Size exclusion chromatography peptides

Peptides are much more difficult to run by size exclusion chromatography than proteins because their solubilities differ greatly. Their shapes also vary from linear to semidefined. For a three peptide standard, Mant and Hodges... 

This section discusses in detail the column types that are available for the size exclusion chromatography of both polar and nonpolar analytes. It first discusses the various columns available for standard nonaqueous size exclusion chromatography. It then reviews the columns available for general size exclusion chromatography using aqueous mobile phases. Finally, it examines the columns designed for size exclusion chromatography of proteins and peptides. 

Separation methods based on size include size exclusion chromatography, ultra-filtration, and ultracentrifugation (see Chapter Appendix). The ionic properties of peptides and proteins are determined principally by their complement of amino acid side chains. Furthermore, the ionization of these groups is pH-dependent. 

Other groups have also used EC and CE to perform non-comprehensive multidimensional separations (15, 16). A three-dimensional separation was performed by Stromqvist in 1994, where size exclusion chromatography (SEC), reverse-phase HPLC, and CZE were used in an off-line manner to separate peptides (17). The most useful information gained from all of these non-comprehensive studies was knowledge of the orthogonality and compatibility of EC and CE. 

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

A. W. Moore, Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/ optically gated capillary zone electrophoresis . Anal. Chem. 67 3456-3463 (1995). 

Detection in 2DLC is the same as encountered in one-dimensional HPLC. A variety of detectors are presented in Table 5.2. The choice of detector is dependent on the molecule being detected, the problem being solved, and the separation mode used for the second dimension. If MS detection is utilized, then volatile buffers are typically used in the second-dimension separation. Ultraviolet detection is used for peptides, proteins, and any molecules that contain an appropriate chromophore. Evaporative light scattering detection has become popular for the analysis of polymers and surfactants that do not contain UV chromophores. Refractive index (RI) detection is generally used with size exclusion chromatography for the analysis of polymers. 

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. 

Aurora Biomolecules dedicates to peptide synthesis (and polyclonal antibody production) for any small quantity purpose. FMOC chemistry (on Perceptive Biosystems Pioneer instruments) is used for peptides synthesis Online monitoring of the coupling efficiencies and HATU activation helps insure that the major component of the synthesis is the correct oligopeptide. Purification is firstly carried out by size exclusion chromatography, and then by HPLC on a PE vision purification workstation. Typically, 20 mg of pure peptide are obtained. The molecular weight of the purified peptide is determined as a final confirmation of quality. 

Figure 9. The effect of storage on protein and peptide composition in cooked ground beef stored in a refrigerator of 4 days (adapted from 7). Upper graph represents the size exclusion chromatography of acidic extracts of fresh, cooked, and cooked-stored beef. Lx)wer graph represents the reverse phase HPLC of peak II from the size exclusion chromatography.

Gonzalez de Llano et al. (47) separated amino acids from low-molecular-weight peptides by means of size-exclusion chromatography on Sephadex G-10, with water as the solvent, as a preparatory step before RP-HPLC analysis of peptides from blue cheeses soluble in 5% PTA (Fig. 1). This technique has also been used (51a) to eliminate the amino acids from the ethanol-... 

The syntheses of heterostranded coiled coils are presented (Section 13.2.2.4), as are templates and conformationally defined peptide libraries (Sections 13.2.4 and 13.2.4.1). Finally, Section 13.1 concludes with a presentation of the methods for characterization of the coiled coils including circular dichroism, microcalorimetry, size-exclusion chromatography, and analytical ultracentrifugation (Section 13.2.5). 

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