"Orthogonal" separations for reversed-phase liquid chromatography.

A general procedure is proposed for the rapid development of a reversed-phase liquid chromatographic (RP-LC) separation that is "orthogonal" to a pre-existing ("primary") method for the RP-LC separation of a given sample. The procedure involves a change of the mobile-phase organic solvent (B-solvent), the replacement of the primary column by one of very different selectivity, and (only if necessary) a change in mobile phase pH or the use of a third column. Following the selection of the "orthogonal" B-solvent, column and mobile phase pH, further optimization of peak spacing and resolution can be achieved by varying separation temperature and either isocratic %B or gradient time. The relative "orthogonality" of the primary and "orthogonal" RP-LC methods is then evaluated from plots of retention for one method versus the other. The present procedure was used to develop "orthogonal" methods for nine routine RP-LC methods from six pharmaceutical analysis laboratories. The relative success of this approach can be judged from the results reported here.