3.2 Optimization of the HPLC Condition
The chromatographic conditions were optimized so as to obtain a good separation between benzoic acid, sorbic acid and matrix interferences in noodles. Acetate and phosphate aqueous solutions with different pH and organic solvent (methanol and acetonitrile) were investigated and system suitability test (SST) parameters were performed during the development and optimization of method. Firstly, the separation was attempted and compared on a C18 column (150 mm × 4.6 mm, 5 μm) and a C18 column (50 mm × 4.6 mm, 5 μm) with a wavelength at 224 nm (for sorbic acid), 234 nm (for benzoic acid) and 254 nm using acetonitrile and 5 mM phosphate buffer (pH 7) in the ratio range from 5:95 - 30:70. Both acids were eluted quickly and could not separate from matrix interferences. Methanol was then replaced in the same ratio range but the peaks of both acids were co-eluted. Increasing of phosphate buffer concentration (5-30 mM) led to increase in analytes’ retention, decrease in tailing factor and resolution value. Consequently, ammonium acetate aqueous solution was employed instead of phosphate to achieve the best resolution. Poor resolution and peak tailing was obtained when acetonitrile was used. Separation of benzoic acid and sorbic acid was accomplished by varying the concentration of ammonium acetate (5-30 mM) and the ratio of methanol. An increase in retention and peak broadening of benzoic acid and sorbic acid was observed when higher concentration of ammonium acetate at higher pH was utilized. Hence, the aqueous component of the mobile phase was selected to be ammonium acetate 5 mM, pH 4.4.