COMPARISON OF REVERSED-PHASE AND NORMAL-PHASE COLUMN LIQUID CHROMATOGRAPHIC TECHNIQUES FOR THE SEPARATION OF LOW AND HIGH MOLECULAR WEIGHT COMPOUNDS

Abstract

ABSTRACT Liquid chromatography of complex samples often requires gradient elution to separate components with great differences in retention properties. The theory of gradient elution has now been elaborated so that it allows predicting the retention and the resolution of sample compounds not only in reversed-phase, but also in various normal-phase (adsorption chromatography) systems, for almost any combination of gradient profile and relationship between the retention and mobile phase composition. Using this knowledge, parameters of binary or ternary linear and non-linear gradients can be adjusted by predictive calculations for the desired resolution and minimum separation time in various chromatographic systems. In addition to accurate calculations of the gradient elution data, simple procedures can be employed for rapid estimation of the effects of the gradient program on the retention, both in reversed-phase and in normal-phase gradient chromatography. However, these procedures cannot be used for fine-tuning of separation, as they do not take into account possible effects of the gradient program on the elution order. Non-ideal instrumental or phase system effects impair the reproducibility of the retention data and the accuracy of the retention prediction and separation optimization. The most important, are the migration of some compounds during the dwell period, before the start of the gradient and the preferential adsorption of strong solvents, or of the traces of water from the mobile phase on to the column during gradient run. These effects should be accounted for to increase the accuracy of the predicted gradient retention data and of the gradient optimization. Both in reversed-phase and in normal-phase liquid chromatography, the strong solvent affects the retention of macromolecules much more strongly than the retention of small molecules. Consequently, less steep gradients, and much narrower gradient concentration ranges, are usually required for the separation of polymers and oligomers according to the molar mass distribution, than for the gradient-elution separation of small molecules.