Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: Theory and methodology

The use of isoelectric buffers in capillary zone electrophoresis is reviewed. Such buffers allow application of extremely high voltage gradients (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 100 μm internal diameter), permitting separations of the order of a few minutes and thus favoring high resolution due to minimal, diffusion‐driven peak spreading. The fundamental properties of ampholytes are first discussed, such as buffering power (β) as a function of Δp K. i.e. of the distance between the p I value and neighboring protolytic groups. The highest possible relative β value (= 2) is obtained for amphoteres possessing a Δp K = 0.6, a condition not met by existing amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the β/λ ratio, i.e. the ratio between the β power and conductivity at the p I value. It is additionally shown that the p I is not a constant value, but depends on ampholyte concentration in solution. In addition, at constant concentration, the theoretical p I can change as a function of Δp K . Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absense of sieving media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (p I 2.77 at 50 m M concentration) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values ( e.g. pH 3.0 for 30 m M Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human β‐globin chains. Also imino diacetic acid (p I 2.33 at 50 m M concentration) allows generation of high resolution peptide maps.