Impact of the probe solutes set on orthogonality evaluation in reverse phase chromatographic systems

Two-dimensional liquid chromatography (2DLC) is a very attractive technique for the characterization of complex samples due to its separation power obtained via the coupling of two separation modes exhibiting different mechanisms, i.e. orthogonality. In reverse phase, orthogonality is mainly governed by three factors: the pH of the mobile phase, the structure of the stationary phase and the nature of the organic modifier. In the present paper, we studied the impact of the nature of the probe solutes on orthogonality evaluation. A set of 63 compounds with various physicochemical properties was used to evaluate 32 reverse phase chromatographic systems (2 pH × 8 stationary phases × 2 organic modifiers). Principle component analysis revealed that the solutes could be split into three subsets according to their charge in the experimental conditions. The factors affecting orthogonality and the magnitude of their effect were shown to depend on the charge of the compounds. For positively charged (basic) compounds, the pH was the most important factor, followed by the nature of the stationary phase. For negatively charged (acidic) compounds, the nature of the stationary phase had the highest impact. For neutral compounds, only the nature of the stationary phase and, to a smaller extent, the organic modifier had an influence. The present study also showed that a reduced set of only 9 test compounds instead of whole set of the 63 could enable an appropriate orthogonality evaluation.