Selectivity of the Separation of Isomeric Chlorogenic Acids under the Conditions of Reversed-Phase HPLC

Abstract

The selectivity of the separation of isomeric chlorogenic acids often found in natural samples (3, 4, and 5-caffeoylquinic acids, 3CQA, 4CQA, and 5CQA, respectively) under the conditions of reversed-phase chromatography on stationary phases of different brands, Kromasil (C18, C8, and C4), Symmetry C18, Reprosil-Pur C18-AQ, Nucleosil RPC18, and Diasfer C18, is studied. The results obtained favor the retention mechanism with predominant surface adsorption. The change in the selectivity of separation of a pair of 5CQA and 4CQA correlates with a change in the activity of residual silanol groups. At their low activity, the order of elution 3CQA < 5CQA < 4CQA does not depend on the mode (isocratic or gradient), the type of organic modifier, and the addition of an acid (formic, orthophosphoric) to the mobile phase. With an increase in the activity of silanol groups, the separation of the pair of 5CQA and 4CQA deteriorates up to the inversion of retention times. A Kromasil 100-5C18 column (250 × 4.6 mm) was chosen for a reliable separation of three acids in plant samples (instant coffee, juice, and “Mate” tea).