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.