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

• Speed-resolution limits of SFC are investigated for inlet pressures up to 1033 bar.
• 2.7 μm superficially porous particles provide very high efficiencies (200,000 plates)
• 1.8 μm fully porous particles provide fast and efficient separations (85,000 plates).
• Coupling columns results in a decrease in optimal flow rate and plate count.
• When using high modifier content, increased inlet pressures may be beneficial.

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 6 min using superficially porous particles (2.7 and 4.6 μm) while remaining within the pressure limits of current commercial instrumentation and columns. If lower efficiencies are sufficient (<100, 000 plates), smaller particles (e.g. 1.8 μm) 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 1 min by pushing the pressure limits up to 1050 bar on a column length of 500 mm. As the optimal mobile phase velocity for these small particle columns is even higher, it is required to use narrow-bore columns (2.1 mm 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 250 mm. It is also illustrated that when using sub-2 μm 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%v% methanol on a 300 mm 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 × 150 mm column (2.7 μm particles) has an optimal flow rate of 1.5 ml/min and minimal plate height of 5.66 μm, a 3 × 1050 mm column has an optimal flow rate of 1.2 ml/min and a minimal plate height of 6.25 μm. Nevertheless, an increase in operating pressure drop in SFC results in a significant gain in kinetic performance.