Criteria for optimizing the separation of target analytes in complex chromatograms

Optimization procedures require adequate criteria to assess the quality of the separation. So far, the vast majority of reports on the subject deal with the situation in which all peaks need to be separated (‘complete optimization’). In practice one of the most important requirements for optimization criteria is their ability to deal with limited optimization, i.e. with situations in which only a limited number of solutes in a sample is relevant. In this paper, the adaptation of the most widely used optimization criteria to limited optimization is discussed. Among the most useful criteria are the minimum resolution and the calibrated normalized resolution product. The characteristics and objectives of these two criteria are discussed in detail. The case of non-ideal separations (featuring asymmetrical peaks and/or peaks of vastly different areas) is also investigated. The discussion and evaluation of optimization criteria are based on simulated and experimental data obtained during HPLC optimization studies.