Low-Cost Liquid Chromatography (LC-LC). IV. “Pulsed Open Tube Gradient Generation”, A New Approach for Generating Nanoliter Volume Linear and Tailored Gradients for Capillary Electrophoresis and Micro-LC

Abstract

There is a need to generate gradients of nanoliter volumes compatible with 25–100 micron i.d. capillaries used in capillary electrophoresis (CE). Balchunas and Sapaniak [1] showed that manually produced step gradients of increasing propanol and surfactant concentration with micellar electrokinetic chromatography gave an improved separation. And while SDS-PAGE has shown remarkable results in capillaries e.g. [2], gradients in gel crosslinking to extend the molecular weight range that can be resolved has not yet been shown in capillaries. Gradients in pH in polyelectrolytes for isoelectric focusing show some of the highest resolution known. These have been generated in-place by electromigration. Small gradients are also needed in micro-liquid chromatography, a field receiving renewed interest today because of the high costs of solvent disposal.

Previous papers on “low cost liquid chromatography” (LC-LC) described how to eliminate the injection valve for “weak-eluent sariple-loading” [3, 41 and how a flow-through system can be used to take eluents to a fixed and reproducible pH., eliminating the time and effort to prepare eluents [5, 6].

Our previous paper on LC-LC described a simple-gradient method [7, 8, 91 in which an abrupt discontinuous interface between the weak and strong eluent can be spread into tin S-shaped gradient by passing the interface through an open tube. Volumes were in the microliter range (20 to 110 uL), determined by the open tube dimensions (0.53 m i.d.) and the flow rate through the open tube. Here we extend this simple-gradient approach to the high nanoliter range using 0.10 mm i.d. tubing.

This paper describes a new, very flexible method for producing shaped nanoliter gradients (“nano-gradients”) og a broad vo1ume range, independent of tube dimensions and flow rates. In the pulsed open tube generator, (I) the gradient can be changed in volume over a broad range, and (2) the yadient shape can be tailored. The new approach tailors the shape of the gradient by using a multiport valve to pulse in alternating slugs of weak and strong eluent; the timing determines the composition; and the open tube mixes the segments together.

The pulsed open tube generator described here should permit nanoliter and lower (picoliter) gradient generation when narrower bore open tubes are used (e.g. 50 to 5 i.d. micron open tubes).