A Single, UV and Mass Spec Compatible, Method Demonstrating the Influence of pH on the Elution Order of Peptides on Reversed-Phase Columns

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Paul Rainville, Kefei Zheng, Claude Mallet, Uwe Neue, Jeff Mazzeo, Reb Russell II
HPLC 2002; Montreal; 5-6 June 2002
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An evaluation of different reversed-phase stationary phases was carried out to determine and compare retention time, peak shape and peak capacity for peptide samples obtained from a tryptic digest of cytochrome c. Symmetry, Spherisorb, Resolve, Nova-Pak and XTerra packings were all tested. We developed a single unique UV and MS chromatography approach where high pH and low pH mobile phases are used to create orthogonal separations on the same column chemistry. The advantage of this technique is a salt-free, high capacity, high resolving separation technique using a single chemistry that can readily be used on either a UV detector or introduced into the mass spectrometer using the same method. It provides the end user a single column and method for determining impurities in the development of well-characterized biologics.

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