The separation of constituents is carried out in such a way that a mixed amino acid sample is supplied to a separation column packed with a cation exchange resin and that, during the analysis of the single sample, five sorts of elutes of different compositions are supplied to the separation column in succession and by stages. The pH of the elute at the second stage is held higher than that of the elute at the first stage, and the pH's of the elutes at the third to fifth stages are held successively higher. However, the pH of the elute at the third stage is held lower than that of the elute at the second stage. On the other hand, the concentrations of counter ions contained in the elutes at the first to fifth stages are held successively higher inversely to the order in which the elutes are supplied. Notwithstanding that the pH of the elute at the third stage is lowered, the broadening of a component peak can be prevented by the increase of the counter ion concentration. Moreover, the analytical time is shortened as a whole.