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.

Online 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 a combination of hydrophilic interaction and reversed-phase liquid chromatography. The modified LC × LC system enabled us 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).

Stationary-phase-assisted modulation is used to overcome one of the limitations of contemporary comprehensive two-dimensional liquid chromatography, which arises from the combination of a first-dimension column that is typically narrow and long and a second-dimension column that is wide and short. Shallow gradients at low flow rates are applied in the first dimension, whereas fast analyses (at high flow rates) are required in the second dimension. Limitations of this approach include a low sample capacity of the first-dimension column and a high dilution of the sample in the complete system. Moreover, the relatively high flow rates used for the second dimension make direct (splitless) hyphenation to mass spectrometry difficult. In the present study we demonstrate that stationary-phase-assisted modulation can be implemented in an online comprehensive two-dimensional LC (LC × LC) setup to shift this paradigm. The proposed active modulation makes it possible to choose virtually any combination of first- and second-dimension column diameters without loss in system performance. In the current setup, a 0.30 mm internal diameter first-dimension column with a relatively high loadability is coupled to a 0.075 mm internal diameter second-dimension column. This actively modulated system is coupled to a nanoelectrospray high-resolution mass spectrometer and applied for the separation of the tryptic peptides of a six-protein mixture and for the proteome-wide analyses of yeast from Saccharomyces cerevisiae. In the latter application, about 20000 MS/MS spectra are generated within 24 h analysis time, resulting in the identification of 701 proteins.

Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (∼1.5 μg).

Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid chromatographic separation of sugar phosphates, such as hexose and pentose phosphates, 2- and 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, as well as glucosamine 1- and 6-phosphate utilizing mixed-mode chromatography with reversed-phase/weak anion-exchangers and a charged aerosol detector. The best results were obtained when the reversed-phase/weak anion-exchanger column was operated under hydrophilic interaction liquid chromatography elution conditions. The effects of various chromatographic parameters were examined and are discussed on the basis of a simple stoichiometric displacement model for explaining ion-exchange processes. Employed acidic conditions have led to the complete separation of α- and β-anomers of glucose 6-phosphate at low temperature. The anomers coeluted in a single peak at elevated temperatures (>40°C) (peak coalescence), while at intermediate temperatures on-column interconversion with a plateau in-between resolved anomer peaks was observed with apparent reaction rate constants between 0.1 and 27.8×10(-4) s(-1). Dynamic HPLC under specified conditions enabled to investigate mutarotation of phosphorylated carbohydrates, their interconversion kinetics, and energy barriers for interconversion. A complex mixture of six hexose phosphate structural isomers could be resolved almost completely.