Influence of sample mass on the performance of reversed-phase columns in the analysis of strongly basic compounds by high-performance liquid chromatography

The overloading behaviour of two different silica-based ODS columns was investigated using six basic probe compounds with methanolic mobile phases buffered at pH 3.0 and pH 7.0. At pH 3.0, peak shape deteriorated seriously in most cases when the sample mass was increased above about 0.5 mu g, as has been reported previously. The rate at which efficiency loss occurred varied with the solute and column, as reflected in values for the saturation capacity w(s) calculated for the solute/column combination. The model proposed by Snyder and co-workers was shown to allow prediction of the column overload profile for five of the basic solutes, although pyridine gave anomalous behaviour. Whereas peak shapes at pH 7.0 were generally worse than at pH 3.0, no deterioration was shown with sample masses up to at least 10 mu g. In some cases peak shape showed significant improvement as sample mass was increased. Thus, at high sample loading, analysis at pH 7.0 may be preferred for some solutes. It was shown that while overloading has not influenced our previous column evaluation studies, great care is needed to avoid confounding the results of such studies by these effects. (C) 1998 Elsevier Science B.V.