Purification of micromolar quantities of nucleotide analogs by reverse-phase high-performance liquid chromatography...

The separation of 5'-adenosine di- and triphosphates from inorganic pyrophosphate or imidodiphosphate can be accomplished with reverse-phase HPLC by using a solvent system buffered by triethylammonium bicarbonate (pH 6.7). This buffer was used because it was neutral, readily volatile at 20 degrees C, and formed ion pairs with phosphate compounds to allow their separation by reverse-phase chromatography. Micromolar amounts of radioactive or fluorescent nucleotide analogs have been purified using C-18 columns or a polystyrene divinylbenzene column (PRP-1, Hamilton) with the solvent system described. The method is particularly advantageous in preparing salt-free acid-, base-, or thermally-labile nucleotide analogs. It is possible with this method to remove 32Pi (173 mumol) from ATP (50 mumol, 30 mg) in one run using a C-18 analytical column demonstrating that this approach can be useful for selected semipreparative purifications.