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Separation by capillary electrophoresis followed by dynamic elution
P Camilleri1, G N Okafo, C Southan
1SmithKline Beecham Pharmaceuticals, Research and Development, The Frythe, Welwyn, Herts, United Kingdom.
Analytical Biochemistry
|July 1, 1991
Summary
Using deuterium oxide (D2O) buffers in capillary electrophoresis (CE) minimizes peptide diffusion after separation. This method enables the isolation of peptides for sequencing with high resolution.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Separation Science
Background:
- Capillary electrophoresis (CE) is a powerful separation technique.
- Peptide diffusion after CE can lead to loss of resolution during subsequent elution.
- Efficient isolation of peptides is crucial for downstream applications like sequencing.
Purpose of the Study:
- To investigate the behavior of peptides after capillary electrophoresis followed by pressure-elution.
- To determine if deuterium oxide (D2O) buffers can improve peptide retention and resolution post-CE.
- To establish a simple, effective method for isolating picomole quantities of peptides for sequence analysis.
Main Methods:
- Capillary electrophoresis (CE) was performed using D2O-based buffer solutions.
- Peptides were eluted under pressure using a dynamic flow system.
- A two-step process involving low-voltage CE, power-off, and syringe pump elution was developed.
Main Results:
- D2O-based buffers significantly restricted peptide diffusion compared to H2O-based buffers.
- The developed two-step method retained excellent separation characteristics.
- Picomole quantities of peptides were successfully isolated with minimal loss of resolution.
Conclusions:
- The use of D2O buffers in CE is effective in minimizing post-separation peptide diffusion.
- A simple two-step CE and dynamic flow elution process allows for high-resolution peptide isolation.
- This technique is suitable for the preparation of peptides for sensitive sequence determination.