Effect of the temperature and mobile phase composition on the retention behavior of nitroanilines on ligand-exchange stationary phase

This paper deals with the separation of isomers of nitroaniline by liquid chromatography using the ligand-exchange technique. The chromatographic separations were performed on the ligand-exchanger sporopollenin. The sporopollenin used as support of stationary phase was modified with carboxylated-ethylenediamine matrix and was loaded with cobalt(II) ions. Using the column packed with cobalt(II) loaded carboxylated diaminoethyl sporopollenin [Co(II)-CDAE-S], the retention behavior of 3- and 4-nitroanilines was investigated. The mobile phase used, was a mixture of 0.05 M NH4OH in ethanol–water. The resolution was strongly affected by the presence of ammonium hydroxide in the mobile phase and a concentration of 0.05 M was shown to be necessary for the separation of analytes. To study the effects of temperature on the resolution, column runs were also performed at various temperatures (15–60 °C). With increasing temperature, a decreased interaction between the solutes and the ligand-exchanger was observed. Consequently, the best results were obtained using a mixture of 0.05 M NH4OH in ethanol–water (10:90, v/v) as the mobile phase at a column temperature of 35 °C. Ligand-exchange chromatography on the Co(II)-CDAE-S could be a useful alternative method for the separation of nitroaniline.