Initial experiments in high-performance liquid chromatographic method development .2. Recommended approach and conditions for isocratic separation

Initial method development experiments for both neutral and ionic samples are best carried out with reversed-phase high-performance liquid chromatography (HPLC) using acetonitrile-methanol-buffer mobile phases. The preceding paper (Part I) suggests the use of an acetonitrile-buffer gradient to start method development. In this paper, optimum conditions for this first separation are discussed. Sequential method development experiments plus computes simulations are then used to obtain a final HPLC method. In this connection, we have examined how many experiments are required for reliable predictions of ternary-solvent retention. Three experiments are sufficient to predict isoeluotropic retention for methanol-acetonitrile-buffer ternaries where solvent strength does not vary, but five experiments are required for ternaries that contain tetrahydrofuran.