Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column

Area of Science:

• Analytical Chemistry
• Separation Science
• Chromatography

Background:

• Capillary electrochromatography (CEC) offers unique separation capabilities.
• Hydrophobic monolithic columns provide a distinct stationary phase for chromatography.
• Optimizing CEC for peptide separation requires careful consideration of column properties and mobile phase composition.

Purpose of the Study:

• To develop a capillary electrochromatography (CEC) method for ionic compound separation using a neutrally hydrophobic monolithic column.
• To investigate the separation mechanism of peptides based on electrophoretic mobility and hydrophobic interactions.
• To compare the separation selectivity of CEC with capillary zone electrophoresis (CZE).

Main Methods:

• Preparation of a monolithic column via in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate, creating a C12 hydrophobic stationary phase.
• Development of a CEC mode utilizing electrophoretic mobility for separation on the hydrophobic monolithic column.
• Investigation of mobile phase composition effects on peptide retention at low pH.

Main Results:

• Poor electroosmotic flow (EOF) was observed in the hydrophobic monolithic column, even at pH 8.0.
• Peptides were successfully separated based on electrophoretic mobility and hydrophobic interactions, yielding high column efficiency (up to 150,000 plates/meter) and good reproducibility (RSD <0.5%).
• Significant differences in selectivity were observed between CEC and CZE, with CEC successfully separating peptide isomers intractable by CZE.

Conclusions:

• The developed CEC method provides high efficiency and reproducibility for peptide separation.
• The hydrophobic monolithic column enables separation selectivity distinct from CZE, particularly for challenging peptide isomers.
• This approach offers a valuable alternative for complex peptide mixture analysis.