Graduate Thesis Or Dissertation
 

Preparative purification of chemotactic peptides by gradient elution in reversed-phase chromatography

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/j9602339z

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  • Gradient elution chromatography is frequently used for the preparative separation of peptides and proteins. Separations at high loadings are often avoided because peaks become asymmetrical and saturate the detector. However, non-linear interactions which become important at high loadings may actually improve the separation with greater concentrations of the product being extracted. In this study, the separation of a mixture of two chemotactic peptides N-formyl-met-phe (X~phe) and N-formyl-met-trp (X~trp) was considered using reversed-phase (RP) chromatography. These runs were limited by the solubility of the peptides. The effects of solubility and feed conditions on the chromatograms were examined. Because of the poor solubility of the peptides in low organic solvent concentration, runs where feed conditions are different from the column inlet conditions were examined. Since these two components exhibit low selectivity on the RP column, separation is difficult. Productivities were obtained and compared for the preparative purification of these compounds using isocratic, stepwise and gradient elution with acetonitrile/buffer and methanol/water mobile phase conditions. Selectivity reversal was present in methanol/water conditions. Selectivity reversal implies that the order of retention of the feed compound changes as the mobile phase composition varies. Curved gradient shapes were used in an attempt to improve the separation under selectivity reversal limitation. Using a combination of solubility methods and non-linear interactions at high loadings, simultaneous concentration and purification was achieved.
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