HPLC Separation of Dequalinium and Benzonitrile on Different Columns

Dequalinium, also known as Dequadin, is a disinfectant and antiseptic composed of two quaternary ammonium groups linked by a C10 hydrophobic chain. Due to its strong basicity and the presence of quaternary amines, dequalinium often exhibits poor peak shape and significant tailing (symmetry factor < 0.5, USP tailing > 2) when analyzed on traditional reverse-phase (RP) HPLC columns. These issues arise from interactions between the quaternary groups and residual silanol sites on the silica surface.

In this HPLC application, dequalinium is effectively analyzed using the Zodiac HST RP mixed-mode column, which provides excellent peak symmetry and high efficiency. The Zodiac HST RP column incorporates a hydrophobic chain and a basic functional group near the silica surface. This configuration creates a shielding effect that minimizes interactions between the analyte and acidic silanol groups, resulting in sharper peaks for basic compounds like dequalinium.

This method is particularly suitable for analyzing and quantifying hydrophobic amines, especially when conventional RP columns fail to deliver acceptable peak shapes. A comparative evaluation with several modern LC columns—such as Zorbax (Agilent), XTerra (Waters), and Gemini (Phenomenex)—demonstrates that the Zodiac HST RP column performs reliably for the separation of hydrophobic quaternary amines and diamines.

Additionally, benzonitrile is included in the mobile phase to enhance packing performance and peak shape for neutral compounds. The method employs trifluoroacetic acid (TFA) as a mobile phase additive, although other acids and buffers may be used based on the detection requirements.