Separation Optimization of a Mixture of Ionized and Non-Ionized Solutes under Isocratic and Gradient Conditions in Reversed-Phase HPLC by Means of Microsoft Excel Spreadsheets

Abstract

Τhe crucial role of mobile phase pH for optimizing the separation of a mixture of ionized and non-ionized compounds on a Phenomenex extended pH-range reversed-phase column (Kinetex 5 µm EVO C18) was examined. A previously developed Excel-spreadsheet-based software was used for the whole separation optimization procedure of the sample of interest under isocratic conditions as well as under single linear organic modifier-gradients in different eluent pHs. The importance and the advantages of performing a computer-aided separation optimization compared with a trial-and-error optimization method were realized. Additionally, this study showed that the optimized separation conditions for a given stationary phase may be used to achieve successful separations on new columns of the same type and size. In general, the results of this work could give chromatographers a feel of confidence to establish desired separations of a mixture of ionizable and neutral compounds in reversed-phase columns.