Effect of the packing pressure on the performance of C-18 reversed-phase liquid chromatographic columns

Early evidence has shown that HPLC columns packed with a given ODS phase give different performance, related to differences in their packing densities and external porosities. This effect was systematically studied on two groups of ten analytical (10X0.46 cm) columns packed with Kromasil and Zorbax 10 mu m spherical C-18, respectively, using chloroform as the slurry solvent and methanol as the pushing solvent. The packing pressure used for column j in each group was 1000j p.s.i. The mass of packing material inside each column increased with increasing packing pressure. The column external porosity, determined by inverse size-exclusion chromatography method, decreased as the packing pressure increased, while the internal porosity remained constant. With a methanol-water (45:55, v/v) solution as the mobile phase, the retention factors and column efficiencies were measured at infinite dilution for acetone, benzyl acetate, benzyl alcohol, m-cresol, 2,6-dimethyl phenol, methyl benzoate, 3-phenyl-1-propanol, and uracil (also used as non-retained compound to determine the hold-up times). The retention factor and the column efficiency of each compound increased linearly with increasing packing pressure. The isotherm of 5-phenyl-1-propanol was determined on each column by elution by characteristic points and frontal analysis methods. These data were normalized by the column geometrical volume (V-k: ml), by the stationary phase volume (V-p: ml), and by the mass of packing (W-p: g). The parameters obtained from the isotherm normalized by V-k cannot predict the band profile accurately. Better results were obtained with the parameters obtained from the isotherm normalized by V-p. Close agreement was obtained between the parameters obtained by mass normalization. This allowed a reasonable prediction of the band profiles recorded on one column using isotherm data measured on another column. Considering the difficulties encountered in volume determinations, normalization of isotherm data by the packing weight seems to be the most practical solution.