Validation of a High-Performance Liquid Chromatography Separation of Weak Acids, Neutral Compounds, and Permanent...

Sequential elution-liquid chromatography (SE-LC) employs conventional high-performance LC instrumentation to separate compounds by class and within each class using two or more sequential elution modes. The aim of this work was to validate a novel SE-LC method used to separate weak acids, permanently charged ions (anions), and neutral compounds by applying first a low pH isocratic elution to elute the weak acids, then an acetonitrile gradient to elute the neutral compounds, and lastly a sodium methanesulfonate gradient to elute the anionic compounds using a tandem-column arrangement comprised of a C18 column and a strong anion exchange column. The effects of flow rate, starting mobile phase organic content, column temperature, mobile phase composition, and gradient start time were investigated using a 2^5-1 fractional factorial design with center points for robustness. The method was demonstrated to be robust, specific, linear, accurate, and precise (repeatable) over the range of 6%-120% of the target analyte concentrations.