Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions

J Sep Sci. 2005 Sep;28(14):1694-703. doi: 10.1002/jssc.200500116.


Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis.

Publication types

  • Comparative Study

MeSH terms

  • Chromatography, High Pressure Liquid / instrumentation
  • Chromatography, High Pressure Liquid / methods
  • Hydrogen-Ion Concentration
  • Indicators and Reagents
  • Peptides / chemistry*
  • Peptides / isolation & purification*
  • Proteome / chemistry*


  • Indicators and Reagents
  • Peptides
  • Proteome