Anomalous Retention Prediction Using Modelling Software in Gradient Reversed-Phase Liquid Chromatography: Why it Can Occur and How to Prevent It

This article presents the benefits of screening column and mobile phase combinations that generate differing chromatographic selectivities in reversed-phase gradient ultrahigh-pressure liquid chromatographic (UHPLC) method development strategies. Photodiode array (PDA) and mass spectrophotometric (MS) detection was necessary to facilitate peak tracking or identification of components in the sample mixture to build retention models. Retention time prediction accuracies of < 0.3% were obtained from a two-dimensional gradient time vs. temperature model when the initial gradient conditions were maintained. However, anomalous retention predictions were observed when higher %B initial gradient conditions were employed. Polar analytes in the sample mixture that started to migrate down the column in the dwell volume of the UHPLC system produced inaccurate retention time predictions if an inappropriate dwell volume was used in the retention model. An iterative dwell volume estimation was demonstrated to generate more accurate retention time predictions than when a practically determined dwell volume was used. However, to obtain good predictions the analyst should endeavour to use initial chromatographic conditions that promote focusing of all analytes on top of the column (that is, retention factor > 10).