Extending the separation space in online comprehensive two-dimensional liquid chromatography-mass spectrometry...

The complete characterization of food samples is often hampered by the polarity range of their constituents. Although comprehensive two-dimensional liquid chromatography (LC × LC) offers an enhanced separation space compared to one-dimensional LC, a single pair of columns is often unable to retain compounds across this entire range, leaving part of the separation space unused. In this work, an online multi-two-dimensional (²D) LC × LC method was developed to extend the separation space achievable with LC × LC, using tamarind (Tamarindus indica L.) pericarp as a chemically diverse model sample. An orthogonality study, screening nine reversed-phase liquid chromatography (RPLC) and hydrophilic liquid interaction chromatography (HILIC) columns under various mobile-phase conditions, was used to select the most promising conditions. A HILIC × RPLC method was able to separate moderately polar constituents well, however, leaving the most polar compounds poorly retained in the second dimension. A second, HILIC-based second-dimension method was therefore developed for this polar fraction. Both second-dimension columns were combined in a single analysis through a zero-flow column-switching strategy, allowing each column to be operated under its optimal conditions. The multi-²D configuration increased the fraction surface coverage from 0.507 to 0.533 for the individual LC × LC methods to 0.868, and yielded the highest effective peak capacity (339).