A HSCCC method combined with the consecutive injection and the economical two-phase solvent system preparation using UNIFAC mathematical model was successfully developed for increasing the yield of target compound. HSCCC was performed with a two-phase solvent system composed of n-hexane ethyl acetate-methanol-water (3:5:3:5, v/v) at a flow rate of 6 mL/min. The components of the upper and lower phases were calculated according to UNIFAC mathematical model to prepare the stationary and mobile phases, separately. Only 300 mL stationary phase was filled into the column, and the crude sample (200 mg, ten times) was consecutive loaded into the column at the given interval time and eluted using the individually prepared mobile phase without reestablishing hydrodynamic equilibrium. After recrystallized, 168 mg tiliroside with the purity of 95.8% was obtained from 2 g crude sample of Pinus massoniana L. The structure was identified by H-1 NMR and C-13 NMR. The present method not only avoided the time consuming of exchanging the new solvent system and reestablishing hydrodynamic equilibrium, but also coped with a large amount of organic solvent waste. (c) 2016 Elsevier B.V. All rights reserved.