Computer simulation (DryLab software) as a means of facilitating the development of isocratic high-performance liquid chromatographic methods is reviewed. The various features of computer simulation are discussed and several examples of its application are presented.

Computer simulations can be used to develop high-performance liquid chromatographic gradient elution methods. However, the usefulness of this approach depends on the accuracy of the resulting predictions. Possible sources of error in computer simulation for the prediction of separation based on gradient elution have been investigated. This has in turn led to recommendations for minimizing such errors. With suitable precautions it appears possible to make adequately reliable predictions of separation by gradient elution. Several examples with protein mixtures as samples are reported.

The effect of the gradient on high-performance liquid chromatographic separations has been examined from a theoretical standpoint, using computer simulations to visualize the effects of different variables. Samples to be separated by gradient elution can be classified according to their separation characteristics into three groups (referred to here as cases I, II and III). Each of these sample types responds differently to a change in gradient conditions.

Case III samples exhibit changes in band spacing when the gradient conditions are varied, and gradients composed of multiple linear segments are especially useful for controlling band spacing and resolution for such samples. The effect of different gradient conditions (starting %B, gradient steepness and gradient shape) on the separation of case III samples is examined in detail.

Application of principles of DryLab G.

The commercially available computer program, DryLab, for optimization of separations by high-performance liquid chromatography (HPLC) using binary solvent mixtures is used to improve an HPLC method for separation of the bitter principle, limonin, in grapefruit and navel orange juices. Best conditions for separation of limonin in a reasonable time are 30 to 32% acetonitrile in water at 0.9 mL/min using a 5-μm C18 column 10 cm long. These conditions are used to analyze grapefruit and navel orange juice samples, and these HPLC results are compared with values determined by enzyme immunoassay or thin-layer chromatography (TLC) on the same samples.

Application of DryLab G method development strategy to a real sample.