Conical precolumn as loading buffer for the main column.

Abstract

A conical high-performance liquid chromatographic precolumn was developed to cope with the problems that arise during the processing of large volumes of biological samples. The shape of the column was designed so as to offer a large loading capacity at the front of the precolumn. The stainless-steel construction, which is pressure resistant up to 40 MPa, can be fully integrated into high-performance systems. In the present work, the precolumn arrangement was used in the assay of pamoic acid in human plasma and in the isolation of radioactive metabolites from pools of animal urine and of supernatants from liver homogenates. Apart from extremely polar compounds, which were not retained on the precolumn, recovery of metabolites was practically complete. Almost the same resolution was obtained with the equivalent of 900 ml of urine, purified and enriched on the precolumn, as with a 2-ml sample of the original urine. Likewise, the chromatographic metabolite pattern of 650 ml of supernatant from homogenized liver was similar to that of a deproteinized sample of 2 ml. It is suggested that the precolumn is usable for all chromatographic problems involving enrichment of small amounts of substances in large amounts of complex matrices.