Maximizing peak capacity and separation speed in liquid chromatography

The practical effects of gradient time and flow rate on the peak capacities of a range of analytes of differing molecular weights (MWs) and physico-chemical properties have been evaluated using ultra high pressure LC instrumentation with sub-2 mum and superficially porous particle phases. Optimum peak capacity, in RP gradient LC, for small molecules, including typical pharmaceutical drugs and peptides with MWs up to 1300, was demonstrated at a maximum flow rate for a given gradient time (i.e. up to 40 min). Flow rates significantly higher than the optimum in the van Deemter plots and also higher than those typically employed by the majority of the chromatographers today are recommended for gradient LC (i.e. up to 1.0 mL/min on 50-150x2.1 mm 1.7 mum columns). This recommendation is applicable for temperatures above 40 degrees C, i.e. temperatures typically utilized for separations employing sub-2 mum particles to reduce column back pressure. Van Deemter and pseudo van Deemter plots were determined and combined with chromatographic gradient elution theory to explain our unexpected observations. The derived models exhibited good agreement between experimental and predicted peak capacities (absolute average error 4%, max. error 12%).