Comparison of ultrahigh- and low-pressure packing methods for preparation of 50 cm long capillary UHPLC columns.

Interdependent variables including pressure, slurry concentration, and slurry solvent contribute to producing high-efficiency liquid chromatography columns by slurry packing. Most commonly, ultrahigh-pressure (over 2000 bar) slurry packing methods have been observed to generate the highest performing column packed beds. However, low-pressure packing (<200 bar) methods are also commonly used but have not been examined in depth or compared to ultrahigh-pressure packed columns for kinetic performance. In this work, we compare methods that employed low- (100 bar) or ultrahigh-pressures (2070 bar) to slurry pack 50 cm long × 75 µm inner diameter (i.d.) capillaries with 1.7 µm diameter ethylene-bridged hybrid (BEH) C18 particles. Both packing methods utilized a high slurry concentration, with low-pressure packing using a settled slurry (3 mm tall layer of particles in the bottom of a tube with slow stirring) and ultrahigh-pressure packing using a suspended (fast stirring to prevent settling) 200 mg/mL slurry. Columns were evaluated with isocratic separations of small molecules and gradient separations of a peptide standard mixture. Low-pressure packed columns saw an improvement in performance when sonication was used during packing. Low-pressure packed columns achieved a minimum reduced plate height as low as 1.18 and a peak capacity of 534 in <2 h, thus generating comparable separation performance to columns packed at ultrahigh-pressures. Similar column-to-column and run-to-run performance repeatability was also observed for both packing methods. The low-pressure packing technique provides a less technically demanding and more cost-effective approach for preparing high-efficiency capillary columns.