[Optimum separation conditions of catechin compounds by HCI program in reversed-phase high performance liquid...

An efficient optimization method was used to separate catechin compounds by reversed-phase high performance liquid chromatography (RP-HPLC). The binary mobile phase of water and methanol was utilized with the buffer of acetic acid (AA). The elution profiles were calculated by the plate theory based on the linear and quadratic equations of retention factor, In k = ln kw + SF, k = A + B/F, ln k = L + MF + NF2, where F is the volume fraction of methanol in the mobile phase. The retention theory was modified to calculate the elution profile in both isocratic and gradient modes. Based on the retention theory, elution profiles were predicted by introducing the concept of solute migration in the mobile phase with the linear and quadratic dependence of ln k in terms of the organic modifier content. Using the HCI program (a software designed by Inha University), the recommended experimental conditions of mobile phase composition and gradient step were suggested, and the elution profiles calculated by the quadratic relationship of ln k showed better coincidence with the experimental data than the linear correlation did. The calculated results of mobile phase condition for separation of catechin compounds suggested that the mobile phase composition was 0.1% AA in water/0.1% AA in methanol, 75/25 (v/v), then after 15 min, the composition was linearly changed to 50/50 (v/v) in 10 min and held at the isocratic mode to the end. In the experimental conditions, the agreement between the experimental elution profiles and the calculated values of eluted concentration was relatively good.