Peptide Purification by Reversed‐Phase Chromatography

Abstract

Reversed-phase high-performance liquid chromatography (RP-HPLC) has dominated peptide purification since the late 1970s due to its efficient separation and ease of use with volatile compatible buffer systems for numerous applications [1,2]. RP-HPLC separation of chains of amino acids (peptides and proteins) is based predominantly on reversible hydrophobic interactions between the amino acid side chains with the hydrophobic surface of the chromatographic stationary phase (solid particles or packings) in competition with the flowing mobile phase (chromatographic buffer or solvent). Separation is performed by binding peptides onto the hydrophobic RP stationary phase in polar conditions (e.g., water) and eluting in nonpolar solvents (e.g., organic solvent such as acetonitrile) in the presence of a pH modifying agent. As peptides bind to the stationary phase, the amount of hydrophobic area on the surface of the peptide(s) exposed to the solvent is minimized. Thus, the degree of organized water is decreased with a concomitant favorable increase in entropy of the system. For this reason, it is advantageous, under these solvent conditions, for peptides to associate with the stationary phase upon loading onto the RP-HPLC column. Mobile phase composition is then subsequently modified so that bound peptides are differentially eluted (desorbed) back into the mobile phase. The order of peptide desorption is simply based on their relative hydrophobicity (i.e., least hydrophobic proteins elute first, followed by peptides in increasing order of heir surface hydrophobicity). Peptide elution is usually performed by increasing the organic solvent concentration (either in a stepwise fashion or in a gradient manner). By these means peptides, which are concentrated or trace enriched during the binding and separation process, are eluted in a purified and concentrated form ready for collection or subsequent analysis using hyphenated techniques such as mass spectrometry.

Patchwork Protein Chemistry: A Practitioner's Treatise on the Advances in Synthetic Peptide Stitchery

“…[27] Even purified products can therefore remain heterogeneous, despite appearing homogeneous on evaluation by reversed-phase HPLC. [28][29][30][31] From silver couplings… Many contemporary tools are available nowadays to the peptide chemist, with notable developments including the use of pseudoproline residues or microwave-assisted procedures. [32][33][34] However, the concerns described above argue against the preparation of species of the size of proteins by linear solidphase coupling of amino acid monomers.…”