Most chromatographic methods, including capillary electrochromatography (CEC), require gradient elution for high resolution of proteins. The gradients used in the CEC experiments described herein were generated by an HPLC instrument and pumped past one end of the capillary column. Part of the gradient was at the same time transported into the capillary solely by electroendosmosis. Employing these gradients, positively charged proteins were separated on a column filled with a continuous bed derivatized with C18 groups (for reversed-phase separation) and with ammonium groups (for generation of electroendosmotic flow (EOF)). Both the proteins and the EOF-generating ligands thus had positive charges to eliminate electrostatic interactions. The gradient and the sample were introduced at the same end of the capillary as in conventional (electro)chromatography or in a new approach, at different ends. In the former mode, the electroendosmotic velocity must be higher than the electrophoretic velocity, whereas in the latter mode, it must be lower. Accordingly, gradient elution in electrochromatography can be used for many CEC columns since the magnitude of their EOF is not critical. The EOF is a function of the concentration of the gradient constituents and may, therefore, be different in different segments of the capillary. The possible attendant effects on zone broadening have been treated, as well as the electrophoretic zone broadening and zone sharpening caused by the gradient. Special precaution was taken in order to ensure that the electrophoretic contribution to the recorded separation did not dominate over the chromatographic one. We used a new approach to synthesize continuous beds with ligands of high concentration. It can briefly be described as follows. By a suspension−polymerization off-capillary procedure (in the absence of stabilizers and surfactants), very small gel particles derivatized with C18 ligands are prepared under ultrasonication for 45 min. Then, piperazine diacrylamide (cross-linker) and dimethyl diallylammonium chloride (both EOF-generating ligand and cross-linker) are added. This suspension is propelled into the capillary (with a methacryloyl-activated inner surface). At this stage, the concentration of nonterminated polymer chains on the surface of the gel particles is sufficiently high for further polymerization reactions. The polymer bed becomes attached covalently to the capillary wall concomitantly with the formation of channels in the bed.