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Application of capillary reversed-phase high-performance liquid chromatography to high-sensitivity protein sequence
1Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Parkville, Victoria, Australia.
Journal of Chromatography
|May 22, 1992
Summary
A new continuous gradient elution method was developed for capillary liquid chromatography. This method enables accurate separation and detection of proteins and phenylthiohydantoin-amino acids with high sensitivity.
Area of Science:
- Analytical Chemistry
- Chromatography
- Biochemistry
Background:
- Capillary liquid chromatography (LC) offers advantages in sensitivity and solvent consumption.
- Developing robust gradient elution methods for capillary LC is crucial for complex sample analysis.
Purpose of the Study:
- To develop and evaluate a continuous gradient elution method for capillary LC (0.32 mm I.D.).
- To assess the method's performance in resolving proteins, peptides, and phenylthiohydantoin (PTH)-amino acid derivatives.
Main Methods:
- A continuous gradient elution was achieved by splitting the eluent from a microbore LC system.
- Fused-silica capillary columns (0.32 mm I.D.) were slurry-packed with C8 bonded-phase particles.
- The column outlet was connected to a fused-silica capillary and a capillary flow cell for detection.
Main Results:
- The method achieved efficient separation of proteins, peptides, and PTH-amino acids.
- Minimum detectable amounts were 50 pg for proteins and 25 fmol for PTH-amino acids.
- Protein and peptide recovery was high (2-8 µL) at a flow rate of 3.6 µL/min.
Conclusions:
- The developed continuous gradient elution method is effective for capillary LC.
- The method provides high sensitivity and resolution for analyzing biomolecules.
- It is suitable for identifying tryptophan- and tyrosine-containing peptides using a multiple-wavelength detector.