Efficient Method Development: Exploring pH, Solvent and Stationary Phase

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Diane Wagrowski-Diehl, Uwe Neue, KimVan Tran, Eric Grumbach, Jeff Mazzeo
HPLC 2002; Montreal; 3-4 June 2002
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The efficient development of new reversed-phase HPLC methods has been a topic of discussion since the early times of the technology. In this presentation, we outline an efficient starting point for the development of an HPLC method based on our evaluation of the major selectivity differences between the three major variables of pH, organic solvent and stationary phase. We found that the largest selectivity differences for ionizable compounds were caused by pH changes. The second largest selectivity changes were produced by the organic modifier, methanol or acetonitrile. The third group of selectivity changes was due to the stationary phase itself. However, only very small changes were found when stationary phases of different chain lengths were compared. Larger selectivity effects were found when a classical stationary phase was compared to one with an incorporated polar group. In our general method development set-up, we combined both phases with a phase with a phenyl ligand to maximize the selectivity differences between different stationary phases. We also provide some general guidelines on how to fine-tune a method based on the data generated in the initial screening.

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