General experimental aspects of the use of isoelectric buffers in capillary electrophoresis

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cys teic acid [Cys-A, isoelectric point (pl) 1.85], iminodiacetic acid (IDA, pi 2.23), aspartic acid (Asp, pi 2.77) and glutamic acid (Glu, pi 3.22). Thes e four buffers allow to explore an acidic portion of the titration curves o f macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus p ermitting resolution of compounds having coincident titration curves at a g iven pH value. Given the rather acidic pi values of these buffers, their lo ng-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all f our buffers appear to give constant pH and conductivity readings up to 15 d ays; after that, the conductivity keeps steadily increasing in a similar fa shion. The same parameters, when the same buffers are dissolved in 6 M urea , appear to be stable for only one week, with the conductivity progressivel y augmenting after this period. A similar behaviour is exhibited by histidi ne (pl 7.70), a neutral, isoelectric buffer adopted for separation of DNA f ragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a de gradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in LD A and Asp solutions. It is additionally shown that the acidic buffers are n ot quite stationary in the electric field, but can be transported at progre ssively higher rates (according to the pi value) from the cathodic to the a nodic vessel. This is due to the fact that, at their respective pi values, a fraction of the amphotere has to be negatively charged in order to provid e counterions to the excess of protons due to bulk water dissociation. Guid elines are given for the proper use and storage of such buffers. (C) 1999 P ublished by Elsevier Science BN. All rights reserved.