Capillary electrophoretic separation of nucleotide isomers via complexation with cyclodextrin and borate.

The electrophoretic behaviour of monophosphorylated nucleotide isomers can be manipulated using complex-forming reactions with beta-cyclodextrin (beta-CD) and borate. Resolution of the 2'- and 3'-isomers of nucleotides is possible when the electrophoresis buffer contains 10 mM CD. The effect of beta-CD concentration on electrophoretic mobility is used to calculate the formation constant, K, of beta-CD-nucleotide complexes. The 3'-isomer of adenosine monophosphate (AMP) forms the strongest complex with beta-CD probably as a result of hydrogen bonding between the phosphate group of AMP and hydroxyls of beta-CD. In addition, complexation of 5'-nucleotides with borate increases the migration time window and leads to better separation. Complex-forming reactions of guanosine monophosphate and uridine monophosphate are shown to be strongly dependent on buffer pH. A mixture of 12 monophosphorylated nucleotides can be separated in less than 15 min using a buffer of 20 mM borate-10 mM beta-CD.