HPLC Separation and Indirect Ultraviolet Detection of Phosphorylated Sugars

An HPLC method for the separation and analysis of phosphorylated sugars is presented. Ion-exchange chromatography coupled to indirect ultraviolet detection has produced good resolution and sensitivity. Fructose 6-P, glucose 6-P, ribose 5-P, 3-phosphoglyceric acid, ribulose 1,5-P(2), fructose 1,6-P(2), and sedoheptulose 1,7-P(2) can be separated at a sensitivity down to 10 nanomoles. The system resolves 2-carboxy-D-arabinitol 1,5-P(2) from 2-carboxy-D-ribitol 1,5-P(2). The natural inhibitor of ribulose bisphosphate carboxylase, 2-carboxy-D-arabinitol 1-P, has been separated from its 5-P isomer and most other phosphorylated compounds. This method is applied to identification of the products obtained upon ion-exchange purification of synthetic 2-carboxyarabinitol 1-P.