Analysis of packing microstructure and wall effects in a narrow-bore ultrahigh pressure liquid chromatography column using focused ion-beam scanning electron microscopy

- Packing microstructure in a 2.1 mm inner-diameter UHPLC column is reconstructed.
- Two wall effects, a geometrical and a frictional, are identified and analyzed.
- The region affected by the wall effects extends by ∼125 μm (∼62 dp).
- Flow simulations imply a significant velocity heterogeneity due to the wall effects.
- Efficiency gains of 0.5-1.0 hmin are envisioned by minimization of wall effects.

Column wall effects are well recognized as major limiting factor in achieving high separation efficiency in HPLC. This is especially important for modern analytical columns packed with small particles, where wall effects dominate the band broadening. Detailed knowledge about the packing microstructure of packed analytical columns has so far not been acquired. Here, we present the first three-dimensional reconstruction protocol for these columns utilizing focused ion-beam scanning electron microscopy (FIB-SEM) on a commercial 2.1 mm inner diameter × 50 mm length narrow-bore analytical column packed with 1.7 μm bridged-ethyl hybrid silica particles. Two sections from the packed bed are chosen for reconstruction by FIB-SEM: one from the bulk packing region of the column and one from its critical wall region. This allows quantification of structural differences between the wall region and the center of the bed due to effects induced by the hard, confining column wall. Consequences of these effects on local flow velocity in the column are analyzed with flow simulations utilizing the lattice-Boltzmann method. The reconstructions of the bed structures reveal significant structural differences in the wall region (extending radially over approximately 62 particle diameters) compared to the center of the column. It includes the local reduction of the external porosity by up to 10% and an increase of the mean particle diameter by up to 3%, resulting in a decrease of the local flow velocity by up to 23%. In addition, four (more ordered) layers of particles in the direct vicinity of the column wall induce local velocity fluctuations by up to a factor of three regarding the involved velocity amplitudes. These observations highlight the impact of radial variations in packing microstructure on band migration and column performance. This knowledge on morphological peculiarities of column wall effects helps guiding us towards further optimization of the packing process for analytical HPLC columns.