NEW RETENTION MECHANISM OF MONONUCLEOTIDES WITH BUFFER CONCENTRATIONS IN ION-SUPPRESSING RP-HPLC

초록

HPLC separation of ionic samples tends to be more complicated and difficult to understand than separation of non-ionic compounds. On the other hand, band spacing is much more easily manipulated for ionic than for neutral samples. For ionic samples, we have a choice of three HPLC methods: reversed-phase, ion-pair or ion-exchange chromatography. Because of its simplicity and better column performance reversed-phase chromatography is frequently considered. The ion-suppressing RP-HPLC method by organic modifier and aqueous buffer solution was applied. In this work, the samples were five mononucleotides of cytidine-5-monophosphate (CMP) disodium salt, uridine-5-monophosphate disodium salt (UMP), guanosine-5-monophosphate disodium salt (GMP), inosine-5-monophosphate disodium salt (IMP), and adenosine-5-monophosphate disodium salt (AMP). Acetic acid and sodium phosphate were used as buffers, and methanol as an organic modifier. The new relationship of the retention factor and the buffer concentration at fixed modifier contents could be expressed by following : k = (A + B [buffer])/(1+C [buffer]), where [buffer] was concentration of buffer. This equation showed that as the buffer concentration increased, k approached to a certain value. Also the values were different with the buffers of acetic acid and sodium phosphate.