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 dete ction. The influence of specific analyte properties, such as nucleotide bas e structure, sugar structure, and degree of phosphorylation, is examined. T he pK(a) values and berate complexation with vicinal diols are important fa ctors that caused the focusing. Therefore, the pH and berate 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 (simil ar to 300 nL), resulting in at least 50-fold improvement in concentration s ensitivity. The detection limit of 4.0 x 10(-8) M for nucleotides can be ac hieved in favorable conditions. V-DIF can be also applied to nucleotide poo l analysis from cell extracts to improve the concentration sensitivity of C E and to reduce the time-consuming steps of desalting and off-line preconce ntration that are often required for assays of nucleotides from biological samples.