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

Abstract

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 75 µm i.d. fused-silica capillary columns packed with 1.9 µm, C18-modified, bridged-ethyl hybrid silica particles slurried in acetone to concentrations ranging from 5 to 200 mg/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.

FlashPack: Fast and simple preparation of ultra-high performance capillary columns for LC-MS

“…The quantitative proteomics data obtained using FlashPack columns was in accordance with previously published results [22]. Our results reconfirmed that column packing from high sorbent slurry concentrations did not negatively affect chromatographic resolution [12,21,23].…”

FlashPack: Fast and Simple Preparation of Ultrahigh-performance Capillary Columns for LC-MS*

“…The quantitative proteomics data obtained using FlashPack columns were in accordance with previously published results (22). Our results reconfirmed that column packing from high sorbent slurry concentrations did not negatively affect chromatographic resolution (12,21,23).…”

Single-Molecule Dynamics Reflect IgG Conformational Changes Associated with Ion-Exchange Chromatography

“…Factors such as slurry solvent and concentration, packing density, and packing method can significantly affect the quality of a separation. Better efficiency, for example, can be achieved by using higher slurry concentrations resulting in suppressing particle size segregation inside the column . Conversely, there are no special techniques and optimization procedures used to pack the present flow chamber.…”

“…Better efficiency, for example, can be achieved by using higher slurry concentrations resulting in suppressing particle size segregation inside the column. 70 Conversely, there are no special techniques and optimization procedures used to pack the present flow chamber.…”