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Capillary zone electrophoresis of oligonucleotides in isoelectric buffers and against a stationary pH gradient
Author(s) -
Stoyanov Alexander V.,
Gelfi Cecilia,
Righetti Pier Giorgio
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150180510
Subject(s) - chemistry , capillary electrophoresis , isoelectric focusing , electrolyte , immobilized ph gradient , chromatography , analytical chemistry (journal) , protonation , isoelectric point , electrophoresis , oligonucleotide , stacking , electrode , ion , organic chemistry , biochemistry , dna , enzyme
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 (β) at the p I (9.74) than the latter (p I 7.47), due to the favorable Δp K 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 β/λ ratio, i.e. , the ratio between the buffering power and its conductivity. Ideal buffers are those with the highest β/λ ratio, since this allows delivering very high voltage gradients with minimal Joule effects. Since the p I of Lys is situated in a pH region (9.74) where bulk water begins to conduct, whereas His has a p I close to neutrality, the β/λ 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 β/λ ratio reaches a maximum and is constant as well. This last condition allows high voltage gradients (typically 1000 V/cm, even in 75 μm capillaries) to be delivered, thus greatly reducing the analysis time and maintaining peak sharpness, due to limited diffusion.