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

✍ Scribed by Martin Gilar; Petra Olivova; Amy E. Daly; John C. Gebler

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 442 KB
• Volume: 28
• Category: Article
• ISSN: 1615-9306
• DOI: 10.1002/jssc.200500116

✦ Synopsis

Abstract

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.