Fabrication of stable packed capillary reversed-phase columns for protein structural analysis
- Author(s)
- Tong, DX; Moritz, RL; Eddes, JS; Reid, GE; Rasmussen, RK; Dorow, DS; Simpson, RJ;
- Details
- Publication Year 1997-07,Volume 16,Issue #5,Page 425-431
- Journal Title
- JOURNAL OF PROTEIN CHEMISTRY
- Publication Type
- Journal Article
- Abstract
- Capillary column (less than or equal to 320-mu m ID) liquid chromatography is an essential tool for the separation and concentration of low-picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrication of stable and efficient 50- to 180-mu m ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300-400 bar) enabled their use for rapid chromatography (>3400 cm/hr; i.e., similar to 40 mu l/min for 200-mu m ID columns) and the loading of large sample volumes (up to 500 mu l) The accurate low flow rates (0.4-4.0 mu l/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119-130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.
- Publisher
- PLENUM PUBL CORP
- Keywords
- PERFORMANCE LIQUID-CHROMATOGRAPHY; MASS-SPECTROMETRY; SEQUENCE-ANALYSIS; PEPTIDES
- Publisher's Version
- https://doi.org/10.1023/A:1026345023941
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 1997-07-01 12:00:00