Preparative liquid chromatography with analytical separation quality : Internal injection/displacement reversed-phase high-performance liquid chromatography

Abstract

A method for the enrichment and separation of compounds, called interval injection/displacement reversed-phase high-performance liquid chromatography (HPLC), was applied to several analytical and preparative separations. In principle, the surface of a reversed-phase stationary phase, equilibrated with a weak mobile phase, is “coated” with compounds by stepwise injection of small samples. Distinct time intervals between injections allow the stationary phase to re-equilibrate. In this way, sample enrichment can be achieved in ranges comparable to those in overload elution or displacement HPLC. Sample fractionation proceeds in a similar manner to conventional displacement modes. Also under full mass load conditions, a quality of separation similar to that in analytical elution HPLC is achieved. The results obtained indicate that a very large number of samples can be applied to conventionally sized columns without overload problems, the full mass capacity of a column can be utilized and the method is useful for enriching and separating compounds with a wide range of polarities. Interval injection/displacement reversed-phase HPLC was successfully applied to complex mixtures of natural products and enzyme assay mixtures. Further, this technique is likely to be useful for the analysis of trace compounds and quality control of chemicals. The experiments reported were performed on highly unstable natural products (thiophenic and benzofuran compounds) from Tagetes plants.