Efficiency enhancements in micellar liquid chromatography through selection of stationary phase and mobile phase organic modifier.

摘要

Micellar liquid chromatography (MLC) as a separation science technique remains hindered by reduced chromatographic efficiency compared to reversed phase liquid chromatography using hydro-organic mobile phases. The reduced efficiency is linked to the adsorption of surfactant monomers onto the stationary phase, resulting in a slow mass transfer of the analyte within the interfacial region of the mobile phase and stationary phase. The effect of various bonded stationary phases and silica pore sizes on efficiency in MLC was evaluated using an array of twelve liquid chromatography columns, including large-pore short alkyl chain, non-porous, superficially porous, and perfluorinated stationary phases. The effect of organic micellar mobile phase additives was also evaluated using combinations of 1-propanol, 1-butanol, 1-pentanol, and triethylamine. A simplified equation for calculation of A' and C' terms from reduced plate height (h) versus reduced velocity (nu) plots was developed to compare efficiency data obtained with different columns and mobile phases. Analyte diffusion coefficients needed for the h versus nu plots were determined by the Taylor-Aris dispersion technique. Surfactant adsorption isotherms were measured for five columns with three micellar mobile phases to further understand the relationship between adsorbed surfactant, mobile phase additive, and column efficiency. Clear improvements in efficiency were observed with addition of 2% (v/v) triethylamine to 1-butanol modified aqueous micellar mobile phase in combination with the use of short alkyl chain, wide-pore silica columns, specifically, Nucleosil C4, 1000 A pore size. This finding is supported by lower amounts of surfactant adsorbed onto the stationary phase when triethylamine is present in the mobile phase compared to surfactant only, or 1-butanol modified mobile phase. In a separate series of experiments, elevated column temperatures were evaluated to determine the effect of temperature on efficiency. Efficiency improvements from 9% to 58% were observed for different columns over the temperature range of 40 to 70°C. Finally, a quantitative method of direct injection of equine serum for detection of banned non-steroidal anti-inflammatory drugs in equestrian events was developed to take advantage of the observed enhancements in efficiency in the area of greatest benefit for MLC, the direct injection of physiological fluids.