Different compositions of mobile phase were applied on Zorbax Extend-C18 for the separation of studied sulfonamide residues in least amount of time possible. The best separation was achieved at pH 4.50 using 20 mM acetate buffer solution at flow rate of 0.7 mL/min in less than 30 min. From the bibliographic study, it was discovered that a high temperature (40–55◦C) is necessary to separate the derivatized sulfonamide compounds, however, in our laboratory; the chromatography was conducted at 28◦C. Moreover,the short and long analytical columns were cleaned and regenerated for 10 min individually at a valve position: A (1–4) according to Fig. 2(a).