Heart-cutting two-dimensional electrophoresis in a single capillary

The possibility of performing two-dimensional capillary electrophoresis (2D-CE) separations in a single capillary was investigated. For this purpose, a fraction stemming from the first dimension of the separation was selected and isolated in the capillary by evacuating out of the capillary the other undesirable compounds. Next, the isolated fraction was submitted to a second separation medium that was introduced in the capillary by electroosmotic flow. The second separation medium was able to reach the isolated fraction since the solutes were migrating in counter-electroosmotic mode. Since only one fraction is submitted to the second dimension of the separation, this new methodology is closer to a heart-cutting approach than to a true comprehensive 2D separation. However, it has the advantage of not requiring any special coupling device between capillaries since the two dimensions of the separation are performed in the same capillary. In this work, a simple mixture of synthetic polymers taken as model compounds was separated according to: (i) the charge density by free solution CE in the first dimension, and (ii) the molar masses by CE in the presence of an entangled polymer solution in the second dimension. The different strategies that were investigated to isolate the fraction at the end of the first dimension are described in detail. The influence of important experimental parameters (capillary diameter, applied pressure for mobilizing the solutes, diffusion coefficients of the solutes) on the performances of the two-dimensional separations were studied. A careful attention was paid to the influence of these parameters on the efficiency of the separations. The experimental results demonstrate that heart-cutting 2D electrophoretic separations can be performed in a capillary format using a single capillary.