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 (F) is defined herein as the solvent volumetric flow rate (F) maximizing the separation (Δs) of a predetermined peak-pair or the separation capacity (s) of the entire LC analysis. The theoretical background and the experimental technique of measurement of F in gradient elution analysis were considered and experimentally demonstrated. The technique of measuring F is based on translatable changes of F where the product Ft (t is the gradient time) was the same for all values of F. The F was found as F corresponding to the maximum in Δs or in s.