3DHPLC

3DHPLC, or three-dimensional high-performance liquid chromatography, is a multidimensional separation technique that integrates three distinct chromatographic separations in series to improve resolution and peak capacity when analyzing complex sample mixtures. By combining orthogonal separation mechanisms, 3D-HPLC seeks to separate components that are challenging to resolve with a single dimension or even with two dimensions.

A typical 3D-HPLC setup uses three columns connected through valve modules and a modulation strategy. The sample first undergoes separation on the first dimension, with fractions being directed to a second dimension; fractions from the second are then injected into the third dimension for final separation. The chemistries of the three dimensions are chosen to maximize orthogonality, such as reversed-phase (RP) with ion-exchange (IEX) with size-exclusion (SEC) or with HILIC, etc.

Applications include proteomics and peptidomics, metabolomics, lipidomics, natural products, and pharmaceutical analysis, where high complexity requires resolving coeluting species, congeners, isomers.

The approach offers higher peak capacity and improved separation power, aiding detection and identification of trace components. Limitations include longer run times, greater solvent consumption, increased instrumentation complexity, potential sample loss during transfers, and more demanding data analysis and method development.