Influence of the solvent strength determined by the addition of a mobile-phase organic modifier and pH on chromatographic separation of sorbic acid and vanillin has been investigated by the relatively new technique, liquid sequential injection chromatography (SIC). This technique uses reversed-phase monolithic stationary phase to execute fast separations (less than 1 min), allowing the students to quickly investigate the effect of analytes distribution (protonated and unprotonated) on the separation of compounds having acid-base properties. Design of experiments (DOE) was used to find the suitable condition for optimal separation in short chromatographic runs. The experiment is suitable for any chemistry student who has completed a minimum of one year of general chemistry, having a solid background in fundamentals of analytical chemistry. It is appropriate for an instrumental analysis course either for upper-division undergraduate or graduate students. (Contains 1 table and 2 figures.)