Basis of the separation process

As explained in the introductory section, chromatographic separation process based on the difference in the surface interactions of the analyte and eluent molecules.

Let us consider a separation of a two component mixture dissolved in the eluent. Assume that component A has the same interaction with the adsorbent surface as an eluent, and component B has strong excessive interaction. Being injected into the column, these components will be forced through by eluent flow. Molecules of the component A will interact with the adsorbent surface and retard on it by the same way as an eluent molecules. Thus, as an average result, component A will move through the column with the same speed as an eluent.

Molecules of the component B being adsorbed on the surface (due to their strong excessive interactions) will sit on it much longer. Thus, it will move through the column slower than the eluent flow.

Figure below represents the general shape of the chromatogram for this mixture.

Usually a relatively narrow band is injected (5 - 20 ul injection volume). During the run, the original chromatographic band will be spread due to the noneven flows around and inside the porous particles, slow adsorption kinetics, longitudinal diffusion, and other factors. These processes together produce so called band broadening of the chromatographic zone. In general, the longer the component retained on the column, the more broad its zone (peak on the chromatogram).

Separation performance depend on both component retention and band broadening. Band broadening is, in general, a kinetic parameter, dependent on the adsorbent particle size, porosity, pore size, column size, shape, and packing performance. On the other hand, retention does not depend on the above mentioned parameters, but it reflects molecular surface interactions and depends on the total adsorbent surface.