Velocity-difference induced focusing of nucleotides in capillary electrophoresis with a dynamic pH junction

Velocity-difference induced focusing (V-DIF) of nucleotides was achieved by using a dynamic pH junction in capillary electrophoresis (CE) with UV detection. The influence of specific analyte properties, such as nucleotide base structure, sugar structure, and degree of phosphorylation, is examined. The pKa values and borate complexation with vicinal diols are important factors that caused the focusing. Therefore, the pH and borate content in the sample and background electrolyte can be adjusted to optimize the focusing effect. This method allows the injection of large volumes of sample (approximately 300 nL), resulting in at least 50-fold improvement in concentration sensitivity. The detection limit of 4.0 x 10(-8) M for nucleotides can be achieved in favorable conditions. V-DIF can be also applied to nucleotide pool analysis from cell extracts to improve the concentration sensitivity of CE and to reduce the time-consuming steps of desalting and off-line preconcentration that are often required for assays of nucleotides from biological samples.