HPLC Analysis of Benzonitrile, Methyl Paraben, Propyl Paraben and Toluene on Mixed-Mode and Reverse Phase Columns

Parabens, widely used as preservatives in pharmaceutical and cosmetic formulations, are esters of p-hydroxybenzoic acid. A method for separating methyl paraben, propyl paraben, benzonitrile, and toluene was developed using the Zodiac HST TH column. These neutral compounds are primarily retained via a reversed-phase mechanism. Unlike traditional reversed-phase columns, the Zodiac HST TH column exhibits pH-dependent retention due to the ionizable groups embedded in the stationary phase.

The column incorporates a C12 carbon chain and a carboxylic acid group with a pKa of 4. At low pH (pH 2, using TFA), the carboxylic acid group remains non-ionized, increasing the column’s hydrophobic character and influencing retention. This pH responsiveness allows fine-tuning of separation for basic, acidic, and neutral compounds. The method is compatible with multiple detection techniques including UV, ELSD, CAD, and LC/MS, making it a versatile approach for comprehensive paraben and solvent analysis.