The relation between retention time or peak width and composition of mobile phase has been set up by polynomial-fitting least square according to experimental data. The concentration of methanol in mobile phase, which is the main factor affecting the separation of water-sol. vitamins, is considered to be a variable. In definite range of the variable, the optimum composition of mobile phase is selected to result in the shortest anal. time and to keep the Rs,min (the resoln. of two of the most difficult to sep. compounds) ?.5. The method has been used to optimize concentration of methanol in mobile phase for separation of six water-sol. vitamins including vitamins C, B1, B2, B6, nicotinic acid, and nicotinamide.

Key words: VITAMIN B1, HIGH SPEED LIQUID CHROMATOGRAPHY, REVERSED PHASE CHROMATOGRAPHY, NICOTINAMIDE, VITAMIN, VITAMIN C, REGRESSION ANALYSIS, OPTIMIZATION, LEAST SQUARE METHODS, NIACIN