Exploring the speed-resolution limits of supercritical fluid chromatography at ultra-high pressures.

The limits of supercritical fluid chromatography have been experimentally explored using inlet pressures at the limits of the current commercial instrumentation (400-600 bar), as well as pressures significantly surpassing this (up to 1050 bar). It was found that efficiencies in the range of 200,000 theoretical plates can be achieved for a void time t0 of roughly 6min using superficially porous particles (2.7 and 4.6µm) while remaining within the pressure limits of current commercial instrumentation and columns. If lower efficiencies are sufficient (<100, 000 plates), smaller particles (e.g. 1.8µm) provide the best trade-off between analysis time and efficiency. Apparent efficiencies of 83,000 (k'=2.2) to 76,000 (k'=6.6) plates could be achieved for void times around 1min by pushing the pressure limits up to 1050 bar on a column length of 500mm. As the optimal mobile phase velocity for these small particle columns is even higher, it is required to use narrow-bore columns (2.1mm ID) to remain within the instrument limits of flow rate. The smaller column volume however puts a strain on the separation efficiency due to extra-column band broadening, resulting in losses up to 50% for weakly retained compounds for column lengths below 250mm. It is also illustrated that when using sub-2µm particles, especially for separations where a significant amount of organic modifier is required, the current pressure limits of state-of-the-art instrumentation can sometimes be insufficient. For a gradient running from 4 to 40 v% methanol on a 300mm column at the optimal flow rate the pressure increases from 420 to 810 bar. Operating SFC-columns with a large pressure gradient induces several (undesired) side effects which have been investigated as well. It has been found that, since the viscosity increases strongly with pressure in SFC, the optimal flow rate and the minimal plate height can significantly change when the column length is changed. Whereas e.g. a 3×150mm column (2.7µm particles) has an optimal flow rate of 1.5ml/min and minimal plate height of 5.66µm, a 3×1050mm column has an optimal flow rate of 1.2ml/min and a minimal plate height of 6.25µm. Nevertheless, an increase in operating pressure drop in SFC results in a significant gain in kinetic performance.

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

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 (2.1mm ID) could be increased beyond those of commercially available (1300bar) instrumentation without performance loss. Whereas previous studies applying pressures higher than 2000bar were limited to the use of columns with a diameter smaller or equal to 1mm, it is a difficult feat to expand this to 2.1mm 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 2600bar (260MPa) for large separation volumes (>5mL). The performance of an in-house-built injector was compared at 800bar 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 2400bar and scaling the column length proportionally (from 150mm to 450mm), 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 600mm long (coupled) column at 2600bar. Viscous-heating effects were diminished by insulating coupled columns and applying an intermediate-cooling strategy in a forced-air oven.