Optimization of peptide separations in reversed-phase HPLC: Isocratic versus gradient elution |
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Authors: | C T Mant T W Lorne Burke R S Hodges |
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Institution: | (1) Department of Biochemistry and the Medical Research Council of Canada Group in Protein Structure and Function, University of Alberta, T6G 2H7 Edmonton, Alberta, Canada |
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Abstract: | Summary We have determined the interaction behaviour of peptides with hydrophobic stationary phases on analytical columns using isnocratic
or shallow gradient elution for the purpose of developing procedures for rapid optimization of conditions for preparative
reversed-phase chromatography of peptides. From our investigation of the separation of two closely related decapeptides (differing
by one methyl group), in a 1:1 molar ratio on an analytical C8 column, we have found that shallow gradients of 0.1% acetonitrile/min
appeared to be the best compromise between resolution and a practical run time for preparative peptide separations. Up to
20mg of the two-peptide mixture was efficiently resolved on the analytical column, with >97% recovery of homogeneous peptides. |
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Keywords: | Preparative reversed-phase chromatography Peptides Isocratic and gradient elution Analytical columns |
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