Assessing effects of ultra-high-pressure liquid chromatography instrument configuration on dispersion, system...

This study involves the systematic assessment of the effects of system configuration on dispersion, pressure, and retention characteristics while operating a 1500 bar UHPLC system with 2.1 mm i.d. × 100 mm long columns packed with 1.5 µm core-shell particles in isocratic and gradient mode. Altering the system configuration by changing the i.d. of connection tubing and flow cells affects the elution time, dispersion characteristics, and the kinetic performance limits of the system. The gain in separation efficiency when decreasing tubing i.d. from 100 to 75 µm was found to contribute more to the decrease in separation impedance and the position of the kinetic performance curve than the loss in available column pressure induced by the narrower tubing. When applying steep gradients, characterized by gradient-to-column dead-time ratio < 7, optimizing instrument configuration leads to either a significant time gain factor of 3.9 without compromising peak capacity, or a gain in peak capacity with a gain factor of 1.3 while maintaining the analysis time constant. Due to the reduced fluidic volume of connection tubing of smaller i.d., a decrease in residence time is obtained. At the same time, an increase in k was observed due to a pressure-induced retention effect, and this effect is significant for late-eluting analytes.