APPLICATION OF CAPILLARY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY TO HIGH-SENSITIVITY PROTEIN-SEQUENCE ANALYSIS
- Author(s)
- Moritz, RL; Simpson, RJ;
- Details
- Publication Year 1992-05-22,Volume 599,Issue #1-2,Page 119-130
- Journal Title
- JOURNAL OF CHROMATOGRAPHY
- Publication Type
- Journal Article
- Abstract
- A continuous gradient elution method for capillary column (<0.32 mm I.D.)liquid chromatography was developed. Gradient eluent from a microbore liquid chromatograph was split ahead of the injector so that an accurate percentage (2-3%) of the mobile phase delivered by the pump flowed through the capillary column. The outlet of the column was connected to a length of 0.075 mm I.D. fused-silica capillary tubing which. in turn. was connected to a 6-mm optical path length longitudinal capillary flow cell. Fused-silica capillary columns of 0.32 mm I.D. were slurry-packed efficiently with 7-mu-m spherical, 300 angstrom pore size, C-8 bonded-phase particles, and evaluated in terms of their ability to resolve mixtures of proteins. peptides or phenylthiohydantoin (PTH)-amino acid derivatives. The gradient elution profiles agreed with those obtained using microbore (<2.1 mm I.D.) and larger bore columns. The minimum detectable amounts for proteins and PTH-amino acids on 0.32 mm I.D. capillary columns were 50 pg and 25 fmol, respectively. At a flow-rate of 3.6-mu-l min. proteins and peptides were recovered from the capillary columns in volumes of about 2-8-mu-l. The use of a multiple-wavelength, forward-optics detector for identifying tryptophan- and tryosine-containing peptides is discussed.
- Publisher
- ELSEVIER SCIENCE BV
- Keywords
- BOMBARDMENT MASS-SPECTROMETRY; AMINO-ACID-SEQUENCE; POLYVINYLIDENE DIFLUORIDE MEMBRANES; RECOVERING SUBNANOMOLE AMOUNTS; TWO-DIMENSIONAL GELS; CELL ANTIGEN PC-1; MICROBORE COLUMNS; GRADIENT ELUTION; SEPARATING PROTEINS; GROWTH-FACTOR
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Creation Date: 1992-05-22 12:00:00