low. This behavior is explained by the formation of a low-porosity layer of highly compressed particles at the downstream end of the column during high pressure packing so that the assumption of axially uniform column properties used in the theoretical approach leads to very large errors.