Recent advances in applications of gradient elution in two-dimensional liquid chromatography are reviewed. Benefits of simultaneous programmed elution in two-dimensional liquid chromatography are shown, with emphasis on fast gradient elution in second dimension using various gradient types and profiles. The theory of gradient elution is shortly overviewed with attention to its impacts on two-dimensional separations. Various two-dimensional gradient systems are compared in terms of peak capacity and peak production rate: off-line, stop-and-go and real-time comprehensive approaches. Selection of stationary phases in combination with gradient elution and the effects of gradient elution on improved regularity of two-dimensional space coverage, orthogonality and peak capacity are discussed. Not only two-dimensional reversed-phase systems, but also other HPLC modes such as HILIC, ion-exchange or normal-phase chromatography with organic mobile phases are included. Fraction modulation in gradient elution setups, mobile phase compatibility and solvent effects in the fraction transfer between the first and the second dimension are addressed. Design and optimization of two-dimensional gradient separation systems for particular ordered and non-ordered samples are dealt with in detail. The discussed principles are illustrated by practical examples.
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