1A-O1-1620 PDF
Hydrophilic Interaction Chromatography Using Meter-scale Monolithic Silica Capillary Column for Proteomics LC-MS
A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10-13 m2, which are comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m-long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m-long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we obtained a separation efficiency comparable with that of nanoLC-MS systems using C18-modified monolithic silica columns in reversed-phase liquid chromatography (RPLC) mode when a shallow gradient was applied. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS as well as over 5-fold increase in the peak response on average for commonly identified tryptic peptides due to the higher organic content of the mobile phase of HILIC than RPLC, it is useful as a complementary tool to increase proteome coverage in proteomics studies.