Retention mechanism of mononucleotides with buffer in RP-HPLC

Abstract

To estimate the retention equilibrium constant of ionized solute by the buffers in the mobile phase, RP-HPLC experiments were performed under isocratic conditions. Four nucleotides (5'-UMP, 5'-GMP, 5'-IMP, 5'-TMP) were used as solutes. The first absolute moment and second central moment were determined from the elution curves by means of moment analysis. The retention equilibrium constant decreases upon increasing the concentration of the buffers. Three different mass transfer processes (i.e., delta(ax), delta(f), and delta(d)) that take place in the column were investigated using different buffers. The results indicate that delta(ax) and delta(f) had large effects on mu'(2), but the contribution of delta(d) to mu'(2) was negligible. In addition, we found that surface diffusion plays a dominant role in intraparticle diffusion.