Abstract
The electrophoretic behaviour of monophosphorylated nucleotide isomers can be manipulated using complex-forming reactions with β-cyclodextrin (β-CD) and borate. Resolution of the 2'- and 3'-isomers of nucleotides is possible when the electrophoresis buffer contains 10 m M CD. The effect of β-CD concentration on electrophoretic mobility is used to calculate the formation constant, K, of β-CD—nucleotide complexes. The 3'-isomer of adenosine monophosphate (AMP) forms the strongest complex with β-CD probably as a result of hydrogen bonding between the phosphate group of AMP and hydroxyls of β-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 m M borate—10 m M β-CD.
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