ABSTRACT
More than 20 years ago, the author was attempting the separation of the cinchona alkaloids, a group of naturally occurring bases, by reversed-phase high-performance liquid chromatography (RP-HPLC). Quinine, used for the treatment of malaria, and quinidine, used as a cardiac antiarrhythmic drug are important members of the group. A published method [1] had achieved some separation, but with rather broad peaks, using a 10µm µ-Bondapak column and a methanolic mobile phase containing acetic acid. Surely, a much higher resolution separation would be obtained with a 5µm column. However, the same mobile phase with a 5µm column from a different manufacturer (Hypersil ODS) showed excessive or complete retention of the alkaloids (even with increase in the methanol concentration) and serious tailing. Other authors had also failed to reproduce the alkaloid separation on a variety of RP columns [2]. Here was an early example of the difficulties posed by the analysis of basic compounds. Tailing peaks with low efficiency and the poor reproducibility of separations on columns made from different silica are typical consequences of undesirable interactions of bases with ionized silanol groups on RP columns. These interactions are most serious on the relatively impure silica phases in use at the time and lead not only to problems of low resolution but also to poor quantitation. While more modern high-purity silica phases are now available, which give far superior results at low pH, another problem that leads to broad peaks and tailing is overloading, which occurs even on these modern phases. Further, the poor retention of hydrophilic bases in RP chromatography is yet another difficulty. Use of higher pH, where the ionization of bases is partially suppressed, is an attractive possibility and can give rise to useful separation selectivity. However, interaction with ionized silanols is still an almost inevitable consequence of working at neutral or higher pH, and thus different strategies may need to be employed. Many workers have attempted the use of alternative materials to silica such as polymeric-, zirconia-, and carbon-based phases, but so far these have not produced a generally accepted substitute for silica in the analysis of basic compounds.
