Extending the limits of operating pressure of narrow-bore column liquid chromatography instrumentation

• A complete liquid chromatography set-up is described for pressures up to 2600 bar.
• System performance and repeatability is as good as for commercial systems.
• 2.1 mm ID columns are investigated at these challenging conditions.
• Temperature profiles are monitored while using intercooling at these pressures.
• Influence of pressure and temperature is studied on retention.

The increase of the operating pressure in Liquid Chromatography, has been one of the crucial steps toward faster and more efficient separations. In the present contribution, it was investigated if the pressure limits for narrow-bore columns (2.1 mm ID) could be increased beyond those of commercially available (1300 bar) instrumentation without performance loss. Whereas previous studies applying pressures higher than 2000 bar were limited to the use of columns with a diameter smaller or equal to 1 mm, it is a difficult feat to expand this to 2.1 mm ID given that viscous-heating effects increase according to the fifth power of the column radius. A prototype LC set-up was realized, allowing to operate at pressures up to 2600 bar (260 MPa) for large separation volumes (>5 mL). The performance of an in-house-built injector was compared at 800 bar to commercially available injectors, yielding equal performance but twice the maximum pressure rating. The performance of (coupled) custom columns packed with fully porous and superficially porous particles were assessed at ultra-high-pressure conditions. Increasing the inlet pressure from 800 to 2400 bar and scaling the column length proportionally (from 150 mm to 450 mm), resulted in the theoretically expected linear increase in plate count from 20,000 to 59,000. A maximum plate number of 81,000 was realized using a 600 mm long (coupled) column at 2600 bar. Viscous-heating effects were diminished by insulating coupled columns and applying an intermediate-cooling strategy in a forced-air oven.