Plant Growth Regulators

An example of the use of a C18 reverse phase column that separates substances purely on the basis of dispersive interactions is shown in figure 47. The packing is incorporated in a short column 3.3 cm long, 4.6 mm in diameter and packed with particles 3 mm in diameter. The example of its use is in the separation of mixture of growth regulators The efficiency expected from such a column when operated at the optimum velocity would be about 5,500 theoretical plates. This is not a particularly high efficiency and, as a consequence, the separation relied heavily on the phases selected and the gradient employed.

Courtesy of Supelco Inc.

Figure 47 The Separation of a Mixture of Growth Regulators on a C18 Reverse Phase C18 Column

The separation was achieved by a complex mixture of competitive interactions, ionic and dispersive interactions between the solutes and the stationary phase and ionic, polar and dispersive between the solutes and the mobile phase. The gradient started with an initial solvent mixture of 1% acetic acid and 1 mM tetrabutyl ammonium phosphate buffered to a pH of 2.8. Initially 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 increased, however, the concentration of adsorbed tetrabutyl ammonium salt would also be reduced and it would be eluted from the reverse phase with a resulting reduction in the ionic interactions of the solutes with the stationary phase. At higher concentrations of acetonitrile, the tetrabutyl ammonium salt would be completely removed and the interactions of the solutes with the stationary phase would become almost exclusively dispersive. This is an example where the phase system is complex and is required to be so, because limited column efficiency demanded clever phase selection to obtain the necessary differential retention.