The challenge of fully optimizing LC×LC separations is horrendous. Yet, it is essential to address this challenge if sophisticated LC×LC instruments are to be utilized to their full potential in an efficient manner. Currently, lengthy method development is a major obstacle to the proliferation of the technique, especially in industry. A program was developed for the rigorous optimization of LC×LC separations, using gradient-elution in both dimensions. The program establishes two linear retention models (one for each dimension) based on just two LC×LC experiments. It predicts LC×LC chromatograms using a simple van-Deemter model to generalize band-broadening. Various objectives (analysis time, resolution, orthogonality) can be implemented in a Pareto-optimization framework to establish the optimal conditions. The program was successfully applied to a separation of a complex mixture of 54 aged, authentic synthetic dyestuffs, separated by ion-exchange chromatography and ion pair chromatography. The main limitation experienced was the retention-time stability in the first (ion-exchange) dimension. Using the PIOTR program LC×LC method development can be greatly accelerated, typically from a few months to a few days.
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