Peptides reversed-phased chromatography

M. Stromqvist, Peptide mapping using combinations of size-exclusion chromatography, reversed-phase chromatography and capillary electrophoresis , 7. Chromatogr. 667 304-310(1994). 

Recently, new approaches of sorbent construction for reversed-phase chromatography have been developed. Silicas modified with hydrocarbon chains have been investigated the most and broadly utilized for these aims. Silica-based materials possess sufficient stability only in the pH 2-8 range. Polymeric HPLC sorbents remove these limitations. Tweeten et al. [108] demonstrated the application of stroongly crosslinked styrene-divinylbenzene resins for reversed-phase chromatography of peptides. 

Zhou, N. E., Mant, C. T., and Hodges, R. S., Effect of preferred binding domains on peptide retention in reversed-phase chromatography amphipathic... 

Figure 2.6. LC-tandem mass spectrometry to examine complex mixtures. The mixture of many different proteins is digested to yield peptides and the peptides are resolved into fractions hy cation exchange chromatography followed by reverse phase chromatography. The fractionation steps resolve the peptides into fractions that he processed hy tandem mass spectrometry to yield sequence information suitable for database searching.

Analysis using reversed-phase chromatography at low pH was carried out under conditions typically used for 2DLC-MS/MS analysis of peptides. The mobile phases for other chromatographic conditions were also chosen to be compatible with MS detection, including SCX LC, where the peptides were eluted with volatile ammonium formate buffer. 

These systems rely on various combinations of size-exclusion chromatography, reversed-phase chromatography, and zone electrophoresis to characterize amines, peptides, and proteins (Yamamoto etal., 1989 Bushey and Jorgenson 1990 Larmann et al., 1993, Moore and Jorgenson, 1995 Optick and Jorgenson, 1997). Haleem Issaq reviews these separations in Chapter 16 of this book. 

Since amino acids and nucleotides are all polar and hydrophilic, they will be eluted quickly by the column. The mobile phase (see below) is also selected on the basis of polarity, with a medium- to high-polarity solvent required. The opposite of reverse phase chromatography is normal phase, where the column packing is medium to high polarity and the mobile phase is nonpolar. This technology is generally not applied to the analysis of polar molecules such as amino acids or nucleotides. Some peptides are more hydrophobic, making this method potentially more useful for peptides than for amino acids or nucleotides. 

Figure 4.1 Correlation of predicted and observed retention times in reversed-phase chromatography. The predicted retention times for 58 peptides of 2 to 16 residues in length were obtained by summation of retention coefficients for each residue in the peptide. Retention coefficients were determined from the retention of model synthetic peptides with the structure Ac-Gly-XX-(Leu)3-(Lys)2-amide, where X was substituted by the 20 protein amino acids. (Reproduced from D. Guo, C.T. Mant, A.K. Taneja, and R.S. Hodges, J. Chromatogr., 359 519 [1986]. With permission from Elsevier Science.)...

K. Kalghatgi and C. Horvath, Micropellicular sorbents for rapid reversed-phase chromatography of proteins and peptides, in Analytical Biotechnology, Capillary Electrophoresis, and Chromatography, C. Horvath and J.G. Nikelly (Eds.), American Chemical Society, Washington, D.C., 1990, p. 162. 

Module 3, Column and Mobile Phase Design (CMP). This is the core module for ECAT. It can currently specify i) analytical column and mobile phase constituents for reverse phase chromatography of common classes of organic molecules ii) reverse phase, ion exchange phase and hydrophobic interaction chromatography of proteins and peptides iii) a limited set of specialty classes of molecules best treated by straight phase chromatography (e.g., mono- and disaccharides). The rules for selection of the HPLC detector are under development within Module 3. Some of the rules for detector mobile phase compatibility are already encoded. A set of rules for detector selection is ready but not yet encoded. 

Several researchers have used liquid chromatography in its different modes, such as reversed phase chromatography (RPC), hydrophobic interaction chromatography (HIC), lEC, SEC, and AC, to analyze and fractionate food proteins and peptides. 

This chapter commences with Section 7.1 which deals with reversed-phase chromatography (RPC) and related techniques as applied to synthetic peptides 1-3 A detailed discussion on RPC is presented. Basic considerations are covered as are issues of fundamental physical chemistry. Many examples of chromatography and quantitative relationships are described for peptides and peptide derivatives. There is also an extensive table of naturally occurring peptides that have been isolated and purified by RPC techniques. The section includes many examples of RPC and HPLC profiles of peptidic systems. 

Hydrophobic Effects and Solvophobic Considerations for the Isolation of Peptides by Reversed-Phase Chromatography Methods... 

The Nature of the In k versus [S]m Dependencies and Their Impact on Peptide Separation in Reversed-Phase Chromatography... 

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