Two-dimensional liquid chromatography with reversed phase in both dimensions: A review

Two-dimensional liquid chromatography (2D-LC), and in particular comprehensive two-dimensional liquid chromatography (LC×LC), offers increased peak capacity, resolution and selectivity compared to one-dimensional liquid chromatography. It is commonly accepted that the technique produces the best results when the separation mechanisms in the two dimensions are completely orthogonal; however, the use of similar separation mechanisms in both dimensions has been gaining popularity as it helps avoid difficulties related to mobile phase incompatibility and poor column efficiency. The remarkable advantages of using reversed phase in both dimensions (RPLC×RPLC) over other separation mechanisms made it a promising technique in the separation of complex samples. This review discusses some physical and practical considerations in method development for 2D-LC involving the use of RP in both dimensions. In addition, an extensive overview is presented of different applications that relied on RPLC×RPLC and 2D-LC with reversed phase column combinations to separate components of complex samples in different fields including food analysis, natural product analysis, environmental analysis, proteomics, lipidomics and metabolomics.