Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time III. Improving the accuracy of computer simulation.

Previous studies have shown that four experimental runs, where both temperature T and gradient time tG are varied, can be used for the reliable prediction of separation as a function of these two variables (two-dimensional optimization). Computer simulation (e.g., DryLab) can then be used to predict "optimized" conditions for maximum sample resolution using either isocratic or gradient elution. Samples that contain a large number of components (e.g., n>15-20) present a greater challenge. Resolution for these more complex samples is often quite sensitive to small changes in T or tG in turn requiring greater accuracy in predictions that result from computer simulation. In the present study of several samples, we have examined computer simulation errors that can arise from inexact expressions for retention time as a function of T, tG or isocratic %B. Resulting conclusions are applicable to both complex and simpler samples, in either one- or two-dimensional optimization. Means to anticipate and minimize the impact of these predictive errors are examined.