Micellar and sub-micellar ultra-high performance liquid chromatography of hydroxybenzoic acid and phthalic acid positional isomers

Abstract

Micellar liquid chromatography (MLC) has been used primarily for the separation of neutral analytes of varying polarities, most commonly phenols and polyaromatic hydrocarbons, but does not seem to have been used to study aromatic hydroxy acids in detail. We have studied the separation of hydroxybenzoic acid mixtures, including monohydroxybenzoic and dihydroxybenzoic acid positional isomers by MLC. Sodium dodecylsulfate (SDS) is investigated as the modifying surfactant on a C18 ultra-high performance liquid chromatography (UHPLC) column (100×2.1mm, 1.8μm). The addition of only SDS (no organic solvent) to the mobile phase reduced the influence of hydrophobic interactions while improving the retention times, resolution, and peak shapes, even at concentrations below the critical micellization concentration (CMC). The UHPLC separation of 7 hydroxybenzoic acids, including 6 dihydroxybenzoic acid positional isomers and one trihydroxybenzoic acid, is achieved with high efficiency using 0.1% SDS in 1.84mM sulfuric acid (pH 2.43) mobile phase, in less than 6min with a flow rate of 0.3mLmin−1, and in less than four min with a flow rate of 0.7mLmin−1. Six monohydroxybenzoic acid isomers are also effectively separated by MLC, using a 0.5% SDS mobile phase modifier, in less than 20min with a flow rate of 0.3mLmin−1, and in less than 14min with a flow rate of 0.7mLmin−1. The 3 phthalic acid isomers could be separated using a similar mobile phase and flow rates in less than 6 and 4min. Solute-micelle equilibrium constants and partition coefficients are calculated for 6 monohydroxybenzoic acids based on a plot of MLC retention factor vs. mobile phase micelle concentration. All aromatic acid isomers studied can be classified as binding solutes in the MLC retention mechanism. Less effective separations are observed with shorter chain surfactants, leading to higher retention times and poor peak shapes. It is concluded that increasing chain length led to more efficient MLC separations, and SDS is the preferred modifying surfactant for the examined separation.