INITIAL EXPERIMENTS IN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHODDEVELOPMENT .2. RECOMMENDED APPROACH AND CONDITIONS FOR ISOCRATIC SEPARATION

Initial method development experiments for both neutral and ionic samp les are best carried out with reversed-phase high-performance liquid c hromatography (HPLC) using acetonitrile-methanol-buffer mobile phases. The preceding paper (Part I) suggests the use of an acetonitrile-buff er gradient to start method development. In this paper, optimum condit ions for this first separation are discussed. Sequential method develo pment experiments plus computes simulations are then used to obtain a final HPLC method. In this connection, we have examined how many exper iments are required for reliable predictions of ternary-solvent retent ion. Three experiments are sufficient to predict isoeluotropic retenti on for methanol-acetonitrile-buffer ternaries where solvent strength d oes not vary, but five experiments are required for ternaries that con tain tetrahydrofuran.