New retention mechanism of mononucleotides with buffer concentrations in ion-suppressing RP-HPLC

Abstract

HPLC separation of ionic samples tends to be more complicated and difficult to understand than that of non-ionic compounds. On the other hand, band spacing is much more easily manipulated for ionic than for neutral samples. Ion-suppressing RP-HPLC method was used with organic modifier and aqueous buffer solution. In this work, five mononucleotides of cytidine-5-monophosphate (5′-CMP) disodium salt, uridine-5-monophosphate disodium salt (5′-UMP), guanosine-5-monophosphate disodium salt (5′-GMP), inosine-5-monophosphate disodium salt (5′-IMP), and adenosine-5-monophosphate disodium salt (5′-AMP) were examined. Acetic acid and sodium phosphate were used as buffers, and methanol as an organic modifier. A new relationship between the retention factor and the buffer concentration at a fixed modifier content (5% of methanol) could be expressed by fol|lowing:k=(k ₋₁+k ₀ (K _B/K _S)C _B ^a)/(1+(K _B/K _S)C _B ^a), whereC _B was the concentration of buffer. Using this relationship, the calculated values closely matched the experimental data. The derived relationship showed that as the buffer concentration increased, the retention factor approached a certain value, and this was buffer dependent.