The Influence of Sample Mass and Flow Velocity on Performance in Preparative Column Liquid Chromatography

Abstract The H/u curves of two HPLC columns of 10 mm i.d., filled with silica of a particle diameter of 5 and 25–40 μm, respectively, have been determined at various amounts of injected sample. Indications of mass overload, Le, peak broadening, occur at a sample load of 10 μg/g for the column with 5 μm silica but not until 100 μg/g for the column with coarse stationary phase. Although H values increase markedly with higher sample mass, the slopes of the H/u curves remain almost constant on both columns up to samples of 1 mg/g. This means the relative increase of the plate height with increasing flow velocity becomes nearly negligible under conditions of moderate mass overload. However, by increasing the sample load up to 5 or 10 mg/g, the H/u curves show a dramatic increase of slope: at high mass overload the mobile phase flow rate cannot be increased without a remarkable decrease in column performance.