Protocols for finding the most orthogonal dimensions for two-dimensional high performance liquid chromatography Academic Article uri icon


  • The selection of two high performance liquid chromatography (HPLC) columns with vastly different retention mechanisms is vital for performing effective two-dimensional (2D-) HPLC. This paper reports on a systematic method to select a pair of HPLC columns that provide the most different separations for a given sample. This was completed with the aid of a HPLC simulator that predicted retention profiles on the basis of real experimental data, which is difficult when the contents of sample matrices are largely-or completely-unknown. Peaks from the same compounds must first be matched between chromatograms to compare the retention profiles and optimised 2D-HPLC column selection. In this work, two methods of matching peaks between chromatograms were explored and an optimal pair of chromatography columns was selected for 2D-HPLC. First, a series of 17 antioxidants were selected as an analogue for a coffee extract. The predicted orthogonality of the standards was 39%, according to the fractional surface coverage 'bins' method, which was close to the actual space utilisation of the standard mixture, 44%. Moreover, the orthogonality for the 2D-HPLC of coffee matched the predicted value of 38%. The second method employed a complex sample matrix of urine to optimise the column selections. Seven peaks were confidently matched between chromatograms by comparing relative peak areas of two detection strategies: UV absorbance and potassium permanganate chemiluminescence. It was found that the optimal combinations had an orthogonality of 35% while the actual value was closer to 30%.


  • Bassanese, DN
  • Holland, BJ
  • Conlan, XA
  • Francis, PS
  • Barnett, NW
  • Stevenson, PG

publication date

  • 2015