Simple 2D-HPLC using a monolithic silica column for peptide separation.
Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.
Kimura
Hiroshi
H
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Tanigawa
Tetsuya
T
Morisaka
Hironobu
H
Ikegami
Tohru
T
Hosoya
Ken
K
Ishizuka
Norio
N
Minakuchi
Hiroyoshi
H
Nakanishi
Kazuki
K
Ueda
Mitsuyoshi
M
Cabrera
Karin
K
Tanaka
Nobuo
N
D000818:Animals; D002412:Cations; D002417:Cattle; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification; D011108:Polymers / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays
D002412:Cations; D010455:Peptides; D011108:Polymers; D012822:Silicon Dioxide; D014357:Trypsin