Characterization and optimization by experimental design of a liquid chromatographic method for the separation of...

Traditionally, the determination of hydroxylated polychlorinated biphenyls (OH-PCBs) has been carried out by gas chromatography (GC). However, the gas chromatographic behavior and sensitivity of this type of hydroxylated compounds are not always satisfactory, hence a prior derivatization of the OH-PCBs must be performed. Therefore, the development of liquid chromatographic methods should prove to be a very interesting task aimed at dealing with the instrumental determination of OH-PCBs. Taking into account that octadecylsilane stationary phases are not the most adequate for the separation of isobaric compounds, an amide-type column has been tested. For the development of the method, the Response Surface Methodology was used, based on a Box-Wilson Central Composite experimental design. The initial content of methanol in the mobile phase, the gradient time, and the concentration and the pH value of the buffer were chosen as relevant experimental parameters. A global optimum was obtained by selecting the elution time, the sensitivity and the overall resolution as responses to optimize. The developed method for liquid chromatography presented a very good resolution and sensitivity, and a reasonably short analysis time. In addition, a retention study was conducted in order to survey the different interactions that take place in the separation process, showing that hydrogen bonding is the main interaction between OH-PCBs and the amide-type stationary phase. However, a substantial contribution of dispersion forces was present in methanol contents in the mobile phase below 65%. Besides, the pH value of the mobile phase was found to be the most important parameter to control the hydrogen bond forces and therefore, to regulate the OH-PCBs separation.