Fundamental issues in the implementation of a two-dimensional LCxLC separation - Candice Grivel, Amélie Dechenaux, Sabine Heinisch

This article aims at presenting the fundamental steps when implementing a two-dimensional on-line separation (2D-LC) of complex samples.

Firstly, an approach is proposed to make the search for orthogonal conditions easy. It is based on the treatment of retention data acquired from generic gradient separations of test mixtures in various systems (stationary phase / mobile phase / temperature). The regression coefficient of 2D retention plots and the practical peak capacity are both determined. The instrumental design and the choice of analytical conditions are also discussed. This article demonstrates that an ultra-fast second dimension combining high temperature and ultra high pressure (HT-UHPLC) is very attractive. Finally, relevant choices are illustrated by a 2D separation of a protein digest.