Optimized HPLC Separation of Structural Analogs

While it is difficult to separate naringin and hesperidin, honokiol and magnolol, aloe emodin, rhein, emodin and chrysophanol as they are structurally similar, separation was improved when 0.2% (1:500, v/v) aqueous acetic acid was added to the sample solution. A mixture of water and methanol was chosen for the separation as all eight markers dissolve in both water and methanol. The ratio 38:62 (v/v) of methanol and water was optimal for the separation of hesperidin and naringin, and 65:35 (v/v) for the separation of the other six markers, hence the above mentioned gradient elution program. The HPLC column of C₁₈ (150 × 4.6 mm) was chosen to ensure the run time to be within 60 min.