Peptides size exclusion chromatograph

Hearn, M.T., M.I. AgnUar, T. Nguyen, and M. Fridman, High-performance liquid chromatography of amino acids, peptides and proteins. LXXXTV. Application of derivative spectroscopy to the study of column residency effects in the reversed-phase and size-exclusion liquid chromatographic separation of proteins. J Chromatogr, 1988. 435(2) 271-84. 

Provided ionic adsorption and size-exclusion effects are kept minimal, the most polar peptide in a mixture will have the shortest chromatographic retention on nonpolar stationary phases with the remainder eluting... 

Reversed phase (RP) chromatography has been established as the superior separation technique for peptides. The chromatographic resolution of this method is higher compared with other methods like ion-exchange (lEX) or size exclusion chromatography (SEC). 

Figure 18 Size exclusion chromatography pattern of a wheat protein hydrolysate (left) and percent composition of the fractions having molecular mass within the known values of standard substances (right). Molecular weights of peptides are only approximately determinable, since their hydrodynamic properties are not necessarily identical with those of the marker substances. Chromatographic conditions stationary phase TSKgel Toyopearl HW-40F (Tosoh Co., Japan) 10 X 500 mm eluant 0.1 M NaCl in 0.1 M phosphate buffer pH 7.1 flow 0.4 ml/min detection UV 220 nm. (Unpublished data.)...

Because electrostatic, size exclusion, and other interactive effects can also be manifested over different ranges of mobile phase conditions with a particular type of sorbent, then these log k versus ij/ or log k versus C plots can take on the characteristic U-shaped dependences [3,72] illustrated in Fig. 16 for several peptides chromatographed with a RPC system. This behavior with biomacromolecules such as peptides and proteins usually necessitates the use of gradient elution conditions for the separation of protein mixtures, unless closely related or homologous proteins are being separated. Moreover, this behaviour indicates that special care must be taken to avoid that self-association or... 

The majority of reports in the literature concerning SEC of peptides and proteins tend to describe chromatographic conditions designed to ensure a pure size exclusion process, although it is often overlooked that the nonideal properties of size exclusion columns can be advantageous in the separation of peptides [37,77]. Excellent examples of this were reported by Yasukawa et al. [78], who separated various hydrophilic and hydrophobic dipeptides on a non-silica-based column by isocratic elution with 50 mM sodium phosphate buffer (pH 7.0). The peptides, retained by adsorption, were eluted in the order of increasing hydrophobicity. Similarly, Mant et al. [37] demonstrated that the separation of five peptide standards of 10, 20, 30, 40, and 50 residues was markedly improved on a SynChropak GPC-60 column when advantage was taken of the nonspecific adsorptive properties of the column rather than when the column was utilized under conditions of ideal SEC. Thus, what is occasionally viewed as a column limitation may become a useful analytical tool. 

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