Interpretive Approaches to Optimize Serially-Coupled Columns in Reversed-Phase Liquid Chromatography

The serial coupling of chromatographic columns of controlled lengths containing different stationary phases multiplies the available selectivity in reversed-phase liquid chromatography, which expands the probabilities of success in the separation of complex samples. However, the full exploitation of the separation capability of coupled columns requires a reliable predictor system of solute retention, peak profile and resolution, for both isocratic and gradient elution. This work summarizes the modelling and optimization strategies needed to implement a separation with serially-coupled columns, offering as example the analysis of a mixture of 15 sulfonamides with four columns of different nature (two C18 columns, a phenyl phase and a cyano phase). None of these columns was able to reach good resolution, but when properly combined, all compounds were resolved in an acceptable analysis time.