Characterization of radial and axial heterogeneities of chromatographic columns by flow reversal

The impact of the column length (5, 10, and 15 cm) and packing mode (constant pressure and constant flow rate up to 15,000 psi) on the radial and axial heterogeneities of 3.0 mm i.d. research prototype columns packed with the same batch of 2.4 μm BEH-C18 particles was investigated by the flow reversal technique. The data were gathered for a non-retained marker (uracil, acetonitrile/water eluent mixture, 80/20, v/v, flow rate 0.5 mL/min, T = 297 K) and revealed that the radial heterogeneity of the packed bed, characterized by the center-to-wall relative velocity bias (ωβ) and its length scale, is nearly independent on the packing mode: the velocity biases extend over a same length scale estimated at 154 μm while ωβ is in between 4% and 6% for all columns. Secondly, the data revealed that the column length has a slight impact on ωβ: assuming a two-region (wall and center regions) stochastic model of transcolumn eddy dispersion, ωβ increases from 4.6% to 5.1% and to 6.1% for 5, 10, and 15 cm long columns, respectively, when packed at constant flow rate. For columns packed at constant pressure, ωβ increases from 5.0% to 5.2% and to 5.6%, respectively. Finally, it is found that all columns are axially heterogeneous: the bottom half, which is packed first (column inlet), is slightly more uniform than the top half (column outlet) which is packed last. Overall, the results of the flow reversal experiments corroborate recent observations (130 μm thick wall region and ωβ = 5.0%) based on flow simulations in a focused-ion-beam scanning electron microscopy (FIB-SEM) based 3D reconstruction from a 2.1 mm × 50 mm column packed with 2 μm BEH-C18 particles.