SEQUENTIAL MULTIMODAL ELUTION FOR PSEUDOMULTIDIMENSIONAL LIQUID-CHROMATOGRAPHY ON A SINGLE COLUMN

A method of elution is described that yields significantly higher (2-3 times) peak capacities and facilitates the separation of compounds by class (e.g., acids versus neutrals), thereby increasing the informing power and the selectivity. The method is based on the sequential application of two or more elution modes, all of which, except the last one, are selective, i.e., designed to elute only a certain class of compounds. The total peak capacity is given approximately by r-phi-1, where r is the number of elution modes and phi-1 is the peak capacity of an individual elution mode, roughly equal to that obtained in a conventional one-dimensional separation. The increase in information content is proportional to log r but is also dependent on the relative proportion of sample components eluted during individual separation modes. Several reversed-phase examples utilizing sequential pH and solvent gradients are presented, including separations of benzoic acids from neutral aromatic compounds, peptides from neutral species, and phenols from polyaromatic hydrocarbons. Solute bandwidths, retention time, and area reproducibility are comparable to that of conventional reversed-phase separations. Although the resolving power of this multimodal, single-column elution method is somewhat less that that of a true two-dimensional (multiple column) method, it can be an effective and simple alternative for the analysis of moderately complex samples.