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.
Highlights
Reversed-phase liquid chromatography (RPLC) is one of the most widely used chromatographic techniques
Excel-spreadsheet-based a pH: user-friendly andwas widespread software based on few initial experiments forsoftware, each eluent three isocratic runs and two conditions asplatform, wellbyasusing under linear φ-gradients in different eluent pHs successfully software platform, based on a few initial experiments for each pH: three isocratic runsφ
Two single linear φ-gradient runs performed in the studied range of mobile phase strength, In software the implemented by using an Excel-spreadsheet-based software, aeluent user-friendly and widespread single linear φ-gradient runs performed in the studied range of mobile phase strength, φ
Summary
Reversed-phase liquid chromatography (RPLC) is one of the most widely used chromatographic techniques. It is a consequence of its universality, relatively low costs, and general simplicity of analytical procedures. Model-based techniques (fully or semi-automated software programs) can be used in the process of searching for desired RPLC separations [1,2,3,4,5]. These methods usually provide very successful separations based on a series of preliminary experiments. We believe that the number of experimental data may be reduced by utilizing the optimal separation conditions predicted for a specific column by an optimization software to other new columns of the same type and size
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