Reversed-phase electrochromatography of proteins on modified continuous beds using normal-flow and counterflow gradients. Theoretical and practical considerations

Most chromatographic methods, including capillary electrochromatography (CE C), require gradient elution for high resolution of proteins. The gradients used in the CEC experiments described herein were generated by an HPLC ins trument and pumped past one end of the capillary column. Part of the gradie nt was at the same time transported into the capillary solely by electroend osmosis, Employing these gradients, positively charged proteins were separa ted on a column filled with a continuous bed derivatized with C18 groups (f or reversed-phase separation) and with ammonium groups (for generation of e lectroendosmotic now (EOF)). Both the proteins and the EOF-generating ligan ds thus had positive charges to eliminate electrostatic interactions. The g radient 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 tha n the electrophoretic velocity, whereas in the latter mode, it must be lowe r. Accordingly, gradient elution in electrochromatography can be used for m any CEC columns since the magnitude of their EOF is not critical. The EOF i s a function of the concentration of the gradient constituents and may, the refore, be different in different segments of the capillary. The possible a ttendant effects on zone broadening have been treated, as well as the elect rophoretic zone broadening and zone sharpening caused by the gradient. Spec ial precaution was taken in order to ensure that the electrophoretic contri bution 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 pre pared under ultrasonication for 45 min. Then, piperazine diacrylamide (cros s-linker) and dimethyl diallylammonium chloride (both EOF-generating ligand and cross-linker) are added. This suspension is propelled into the capilla ry (with a methacryloyl-activated inner surface). At this stage, the concen tration 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 f ormation of channels in the bed.