Potential advantage of constant pressure versus constant flow gradient chromatography for the analysis of small molecules

A recent model designed to predict the variation of the flow rate with time in constant pressure (cP) gradient chromatography was validated from an experimental viewpoint for non-retained gradients (methanol–water), incompressible eluent (P < 250 bar), and in absence of pressure effects on the analyte retention pattern (small molecules). Experimental data confirmed that cP and constant flow (cF) gradients are strictly equivalent if the analysis time is kept constant. The same model was also used to predict the gradient kinetic performance of cP versus cF gradients when the constraint was the maximum inlet pressure at which the column and/or the HPLC system can safely be run. For linear volume gradients of methanol in water (5–95% in volume) and a maximum pressure of 250 bar, the same peak capacity as that in cF mode is predicted in cP mode. Also, a reduction of the analysis time by 17.3% was expected. These theoretical results were confirmed by separating a real mixture of about twenty small molecules on either one or two 4.6 mm × 150 mm columns packed with 3.5 μm Bridge Ethylene Hybrid (BEH) C18 particles and run at flow rates smaller than 0.8 mL/min and at a maximum inlet pressure of 250 bar. The experimental gain in analysis time was 17.6% (1 column) and 20.1% (2 columns in series) for a virtually insignificant loss of peak capacity (−4%).