Micro‐HPLC 1: Linear “breakthrough‐gradients” using the discontinuous interface between eluents in a packed gradient‐generator column

Abstract

AbstractA new approach is described for micro‐HPLC for generating nearly linear gradients in the 40 to 550 μl volume range. These “linear” gradients have a straight line middle portion with a gentle curved onset of the gradient andd rapid attainment of the final 100% composition. Such “breakthrough‐gradients” are produced by the interface between weak and strong eluent when a small (3 × 0.46 cm) packed “gradient‐generator” column is switched abruptly from weak to strong eluent. The model system described here, without an analytical column, shows gradients from weak eluent (water) to strong eluent (modele with water plus acetone) using a 12‐port high pressure valve to 1) vent the “flush” eluent, and simultaneously 2) begin the gradient and 3) make the sample injection.The volume of the gradient can be changed in two ways. If the breakthrough‐generator is packed with very large (300 to 1600 μm nonporous particles, the gradient volume is nearly independent of the flush flow rate. The flush flow is used to quickly move the breakthrough‐interface to the head of the micro‐HPLC column. However, the gradient volume can be increased by using larger solid particles in the gradient‐generator column because of increased eddy diffusion. If the gradient‐generator is packed with porous particles (75–150 μm), the gradient volume can be changed over a broad range by simply changing the flush flow rate. This simple control over the gradient volume is due to the increased mass transfer of weak eluent out of the porous particles into the strong eluent at higher flush flows.The breakthrough‐interface gradient‐generator column approach provides the following advantages for micro‐HPLC: – Gradient volumes can be readily varied over the micro‐HPLC range 40–550 μl. – Gradient are linear with smooth and rapid onset of intial and final concentrations. – Gradient are produced inexpensively with a single high‐pressure precision pump. – Gradient can be automated with a timer and single 12‐port valve that provides both column regeneration and sample injection.