Liquid Chromatography - Liquid Chromatography Applications 4

The packing is silica based but is contained in a short column 3.3 cm long, 4.6 mm in diameter and packed with particles 3 mm in diameter. The expected efficiency of the column (when operated at the optimum velocity) would be about 5,500 theoretical plates. This is not a particularly high efficiency and so the separation depends on the phases chosen to provide the necessary selectivity and an appropriate gradient program.

The selectivity was achieved using a complex mixture of ionic and dispersive interactions between the solutes and the stationary phase and ionic, polar and dispersive forces between the solutes and the mobile phase. The initial solvent in the gradient program was a 1% acetic acid and 1 mM tetrabutyl ammonium phosphate buffered to a pH of 2.8. The tetrabutyl ammonium salt would be adsorbed strongly on the reverse phase and thus acted as an adsorbed ion exchanger. During the program, acetonitrile was added to the solvent and initially this increased the dispersive interactions between the solute and the mobile phase.

As the acetonitrile concentration became higher, however, the tetrabutyl ammonium salt would be desorbed from the reverse phase reducing the ionic interactions of the solutes with the stationary phase. At even higher concentrations of acetonitrile, the tetrabutyl ammonium salt would be completely desorbed and the interactions of the solutes with the stationary phase would become almost exclusively dispersive. This is an example where a complex phase system was necessary because there was limited column efficiency available. It is likely that a column with intrinsically more efficiency might achieve the separation with a much simpler solvent system and a more straightforward solvent program.

An example of the use of native silica is given by for the analysis of Darvocet® and its generic equivalent formulation. Darvocet® is an acetaminophen product in which the active ingredient (and other material in the medicine) are weakly polar and, consequently, lend themselves to separation on a strongly polar stationary phase such as silica gel. The analysis is depicted in figure 59. The analysis is completed in less than 4 minutes using a short column 3.3 cm long and 4.6 mm in diameter. The silica packing had a particle size of 3 m providing a maximum efficiency of about 5,500 theoretical plates.