Evaluation of retention and selectivity for positional isomers in hydrophilic interaction liquid chromatography using...

Hydrophilic interaction chromatography has been shown to be able to separate positional isomers on polar stationary phases. However, detailed mechanisms for positional isomer separation are not clearly understood due to the lack of methodologies that can differentiate different mechanisms (i.e., partitioning, adsorption, electrostatic interactions) involved in HILIC. In addition, the driving force for selectivity has not been investigated due to unclear retention mechanisms. In this study, we have applied the quantitative retention assessment methodology to evaluate the retention mechanisms and separation of various positional isomers of hydroxybenzenes and dihydroxybenzoic acids. The study results indicate that adsorption is the main retention mechanism for hydroxybenzene isomers, but partitioning becomes more significant at high ammonium acetate concentrations. For dihydroxybenzoic acid isomers, partitioning is the main retention mechanism in the entire range of ammonium acetate concentration used in this study and adsorption is a minor mechanism. On the positively charged stationary phase, electrostatic attraction makes a moderate contribution to the observed retention at low salt concentration. On the negatively charged stationary phase, electrostatic repulsion significantly reduces retention below 10 mM ammonium acetate. Quantitative assessment of retention mechanisms makes it feasible to identify the driving force for selectivity for the positional isomers. Mechanistic-based evaluation indicates that adsorption underscores the selectivity for resorcinol/catechol and phloroglucinol/1,2,4-trihydroxybenzene isomer pairs, but partitioning drives the selectivity for 1,2,4-trihydroxybenzene and pyrogallol. Selectivity for dihydroxybenzoic acid isomers is driving primarily by adsorption on the negatively charged zwitterionic phase; however, electrostatic repulsion plays a significant role at low ammonium acetate concentrations. The driving force for selectivity on the positively charged mixed-mode phase is not clear, but adsorption is likely to play an important role. In addition, the log P data for these isomers found in public databases is often based on calculation and it is difficult to use to evaluate the relative polarity of these isomers. The partitioning coefficients measured in this study can potentially be used for this purpose.