Selection of Reversed-Phase C18 Separation Media for Bottom-Up Proteomics: Multifactor Comparison of Fully Porous Sorbents Including Separation Selectivity and Carryover Characteristics

Seven popular C18 stationary phases for peptide separation in bottom-up proteomics have been evaluated based on overall peptide retention, column backpressure, separation selectivity and efficiency, number of peptide identifications, and carryover statistics. Under standard 0.1% formic acid water-acetonitrile elution conditions, peptide retention varies within an elution range of ∼3.4% acetonitrile and increases in the following order: XSelect CSH C18 2.5 μm, 130 Å < Luna C18(2) 3 μm, 100 Å < PepMap RSLC C18 2 μm, 100 Å ≪ Reprosil-Pur ODS-3 3 μm, 120 Å < Reprosil-Pur C18 AQ 3 μm, 120 Å < Magic C18 AQ 3 μm, 200 Å < XBridge BEH C18 2.5 μm, 130 Å. While the lower retention on the XSelect was expected due to the embedded positively charged groups, the behavior of Luna C18(2) and PepMap C18 can be explained by the reduction of surface accessibility for small pore size matrices. Pore size was a major contributor to varying separation selectivity. The number of unique peptide identifications is driven by peptide separation efficiency and retentivity. These factors favor sorbents with smaller particle sizes and higher retentivity, such as XBridge BEH C18. Low peptide retentivity and average separation efficiency diminish identification output for Luna C18(2) and PepMap RSLC with 100 Å pore size.