Acid Effect on Retention of Acidic Analytes


In mixed-mode chromatography, retention time and elution order can be changed for acidic analytes based on the pH of the mobile phase. In this application, the order of elution for benzoic acid and benzonitrile is changed by changing the pH of the mobile phase. At lower pH (pH-2, TFA), ionization of carboxylic acid fragment of benzoic acid is totally suppressed (not ionized), and benzoic acid does not show any anion-exchange properties. As the pH of the mobile phase increases (pH 4, formic acid), the carboxylic acid fragment of benzoic acid is ionized and participates in ion-exchange interaction with positively charged sites of the mixed-mode column. In most cases, changing pH does not change retention of neutral analytes. Mixed-mode chromatography is compatible with all detection techniques (UV, ELSD, CAD, LC/MS, etc.)
SIELC Technologies usually develops more than one method for each compound on our website. Therefore, this particular method may not be the best available method from our portfolio for your specific application. Before you decide to implement this method in your research, please send us an email to so we can ensure you get optimal results for your compound of interest.
Application Analytes:
Benzoic Acid

Application Detection:
UV Detection