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Peptide separation by pH-zone-refining countercurrent chromatography

Y Ma1, Y Ito

  • 1Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 1676, USA.

Journal of Chromatography. A
|May 30, 1997
PubMed
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This study demonstrates effective peptide separation using countercurrent chromatography (CCC) with a novel stationary phase modifier. The method successfully isolated hydrophobic and hydrophilic peptides, including complex biomolecules like insulin.

Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Separation Science

Background:

  • Countercurrent chromatography (CCC) is a liquid-liquid separation technique.
  • Separating peptides without protecting groups presents challenges in chromatography.
  • Ion-pair reagents can modify stationary phases to enhance separation.

Purpose of the Study:

  • To develop a method for separating unprotected peptides using CCC.
  • To investigate the use of di-(2-ethylhexyl)phosphoric acid (DEHPA) as a stationary phase modifier.
  • To optimize CCC conditions for separating hydrophobic and hydrophilic peptides.

Main Methods:

  • pH-zone-refining countercurrent chromatography (CCC) was employed.
  • Di-(2-ethylhexyl)phosphoric acid (DEHPA) was used as an ion-pair reagent in the stationary phase.

Related Experiment Videos

  • Solvent system hydrophobicity and DEHPA concentration were optimized based on analyte hydrophobicity.

    • Main Results:

    • Successful separation of peptides lacking protecting groups was achieved.
    • Hydrophobic and hydrophilic dipeptides were effectively separated under optimized conditions.
    • The method demonstrated scalability for gram-quantity separations of bacitracin complex and bovine insulin.

    Conclusions:

    • DEHPA-modified CCC is a viable technique for separating unprotected peptides.
    • Optimizing DEHPA concentration and solvent hydrophobicity is crucial for effective separation.
    • This method offers a promising approach for large-scale peptide purification.