Capillary zone electrophoresis of oligonucleotides in isoelectric buffers and against a stationary pH gradient.

Abstract

Capillary zone electrophoresis of oligonucleotides in a background electrolyte of two different types of stationary buffers is proposed: single, isoelectric amphoteres and focused carrier ampholytes. In the first case, two zwitterionic molecules are evaluated: lysine and histidine. Although the former has a five times higher buffering power (beta) at the pI (9.74) than the latter (pI 7.47), due to the favorable delta pK value (1.6 vs. 3) and thus should be the preferred species, a new parameter for evaluating the performance of isoelectric buffers is proposed: the beta/lambda ratio, i.e., the ratio between the buffering power and its conductivity. Ideal buffers are those with the highest beta/lambda ratio, since this allows delivering very high voltage gradients with minimal Joule effects. Since the pI of Lys is situated in a pH region (9.74) where bulk water begins to conduct, whereas His has a pI close to neutrality, the beta/lambda ratio is more favorable for His than for Lys. In the second case (zone electrophoresis of oligonucleotides against a preformed pH gradient), it is shown that migration against a pH 6.5-10 Pharmalyte carrier ampholyte pH gradient offers a unique analyte resolution. This is possibly due to two effects: (i) When injected at the alkaline extreme (ca. pH 10) of the pH gradient, the oligonucleotide zones undergo a stacking effect, with consequent zone sharpening, due to modulation of their free mobility via protonation of the -OH group (enolate ion) in the hetero aromatic rings of G and T, which undergo a lactam-lactim transition. (ii) As the zones migrate down the pH gradient, they transit through a pH 6.5-8.5 zone where, for Pharmalytes, the beta/lambda ratio reaches a maximum and is constant as well. This last condition allows high voltage gradients (typically 1000 V/cm, even in 75 microm capillaries) to be delivered, thus greatly reducing the analysis time and maintaining peak sharpness, due to limited diffusion.