[Study on reversed-phase high performance liquid chromatography separation condition and determination method of...

The pH values of the mobile phase which affect the chromatographic behavior of organic acids in RP-HPLC have been studied. It was considered that the problem of organic acids on an RP-HPLC column was dissociation. The equation for finding the optimum pH of mobile phase was pHlimit of colunm < or = pH < or = pKa -2. In addition the effects of contents of methanol and acetonitrile in mobile phase were studied. By using the formula the mobile phases used for the analysis of organic monobasic acids--formic, acetic, propionic and butyric acid and of organic dibasic acids--propane diacid, butane diacid, pentane diacid and hexane diacid were proposed. For the analysis of the monobasic acids, the wavelength of UV-detector was set at 210 nm and the mobile phase was a mixture of phosphate buffer solution(15 mmol/L, pH 2.1)-methanol(85:15, V/V) and at a rate of 1.5 mL/min and the column was Diamond C18, 150 mm x 4.6 mm i.d.. The average coefficient of correlation was 0.9997 and RSD was less than 0.90%. The mobile phase for organic dibasic acids was a mixture of phosphate buffer solution (20 mmol/L, pH 2.1)-methanol(75:25, V/V). The other conditions were the same as for organic monobasic acids. The average coefficient of correlation was 0.9998 and RSD was less than 0.70%.