NEW APPROACHES TO CONCENTRATION ON A MICROLITER SCALE OF DILUTE SAMPLES, PARTICULARLY BIOPOLYMERS WITH SPECIAL REFERENCE TO ANALYSIS OF PEPTIDES AND PROTEINS BY CAPILLARY ELECTROPHORESIS .1. THEORY

New methods are described for the concentration of ionic analytes, particularly ampholytes, such as peptides and proteins. In most of these methods the sample is depleted (partially) of strong electrolytes concomitantly with the concentration. The methods are based on the fact that electrophoretic migration velocities decrease upon decreasing the absolute value of the zeta potential of a solute and the pore size of the electrophoresis medium and upon increasing the cross section of the electrophoresis chamber, the viscosity and the electrical conductivity of the electrophoresis medium. We have also utilized the zone-sharpening properties of displacement electrophoresis in combination with a hydrodynamic counter flow to create a stationary zone where the sample solutes can be collected continuously. In practice, the whole electrophoresis tube is filled with the sample solution to be concentrated. When a voltage is applied the solutes begin to migrate, but finally cease to move as they approach the end of the tube, provided that the above-mentioned parameters in that section of the tube have been given appropriate values. By means of this technique the sample can be concentrated into a zone of a width of 0.2-0.5 mm. Accordingly, a 400-1000 fold concentration is obtained when a 200 mm long tube is filled completely with the sample and still more if also an electrode vessel (or a vessel connected to this electrode vessel) is loaded with sample. The narrow sample zone can be withdrawn from the tube and subjected to further studies or used as a starting zone for an in-tube zone electrophoresis. The tendency for broadening of the very narrow starting zone during the initial phase of this electrophoresis step can be counteracted by a short mobilization step involving displacement electrophoresis, electrophoresis in a steep pH gradient, or on-tube dialysis against a (diluted) buffer. This step can be omitted when the concentration is accomplished by a combination of displacement electrophoresis and a counter flow. In Part II we show how the theory developed in this paper can be utilized practically.