The effect of beta-cyclodextrins as buffer additives on the (capillary) electrophoretic separation of peptides and proteins.

Electrophoretic and chromatographic experiments performed under straightforward conditions do not always provide satisfactory resolution. An obvious approach, then, is to manipulate the magnitude of relevant separation parameters, such as charge (zeta potential), size, and hydrophobicity, all of which can be accomplished by complex formation. This alternative has been studied with both a neutral, an anionic, and a cationic derivative of beta-cyclodextrin in an attempt to increase the resolution of peptides and proteins in free-zone electrophoresis utilizing the capillary format. The investigation showed that charged beta-cyclodextrins are suitable for this purpose. As expected, the effect seems to be most significant for substances with a small net surface charge, i.e., low mobility. Consequently, it may be advantageous to choose a pH of the buffer that is not far from the isoelectric point of the solutes. It should be emphasized that changes in the electropherograms observed upon addition of any complexing agent to the buffer may involve improvement or worsening of the resolution. Only by experimentation can one determine whether complexation with cyclodextrins favors resolution, since our knowledge about the interactions taking place is limited. However, if a positively (negatively) charged beta-cyclodextrin decreases the resolution of an acidic (basic) protein, one can expect theoretically, a negatively (positively) charged beta-cyclodextrin to increase the resolution, as was verified experimentally. The difference in mobility between two peaks caused by the complexation with cyclodextrins need not be larger than 2-3% for satisfactory resolution because the peaks are sharp. We have introduced a new definition for the resolution of two very adjacent peaks--the most common and interesting case in real-world analyses--that does not require measurement of peak widths.