Optimization of Fat-Soluble Vitamins Separations by Reversed-Phase Liquid Chromatography with the Use of Aliphatic Alcohols as Mobile Phase Additives

In an effort to reduce the total amount of organic solvent required for the elution of fat-soluble vitamins (FSVs) in reversed-phase liquid chromatography separation, a systematic approach was developed using a mixture of methanol, water, and an alkanol as the mobile phase. In the optimization procedure, a five-parameter retention model was developed and the statistical evaluation of model, revealed that the variations of retention times of each vitamins caused by addition of alkanols to the mobile phase were predicted with an average relative deviation of 1.83%. For this model, all the [GRAPHICS] and [GRAPHICS] values for all vitamins were higher than 0.9839 and 0.9798, respectively, and lack of fit (LOF) values were not statistically significant. A mobile phase containing methanol/octanol/water (86:9:5) was proposed as the optimum mobile phase composition using grid search method and a good agreement was observed between predicted and experimental response values (relative error=-0.734%). Using this optimum condition, the simultaneous separation of six FSVs was achieved in 9.94min. The results showed an improvement in the separation of FSVs and in reduction of the total amount of organic solvent used for separation with addition of low amount of alkanols as additive to the mobile phase.