An approach to ideal separation media for (electro)chromatography.
In packed chromatographic beds, both Eddy diffusion and the relatively long time the analytes stay in the mobile phase until they collide and interact with the ligands attached to the beads (the residence time in the mobile phase) contribute considerably to zone spreading. One should not expect Eddy diffusion to occur in macroscopically homogeneous separation media, such as gels or polymer solutions, and residence time should be shorter, since the pore size of these media is much smaller than the average distance between the beads in a packed bed. Accordingly, these separation media (which can be regarded as homogeneous continuous beds [monoliths]) should theoretically give very high resolution, which has been verified experimentally: Frontal analysis of a neutral marker, acetone, showed that electroendosmosis in a homogeneous gel displaced the boundary without any distortion except that caused by diffusion (the marker was selected not to interact with the gel). All other common disturbing phenomena in chromatography (for instance, Eddy diffusion and nonspecific adsorption) were, accordingly, negligible, indicating that electrochromatography in homogeneous gels may be the ideal chromatographic method. However, to fully utilize these desirable chromatographic properties of homogeneous continuous beds, one has to choose analyte/bed interactions with sufficiently high association-dissociation rate constants (i.e., the residence time of the analytes in the stationary phase must be kept very short), which will be the subject of forthcoming studies. The electrophoretic counterpart of capillary electrochromatography (CEC), electrophoresis of noncharged analytes in a solution of charged polymers, seems to give a somewhat larger zone broadening, probably due to their high viscosity and conductivity, with attendant longer analysis (= diffusional) times compared to gels. Since the mobile phase is propelled through the gels by electroendosmosis, the theoretical and experimental requirements for high electroendosmotic flow in gels, i.e., short analysis times, are discussed. The electroendosmotic velocity v(x) can be estimated by the simple equation v(x) = Vmax (1-e-kappa x) (1/kappa = the thickness of the double layer; Vmax = the plug flow velocity) when the distance from the channel wall, x, << the radius R of the channel (pore). For kappa R > or = 5, the equation obtains with good approximation for all R values. An initially straight zone in a gel pore should be heavily distorted by the electroendosmotic flow, according to this equation (see Figure 1). However, due to rapid diffusion and other leveling effects, the zone is transported as in perfect plug flow, as is shown experimentally. A plot of electroendosmotic mobility obtained by frontal analysis against 1/(1 + square root of mu) can be used to estimate roughly the pore size in a gel and permits quantitative examination of the current theory of electroendosmosis. It is not a trivial problem to synthesize ligand-containing gels with pores large enough to allow a high electroendosmotic flow. Therefore, we have described a universal method: a polymer containing phenylboronate and acrylic acid groups was synthesized and entrapped in a standard agarose gel (for automated runs, replaceable methoxylated agarose should be used). Both of these charged groups serve to generate the electroendosmotic flow required in electrochromatography. This gel was designed to have the dual property of separating compounds that contain vicinal OH groups in the cis-configuration (exemplified by ribonucleosides) by reaction with the boronate groups, and aromatic substances by virtue of the acrylic acid residues (and perhaps also the phenyl groups) in the polymer and the agarose chains. The latter interaction, the so-called aromatic adsorption, has the advantage that it does not require a time-consuming attachment of ligands. (ABSTRACT TRUNCATED)
D002847:Chromatography, Agarose / Q000379:methods*; D006801:Humans