Fundamental and Practical Insights on the Packing of Modern High-Efficiency Analytical and Capillary Columns

Abstract

New stationary phases are continuously developed for achieving higher efficiencies and unique selectivities. The performance of any new phase can only be assessed when the columns are effectively packed under high pressure to achieve a stable bed. The science of packing columns with stationary phases is one of the most crucial steps to achieve consistent and reproducible high-resolution separations. A poorly packed column can produce non-Gaussian peak shapes and lower detection sensitivities. Given the ever larger number of stationary phases, it is impossible to arrive at a single successful approach. The column packing process can be treated as science whose unified principles remain true regardless of the stationary phase chemistry. Phenomenologically, the column packing process can be considered as a constant pressure or constant flow high-pressure filtration of a suspension inside a column with a frit at the end. This process is dependent on the non-Newtonian suspension rheology of the slurry in which the particles are dispersed. This perspective lays out the basic principles and presents examples for researchers engaged in stationary phase development. This perspective provides an extensive set of slurry solvents, hardware designs, and a flowchart, a logical approach to optimal column packing, thus eliminating the trial and error approach commonly practiced today. In general, nonaggregating but high slurry concentrations of stationary phases tend to produce well packed analytical columns with small particles. Conversely, C18 packed capillary columns are best packed using agglomerating solvents.

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].…”

“…The rotational speed of the magnet is kept to a minimum speed that is sufficient for cupola destabilization (400-500 RPM). High rotation speed leads to excessive sorbent grinding by a magnet bar [21] and resuspends the settled sorbent layer reducing the effective concentration and packing rate. When the destabilization works properly, the concentrated sorbent suspension moving inside the column can be followed with the naked eye as dense regions occasionally interrupted by transparent gaps (see Supplementary Fig.…”

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).…”

“…The rotational speed of the magnet is kept to a minimum speed that is sufficient for cupola destabilization (400 -500 rpm). High rotation speed leads to excessive sorbent grinding by a magnet bar (21) and resuspends the settled sorbent layer, reducing the effective concentration and packing rate. When the destabilization works properly, the concentrated sorbent suspension moving inside the column can be followed with the naked eye as dense regions occasionally interrupted by transparent gaps (see supplemental Fig.…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…Various improvements such as smaller particle sizes and longer columns have been developed to increase the peak capacity for deeper proteome analysis. Smaller packing particles provide more uniform packing structure in columns, which improves peak symmetry and separation resolution . However, as particle size decreases, the pressure required to provide sufficient flow increases dramatically.…”