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