Electrophoretic behaviour of oligonucleotides and mono-, di- and triphosphate nucleotides by capillary zone electrophoresis

A systematic investigation has been made into the mechanisms of the capilla ry zone electrophoresis (CZE) separation of 12 common nucleotides (mono-, d i- and triphosphorylated) and polydeoxythymidylic acid oligonucleotides (pd (T)(5-18)) using electrophoretic mobility values calculated from migration time data. Relationships between electrophoretic mobility and the physicoch emical characteristics of the analytes (charge, dissociation constants, cha rge-to-mass ratio) acid the background electrolyte conditions (buffer stren gth, percentage organic modifier and buffer pH) were characterised. Nucleot ide migration was dominated by the negatively charged phosphate groups. Add itionally, there were important contributions to migration behaviour from t he ionised amide groups of the nucleobases guanine and uracil at higher buf fer pH values or with the presence of methanol in the electrolyte. Calculat ed electrophoretic mobility values for the nucleotides showed a substantial ly improved (5-fold) inter-run repeatability compared with migration time d ata. These studies show the value of representing nucleotide migration data as electrophoretic mobility in CZE for obtaining a more thorough analysis of separation mechanisms and to compensate for variation in migration time data caused by small changes in electrosmotic flow. Oligonucleotides pd(T)( 5-11) could be adequately resolved from their nearest neighbour, but the li mit of single-base separation was pd(T)(10) from pd(T)(11) under the condit ions used. It was calculated that a difference in charge-to-mass ratio of 2 .64 x 10(-5) was required for resolution under the CZE conditions used.