Online comprehensive two‐dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, life, and polymer sciences). Two‐dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two‐dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high‐molecular‐weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one‐dimensional liquid chromatography, two‐dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two‐dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two‐dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two‐dimensional liquid chromatography separations.

On-line comprehensive two-dimensional liquid chromatography (LC×LC) offers ways to achieve high-performance separations in terms of peak capacity (exceeding 1000) and additional selectivity to realize applications that cannot be addressed with one-dimensional chromatography (1D-LC). However, the greater resolving power of LC×LC comes at the price of higher dilutions (thus reduced sensitivity) and, often, long analysis times (>100 min). The need to preserve the separation attained in the first dimension (1D) causes greater dilution for LC×LC, in comparison with 1D-LC, and long analysis times to sample the 1D with an adequate number of second dimension separations. A way to significantly reduce these downsides is to introduce a concentration step between the two chromatographic dimensions. In this work we present a possible active-modulation approach to concentrate the fractions of 1D effluent. A typical LC×LC system is used with the addition of a dilution flow to decrease the strength of the 1D effluent and a modulation unit that uses trap columns. The potential of this approach is demonstrated for the separation of tristyrylphenol ethoxylate phosphate surfactants, using hydrophilic-interaction liquid chromatography and reversed-phase liquid chromatography. Using this modified LC×LC system we were able to halve the analysis time necessary to obtain a similar degree of separation efficiency with respect to UHPLC based LC×LC and of 5 times with respect to HPLC instrumentation (40 compared with 80 and 200 min, respectively), while at the same time reducing dilution (DF of 142, 299 and 1529, respectively) and solvent consumption per analysis (78, 120 and 800 mL, respectively).