Axial heterogeneities in capillary ultrahigh pressure liquid chromatography columns: Chromatographic and bed morphological characterization.

We study axial heterogeneities in capillary ultrahigh pressure liquid chromatography (UHPLC) columns through kinetic performance and bed morphological analysis. Two columns are used in this work, a 75 µm i.d. × 100 cm column packed with 1.3 µm C18-silica particles and a 75 µm i.d. × 45 cm column packed with 1.9 µm C18-silica particles. The long column is chromatographically characterized and is afterwards sectioned into three segments, each analyzed individually. The column packed with the 1.9 µm particles is subjected to a bed morphological analysis using confocal laser scanning microscopy near the inlet, center, and outlet of the column. Chromatographic and morphological characterizations reveal highest separation efficiency and most homogeneous bed microstructure towards the column outlet. Kinetic performance data for inlet and central packing segments indicate enhanced contributions from wall effects to a transcolumn flow heterogeneity. Bed morphological data reveal systematic changes in geometrical and frictional wall effects along the bed: from inlet to outlet, bed morphologies increasingly reflect packing microstructures associated with concentrated slurries. Variations in separation efficiency and bed morphology can be related to the constant-pressure packing mode; the decrease in packing rate along the bed leaves fewer chances for particle rearrangement and bed consolidation from inlet to outlet. It explains the relatively uniform bed morphology towards the outlet and also the relatively loose wall region near the inlet. Bed microstructural features are discussed in a context of previous observations made in the characterization of capillary UHPLC columns.

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

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 2.1mm inner diameter×50mm length narrow-bore analytical column packed with 1.7µm 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.

Bed morphological features associated with an optimal slurry concentration for reproducible preparation of efficient capillary ultrahigh pressure liquid chromatography columns.

Column wall effects and the formation of larger voids in the bed during column packing are factors limiting the achievement of highly efficient columns. Systematic variation of packing conditions, combined with three-dimensional bed reconstruction and detailed morphological analysis of column beds, provide valuable insights into the packing process. Here, we study a set of sixteen 75µm i.d. fused-silica capillary columns packed with 1.9µm, C18-modified, bridged-ethyl hybrid silica particles slurried in acetone to concentrations ranging from 5 to 200mg/mL. Bed reconstructions for three of these columns (representing low, optimal, and high slurry concentrations), based on confocal laser scanning microscopy, reveal morphological features associated with the implemented slurry concentration, that lead to differences in column efficiency. At a low slurry concentration, the bed microstructure includes systematic radial heterogeneities such as particle size-segregation and local deviations from bulk packing density near the wall. These effects are suppressed (or at least reduced) with higher slurry concentrations. Concomitantly, larger voids (relative to the mean particle diameter) begin to form in the packing and increase in size and number with the slurry concentration. The most efficient columns are packed at slurry concentrations that balance these counteracting effects. Videos are taken at low and high slurry concentration to elucidate the bed formation process. At low slurry concentrations, particles arrive and settle individually, allowing for rearrangements. At high slurry concentrations, they arrive and pack as large patches (reflecting particle aggregation in the slurry). These processes are discussed with respect to column packing, chromatographic performance, and bed microstructure to help reinforce general trends previously described. Conclusions based on this comprehensive analysis guide us towards further improvement of the packing process.

Implementation of high slurry concentration and sonication to pack high-efficiency, meter-long capillary ultrahigh pressure liquid chromatography columns.

Slurry packing capillary columns for ultrahigh pressure liquid chromatography is complicated by many interdependent experimental variables. Previous results have suggested that combination of high slurry concentration and sonication during packing would create homogeneous bed microstructures and yield highly efficient capillary columns. Herein, the effect of sonication while packing very high slurry concentrations is presented. A series of six, 1m×75µm internal diameter columns were packed with 200mg/mL slurries of 2.02µm bridged-ethyl hybrid silica particles. Three of the columns underwent sonication during packing and yielded highly efficient separations with reduced plate heights as low as 1.05.

Larger voids in mechanically stable, loose packings of 1.3µm frictional, cohesive particles: Their reconstruction, statistical analysis, and impact on separation efficiency.

Lateral transcolumn heterogeneities and the presence of larger voids in a packing (comparable to the particle size) can limit the preparation of efficient chromatographic columns. Optimizing and understanding the packing process provides keys to better packing structures and column performance. Here, we investigate the slurry-packing process for a set of capillary columns packed with C18-modified, 1.3µm bridged-ethyl hybrid porous silica particles. The slurry concentration used for packing 75µm i.d. fused-silica capillaries was increased gradually from 5 to 50mg/mL. An intermediate concentration (20mg/mL) resulted in the best separation efficiency. Three capillaries from the set representing low, intermediate, and high slurry concentrations were further used for three-dimensional bed reconstruction by confocal laser scanning microscopy and morphological analysis of the bed structure. Previous studies suggest increased slurry concentrations will result in higher column efficiency due to the suppression of transcolumn bed heterogeneities, but only up to a critical concentration. Too concentrated slurries favour the formation of larger packing voids (reaching the size of the average particle diameter). Especially large voids, which can accommodate particles from>90% of the particle size distribution, are responsible for a decrease in column efficiency at high slurry concentrations. Our work illuminates the increasing difficulty of achieving high bed densities with small, frictional, cohesive particles. As particle size decreases interparticle forces become increasingly important and hinder the ease of particle sliding during column packing. While an optimal slurry concentration is identified with respect to bed morphology and separation efficiency under conditions in this work, our results suggest adjustments of this concentration are required with regard to particle size, surface roughness, column dimensions, slurry liquid, and external effects utilized during the packing process (pressure protocol, ultrasound, electric fields).