Reverse-Phase HPLC for Peptide Purification
High-performance liquid chromatography, HPLC, is the most commonly used technique for peptide purification, especially reverse-phase HPLC, RP-HPLC. In RP-HPLC, peptides are separated based on their hydrophobicity, making it an ideal method for purifying complex peptide mixtures and isolating specific peptide species for further study.
Mechanism of RP-HPLC
In RP-HPLC, peptides are loaded onto a column packed with a hydrophobic stationary phase, typically C18 silica. The mobile phase, consisting of water and an organic solvent like acetonitrile, elutes the peptides based on their hydrophobic interactions with the stationary phase. More hydrophobic peptides elute later than hydrophilic ones. This method provides excellent resolution and is widely used in peptide synthesis, proteomics, and pharmacological research.1
Applications in Peptide Research
RP-HPLC is indispensable in the purification of synthetic peptides, ensuring high purity for biological assays. It is also used in peptide mapping, a technique that involves the enzymatic digestion of proteins into peptides, followed by HPLC separation to analyze protein structure and modifications. This is critical in both basic research and drug development.2
Conclusion
RP-HPLC is a robust and versatile technique for peptide purification and analysis, offering high resolution and reproducibility. It is a key tool in peptide synthesis, proteomics, and quality control for peptide-based therapeutics.
Citations and Links
1. Mant, Colin T., and Robert S. Hodges. “Analysis of Peptides by Reverse-Phase HPLC.” Methods in Molecular Biology, vol. 145, 1999, pp. 3–22. doi:10.1385/1-59259-682-5:3.
2. Zhang, Zhongqi. “Peptide Mapping by Liquid Chromatography-Mass Spectrometry.” Journal of the American Society for Mass Spectrometry, vol. 24, no. 5, 2013, pp. 757–765. doi:10.1007/s13361-012-0565-0.
Illustrations
Reverse-Phase HPLC for Peptide Purification1