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. / Graduation date: 1998
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/34025 |
| Date | 22 May 1997 |
| Creators | Kim, Billy |
| Contributors | Velayudhan, Ajoy |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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