Measurement of optimal flow rate in gradient elution liquid chromatography

Method development in gradient LC relies upon the selection of a solvent time program and a mobile phase flow rate. The flow rate, optimal for gradient separation cannot be inherently predicted by the isocratic value optimal for a given analyte, and rather should be identified independently to ensure the highest separation performance of gradient analysis. The optimal flow rate (Fopt) is defined herein as the solvent volumetric flow rate (F) maximizing the separation (Δs) of a predetermined peak-pair or the separation capacity (sc) of the entire LC analysis. The theoretical background and the experimental technique of measurement of Fopt in gradient elution analysis were considered and experimentally demonstrated. The technique of measuring Fopt is based on translatable changes of F where the product FtG (tG is the gradient time) was the same for all values of F. The Fopt was found as F corresponding to the maximum in Δs or in sc.