On the feasibility to conduct gradient liquid chromatography separations in narrow-bore columns at pressures up to 2000 bar

- A mobile phase delivery system for gradient elution up to 2000 bar is given.
- The influence of operating conditions on the delivered gradient is studied.
- The system allows sample injection with comparable performance as commercial ones.
- Gradient elution separations were performed up to 2000 bar.
- Peak capacity scaled with the square root of the column length.

The limits in operating pressures are extended for narrow-bore columns in gradient elution up to 2000 bar. As the required pumps for these pressures are incompatible with common chromatographic solvents and are not suitable to apply a mobile phase composition gradient, a mobile phase delivery and injection system is described and experimentally validated which allows to use any possible chromatographic solvent in isocratic and gradient elution. The mobile phase delivery and injection system also allows to perform multiple separations without the need to depressurize the column. This system consists out of 5 dual on/off valves and two large volume loops in which the gradient and equilibration volume of initial mobile phase are loaded by a commercial liquid chromatography pump. The loops are then flushed toward the column at extreme pressures. The mobile phase delivery and injection system is first evaluated in isocratic elution and shows a comparable performance to a state-of-the-art commercial flow-through-needle injector but with twice the pressure rating. Distortion of the loaded gradient by dispersion in the gradient storage loop is studied. The effect of the most important parameters (such as flow rate, pressure and gradient steepness) is experimentally investigated. Different gradient steepnesses and volumes can be applied at different flow rates and operating pressures with a good repeatability. Due to the isobaric operation of the pumps, the gradient is monitored in real-time by a mass flow meter installed at the detector outlet. The chromatograms are then converted from time to volume-base. A separation of a 19-compound sample is performed on a 300 × 2.1 mm column at 1000 bar and on a 600 × 2.1 mm column at 2000 bar. The peak capacity was found to increase from 141 to 199 and thus scales with L as is predicted by theory. This allows to conclude that the inlet pressure for narrow-bore columns in gradient elution can be increased up to 2000 bar without fundamental pressure-induced limitations.